cas12a  (New England Biolabs)


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    Structured Review

    New England Biolabs cas12a
    CANTRIP can be performed in a two-step reaction without <t>Cas12a</t> heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.
    Cas12a, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles"

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa020

    CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.
    Figure Legend Snippet: CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Techniques Used: Incubation

    CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).
    Figure Legend Snippet: CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Techniques Used: Fluorescence, Activation Assay

    2) Product Images from "Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1"

    Article Title: Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2842

    Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
    Figure Legend Snippet: Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

    Techniques Used: Expressing, Purification

    Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.
    Figure Legend Snippet: Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.

    Techniques Used:

    3) Product Images from "An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip"

    Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2021.05.005

    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Figure Legend Snippet: Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P

    Techniques Used: Fluorescence, Concentration Assay, Sequencing, Recombinase Polymerase Amplification, Incubation, Modification

    Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.
    Figure Legend Snippet: Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.

    Techniques Used: Real-time Polymerase Chain Reaction, Stripping Membranes, Incubation

    Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.
    Figure Legend Snippet: Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.

    Techniques Used: Fluorescence, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).
    Figure Legend Snippet: Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).

    Techniques Used: Lateral Flow Assay, Stripping Membranes, Incubation, Labeling, Modification, Fluorescence

    Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
    Figure Legend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P

    Techniques Used: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection"

    Article Title: Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection

    Journal: bioRxiv

    doi: 10.1101/2021.10.29.466394

    Assessment of MP1 and MP2 gRNAs efficiency through LbCas12a-mediated restriction of PCR products containing the targeted sequence and subsequent electrophoresis in a 2% agarose gel.
    Figure Legend Snippet: Assessment of MP1 and MP2 gRNAs efficiency through LbCas12a-mediated restriction of PCR products containing the targeted sequence and subsequent electrophoresis in a 2% agarose gel.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Electrophoresis, Agarose Gel Electrophoresis

    Pipeline for CRISPR/Cas12a-mediated detection processes. A nucleic acid extract is used as a template for the LAMP or RT-LAMP amplification of the target sequences. The amplification product is recognized by the RNA-guided endonuclease LbCas12a through base-pair complementarity of the gRNA and the target sequence, thus triggering the collateral activity of the LbCas12a which, in a non-specific manner, degrades the ssDNA probe revealing the presence of the target sequence. The reporter molecule attached to the probe defines the read-out of the assay, either by fluorescence or using a lateral flow strip.
    Figure Legend Snippet: Pipeline for CRISPR/Cas12a-mediated detection processes. A nucleic acid extract is used as a template for the LAMP or RT-LAMP amplification of the target sequences. The amplification product is recognized by the RNA-guided endonuclease LbCas12a through base-pair complementarity of the gRNA and the target sequence, thus triggering the collateral activity of the LbCas12a which, in a non-specific manner, degrades the ssDNA probe revealing the presence of the target sequence. The reporter molecule attached to the probe defines the read-out of the assay, either by fluorescence or using a lateral flow strip.

    Techniques Used: CRISPR, Amplification, Sequencing, Activity Assay, Fluorescence, Stripping Membranes

    5) Product Images from "Characterization of Cme and Yme thermostable Cas12a orthologs"

    Article Title: Characterization of Cme and Yme thermostable Cas12a orthologs

    Journal: Communications Biology

    doi: 10.1038/s42003-022-03275-2

    Cas12a orthologs tolerate mismatches between crRNA and target DNA for activation of trans nuclease activity. Trans nuclease activity generated by Cas12 RNPs was measured using in vitro assays containing target DNA and a 25 nt single-stranded DNA reporter oligonucleotide with 5′ fluorophore (FAM) and 3′ quencher (Q) modifications. Target DNAs were identical in sequence except for nucleotide identity at the indicated positions relative to the first nucleotide of the non-target strand sequence. Compensatory substitutions were present in the target strand of the DNA to maintain the potential for proper base pairing between the two strands of target DNA. Fluorescence was monitored over time and recorded in 1 min increments for 60 min. The time it took for each reaction to reach 25% of the maximum relative fluorescent units (RFU) attained by the fully matched target DNA reaction was determined (see Example graph) and a heatmap was generated where the intensity of the red color corresponds to the time. Reactions were performed at 37 °C for Lba, Asp, and Fno while Cme and Yme reactions were performed at 55 °C. The source data used to make the graph can be found in Supplementary Data 2 .
    Figure Legend Snippet: Cas12a orthologs tolerate mismatches between crRNA and target DNA for activation of trans nuclease activity. Trans nuclease activity generated by Cas12 RNPs was measured using in vitro assays containing target DNA and a 25 nt single-stranded DNA reporter oligonucleotide with 5′ fluorophore (FAM) and 3′ quencher (Q) modifications. Target DNAs were identical in sequence except for nucleotide identity at the indicated positions relative to the first nucleotide of the non-target strand sequence. Compensatory substitutions were present in the target strand of the DNA to maintain the potential for proper base pairing between the two strands of target DNA. Fluorescence was monitored over time and recorded in 1 min increments for 60 min. The time it took for each reaction to reach 25% of the maximum relative fluorescent units (RFU) attained by the fully matched target DNA reaction was determined (see Example graph) and a heatmap was generated where the intensity of the red color corresponds to the time. Reactions were performed at 37 °C for Lba, Asp, and Fno while Cme and Yme reactions were performed at 55 °C. The source data used to make the graph can be found in Supplementary Data 2 .

    Techniques Used: Activation Assay, Activity Assay, Generated, In Vitro, Sequencing, Fluorescence

    Profile of cleavage sites generated by Cas12a orthologs. a Schematic representation of the workflow to determine the position and abundance of cleaved DNA ends. Circular double-stranded DNAs containing a TTTA PAM sequence flanked by a target sequence were incubated with complementary crRNA-Cas12a RNPs and subjected to end repair, adapter ligation and sequencing. The substrate DNA was sequenced end to end in both reads of a paired-end sequencing run and custom scripts were used to map the cleavage site on the non-target strand (NTS) and target strand (TS). b The abundance of cleaved DNA ends was graphed as heatmaps representing the individual summation of all NTS cleavage position frequencies and TS cleavage position frequencies. The intensity of the red color corresponds to the fraction of ends mapped to each position on the NTS and TS. The positions are numbered 1 through 25 relative to the distance from the TTTA PAM sequence. Full data sets with matrices of the frequency of each NTS and TS cleavage site combination are available in Supplementary Data 2 . Sequences of crRNAs and oligonucleotides used to make circular substrate DNAs can be found in Supplementary Data 1 .
    Figure Legend Snippet: Profile of cleavage sites generated by Cas12a orthologs. a Schematic representation of the workflow to determine the position and abundance of cleaved DNA ends. Circular double-stranded DNAs containing a TTTA PAM sequence flanked by a target sequence were incubated with complementary crRNA-Cas12a RNPs and subjected to end repair, adapter ligation and sequencing. The substrate DNA was sequenced end to end in both reads of a paired-end sequencing run and custom scripts were used to map the cleavage site on the non-target strand (NTS) and target strand (TS). b The abundance of cleaved DNA ends was graphed as heatmaps representing the individual summation of all NTS cleavage position frequencies and TS cleavage position frequencies. The intensity of the red color corresponds to the fraction of ends mapped to each position on the NTS and TS. The positions are numbered 1 through 25 relative to the distance from the TTTA PAM sequence. Full data sets with matrices of the frequency of each NTS and TS cleavage site combination are available in Supplementary Data 2 . Sequences of crRNAs and oligonucleotides used to make circular substrate DNAs can be found in Supplementary Data 1 .

    Techniques Used: Generated, Sequencing, Incubation, Ligation

    High temperature trans nuclease activity initiated by DNA target cleavage. Cas12a RNPs were incubated at the temperatures indicated for either 2, 5, 10, or 20 min prior to the addition of target double-stranded DNA and a 25 nt single-stranded DNA reporter with 5′ fluorophore and a 3′ quencher modifications. Reactions were then incubated at the indicated temperature for 10 min and fluorescence was measured at the end of the reaction. The measured values were averaged and divided by the maximum value of fluorescence possible if all reporter DNA was cleaved in the assay. The intensity of the red color in the graphs corresponds to the fraction of maximum fluorescence the Cas12a enzymes were able to generate for each time and temperature combination. Source data used to generate this graph is provided in Supplementary Data 2 .
    Figure Legend Snippet: High temperature trans nuclease activity initiated by DNA target cleavage. Cas12a RNPs were incubated at the temperatures indicated for either 2, 5, 10, or 20 min prior to the addition of target double-stranded DNA and a 25 nt single-stranded DNA reporter with 5′ fluorophore and a 3′ quencher modifications. Reactions were then incubated at the indicated temperature for 10 min and fluorescence was measured at the end of the reaction. The measured values were averaged and divided by the maximum value of fluorescence possible if all reporter DNA was cleaved in the assay. The intensity of the red color in the graphs corresponds to the fraction of maximum fluorescence the Cas12a enzymes were able to generate for each time and temperature combination. Source data used to generate this graph is provided in Supplementary Data 2 .

    Techniques Used: Activity Assay, Incubation, Fluorescence

    Cas12a trans nuclease activity is influenced by target sequence and reporter substrate length. a Schematic representation of Cas12a trans nuclease activity assays. Linear double-stranded DNA containing a TTTA PAM sequence flanked by a target sequence is incubated with a complementary crRNA-Cas12a RNP at 37 °C to allow on-target cleavage. A reporter oligonucleotide with 5′ fluorophore (FAM) and 3′ quencher (Q) modifications is added and cleavage of the reporter via the trans nuclease activity of the activated Cas12a separates the fluorophore and quencher resulting in increased fluorescence. b Fluorescence was measured over time for a reporter that consisted of either a 25 nt defined sequence DNA or 5 nt randomized sequence DNA. The mean relative fluorescent unit (RFU) values for reactions with Target W DNA are plotted as black (25 nt reporter) or blue (5 nt reporter) lines. The mean RFU values for reactions with Target G DNA are plotted as magenta (25 nt reporter) or brown (5 nt reporter) lines. The RFU values represent background normalized signal derived by subtracting the fluorescence of negative control reactions that did not contain target DNA. Individual data points for all experimental replicates are plotted on the graphs and the source data can be found in Supplementary Data 2 .
    Figure Legend Snippet: Cas12a trans nuclease activity is influenced by target sequence and reporter substrate length. a Schematic representation of Cas12a trans nuclease activity assays. Linear double-stranded DNA containing a TTTA PAM sequence flanked by a target sequence is incubated with a complementary crRNA-Cas12a RNP at 37 °C to allow on-target cleavage. A reporter oligonucleotide with 5′ fluorophore (FAM) and 3′ quencher (Q) modifications is added and cleavage of the reporter via the trans nuclease activity of the activated Cas12a separates the fluorophore and quencher resulting in increased fluorescence. b Fluorescence was measured over time for a reporter that consisted of either a 25 nt defined sequence DNA or 5 nt randomized sequence DNA. The mean relative fluorescent unit (RFU) values for reactions with Target W DNA are plotted as black (25 nt reporter) or blue (5 nt reporter) lines. The mean RFU values for reactions with Target G DNA are plotted as magenta (25 nt reporter) or brown (5 nt reporter) lines. The RFU values represent background normalized signal derived by subtracting the fluorescence of negative control reactions that did not contain target DNA. Individual data points for all experimental replicates are plotted on the graphs and the source data can be found in Supplementary Data 2 .

    Techniques Used: Activity Assay, Sequencing, Incubation, Fluorescence, Derivative Assay, Negative Control

    Relatedness of Cas12a orthologs. a Phylogenetic tree of 100 Cas12a protein sequences. The branches of the tree corresponding to the five proteins examined in this study are marked with an orange circle and the table shows their percentage of shared amino-acid sequence. b Cartoon representation of the Cme and Yme loci from metagenomic data and an alignment of Cas12a crRNA sequences. DNA CRISPR repeat sequences shown in green are representative of the crRNA sequence utilized by the corresponding Cas12a protein. A typical crRNA is also represented as a cartoon and has 20 nt of target sequence appended to the 3′-end of the sequence shown in the alignment next to cartoon.
    Figure Legend Snippet: Relatedness of Cas12a orthologs. a Phylogenetic tree of 100 Cas12a protein sequences. The branches of the tree corresponding to the five proteins examined in this study are marked with an orange circle and the table shows their percentage of shared amino-acid sequence. b Cartoon representation of the Cme and Yme loci from metagenomic data and an alignment of Cas12a crRNA sequences. DNA CRISPR repeat sequences shown in green are representative of the crRNA sequence utilized by the corresponding Cas12a protein. A typical crRNA is also represented as a cartoon and has 20 nt of target sequence appended to the 3′-end of the sequence shown in the alignment next to cartoon.

    Techniques Used: Sequencing, CRISPR

    Cas12a thermostability and dsDNA target cleavage activity. a Thermal stability of Cas12a proteins without crRNA (apo) or loaded with crRNA (RNP). Protein unfolding was measured with nano differential scanning fluorometry (NanoDSF) over a temperature range from 20 °C to 80 °C. Fluorescence was monitored as temperature increased at a rate of 1 °C sec −1 . The inflection point of the fluorescent curve is interpreted as the unfolding point of the protein. Data points collected from replicate experiments are plotted and error bars represent mean ±standard deviation. Raw traces of the fluorescence data can be found in Supplementary Figure S2 . The on-target double-stranded DNA (dsDNA) cleavage activities of Cas12 RNPs was measured using in vitro assays containing fluorophore-labeled dsDNA target substrates. Cas12a protein was incubated with crRNA at either 25 °C ( b ) or at varying temperatures ( c ) to form RNPs. RNPs formed at 25 °C were then transferred to the indicated reaction temperature. A cleavage reaction was initiated by adding 5′ fluorescein-labeled target DNA pre-incubated at the reaction temperature to the Cas12a RNPs. Cleaved fragments were quantified and the proportion of the substrate cleaved in each condition is represented in a heatmap where the intensity of the red color corresponds to the extent of target cleavage. Source data used to make the graphs are provided in Supplementary Data 2 .
    Figure Legend Snippet: Cas12a thermostability and dsDNA target cleavage activity. a Thermal stability of Cas12a proteins without crRNA (apo) or loaded with crRNA (RNP). Protein unfolding was measured with nano differential scanning fluorometry (NanoDSF) over a temperature range from 20 °C to 80 °C. Fluorescence was monitored as temperature increased at a rate of 1 °C sec −1 . The inflection point of the fluorescent curve is interpreted as the unfolding point of the protein. Data points collected from replicate experiments are plotted and error bars represent mean ±standard deviation. Raw traces of the fluorescence data can be found in Supplementary Figure S2 . The on-target double-stranded DNA (dsDNA) cleavage activities of Cas12 RNPs was measured using in vitro assays containing fluorophore-labeled dsDNA target substrates. Cas12a protein was incubated with crRNA at either 25 °C ( b ) or at varying temperatures ( c ) to form RNPs. RNPs formed at 25 °C were then transferred to the indicated reaction temperature. A cleavage reaction was initiated by adding 5′ fluorescein-labeled target DNA pre-incubated at the reaction temperature to the Cas12a RNPs. Cleaved fragments were quantified and the proportion of the substrate cleaved in each condition is represented in a heatmap where the intensity of the red color corresponds to the extent of target cleavage. Source data used to make the graphs are provided in Supplementary Data 2 .

    Techniques Used: Activity Assay, Nano Differential Scanning Fluorimetry, Fluorescence, Standard Deviation, In Vitro, Labeling, Incubation

    Tolerance of non-canonical PAMs by Cas12a orthologs. Target-directed double-stranded DNA (dsDNA) cleavage activities of Cas12a RNPs were measured using in vitro assays containing fluorophore-labeled dsDNA target substrates in the presence of a 10-fold mass excess of HeLa genomic DNA. Target dsDNAs were identical except for nucleotide identity at the −2, −3, and −4 positions relative to the first nucleotide of the target sequence. Aliquots from cleavage reactions were removed and quenched after 1, 5, 10 min. Cleaved fragments were quantified and the proportion of the substrate cleaved in each condition is represented in a heatmap where the intensity of the red color corresponds to the extent of target cleavage. Reactions were performed at 37 °C for Lba, Asp, and Fno while Cme and Yme reactions were performed at 55 °C. The source data used to make the graphs can be found in Supplementary Data 2 .
    Figure Legend Snippet: Tolerance of non-canonical PAMs by Cas12a orthologs. Target-directed double-stranded DNA (dsDNA) cleavage activities of Cas12a RNPs were measured using in vitro assays containing fluorophore-labeled dsDNA target substrates in the presence of a 10-fold mass excess of HeLa genomic DNA. Target dsDNAs were identical except for nucleotide identity at the −2, −3, and −4 positions relative to the first nucleotide of the target sequence. Aliquots from cleavage reactions were removed and quenched after 1, 5, 10 min. Cleaved fragments were quantified and the proportion of the substrate cleaved in each condition is represented in a heatmap where the intensity of the red color corresponds to the extent of target cleavage. Reactions were performed at 37 °C for Lba, Asp, and Fno while Cme and Yme reactions were performed at 55 °C. The source data used to make the graphs can be found in Supplementary Data 2 .

    Techniques Used: In Vitro, Labeling, Sequencing

    PAM requirements of Cas12a orthologs. a Schematic representation of the workflow used to determine PAM sequence requirements for double-stranded DNA (dsDNA) cleavage. Single-stranded DNA oligonucleotide libraries with 10 nt of randomized sequence adjacent to a 20 nt target sequence were converted into dsDNA minicircle libraries and exposed to Cas12a RNPs. Linearized dsDNA libraries were subjected to end repair, adapter ligation, and sequencing. A detailed, step-wise diagram of this method is provided in Supplementary Figure S3 . b PAM sequence requirements of Cas12a orthologs. Position weight values were calculated as described in the methods and are shown as graphs where a positive score corresponds to an enrichment of a particular base in the randomized 10 nt region on a log 2 scale and a negative score corresponds to depletion. The position is the distance in nt relative to the first nt of the target sequence. Sequences of oligonucleotides used to make circular substrate DNAs can be found in Supplementary Data 1 1.
    Figure Legend Snippet: PAM requirements of Cas12a orthologs. a Schematic representation of the workflow used to determine PAM sequence requirements for double-stranded DNA (dsDNA) cleavage. Single-stranded DNA oligonucleotide libraries with 10 nt of randomized sequence adjacent to a 20 nt target sequence were converted into dsDNA minicircle libraries and exposed to Cas12a RNPs. Linearized dsDNA libraries were subjected to end repair, adapter ligation, and sequencing. A detailed, step-wise diagram of this method is provided in Supplementary Figure S3 . b PAM sequence requirements of Cas12a orthologs. Position weight values were calculated as described in the methods and are shown as graphs where a positive score corresponds to an enrichment of a particular base in the randomized 10 nt region on a log 2 scale and a negative score corresponds to depletion. The position is the distance in nt relative to the first nt of the target sequence. Sequences of oligonucleotides used to make circular substrate DNAs can be found in Supplementary Data 1 1.

    Techniques Used: Sequencing, Ligation

    6) Product Images from "Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects"

    Article Title: Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects

    Journal: bioRxiv

    doi: 10.1101/2021.04.05.438458

    ONE-seq outperforms existing methods for nominating bona fide Cas12a, CBE, and ABE off-targets in human cells. a, Schematic overview of ONE-seq selections for Cas12a nucleases, CBEs, and ABEs. b , Venn diagrams illustrating identification of previously validated and newly validated LbCas12a off-target sites by ONE-seq; comparisons are shown for four different gRNAs previously assayed by GUIDE-seq or Digenome-seq. c, Venn diagrams illustrating identification of previously validated and newly validated CBE off-target sites by ONE-seq; comparisons are shown for seven different gRNAs previously assayed by Digenome-seq. d , Venn diagrams illustrating identification of previously validated and newly validated ABE off-target sites by ONE-seq; comparisons are shown for five different gRNAs previously assayed by Digenome-seq. b-d, All sites shown as validated by ONE-seq (light colored circles) had ONE-seq scores > 0.01. CBE, cytidine base editor; ABE, adenine base editor.
    Figure Legend Snippet: ONE-seq outperforms existing methods for nominating bona fide Cas12a, CBE, and ABE off-targets in human cells. a, Schematic overview of ONE-seq selections for Cas12a nucleases, CBEs, and ABEs. b , Venn diagrams illustrating identification of previously validated and newly validated LbCas12a off-target sites by ONE-seq; comparisons are shown for four different gRNAs previously assayed by GUIDE-seq or Digenome-seq. c, Venn diagrams illustrating identification of previously validated and newly validated CBE off-target sites by ONE-seq; comparisons are shown for seven different gRNAs previously assayed by Digenome-seq. d , Venn diagrams illustrating identification of previously validated and newly validated ABE off-target sites by ONE-seq; comparisons are shown for five different gRNAs previously assayed by Digenome-seq. b-d, All sites shown as validated by ONE-seq (light colored circles) had ONE-seq scores > 0.01. CBE, cytidine base editor; ABE, adenine base editor.

    Techniques Used:

    ONE-seq outperforms existing methods for nominating bona fide Cas12a nuclease off-targets in human cells. a, Swarm plots showing ONE-seq nuclease scores for Cas12a gRNAs previously characterized by Digenome-seq in HEK293T cells (left) and by GUIDE-seq in U2OS cells (right). Each circle represents an individual ONE-seq library member. Open pink circles and closed pink circles represent previously validated and newly validated Cas12a off-target sites, respectively. Other sites tested in this study by targeted amplicon sequencing are represented as closed yellow circles. Sites with ONE-seq nuclease scores below 0.001 are not shown. ONE-seq scores for matched9, DNMT3 and DNMT4 represent the average of duplicate ONE-seq experiments. b, Cas12a off-targets nominated by ONE-seq and tested and/or validated in HEK293T cells and/or U2OS cells are shown. Nucleotide sequences in bold at the top represent on-target sequences. Off-target sites assessed in cells are shown below the on-target site. Lower case nucleotides in colored boxes represent mismatches to the on-target site. Validation status of off-targets is shown by colored ovals. “Edit %” refers to the mean editing frequency from three independent replicates. 166 previously identified off-target sites for matched6 are not shown.
    Figure Legend Snippet: ONE-seq outperforms existing methods for nominating bona fide Cas12a nuclease off-targets in human cells. a, Swarm plots showing ONE-seq nuclease scores for Cas12a gRNAs previously characterized by Digenome-seq in HEK293T cells (left) and by GUIDE-seq in U2OS cells (right). Each circle represents an individual ONE-seq library member. Open pink circles and closed pink circles represent previously validated and newly validated Cas12a off-target sites, respectively. Other sites tested in this study by targeted amplicon sequencing are represented as closed yellow circles. Sites with ONE-seq nuclease scores below 0.001 are not shown. ONE-seq scores for matched9, DNMT3 and DNMT4 represent the average of duplicate ONE-seq experiments. b, Cas12a off-targets nominated by ONE-seq and tested and/or validated in HEK293T cells and/or U2OS cells are shown. Nucleotide sequences in bold at the top represent on-target sequences. Off-target sites assessed in cells are shown below the on-target site. Lower case nucleotides in colored boxes represent mismatches to the on-target site. Validation status of off-targets is shown by colored ovals. “Edit %” refers to the mean editing frequency from three independent replicates. 166 previously identified off-target sites for matched6 are not shown.

    Techniques Used: Amplification, Sequencing

    7) Product Images from "Fluorescence polarization system for rapid COVID-19 diagnosis"

    Article Title: Fluorescence polarization system for rapid COVID-19 diagnosis

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2021.113049

    CODA system for COVID-19 diagnosis. (a) Assay schematic. When target viral RNA is present, RT-RPA and CRISPR/Cas detection take place together. DNA polymerase recognizes target sequences and displaces double-stranded DNA. Cas12a/gRNA complexes then bind to specific sites (green and orange) in the exposed single strand, get activated, and start to cleave nearby reporter probes. This cleaving process is amplified, as RT-RPA reaction proceeds. As a result, the fluorescence anisotropy ( r ) of the sample decreases (right). (b) CODA device configuration. A compact device integrates rapid sample heating, precision signal processing, and real-time polarization anisotropy detection. A sample tube (50 μL) is inserted into a heated metal block, whereby two photodetectors detect fluorescence light. (c) CODA optics. A linearly polarized light illuminates a sample tube from its bottom side. Two photodetectors measure orthogonal polarization of fluorescence light emitted by the sample. (d) Photograph of the portable CODA device for onsite application. The enclosure was made of a lightweight photopolymer. Optical mounts and the sample holder were machined in aluminum. (e) A partial screenshot of an extended user interface. The CODA device communicates with a computer to present real-time data. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: CODA system for COVID-19 diagnosis. (a) Assay schematic. When target viral RNA is present, RT-RPA and CRISPR/Cas detection take place together. DNA polymerase recognizes target sequences and displaces double-stranded DNA. Cas12a/gRNA complexes then bind to specific sites (green and orange) in the exposed single strand, get activated, and start to cleave nearby reporter probes. This cleaving process is amplified, as RT-RPA reaction proceeds. As a result, the fluorescence anisotropy ( r ) of the sample decreases (right). (b) CODA device configuration. A compact device integrates rapid sample heating, precision signal processing, and real-time polarization anisotropy detection. A sample tube (50 μL) is inserted into a heated metal block, whereby two photodetectors detect fluorescence light. (c) CODA optics. A linearly polarized light illuminates a sample tube from its bottom side. Two photodetectors measure orthogonal polarization of fluorescence light emitted by the sample. (d) Photograph of the portable CODA device for onsite application. The enclosure was made of a lightweight photopolymer. Optical mounts and the sample holder were machined in aluminum. (e) A partial screenshot of an extended user interface. The CODA device communicates with a computer to present real-time data. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Recombinase Polymerase Amplification, CRISPR, Amplification, Fluorescence, Blocking Assay

    8) Product Images from "Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles"

    Article Title: Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

    Journal: The CRISPR Journal

    doi: 10.1089/crispr.2020.0086

    Activity of Cas12a orthologs at varied temperatures. (A) Cas12a/SpCas9 proteins with their respective crRNA/sgRNA and buffer (same as indicated in (B) ) were combined to form the ribonucleoprotein (RNPs) for 5 min at the temperatures indicated. The double-stranded DNA (dsDNA) cleavage reactions were initiated by adding the 5′-FAM-labeled dsDNA containing the target site preincubated at the designated incubation temperatures. Digested fragments were resolved by capillary electrophoresis, and peaks corresponding to cleaved and intact substrates were quantified. Data are shown as the mean ± standard deviation ( SD ) of three experimental replicates. (B) ). The melting temperature ( T m ) of three experimental replicates are shown, with means ± SD indicated.
    Figure Legend Snippet: Activity of Cas12a orthologs at varied temperatures. (A) Cas12a/SpCas9 proteins with their respective crRNA/sgRNA and buffer (same as indicated in (B) ) were combined to form the ribonucleoprotein (RNPs) for 5 min at the temperatures indicated. The double-stranded DNA (dsDNA) cleavage reactions were initiated by adding the 5′-FAM-labeled dsDNA containing the target site preincubated at the designated incubation temperatures. Digested fragments were resolved by capillary electrophoresis, and peaks corresponding to cleaved and intact substrates were quantified. Data are shown as the mean ± standard deviation ( SD ) of three experimental replicates. (B) ). The melting temperature ( T m ) of three experimental replicates are shown, with means ± SD indicated.

    Techniques Used: Activity Assay, Labeling, Incubation, Electrophoresis, Standard Deviation

    9) Product Images from "Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. Thonn"

    Article Title: Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. Thonn

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-00006-1

    Species authentication by Bar-cas12a in admixture samples. ( A ) DNA amplification of DNA admixture in different proportions by RPA using trnL loci. DNA admixture between P. amarus (PA) and P. urinaria (PU) were done in different amount proportions as 100%, 50%, 25%, 10%, 5%, 2% and 0% of PA contaminated with PU which stock concentration of each species used was 10 ng/µl. ( B , D ) In vitro digestion of cas12a using gRNA A and gRNA B and observed under LED transilluminator. ( C , E ) the acquisition of fluorescence signal by realtime PCR over two hours. Initial images of agarose gel electrophoresis are shown in Figure S4 .
    Figure Legend Snippet: Species authentication by Bar-cas12a in admixture samples. ( A ) DNA amplification of DNA admixture in different proportions by RPA using trnL loci. DNA admixture between P. amarus (PA) and P. urinaria (PU) were done in different amount proportions as 100%, 50%, 25%, 10%, 5%, 2% and 0% of PA contaminated with PU which stock concentration of each species used was 10 ng/µl. ( B , D ) In vitro digestion of cas12a using gRNA A and gRNA B and observed under LED transilluminator. ( C , E ) the acquisition of fluorescence signal by realtime PCR over two hours. Initial images of agarose gel electrophoresis are shown in Figure S4 .

    Techniques Used: Amplification, Recombinase Polymerase Amplification, Concentration Assay, In Vitro, Fluorescence, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Accuracy test of Bar-cas12a for species authentication
    Figure Legend Snippet: Accuracy test of Bar-cas12a for species authentication

    Techniques Used:

    Accuracy test for P. amarus determination by Bar-cas12a using trnL . ( A ) DNA amplification by RPA on agarose gel of the different Phyllanthus species including P. amarus (PA), P. urinaria (PU), P. debilis (PD), P. virgatus (PV) , P. airy-shawii (PS) , P. acidus (PAc) , P. emblica (PE) , P. reticulatus (PR), Phyllanthus sp. (Psp). Cas12a assay for detecting PA-specific RPA product by naked eye under LED transilluminator ( B ) and fluorescence signal by a realtime PCR machine ( C ). ( D ) The confusion matrix of actual and predicted species with accuracy rate and precision rate of P. amarus authentication. Initial images of agarose gel electrophoresis are shown in Figure S5 .
    Figure Legend Snippet: Accuracy test for P. amarus determination by Bar-cas12a using trnL . ( A ) DNA amplification by RPA on agarose gel of the different Phyllanthus species including P. amarus (PA), P. urinaria (PU), P. debilis (PD), P. virgatus (PV) , P. airy-shawii (PS) , P. acidus (PAc) , P. emblica (PE) , P. reticulatus (PR), Phyllanthus sp. (Psp). Cas12a assay for detecting PA-specific RPA product by naked eye under LED transilluminator ( B ) and fluorescence signal by a realtime PCR machine ( C ). ( D ) The confusion matrix of actual and predicted species with accuracy rate and precision rate of P. amarus authentication. Initial images of agarose gel electrophoresis are shown in Figure S5 .

    Techniques Used: Amplification, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction

    The preparation of cas12a condition for species authentication. ( A ) gRNA-A and gRNA-B design based on the multiple alignment of trnL loci and the construction of double stranded DNA as temples for gRNA synthesis. ( B ) The synthesis of gRNA-A and gRNA-B by in vitro transcription with T7 RNA polymerase and the synthesized gRNAs were detected by agarose gel electrophoresis. ( C ) The schematic illustrates the mechanism of cas12a to form binary and tertiary complex to cleave the reporter single stranded DNA as result of the fluorescence signal. ( D ) The condition optimization of in vitro digestion of cas12a by varying the concentration ratio of cas12a and gRNA at 1: 1 to produce the fluorescence at 37 °C for an hour. Phyllanthus species were studied including Phyllanthus amarus (PA), Phyllanthus urinaria (PU), Phyllanthus debilis (PD), Phyllanthus virgatus (PV). Initial image of agarose gel electrophoresis is shown in Figure S1 .
    Figure Legend Snippet: The preparation of cas12a condition for species authentication. ( A ) gRNA-A and gRNA-B design based on the multiple alignment of trnL loci and the construction of double stranded DNA as temples for gRNA synthesis. ( B ) The synthesis of gRNA-A and gRNA-B by in vitro transcription with T7 RNA polymerase and the synthesized gRNAs were detected by agarose gel electrophoresis. ( C ) The schematic illustrates the mechanism of cas12a to form binary and tertiary complex to cleave the reporter single stranded DNA as result of the fluorescence signal. ( D ) The condition optimization of in vitro digestion of cas12a by varying the concentration ratio of cas12a and gRNA at 1: 1 to produce the fluorescence at 37 °C for an hour. Phyllanthus species were studied including Phyllanthus amarus (PA), Phyllanthus urinaria (PU), Phyllanthus debilis (PD), Phyllanthus virgatus (PV). Initial image of agarose gel electrophoresis is shown in Figure S1 .

    Techniques Used: In Vitro, Synthesized, Agarose Gel Electrophoresis, Fluorescence, Concentration Assay

    Specificity and sensitivity of Bar-cas12a for P. amarus authentication. ( A ) DNA amplification for the four species including P. amarus (PA), P. urinaria (PU), P. debilis (PD) and P. virgatus (PV) by RPA using universal trnL primer. ( B ) Specificity test by Bar-cas12a using gRNA-A and gRNA-B. ( C , D ) Sensitivity test by PCR and Bar-cas12a using gRNA-A and gRNA-B and the fluorescence signal were monitored for cleavage of ssDNA reporters by realtime PCR within two hours. Initial images of agarose gel electrophoresis are shown in Figure S2 and S3 , respectively.
    Figure Legend Snippet: Specificity and sensitivity of Bar-cas12a for P. amarus authentication. ( A ) DNA amplification for the four species including P. amarus (PA), P. urinaria (PU), P. debilis (PD) and P. virgatus (PV) by RPA using universal trnL primer. ( B ) Specificity test by Bar-cas12a using gRNA-A and gRNA-B. ( C , D ) Sensitivity test by PCR and Bar-cas12a using gRNA-A and gRNA-B and the fluorescence signal were monitored for cleavage of ssDNA reporters by realtime PCR within two hours. Initial images of agarose gel electrophoresis are shown in Figure S2 and S3 , respectively.

    Techniques Used: Amplification, Recombinase Polymerase Amplification, Polymerase Chain Reaction, Fluorescence, Agarose Gel Electrophoresis

    10) Product Images from "Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection"

    Article Title: Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

    Journal: Journal of Analysis and Testing

    doi: 10.1007/s41664-022-00212-4

    Nanoparticle-assisted CRISPR-Cas12-based nucleic acids detection . a Schematic of LFA which works in signal-off mode. Adapted from Ref. [ 48 ]. Copyright 2020 Elsevier. b Schematic of a magnetic pull-down-assisted colorimetric method. Adapted from Ref. [ 51 ]. Copyright 2021 American Chemical Society. c Schematic of colorimetric analysis using DNA-AuNP probes as trans-cleavage substrates. Adapted from ref. [ 53 ]. Copyright 2021 American Chemical Society. d Schematic of CRISPR-Cas12a coupled with PtNP for visual detection on a volumetric bar-chart chip. Adapted from Ref. [ 54 ]. Copyright 2019 American Chemical Society
    Figure Legend Snippet: Nanoparticle-assisted CRISPR-Cas12-based nucleic acids detection . a Schematic of LFA which works in signal-off mode. Adapted from Ref. [ 48 ]. Copyright 2020 Elsevier. b Schematic of a magnetic pull-down-assisted colorimetric method. Adapted from Ref. [ 51 ]. Copyright 2021 American Chemical Society. c Schematic of colorimetric analysis using DNA-AuNP probes as trans-cleavage substrates. Adapted from ref. [ 53 ]. Copyright 2021 American Chemical Society. d Schematic of CRISPR-Cas12a coupled with PtNP for visual detection on a volumetric bar-chart chip. Adapted from Ref. [ 54 ]. Copyright 2019 American Chemical Society

    Techniques Used: CRISPR, Chromatin Immunoprecipitation

    11) Product Images from "Dual-Enzyme-Based Signal-Amplified Aptasensor for Zearalenone Detection by Using CRISPR-Cas12a and Nt.AlwI"

    Article Title: Dual-Enzyme-Based Signal-Amplified Aptasensor for Zearalenone Detection by Using CRISPR-Cas12a and Nt.AlwI

    Journal: Foods

    doi: 10.3390/foods11030487

    Schematic diagram of a magnetic-bead-assisted dual-signal-amplification aptasensor for sensitive ZEN detection based on the Nt.AlwI enzyme and the Cas12a enzyme. Step 1: The aptamer probe recognizes the ZEN toxin and causes Z1 to dissociate into solution by competitive binding. Step 2: After Z1 and Z2 were hybridized, the cutting activity of the Nt.AlwI enzyme was activated, the Z2 chain was cut to release Z3, Z1 was self-shed after the cutting was finished and it hybridized with Z2 again, and a large amount of Z3 was released by the enzyme-cutting signal amplification to achieve the first signal amplification. Step 3: The combination of Z3 and the Cas12a-crRNA complex activates trans-cleavage activity, non-specifically cleaving any ssDNA so that the added fluorescent signal molecule was cleaved and the quenched fluorescence was restored.
    Figure Legend Snippet: Schematic diagram of a magnetic-bead-assisted dual-signal-amplification aptasensor for sensitive ZEN detection based on the Nt.AlwI enzyme and the Cas12a enzyme. Step 1: The aptamer probe recognizes the ZEN toxin and causes Z1 to dissociate into solution by competitive binding. Step 2: After Z1 and Z2 were hybridized, the cutting activity of the Nt.AlwI enzyme was activated, the Z2 chain was cut to release Z3, Z1 was self-shed after the cutting was finished and it hybridized with Z2 again, and a large amount of Z3 was released by the enzyme-cutting signal amplification to achieve the first signal amplification. Step 3: The combination of Z3 and the Cas12a-crRNA complex activates trans-cleavage activity, non-specifically cleaving any ssDNA so that the added fluorescent signal molecule was cleaved and the quenched fluorescence was restored.

    Techniques Used: Amplification, Binding Assay, Activity Assay, Fluorescence

    Characterization of Cas12a. ( A ) Seven small centrifuge tubes were prepared, and to each tube was added 10 μL of 5 μM ssDNA R1 (40 nt) as a substrate for trans-cleavage by the Cas enzyme, and to tubes 2–7 was added the same amount of Cas12a-crRNA-Z3 enzyme, and the reaction was carried out at 37° for 30–180 min, respectively. Lane M was a DNA marker, and the reaction product was subjected to 10% polyacrylamide electrophoresis. ( B ) Under the same conditions, the integrity of Cas enzyme on the effect of digestion. a: Quenched Reporter + Cas12a-crRNA + Z3; b: Quenched Reporter + Cas12a; c: Quenched Reporter + Cas12a-crRNA; d: Quenched Reporter. ( C ) The fluorescence intensity of the trans-cleavage activity of Cas12a-crRNA at four concentration ratios. ( D ) The fluorescence intensity of Quenched Reporter (concentrations of 100 nM, 300 nM,500 nM, 800 nM, and 1000 nM) when the fluorescence signal was restored after it was completely cut by CRISPR-Cas12a. ( E ) The fluorescence intensity of the Cas12a after cutting for 0–40 min.
    Figure Legend Snippet: Characterization of Cas12a. ( A ) Seven small centrifuge tubes were prepared, and to each tube was added 10 μL of 5 μM ssDNA R1 (40 nt) as a substrate for trans-cleavage by the Cas enzyme, and to tubes 2–7 was added the same amount of Cas12a-crRNA-Z3 enzyme, and the reaction was carried out at 37° for 30–180 min, respectively. Lane M was a DNA marker, and the reaction product was subjected to 10% polyacrylamide electrophoresis. ( B ) Under the same conditions, the integrity of Cas enzyme on the effect of digestion. a: Quenched Reporter + Cas12a-crRNA + Z3; b: Quenched Reporter + Cas12a; c: Quenched Reporter + Cas12a-crRNA; d: Quenched Reporter. ( C ) The fluorescence intensity of the trans-cleavage activity of Cas12a-crRNA at four concentration ratios. ( D ) The fluorescence intensity of Quenched Reporter (concentrations of 100 nM, 300 nM,500 nM, 800 nM, and 1000 nM) when the fluorescence signal was restored after it was completely cut by CRISPR-Cas12a. ( E ) The fluorescence intensity of the Cas12a after cutting for 0–40 min.

    Techniques Used: Marker, Electrophoresis, Fluorescence, Activity Assay, Concentration Assay, CRISPR

    12) Product Images from "A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020"

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.02.012

    PAM usage of Lb, Lb2, and Lb2-KY (A) A diagram outlining the mammalian-cell PAM identification assay used in subsequent panels. A library of randomized 5′-NNNNNN-3′ PAMs preceding a crRNA target was stably expressed in HEK293T cells. Plasmids encoding Cas12a proteins, crRNAs, and the fluorescent marker mCherry were transfected to these cells. Cells were sorted for mCherry expression, and a region containing the randomized PAM and the target sequence was deep-sequenced with paired-end reads of 300 base pairs. Indel frequencies were then calculated for each four-nucleotide PAM. Note that the first two nucleotides of the 6-mer randomized PAM sequence had no detectable impact on the activities of any Cas12a variant. (B) Heatmaps showing the result of mammalian-cell PAM screen for Lb2Cas12a, Lb2-KY, LbCas12a, and cells lacking a Cas12a ortholog (control). PAMs and the related editing efficiencies were pooled by the first three nucleotide positions. Each heatmap indicates an average of two or three independent replicates. The range of replicates for each 3-nucleotide PAM sequence is less than 10% of the signal at each position. (C) An expansion of the data generated in (B), except that all 64 four-nucleotide PAMs are plotted. The range of replicates for each 4-nucleotide sequence is less than 25% of the average signal for each four-nucleotide PAM.
    Figure Legend Snippet: PAM usage of Lb, Lb2, and Lb2-KY (A) A diagram outlining the mammalian-cell PAM identification assay used in subsequent panels. A library of randomized 5′-NNNNNN-3′ PAMs preceding a crRNA target was stably expressed in HEK293T cells. Plasmids encoding Cas12a proteins, crRNAs, and the fluorescent marker mCherry were transfected to these cells. Cells were sorted for mCherry expression, and a region containing the randomized PAM and the target sequence was deep-sequenced with paired-end reads of 300 base pairs. Indel frequencies were then calculated for each four-nucleotide PAM. Note that the first two nucleotides of the 6-mer randomized PAM sequence had no detectable impact on the activities of any Cas12a variant. (B) Heatmaps showing the result of mammalian-cell PAM screen for Lb2Cas12a, Lb2-KY, LbCas12a, and cells lacking a Cas12a ortholog (control). PAMs and the related editing efficiencies were pooled by the first three nucleotide positions. Each heatmap indicates an average of two or three independent replicates. The range of replicates for each 3-nucleotide PAM sequence is less than 10% of the signal at each position. (C) An expansion of the data generated in (B), except that all 64 four-nucleotide PAMs are plotted. The range of replicates for each 4-nucleotide sequence is less than 25% of the average signal for each four-nucleotide PAM.

    Techniques Used: Stable Transfection, Marker, Transfection, Expressing, Sequencing, Variant Assay, Generated

    Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. 21 , 22 (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. 11 Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p
    Figure Legend Snippet: Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. 21 , 22 (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. 11 Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p

    Techniques Used: Generated, Binding Assay, Expressing, Construct, Sequencing, Luciferase, Stable Transfection, Non-Homologous End Joining, Activity Assay

    13) Product Images from "Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay"

    Article Title: Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.767315

    Optimizing the RAA reaction time and Cas12a cleavage time. The RAA-CRISPR/Cas12a assay was performed using 1 × 10 4 copies/μL of V. vulnificus genomic DNA as the template, F7/R7 as the primer set, and CR3 + 4 as the crRNA to optimize the RAA reaction time (A) and Cas12a cleavage time (B) , and the fluorescence signal was analyzed using a multifunctional microplate reader (upper) or a UV torch (below).
    Figure Legend Snippet: Optimizing the RAA reaction time and Cas12a cleavage time. The RAA-CRISPR/Cas12a assay was performed using 1 × 10 4 copies/μL of V. vulnificus genomic DNA as the template, F7/R7 as the primer set, and CR3 + 4 as the crRNA to optimize the RAA reaction time (A) and Cas12a cleavage time (B) , and the fluorescence signal was analyzed using a multifunctional microplate reader (upper) or a UV torch (below).

    Techniques Used: CRISPR, Fluorescence

    Screening optimal RAA primers and crRNA for the RAA-CRISPR/Cas12a assay. (A) Gel electrophoresis analysis of RAA products amplified with different primer set. M, 500 DNA marker; lanes 1–9, RAA products amplified by primer set 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively. (B) crRNA sequences designed in this study. (C) Analysis of fluorescence signals triggered by the different crRNA using a multifunctional microplate reader (upper) or a UV torch (below). Data is one representative of three experiments.
    Figure Legend Snippet: Screening optimal RAA primers and crRNA for the RAA-CRISPR/Cas12a assay. (A) Gel electrophoresis analysis of RAA products amplified with different primer set. M, 500 DNA marker; lanes 1–9, RAA products amplified by primer set 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively. (B) crRNA sequences designed in this study. (C) Analysis of fluorescence signals triggered by the different crRNA using a multifunctional microplate reader (upper) or a UV torch (below). Data is one representative of three experiments.

    Techniques Used: CRISPR, Nucleic Acid Electrophoresis, Amplification, Marker, Fluorescence

    Evaluating the specificity of RAA-CRISPR/Cas12a assay in V. vulnificus detection. Ten bacterial strains were used to evaluate the specificity of RAA-CRISPR/Cas12a assay, and the fluorescence sigils were analyzed using a multifunctional microplate reader (upper) or a UV torch (below). V. vulnificus strain 1 was isolated from eel, and strain 2 was isolated from human.
    Figure Legend Snippet: Evaluating the specificity of RAA-CRISPR/Cas12a assay in V. vulnificus detection. Ten bacterial strains were used to evaluate the specificity of RAA-CRISPR/Cas12a assay, and the fluorescence sigils were analyzed using a multifunctional microplate reader (upper) or a UV torch (below). V. vulnificus strain 1 was isolated from eel, and strain 2 was isolated from human.

    Techniques Used: CRISPR, Fluorescence, Isolation

    Evaluating the sensitivity of the RAA-CRISPR/Cas12a, RAA, and qPCR assay in V. vulnificus detection. 2 μL of nuclease-free water and 1 × 10 0 to 1 × 10 6 copies/μL of V. vulnificus genomic DNA were used as templates in these assays. (A) The sensitivity of the RAA-CRISPR/Cas12a assay. The results were detected using a multifunctional microplate reader (upper) or a UV torch (below). (B) The sensitivity of the RAA assay. RAA reaction time was 20 min, consistent with the RAA-CRISPR/Cas12a assay. The results were analyzed using gel electrophoresis. (C) The sensitivity of the qPCR assay. qPCR was conducted with the CFX96 real-time PCR detection systems, and the amplification curves of each sample were shown in this figure. Data is one representative of three experiments.
    Figure Legend Snippet: Evaluating the sensitivity of the RAA-CRISPR/Cas12a, RAA, and qPCR assay in V. vulnificus detection. 2 μL of nuclease-free water and 1 × 10 0 to 1 × 10 6 copies/μL of V. vulnificus genomic DNA were used as templates in these assays. (A) The sensitivity of the RAA-CRISPR/Cas12a assay. The results were detected using a multifunctional microplate reader (upper) or a UV torch (below). (B) The sensitivity of the RAA assay. RAA reaction time was 20 min, consistent with the RAA-CRISPR/Cas12a assay. The results were analyzed using gel electrophoresis. (C) The sensitivity of the qPCR assay. qPCR was conducted with the CFX96 real-time PCR detection systems, and the amplification curves of each sample were shown in this figure. Data is one representative of three experiments.

    Techniques Used: CRISPR, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification

    Analysis the feasibility of RAA-CRISPR/Cas12a assay in the detection of V. vulnificus in spiked samples. The genomic DNA was extracted from 11 shrimps, eight of which were spiked with 1.1 × 10 4 CFU/mL of V. vulnificus , using the NaOH-based DNA extraction method. (A) The RAA-CRISPR/Cas12a assay was performed to detect V. vulnificus in those 11 DNA samples using a multifunctional microplate reader (below) or a UV torch (upper). (B) The qPCR assay was performed as a standard method to detect V. vulnificus in those 11 DNA samples. The amplification curves of each sample were shown. Data is one representative of three experiments. (C) Human blood and stool samples were employed to evaluate the feasibility of RAA-CRISPR/Cas12a assay in diagnosis of human vibriosis. 100 μL of blood or 200 mg of stool was added into the tube containing 1.1 × 10 3 CFU of V. vulnificus , and then these samples were used to extract genomic DNA. 2 μL of genomic DNA extracted from spiked samples were used as templates for RAA-CRISPR/Cas12a assays, while 2 μL of blood DNA or stool DNA was used as a negative control. Fluorescence signals were analyzed using a multifunctional microplate reader (upper) or a UV torch (below). Data is one representative of three experiments.
    Figure Legend Snippet: Analysis the feasibility of RAA-CRISPR/Cas12a assay in the detection of V. vulnificus in spiked samples. The genomic DNA was extracted from 11 shrimps, eight of which were spiked with 1.1 × 10 4 CFU/mL of V. vulnificus , using the NaOH-based DNA extraction method. (A) The RAA-CRISPR/Cas12a assay was performed to detect V. vulnificus in those 11 DNA samples using a multifunctional microplate reader (below) or a UV torch (upper). (B) The qPCR assay was performed as a standard method to detect V. vulnificus in those 11 DNA samples. The amplification curves of each sample were shown. Data is one representative of three experiments. (C) Human blood and stool samples were employed to evaluate the feasibility of RAA-CRISPR/Cas12a assay in diagnosis of human vibriosis. 100 μL of blood or 200 mg of stool was added into the tube containing 1.1 × 10 3 CFU of V. vulnificus , and then these samples were used to extract genomic DNA. 2 μL of genomic DNA extracted from spiked samples were used as templates for RAA-CRISPR/Cas12a assays, while 2 μL of blood DNA or stool DNA was used as a negative control. Fluorescence signals were analyzed using a multifunctional microplate reader (upper) or a UV torch (below). Data is one representative of three experiments.

    Techniques Used: CRISPR, DNA Extraction, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence

    Schematic diagram of RAA-CRISPR/Cas12a assay in the detection of V. vulnificus . FL, fluorescence.
    Figure Legend Snippet: Schematic diagram of RAA-CRISPR/Cas12a assay in the detection of V. vulnificus . FL, fluorescence.

    Techniques Used: CRISPR, Fluorescence

    14) Product Images from "An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip"

    Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2021.05.005

    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Figure Legend Snippet: Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P

    Techniques Used: Fluorescence, Concentration Assay, Sequencing, Recombinase Polymerase Amplification, Incubation, Modification

    Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.
    Figure Legend Snippet: Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.

    Techniques Used: Real-time Polymerase Chain Reaction, Stripping Membranes, Incubation

    Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.
    Figure Legend Snippet: Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.

    Techniques Used: Fluorescence, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).
    Figure Legend Snippet: Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).

    Techniques Used: Lateral Flow Assay, Stripping Membranes, Incubation, Labeling, Modification, Fluorescence

    Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
    Figure Legend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P

    Techniques Used: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    15) Product Images from "An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip"

    Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2021.05.005

    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Figure Legend Snippet: Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P

    Techniques Used: Fluorescence, Concentration Assay, Sequencing, Recombinase Polymerase Amplification, Incubation, Modification

    Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.
    Figure Legend Snippet: Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.

    Techniques Used: Real-time Polymerase Chain Reaction, Stripping Membranes, Incubation

    Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.
    Figure Legend Snippet: Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.

    Techniques Used: Fluorescence, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).
    Figure Legend Snippet: Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).

    Techniques Used: Lateral Flow Assay, Stripping Membranes, Incubation, Labeling, Modification, Fluorescence

    Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
    Figure Legend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P

    Techniques Used: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    16) Product Images from "gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing"

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.110494

    Comparison of Cas9 and Cas12a editing efficiency with T7RNAP variants. Efficiency of ADE2 editing by Cas12- or Cas9-mediated gEL DNA in T7RNAP mutant or overexpression strains: IMX1905 (K276R); IMX2031 (wild-type, wt ); IMX2032 (P266L); IMX2030 (P266L_K276R); IME459 (K276R overexpression, ↗K276R); IME475 (P266L overexpression, P266L). For Cas12a, transformed gDNA corresponds to annealed 15093-15094 oligos. For Cas9, transformed gDNA was obtained by PCR-derived fragment using overlapping primers 16745-16746. Editing efficiency is expressed as percentage of red colonies ( ade2 − ). Values represent the average and standard deviations of data obtained from independent biological duplicates. * P
    Figure Legend Snippet: Comparison of Cas9 and Cas12a editing efficiency with T7RNAP variants. Efficiency of ADE2 editing by Cas12- or Cas9-mediated gEL DNA in T7RNAP mutant or overexpression strains: IMX1905 (K276R); IMX2031 (wild-type, wt ); IMX2032 (P266L); IMX2030 (P266L_K276R); IME459 (K276R overexpression, ↗K276R); IME475 (P266L overexpression, P266L). For Cas12a, transformed gDNA corresponds to annealed 15093-15094 oligos. For Cas9, transformed gDNA was obtained by PCR-derived fragment using overlapping primers 16745-16746. Editing efficiency is expressed as percentage of red colonies ( ade2 − ). Values represent the average and standard deviations of data obtained from independent biological duplicates. * P

    Techniques Used: Mutagenesis, Over Expression, Transformation Assay, Polymerase Chain Reaction, Derivative Assay

    Optimization of Cas9 and Cas12a gDNA design. Editing efficiency of ADE2 in strain IMX1905 transformed with gDNAs for cloning-free, T7RNAP-driven expression of gRNA. A) gDNA configurations for Cas9-mediated genome editing and respective editing efficiencies. B) gDNA configurations for Cas12a-mediated genome editing and their respective editing efficiencies. The size of each gDNA is specified on the right of the respective graph bar. Editing efficiency is expressed as percentage of red colonies ( ade2 ) over the total number of colonies. Values represent the average and standard deviations of data obtained from three independent biological replicates.
    Figure Legend Snippet: Optimization of Cas9 and Cas12a gDNA design. Editing efficiency of ADE2 in strain IMX1905 transformed with gDNAs for cloning-free, T7RNAP-driven expression of gRNA. A) gDNA configurations for Cas9-mediated genome editing and respective editing efficiencies. B) gDNA configurations for Cas12a-mediated genome editing and their respective editing efficiencies. The size of each gDNA is specified on the right of the respective graph bar. Editing efficiency is expressed as percentage of red colonies ( ade2 ) over the total number of colonies. Values represent the average and standard deviations of data obtained from three independent biological replicates.

    Techniques Used: Transformation Assay, Clone Assay, Expressing

    Multiplex genome editing by Cas12a-mediated using the gEL DNA approach. (A) Targeted sites for deletion of ADE2 (ADE2-3, green), HIS4 (HIS4-4, orange), PDR12 (PDR12-3, cyan) and CAN1 (CAN1-4, pink; CAN1-3, violet) genes. (B) Percentage of transformants obtained from double gDNAs delivery: ADE2-3 and HIS4-4. (C) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-4. (D) Verification of single editing efficiency of CAN1 targets expressed from plasmid pUDR717 (CAN1-4) or pUDR718 (CAN1-3), with prediction of gRNAs secondary structure. (E) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-3. Number of verified clones is indicated between brackets and diagnostic PCRs are reported in Supplementary Figures (Fig. S4, S5, S6). Zero (0Δ), single (1Δ), double (2Δ), triple (3Δ) or quadruple (4Δ) deletion are indicated at the outside ends of each fraction. Type of obtained deletions are specified with the respective colour of the target. Number of colonies are also stated next to each depiction. Prediction of the gRNA stem-loop for Cas12a recognition is highlighted by a red square.
    Figure Legend Snippet: Multiplex genome editing by Cas12a-mediated using the gEL DNA approach. (A) Targeted sites for deletion of ADE2 (ADE2-3, green), HIS4 (HIS4-4, orange), PDR12 (PDR12-3, cyan) and CAN1 (CAN1-4, pink; CAN1-3, violet) genes. (B) Percentage of transformants obtained from double gDNAs delivery: ADE2-3 and HIS4-4. (C) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-4. (D) Verification of single editing efficiency of CAN1 targets expressed from plasmid pUDR717 (CAN1-4) or pUDR718 (CAN1-3), with prediction of gRNAs secondary structure. (E) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-3. Number of verified clones is indicated between brackets and diagnostic PCRs are reported in Supplementary Figures (Fig. S4, S5, S6). Zero (0Δ), single (1Δ), double (2Δ), triple (3Δ) or quadruple (4Δ) deletion are indicated at the outside ends of each fraction. Type of obtained deletions are specified with the respective colour of the target. Number of colonies are also stated next to each depiction. Prediction of the gRNA stem-loop for Cas12a recognition is highlighted by a red square.

    Techniques Used: Multiplex Assay, Transformation Assay, Plasmid Preparation, Clone Assay, Diagnostic Assay

    Physiological characterization of S. cerevisiae strains expressing T7RNAP. Maximum specific growth rates ( μ max ) of S. cerevisiae constitutively expressing T7RNAP K276R , Cas9 and Cas12a (IMX1905) and its control strain (CENPK.113-7D), or S. cerevisiae strains overexpressing T7RNAP K276R (IME459) or T7RNAP P266L (IME475) and its control strain carrying a 2μm multi-copy empty vector (IME460). Strains were cultivated in 96-well plate containing chemically defined medium supplemented with glucose as sole carbon source. Data points represent average and mean deviations of four biological replicates. *P
    Figure Legend Snippet: Physiological characterization of S. cerevisiae strains expressing T7RNAP. Maximum specific growth rates ( μ max ) of S. cerevisiae constitutively expressing T7RNAP K276R , Cas9 and Cas12a (IMX1905) and its control strain (CENPK.113-7D), or S. cerevisiae strains overexpressing T7RNAP K276R (IME459) or T7RNAP P266L (IME475) and its control strain carrying a 2μm multi-copy empty vector (IME460). Strains were cultivated in 96-well plate containing chemically defined medium supplemented with glucose as sole carbon source. Data points represent average and mean deviations of four biological replicates. *P

    Techniques Used: Expressing, Plasmid Preparation

    Schematic overview of the gEL DNA approach. 1, in silico design and ordering of gDNA cassettes (87 bp) and repair DNA (120 bp) as oligos. 2, tranformation with the double-stranded (ds) gDNA expression cassettes (2a), the ds repair DNA fragments and an empty, split plasmid carrying a marker of choice. 3, expression of the gRNA by the T7RNAP. 4, targetted DNA editing by Cas12a (Cas12a). 5, repair of the ds DNA break via homologous recombination using the repair DNA fragments.
    Figure Legend Snippet: Schematic overview of the gEL DNA approach. 1, in silico design and ordering of gDNA cassettes (87 bp) and repair DNA (120 bp) as oligos. 2, tranformation with the double-stranded (ds) gDNA expression cassettes (2a), the ds repair DNA fragments and an empty, split plasmid carrying a marker of choice. 3, expression of the gRNA by the T7RNAP. 4, targetted DNA editing by Cas12a (Cas12a). 5, repair of the ds DNA break via homologous recombination using the repair DNA fragments.

    Techniques Used: In Silico, Expressing, Plasmid Preparation, Marker, Homologous Recombination

    17) Product Images from "An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip"

    Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2021.05.005

    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Figure Legend Snippet: Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P

    Techniques Used: Fluorescence, Concentration Assay, Sequencing, Recombinase Polymerase Amplification, Incubation, Modification

    Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.
    Figure Legend Snippet: Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.

    Techniques Used: Real-time Polymerase Chain Reaction, Stripping Membranes, Incubation

    Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.
    Figure Legend Snippet: Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.

    Techniques Used: Fluorescence, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).
    Figure Legend Snippet: Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).

    Techniques Used: Lateral Flow Assay, Stripping Membranes, Incubation, Labeling, Modification, Fluorescence

    Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
    Figure Legend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P

    Techniques Used: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    18) Product Images from "An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip"

    Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2021.05.005

    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Figure Legend Snippet: Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to Cas12a. The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P

    Techniques Used: Fluorescence, Concentration Assay, Sequencing, Recombinase Polymerase Amplification, Incubation, Modification

    Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.
    Figure Legend Snippet: Detection of HPV in clinical samples using lateral flow TESTOR assay. a , Lateral flow strips showing HPV16 TESTOR assay results (upper panel). Ten qPCR-positive and eleven qPCR-negative samples were used for HPV16 detection. The Cas12a detection assays were run on lateral flow strips and imaged after 5 min. Performance characteristics of lateral flow TESTOR assay (bottom panel). A total of 21 clinical samples were evaluated using the lateral flow version of the TESTOR assay. Both the positive agreement and negative agreements are 100%. NTC, no-template control; T, test line; C, control line. b , Lateral flow strip readouts for HPV18 detection using clinical samples. A total of 21 clinical samples were evaluated (10 HPV18 positives and 12 negatives). The reactions were 1:5 diluted after incubation at 37 °C for 30 min and then run on lateral flow strips and imaged after 5 min.

    Techniques Used: Real-time Polymerase Chain Reaction, Stripping Membranes, Incubation

    Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.
    Figure Legend Snippet: Detection of HPV in clinical samples using fluorescence TESTOR assay. a , Heatmaps showing the CT values by qPCR (left panel) and fluorescence at 30 min by TESTOR assay (right panel) for HPV16 detection. Two out of twenty clinical samples were qPCR positive but showed weak signal at 30 min by TESTER assay (Patient ID: 9, 20). b , Fluorescence kinetics of the two samples showing late CT values by qPCR or weak signals by TESTER. c , Results of the qPCR (left panel) and fluorescence TESTOR assay at 30 min (right panel) for HPV18 detection. One out of thirteen clinical samples was positive by qPCR but showed weak signal at 30 min by fluorescence TESTER assay (Patient ID: 44). d , Fluorescence kinetics of the clinical sample (Patient ID: 44) showing late CT value by qPCR or weak signal by TESTOR assay. e , Fluorescence curve of re-examination by Cas12a for one patient negative for HPV18 by qPCR but showing slight signal increase by TESTOR. The yield of TESTOR from the patient (ID: 25) was amplified by PCR and then the PCR product was detected by Cas12a reaction.

    Techniques Used: Fluorescence, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).
    Figure Legend Snippet: Development of the novel lateral flow assay. a , Schematic of conventional strip and reporter used for Cas12a-based nucleic acid detection. b , Lateral flow strip readout of 1:5 diluted TESTOR reactions with 0.5 μM or 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter. Strip was incubated at room temperature for 5 min following 30 min of TESTOR reaction at 37 °C. c , Lateral flow strip readouts of 1:10 diluted TESTOR reactions with 1 μM reporter in the presence or absence of N0 gene target using conventional strip and reporter (left panel). Time course of lateral flow strip readouts using 1:5 diluted TESTOR reactions with 1 μM reporter in the absence of N0 gene target (right panel). d , Schematic of the novel strip and reporter. The reporter is labeled with a biotin on 5′ end, a FAM molecule on its 3′ end and a DIG in the middle. Anti-FAM and Anti-DIG antibodies are immobilized at the control and test line, respectively. e , Lateral flow strip readouts using novel strip and reporter at indicated conditions. The onepot reactions were performed at 37 °C for 10 min or 30 min. Novel reporters with or without the phosphorothioate modification between biotin and DIG were used to perform the lateral flow assay. f , Sequences of novel reporters; * and THO represent phosphorothioate modification. g , Fluorescence obtained at 30 min after reaction using two different reporters with or without phosphorothioate modification (upper panel). Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). h , Representative plot of fluorescence intensity versus time (upper left) and its quantification (upper right) after 30 min of reaction using C nucleotide-rich reporters. Error bars represent the mean ± s.d., where n = 3 replicates. Sequences and modification of reporters; * and THO represent phosphorothioate modification (bottom panel). i , Comparison of cleavage efficiency for C nucleotide-rich and –lacking reporters at specified conditions (upper panel). Sequences and modification of reporters; * and THO represents phosphorothioate modification (bottom panel).

    Techniques Used: Lateral Flow Assay, Stripping Membranes, Incubation, Labeling, Modification, Fluorescence

    Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
    Figure Legend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P

    Techniques Used: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

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  • 97
    New England Biolabs recombinant cas9 protein
    Generation of multiple type II Cdh protein null mutant mice. a Exonic regions encoding the translation start site of each Cdh are targeted by <t>CRISPR/Cas9</t> system to introduce small deletions for the immediate frameshift. Primers for genotyping are indicated by arrows. Positions of the beta-galactosidase ( LacZ ) reporter modified Cdh6 -5′ BAC and Cdh8 -5′ BAC constructs are shown as black bars. Position of the EGFP knock-in at the Cdh11 locus is shown as bright green bar. Arrowheads, single guide (sg) RNA target sites. b Results from western blotting analyses for each Cdh and β-actin are arranged. Note that Cdh single KO mice generated are all protein null. c , d Staining pattern from the Cdh6 -5′ BAC-Tg completely recapitulates Cdh6 mRNA profiles in the early forebrain (Fb) compartment at E8.5. c , d dorsal view; c′ , d′ ventral view. The ratio of embryos exhibiting the Fb-specific expression pattern at E8.5 over the total number of transgenic founders is noted at the bottom left corner of c . e Pax6 whole-mount immunostaining pattern is also identical to Cdh6 mRNA profiles in the early Fb. f A Cdh8 -5′ BAC-Tg embryo at the 22-somite stage (s) harbors strong enhancer activity at the Mb. The ratio of embryos exhibiting the Mb-specific expression pattern at this stage over the total number of transgenic founders is noted at the bottom left corner of the f . Section levels for g – l are indicated by white dashed lines g – l , respectively. Scale bar: 250 μm. g – l Expression patterns of Cdh8 mRNA k , l are recapitulated by Lac Z staining from Cdh8 -5′ BAC-Tg embryo at the 22s g – j . A white arrowhead indicates Cdh8 mRNA or Lac Z negative zone in the roof plate. p, prosomere; rp, roof plate. Scale bar: 100 μm. m – q Cdh8 -5′ BAC-Tg mice show progressive activation of the reporter correlated with neural tube closure. Scale bar: 250 μm. n′ , En face view of the 9s embryo reveals the LacZ expression in the Mb respecting the Fb/Mb compartment boundary (white dotted line). r A section level for s – u are indicated by the black line in a whole-mount stained embryo by Pax6. s , t A Cdh8 -5′ BAC-Tg embryo at the 9s is immunostained with Pax6 and LacZ. u Enlarged views of the Fb/Mb compartment boundary outlined by the white box u in the s , t are arranged. Note that Pax6 and Cdh8::LacZ expressions are mutually exclusive at the Fb/Mb boundary. v Cdh6 and Cdh8 enhancer activities at the nascent Fb/Mb compartment boundary are summarized. Noticeably, these two Cdh enhancer activities oppose each other at the dorsal portion of the compartment boundary.
    Recombinant Cas9 Protein, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs cas12a
    CANTRIP can be performed in a two-step reaction without <t>Cas12a</t> heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.
    Cas12a, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation of multiple type II Cdh protein null mutant mice. a Exonic regions encoding the translation start site of each Cdh are targeted by CRISPR/Cas9 system to introduce small deletions for the immediate frameshift. Primers for genotyping are indicated by arrows. Positions of the beta-galactosidase ( LacZ ) reporter modified Cdh6 -5′ BAC and Cdh8 -5′ BAC constructs are shown as black bars. Position of the EGFP knock-in at the Cdh11 locus is shown as bright green bar. Arrowheads, single guide (sg) RNA target sites. b Results from western blotting analyses for each Cdh and β-actin are arranged. Note that Cdh single KO mice generated are all protein null. c , d Staining pattern from the Cdh6 -5′ BAC-Tg completely recapitulates Cdh6 mRNA profiles in the early forebrain (Fb) compartment at E8.5. c , d dorsal view; c′ , d′ ventral view. The ratio of embryos exhibiting the Fb-specific expression pattern at E8.5 over the total number of transgenic founders is noted at the bottom left corner of c . e Pax6 whole-mount immunostaining pattern is also identical to Cdh6 mRNA profiles in the early Fb. f A Cdh8 -5′ BAC-Tg embryo at the 22-somite stage (s) harbors strong enhancer activity at the Mb. The ratio of embryos exhibiting the Mb-specific expression pattern at this stage over the total number of transgenic founders is noted at the bottom left corner of the f . Section levels for g – l are indicated by white dashed lines g – l , respectively. Scale bar: 250 μm. g – l Expression patterns of Cdh8 mRNA k , l are recapitulated by Lac Z staining from Cdh8 -5′ BAC-Tg embryo at the 22s g – j . A white arrowhead indicates Cdh8 mRNA or Lac Z negative zone in the roof plate. p, prosomere; rp, roof plate. Scale bar: 100 μm. m – q Cdh8 -5′ BAC-Tg mice show progressive activation of the reporter correlated with neural tube closure. Scale bar: 250 μm. n′ , En face view of the 9s embryo reveals the LacZ expression in the Mb respecting the Fb/Mb compartment boundary (white dotted line). r A section level for s – u are indicated by the black line in a whole-mount stained embryo by Pax6. s , t A Cdh8 -5′ BAC-Tg embryo at the 9s is immunostained with Pax6 and LacZ. u Enlarged views of the Fb/Mb compartment boundary outlined by the white box u in the s , t are arranged. Note that Pax6 and Cdh8::LacZ expressions are mutually exclusive at the Fb/Mb boundary. v Cdh6 and Cdh8 enhancer activities at the nascent Fb/Mb compartment boundary are summarized. Noticeably, these two Cdh enhancer activities oppose each other at the dorsal portion of the compartment boundary.

    Journal: Communications Biology

    Article Title: Redundant type II cadherins define neuroepithelial cell states for cytoarchitectonic robustness

    doi: 10.1038/s42003-020-01297-2

    Figure Lengend Snippet: Generation of multiple type II Cdh protein null mutant mice. a Exonic regions encoding the translation start site of each Cdh are targeted by CRISPR/Cas9 system to introduce small deletions for the immediate frameshift. Primers for genotyping are indicated by arrows. Positions of the beta-galactosidase ( LacZ ) reporter modified Cdh6 -5′ BAC and Cdh8 -5′ BAC constructs are shown as black bars. Position of the EGFP knock-in at the Cdh11 locus is shown as bright green bar. Arrowheads, single guide (sg) RNA target sites. b Results from western blotting analyses for each Cdh and β-actin are arranged. Note that Cdh single KO mice generated are all protein null. c , d Staining pattern from the Cdh6 -5′ BAC-Tg completely recapitulates Cdh6 mRNA profiles in the early forebrain (Fb) compartment at E8.5. c , d dorsal view; c′ , d′ ventral view. The ratio of embryos exhibiting the Fb-specific expression pattern at E8.5 over the total number of transgenic founders is noted at the bottom left corner of c . e Pax6 whole-mount immunostaining pattern is also identical to Cdh6 mRNA profiles in the early Fb. f A Cdh8 -5′ BAC-Tg embryo at the 22-somite stage (s) harbors strong enhancer activity at the Mb. The ratio of embryos exhibiting the Mb-specific expression pattern at this stage over the total number of transgenic founders is noted at the bottom left corner of the f . Section levels for g – l are indicated by white dashed lines g – l , respectively. Scale bar: 250 μm. g – l Expression patterns of Cdh8 mRNA k , l are recapitulated by Lac Z staining from Cdh8 -5′ BAC-Tg embryo at the 22s g – j . A white arrowhead indicates Cdh8 mRNA or Lac Z negative zone in the roof plate. p, prosomere; rp, roof plate. Scale bar: 100 μm. m – q Cdh8 -5′ BAC-Tg mice show progressive activation of the reporter correlated with neural tube closure. Scale bar: 250 μm. n′ , En face view of the 9s embryo reveals the LacZ expression in the Mb respecting the Fb/Mb compartment boundary (white dotted line). r A section level for s – u are indicated by the black line in a whole-mount stained embryo by Pax6. s , t A Cdh8 -5′ BAC-Tg embryo at the 9s is immunostained with Pax6 and LacZ. u Enlarged views of the Fb/Mb compartment boundary outlined by the white box u in the s , t are arranged. Note that Pax6 and Cdh8::LacZ expressions are mutually exclusive at the Fb/Mb boundary. v Cdh6 and Cdh8 enhancer activities at the nascent Fb/Mb compartment boundary are summarized. Noticeably, these two Cdh enhancer activities oppose each other at the dorsal portion of the compartment boundary.

    Article Snippet: Recombinant Cas9 protein (EnGen Cas9 NLS) were purchased from NEB.

    Techniques: Mutagenesis, Mouse Assay, CRISPR, Introduce, Modification, BAC Assay, Construct, Knock-In, Western Blot, Generated, Staining, Expressing, Transgenic Assay, Immunostaining, Activity Assay, Activation Assay

    CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Journal: Biology Methods & Protocols

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    doi: 10.1093/biomethods/bpaa020

    Figure Lengend Snippet: CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    Techniques: Incubation

    CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Journal: Biology Methods & Protocols

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    doi: 10.1093/biomethods/bpaa020

    Figure Lengend Snippet: CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    Techniques: Fluorescence, Activation Assay

    GE events in the apple MdPDS gene introduced by Lb Cas12a. The genomic loci ( MdPDS _locus A and D) of different apple regenerates transformed with the CRISPR/ Lb Cas12a construct were sequenced by amplicon deep sequencing. All resulting unique sequences containing GE events were aligned to the reference sequence from wt ‘Gala’ and sorted by deletion length in ascending order. The protospacer adjacent motif (PAM) and the target sites of the crRNAs A (blue) and B (green) are indicated in the wt sequence.

    Journal: International Journal of Molecular Sciences

    Article Title: Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

    doi: 10.3390/ijms222212611

    Figure Lengend Snippet: GE events in the apple MdPDS gene introduced by Lb Cas12a. The genomic loci ( MdPDS _locus A and D) of different apple regenerates transformed with the CRISPR/ Lb Cas12a construct were sequenced by amplicon deep sequencing. All resulting unique sequences containing GE events were aligned to the reference sequence from wt ‘Gala’ and sorted by deletion length in ascending order. The protospacer adjacent motif (PAM) and the target sites of the crRNAs A (blue) and B (green) are indicated in the wt sequence.

    Article Snippet: In vitro DNA cleavage assays were performed in a reaction volume of 30 µL using a final concentration of 30 nM EnGen LbaCas12a (Cpf1) (M0653S, New England Biolabs GmbH, Frankfurt am Main, Germany).

    Techniques: Transformation Assay, CRISPR, Construct, Amplification, Sequencing

    The MdPDS target locus for GE analysis. ( A ) The scheme represents the genomic organization of MdPDS (MD04G1023800) comprising exons one to nine (numbered boxes; 5’-UTR (gray), coding sequence (blue)) and introns (black line). Target sites of crRNAs in coding exon 2 (crRNA_A), exon 5 (crRNA_C), and exon 7 (crRNA_D) are indicated by arrows. ( B , C ) Binding sites of primer pairs used for fluorescent PCR capillary gel electrophoresis ( B ) and DNA cleavage assays ( C ) are represented as triangles. Primers labeled with fluorescent dyes are represented in blue and green. ( D ) For DNA cleavage assays, PCR fragments were amplified from genomic DNA of ‘Gala’ and used as substrates for digestion with pre-assembled Lb Cas12a/crRNA complexes. Experimental controls without the Lb Cas12a/crRNA complex or with only one complex component each were also performed (indicated by + and −). The resulting products were separated by gel electrophoresis and, dependent on the target site of the respective crRNA, the cleavage of the PCR fragment resulted in different products: crRNA_A (fragment EX1-FW/EX6-REV: 2981 bp + 220 bp); crRNA_C (fragment EX1-FW/EX6-REV: 2576 bp + 626 bp); crRNA_D (fragment EX6-FW/EX8-REV: 1031 bp + 628 bp).

    Journal: International Journal of Molecular Sciences

    Article Title: Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

    doi: 10.3390/ijms222212611

    Figure Lengend Snippet: The MdPDS target locus for GE analysis. ( A ) The scheme represents the genomic organization of MdPDS (MD04G1023800) comprising exons one to nine (numbered boxes; 5’-UTR (gray), coding sequence (blue)) and introns (black line). Target sites of crRNAs in coding exon 2 (crRNA_A), exon 5 (crRNA_C), and exon 7 (crRNA_D) are indicated by arrows. ( B , C ) Binding sites of primer pairs used for fluorescent PCR capillary gel electrophoresis ( B ) and DNA cleavage assays ( C ) are represented as triangles. Primers labeled with fluorescent dyes are represented in blue and green. ( D ) For DNA cleavage assays, PCR fragments were amplified from genomic DNA of ‘Gala’ and used as substrates for digestion with pre-assembled Lb Cas12a/crRNA complexes. Experimental controls without the Lb Cas12a/crRNA complex or with only one complex component each were also performed (indicated by + and −). The resulting products were separated by gel electrophoresis and, dependent on the target site of the respective crRNA, the cleavage of the PCR fragment resulted in different products: crRNA_A (fragment EX1-FW/EX6-REV: 2981 bp + 220 bp); crRNA_C (fragment EX1-FW/EX6-REV: 2576 bp + 626 bp); crRNA_D (fragment EX6-FW/EX8-REV: 1031 bp + 628 bp).

    Article Snippet: In vitro DNA cleavage assays were performed in a reaction volume of 30 µL using a final concentration of 30 nM EnGen LbaCas12a (Cpf1) (M0653S, New England Biolabs GmbH, Frankfurt am Main, Germany).

    Techniques: Sequencing, Binding Assay, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Labeling, Amplification

    Distribution of CRISPR/ Lb Cas12a induced GE events in a chimeric regenerate. Different albino shoots originating from tissue transformed with the CRISPR/LbCas12a construct were separated and genotyped by fluorescent PCR capillary gel electrophoresis. The percentages of the total peak heights of each allele detected for locus A (upper panel) and locus D (lower panel) are represented in the diagrams.

    Journal: International Journal of Molecular Sciences

    Article Title: Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

    doi: 10.3390/ijms222212611

    Figure Lengend Snippet: Distribution of CRISPR/ Lb Cas12a induced GE events in a chimeric regenerate. Different albino shoots originating from tissue transformed with the CRISPR/LbCas12a construct were separated and genotyped by fluorescent PCR capillary gel electrophoresis. The percentages of the total peak heights of each allele detected for locus A (upper panel) and locus D (lower panel) are represented in the diagrams.

    Article Snippet: In vitro DNA cleavage assays were performed in a reaction volume of 30 µL using a final concentration of 30 nM EnGen LbaCas12a (Cpf1) (M0653S, New England Biolabs GmbH, Frankfurt am Main, Germany).

    Techniques: CRISPR, Transformation Assay, Construct, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

    Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

    Journal: Bio-protocol

    Article Title: Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1

    doi: 10.21769/BioProtoc.2842

    Figure Lengend Snippet: Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

    Article Snippet: Activity assay using purified Cas12a.

    Techniques: Expressing, Purification

    Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.

    Journal: Bio-protocol

    Article Title: Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1

    doi: 10.21769/BioProtoc.2842

    Figure Lengend Snippet: Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.

    Article Snippet: Activity assay using purified Cas12a.

    Techniques: