cas12a  (New England Biolabs)


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    Name:
    EnGen Lba Cas12a Cpf1
    Description:
    EnGen Lba Cas12a Cpf1 70 pmol
    Catalog Number:
    M0653S
    Price:
    70
    Category:
    Other Endonucleases
    Size:
    70 pmol
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    Structured Review

    New England Biolabs cas12a
    EnGen Lba Cas12a Cpf1
    EnGen Lba Cas12a Cpf1 70 pmol
    https://www.bioz.com/result/cas12a/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas12a - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) 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

    2) Product Images from "An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification"

    Article Title: An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification

    Journal: Theranostics

    doi: 10.7150/thno.49047

    Establishment of the HCR-CRISPR assay. (A) Agarose gel (left) and PAGE (right) images of the HCR nucleic acid amplification assay. Lane 1, 2.0 μM H1; lane 2, 2.0 μM H2; lane 3, 2.0 μM H1 and 2 μM H2 mixture; lane 4, NHCR, 0.5 μM nucleolin H0 mixed with a mixture of 2 μM H1 and 2 μM H2; lane 5, PHCR, 0.5 μM PD-L1 H0 mixed with a mixture of 2 μM H1 and 2 μM H2. (B) Diagram of the H1/H2 sequences within the HCR targeted by Cas12a and the respective crRNA. Highlighted bases indicate the 5′ PAM sequence. The same color represents a paired crRNA and PAM. (C) Observed FI of HCR-CRISPR using different crRNAs to target 1μL of nucleolin HCR (NHCR) products. The nonspecific (NS) crRNA controls showed a low or zero value after subtracting the background FI. The P values were calculated by comparison with the crRNA2 group using a one-way ANOVA followed by a Sidak multiple-comparisons test. *** and **** represent P
    Figure Legend Snippet: Establishment of the HCR-CRISPR assay. (A) Agarose gel (left) and PAGE (right) images of the HCR nucleic acid amplification assay. Lane 1, 2.0 μM H1; lane 2, 2.0 μM H2; lane 3, 2.0 μM H1 and 2 μM H2 mixture; lane 4, NHCR, 0.5 μM nucleolin H0 mixed with a mixture of 2 μM H1 and 2 μM H2; lane 5, PHCR, 0.5 μM PD-L1 H0 mixed with a mixture of 2 μM H1 and 2 μM H2. (B) Diagram of the H1/H2 sequences within the HCR targeted by Cas12a and the respective crRNA. Highlighted bases indicate the 5′ PAM sequence. The same color represents a paired crRNA and PAM. (C) Observed FI of HCR-CRISPR using different crRNAs to target 1μL of nucleolin HCR (NHCR) products. The nonspecific (NS) crRNA controls showed a low or zero value after subtracting the background FI. The P values were calculated by comparison with the crRNA2 group using a one-way ANOVA followed by a Sidak multiple-comparisons test. *** and **** represent P

    Techniques Used: CRISPR, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Amplification, Sequencing

    Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P
    Figure Legend Snippet: Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P

    Techniques Used: Enzyme-linked Immunosorbent Assay, CRISPR, Incubation, Concentration Assay, Labeling, Host-Cell Reactivation, Fluorescence

    Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.
    Figure Legend Snippet: Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.

    Techniques Used: CRISPR, Fluorescence, Concentration Assay

    3) Product Images from "Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection"

    Article Title: Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c00013

    Colorimetric target detection based on Cas12a. (A) Correlation of relative variation ratios and color change image with different concentrations of target strands (0, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 nM). Inset: the linear relationship between the relative variation ratio and target concentrations. (B) Illustration of the detection procedure and the resulting color from different viruses. (C) Specificity of our method against SARS-CoV and MERS-CoV sequence s in ORF1ab. All the target sequences used were 100 fM before RPA amplification. (D) Relative absorption variation ratios at 520 nm in reaction buffer, human serum, and saliva matrixes spiked with different amounts of target sequences. (E) and (F) Sensitivity test. Different numbers of the SARS-CoV-2 sequence in ORF1ab (E) and N gene (F) region were analyzed through Cas12a mediated colorimetric assay after amplification by RPA. Error bars represent the standard deviations of three repetitive experiments. (n.d., not detected; ****, P
    Figure Legend Snippet: Colorimetric target detection based on Cas12a. (A) Correlation of relative variation ratios and color change image with different concentrations of target strands (0, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 nM). Inset: the linear relationship between the relative variation ratio and target concentrations. (B) Illustration of the detection procedure and the resulting color from different viruses. (C) Specificity of our method against SARS-CoV and MERS-CoV sequence s in ORF1ab. All the target sequences used were 100 fM before RPA amplification. (D) Relative absorption variation ratios at 520 nm in reaction buffer, human serum, and saliva matrixes spiked with different amounts of target sequences. (E) and (F) Sensitivity test. Different numbers of the SARS-CoV-2 sequence in ORF1ab (E) and N gene (F) region were analyzed through Cas12a mediated colorimetric assay after amplification by RPA. Error bars represent the standard deviations of three repetitive experiments. (n.d., not detected; ****, P

    Techniques Used: Sequencing, Recombinase Polymerase Amplification, Amplification, Colorimetric Assay

    Clinical standard sample analysis. (A) Schematic illustration of the RT-RPA-coupled Cas12a colorimetric assay for clinical SARS-CoV-2 genome samples. The RNA sample was reverse-transcribed to cDNA before being amplified through RPA. The resulting dsDNA target bound and activated the Cas12a trans-cleavage ability. The dispersed AuNP probes will turn into aggregation states after the cleavage of the capped ssDNA. (B) Relative absorption variation ratios at 520 nm of negative and positive clinical standard samples provided by the hospital laboratory department. The error bars represented the standard deviations of three repetitive experiments. B: blank control, N: negative sample, P: positive sample. (****, P
    Figure Legend Snippet: Clinical standard sample analysis. (A) Schematic illustration of the RT-RPA-coupled Cas12a colorimetric assay for clinical SARS-CoV-2 genome samples. The RNA sample was reverse-transcribed to cDNA before being amplified through RPA. The resulting dsDNA target bound and activated the Cas12a trans-cleavage ability. The dispersed AuNP probes will turn into aggregation states after the cleavage of the capped ssDNA. (B) Relative absorption variation ratios at 520 nm of negative and positive clinical standard samples provided by the hospital laboratory department. The error bars represented the standard deviations of three repetitive experiments. B: blank control, N: negative sample, P: positive sample. (****, P

    Techniques Used: Recombinase Polymerase Amplification, Colorimetric Assay, Amplification

    RT-RPA-Coupled Cas12a for Colorimetric Detection of SARS-CoV-2; (A) Schematic Illustration of the Strategy Design; The Whole Process Consists of Three Steps: RT-RPA of the Selected SARS-CoV-2 Genome Region, Cas12a Activation and Colorimetric Detection; (B) SARS-CoV-2 Genome Alignment of the Selected Target Region in the ORF1ab Gene and the N Protein gene; The Accession Numbers of SARS-CoV-2, SARS-CoV, and MERS-CoV Genomes Were NC_045512.2, AY278741.1, and NC_019843.3, Respectively
    Figure Legend Snippet: RT-RPA-Coupled Cas12a for Colorimetric Detection of SARS-CoV-2; (A) Schematic Illustration of the Strategy Design; The Whole Process Consists of Three Steps: RT-RPA of the Selected SARS-CoV-2 Genome Region, Cas12a Activation and Colorimetric Detection; (B) SARS-CoV-2 Genome Alignment of the Selected Target Region in the ORF1ab Gene and the N Protein gene; The Accession Numbers of SARS-CoV-2, SARS-CoV, and MERS-CoV Genomes Were NC_045512.2, AY278741.1, and NC_019843.3, Respectively

    Techniques Used: Recombinase Polymerase Amplification, Activation Assay

    Feasibility verification of Cas12a-mediated colorimetric detection. (A) PAGE analysis of the ssDNA trans-cleavage ability of Cas12a after activated by target strands. Lane 1: substrate; lane 2: substrate + Cas12a; lane 3: substrate + Cas12a + crRNA; lane 4: substrate + Cas12a + Target; lane 5: substrate + Cas12a + crRNA + Target. Incubation time: 15 min. [Cas12a]: 20 nM, [crRNA]: 40 nM, [Target]: 40 nM, [Substrate]: 1 μM; loading volume: 10 μL. Running at 80 V for 80 min. (B) Schematic illustration of the AuNP aggregation resulting from the trans-cleavage of Cas12a. After activation, the Cas12a will cut and release the nucleic acids from the AuNP surface, leading to AuNP aggregation. The distance-dependent optical properties of the AuNPs can be clearly recorded with UV–vis and observed with naked eyes. (C) UV–vis absorption spectra of AuNP probes in different reaction conditions. (D) Color change and TEM images of AuNP probes in different reaction conditions. Well 1: blank; well 2: Cas12a; well 3: Cas12a + crRNA; well 4: Cas12a + target; well 5: Cas12a + crRNA + target. Scale bar: 100 nm. The variations of the relative variation ratio of absorption at 520 nm for ORF1ab (E) and N (F) gene targets in different reaction conditions, respectively. Bar graph data represent mean ± SD ( n = 3). (****, P
    Figure Legend Snippet: Feasibility verification of Cas12a-mediated colorimetric detection. (A) PAGE analysis of the ssDNA trans-cleavage ability of Cas12a after activated by target strands. Lane 1: substrate; lane 2: substrate + Cas12a; lane 3: substrate + Cas12a + crRNA; lane 4: substrate + Cas12a + Target; lane 5: substrate + Cas12a + crRNA + Target. Incubation time: 15 min. [Cas12a]: 20 nM, [crRNA]: 40 nM, [Target]: 40 nM, [Substrate]: 1 μM; loading volume: 10 μL. Running at 80 V for 80 min. (B) Schematic illustration of the AuNP aggregation resulting from the trans-cleavage of Cas12a. After activation, the Cas12a will cut and release the nucleic acids from the AuNP surface, leading to AuNP aggregation. The distance-dependent optical properties of the AuNPs can be clearly recorded with UV–vis and observed with naked eyes. (C) UV–vis absorption spectra of AuNP probes in different reaction conditions. (D) Color change and TEM images of AuNP probes in different reaction conditions. Well 1: blank; well 2: Cas12a; well 3: Cas12a + crRNA; well 4: Cas12a + target; well 5: Cas12a + crRNA + target. Scale bar: 100 nm. The variations of the relative variation ratio of absorption at 520 nm for ORF1ab (E) and N (F) gene targets in different reaction conditions, respectively. Bar graph data represent mean ± SD ( n = 3). (****, P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Activation Assay, Transmission Electron Microscopy

    Optimization of the detection conditions. (A) Influences of MCH treatment and substrate length to the colorimetric assays. The UV–vis absorption spectra were obtained after 1 h of reaction. 4 μL of Cas12a (1 μM) and 8 μL of crRNA (1 μM) were pre-incubated for 10 min before they were mixed with 8 μL of target (ORF1ab segments, 1 μM) in a 100 μL reaction system including 80 μL AuNP probes. (B) Concentration-dependent effect of Cas12a/crRNA on the readout signals. (C) and (D) Reaction time optimization. UV–vis absorption spectra were recorded at an interval of 20 min. The error bars represented the standard deviations of three repetitive experiments.
    Figure Legend Snippet: Optimization of the detection conditions. (A) Influences of MCH treatment and substrate length to the colorimetric assays. The UV–vis absorption spectra were obtained after 1 h of reaction. 4 μL of Cas12a (1 μM) and 8 μL of crRNA (1 μM) were pre-incubated for 10 min before they were mixed with 8 μL of target (ORF1ab segments, 1 μM) in a 100 μL reaction system including 80 μL AuNP probes. (B) Concentration-dependent effect of Cas12a/crRNA on the readout signals. (C) and (D) Reaction time optimization. UV–vis absorption spectra were recorded at an interval of 20 min. The error bars represented the standard deviations of three repetitive experiments.

    Techniques Used: Incubation, Concentration Assay

    4) Product Images from "Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria"

    Article Title: Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.2010196117

    Schematic of one-pot SHERLOCK assay. RT-RPA amplifies Plasmodium species target sequences and occurs in parallel with programmed Cas12a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore-labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout using Au-NP, gold nanoparticles.
    Figure Legend Snippet: Schematic of one-pot SHERLOCK assay. RT-RPA amplifies Plasmodium species target sequences and occurs in parallel with programmed Cas12a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore-labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout using Au-NP, gold nanoparticles.

    Techniques Used: Recombinase Polymerase Amplification, Labeling

    5) 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

    6) Product Images from "Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection"

    Article Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18615-1

    The trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see Supplementary Figs. 1 – 4 . a Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3′- or 5′-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. b A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. c , d , and e 3′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. f , g , and h 5′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signal of a sample with the activator to the corresponding sample without activator. For c – h , error bars represent mean ± SEM, where n = 6 replicates (three technical replicates examined over two independent experiments). Statistical analysis was performed using a two-way ANOVA test with Dunnett’s multiple comparison test, where ns = not significant with p > 0.05, and the asterisks (*, **, ***, ****) denote significant differences with p values listed above. Source data are available in the Source Data file.
    Figure Legend Snippet: The trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see Supplementary Figs. 1 – 4 . a Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3′- or 5′-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. b A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. c , d , and e 3′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. f , g , and h 5′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signal of a sample with the activator to the corresponding sample without activator. For c – h , error bars represent mean ± SEM, where n = 6 replicates (three technical replicates examined over two independent experiments). Statistical analysis was performed using a two-way ANOVA test with Dunnett’s multiple comparison test, where ns = not significant with p > 0.05, and the asterisks (*, **, ***, ****) denote significant differences with p values listed above. Source data are available in the Source Data file.

    Techniques Used: Activity Assay, Modification, Fluorescence, Reporter Assay

    Characterization of ENHANCE with various crRNA modifications and different Cas12a systems. a Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5′-end of the crRNA is cleaved by LbCas12a in the absence of the activator). b Comparison of trans-cleavage activity between AT-rich extensions and GC-rich 7-nt DNA 3′-end extensions on the crRNA + 3′DNA7. For a , b ), error bars denote mean ± SD, where n = 3 technical replicates. c Trans-cleavage activity of LbCas12a with non-fully phosphorothioate (PS) modified crRNA targeting GFP fragment. Sequence representation of 6 non-fully PS extension on the 3′-end of crGFP ranging from 1 to 6 PS. The asterisk symbol (*) signifies the phosphorothioated nucleotide. The graph below the sequence representation shows fold change of the LbCas12a fluorescence-based reporter assay with the activator normalized to the corresponding samples without the activator at t = 20 min. d kinetics of the LbCas12a fluorescence-based reporter assay in c , e Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. For c , d , and e , n = 6 replicates (three technical replicates examined over two independent experiments), where error bars in e represent mean ± SEM. Source data are available in the Source Data file.
    Figure Legend Snippet: Characterization of ENHANCE with various crRNA modifications and different Cas12a systems. a Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5′-end of the crRNA is cleaved by LbCas12a in the absence of the activator). b Comparison of trans-cleavage activity between AT-rich extensions and GC-rich 7-nt DNA 3′-end extensions on the crRNA + 3′DNA7. For a , b ), error bars denote mean ± SD, where n = 3 technical replicates. c Trans-cleavage activity of LbCas12a with non-fully phosphorothioate (PS) modified crRNA targeting GFP fragment. Sequence representation of 6 non-fully PS extension on the 3′-end of crGFP ranging from 1 to 6 PS. The asterisk symbol (*) signifies the phosphorothioated nucleotide. The graph below the sequence representation shows fold change of the LbCas12a fluorescence-based reporter assay with the activator normalized to the corresponding samples without the activator at t = 20 min. d kinetics of the LbCas12a fluorescence-based reporter assay in c , e Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. For c , d , and e , n = 6 replicates (three technical replicates examined over two independent experiments), where error bars in e represent mean ± SEM. Source data are available in the Source Data file.

    Techniques Used: Activity Assay, Modification, Sequencing, Fluorescence, Reporter Assay

    7) 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

    8) Product Images from "CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects"

    Article Title: CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.012933

    High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.
    Figure Legend Snippet: High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.

    Techniques Used: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control

    Effect of double mismatches in the target sequence on Cas12a cleavage activity. Heatmaps show the relative abundance of target sequences with two mismatches over time for the supercoiled ( left ) and nicked ( right ) pools in pLibrary PS4 ( A ), EMX1 ( B ), and CCR5 ( C ) upon cleavage by Cas12a as a function of distance between the two mismatches and time. Time points indicated on the left by the arrow are 1 min, 5 min, 30 min, 1 h, and 3 h. Values plotted represent the average of two (for LbCas12a, pLibrary PS4) and three (all other Cas12a and libraries) replicates.
    Figure Legend Snippet: Effect of double mismatches in the target sequence on Cas12a cleavage activity. Heatmaps show the relative abundance of target sequences with two mismatches over time for the supercoiled ( left ) and nicked ( right ) pools in pLibrary PS4 ( A ), EMX1 ( B ), and CCR5 ( C ) upon cleavage by Cas12a as a function of distance between the two mismatches and time. Time points indicated on the left by the arrow are 1 min, 5 min, 30 min, 1 h, and 3 h. Values plotted represent the average of two (for LbCas12a, pLibrary PS4) and three (all other Cas12a and libraries) replicates.

    Techniques Used: Sequencing, Activity Assay

    Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.
    Figure Legend Snippet: Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.

    Techniques Used: Plasmid Preparation, Incubation

    Cas12a has pervasive nicking activity against mismatched sequences in pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the nicked pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides to the left of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents the normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.
    Figure Legend Snippet: Cas12a has pervasive nicking activity against mismatched sequences in pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the nicked pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides to the left of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents the normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.

    Techniques Used: Activity Assay, Sequencing

    Sequence determinants of Cas12a cleavage activity for pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the supercoiled pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides on the left side of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.
    Figure Legend Snippet: Sequence determinants of Cas12a cleavage activity for pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the supercoiled pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides on the left side of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.

    Techniques Used: Sequencing, Activity Assay

    Cas12a has cis and activated, trans nuclease activities. Cas12a-crRNA complex can bind complementary target dsDNA and cleave it. Some mismatched targets can be nicked rapidly by Cas12a but undergo slow linearization. After this cleavage event, Cas12a releases the PAM-distal cleavage product and remains bound to at least the PAM-proximal cleaved target strand, thus remaining in an activated confirmation. The active RuvC domain can then accept other nucleic acid substrates. Target-activated Cas12a can further nick, linearize, and degrade nonspecific dsDNA, ssDNA, and ssRNA substrates.
    Figure Legend Snippet: Cas12a has cis and activated, trans nuclease activities. Cas12a-crRNA complex can bind complementary target dsDNA and cleave it. Some mismatched targets can be nicked rapidly by Cas12a but undergo slow linearization. After this cleavage event, Cas12a releases the PAM-distal cleavage product and remains bound to at least the PAM-proximal cleaved target strand, thus remaining in an activated confirmation. The active RuvC domain can then accept other nucleic acid substrates. Target-activated Cas12a can further nick, linearize, and degrade nonspecific dsDNA, ssDNA, and ssRNA substrates.

    Techniques Used:

    9) 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

    10) Product Images from "CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects"

    Article Title: CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.012933

    High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.
    Figure Legend Snippet: High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.

    Techniques Used: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control

    Effect of double mismatches in the target sequence on Cas12a cleavage activity. Heatmaps show the relative abundance of target sequences with two mismatches over time for the supercoiled ( left ) and nicked ( right ) pools in pLibrary PS4 ( A ), EMX1 ( B ), and CCR5 ( C ) upon cleavage by Cas12a as a function of distance between the two mismatches and time. Time points indicated on the left by the arrow are 1 min, 5 min, 30 min, 1 h, and 3 h. Values plotted represent the average of two (for LbCas12a, pLibrary PS4) and three (all other Cas12a and libraries) replicates.
    Figure Legend Snippet: Effect of double mismatches in the target sequence on Cas12a cleavage activity. Heatmaps show the relative abundance of target sequences with two mismatches over time for the supercoiled ( left ) and nicked ( right ) pools in pLibrary PS4 ( A ), EMX1 ( B ), and CCR5 ( C ) upon cleavage by Cas12a as a function of distance between the two mismatches and time. Time points indicated on the left by the arrow are 1 min, 5 min, 30 min, 1 h, and 3 h. Values plotted represent the average of two (for LbCas12a, pLibrary PS4) and three (all other Cas12a and libraries) replicates.

    Techniques Used: Sequencing, Activity Assay

    Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.
    Figure Legend Snippet: Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.

    Techniques Used: Plasmid Preparation, Incubation

    Cas12a has pervasive nicking activity against mismatched sequences in pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the nicked pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides to the left of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents the normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.
    Figure Legend Snippet: Cas12a has pervasive nicking activity against mismatched sequences in pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the nicked pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides to the left of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents the normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.

    Techniques Used: Activity Assay, Sequencing

    Sequence determinants of Cas12a cleavage activity for pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the supercoiled pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides on the left side of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.
    Figure Legend Snippet: Sequence determinants of Cas12a cleavage activity for pLibrary PS4. Heatmaps show the relative abundance of different mismatched sequences over time for the supercoiled pool in pLibrary PS4 upon cleavage by Cas12a orthologs. The crRNA sequence is indicated at the top . The nucleotides on the left side of the heatmaps indicate the potential base pair or mismatches formed. The crRNA complementary nucleotides are highlighted by boldface black boxes in the heatmap, which result in Watson–Crick base pairs. The PAM-proximal “seed” sequence is highlighted by the gray box . Each box represents normalized proportion of sequences containing each nucleotide at a given position across time. Values plotted represent average of two replicates for LbCas12a and three replicates for FnCas12a and AsCas12a.

    Techniques Used: Sequencing, Activity Assay

    Cas12a has cis and activated, trans nuclease activities. Cas12a-crRNA complex can bind complementary target dsDNA and cleave it. Some mismatched targets can be nicked rapidly by Cas12a but undergo slow linearization. After this cleavage event, Cas12a releases the PAM-distal cleavage product and remains bound to at least the PAM-proximal cleaved target strand, thus remaining in an activated confirmation. The active RuvC domain can then accept other nucleic acid substrates. Target-activated Cas12a can further nick, linearize, and degrade nonspecific dsDNA, ssDNA, and ssRNA substrates.
    Figure Legend Snippet: Cas12a has cis and activated, trans nuclease activities. Cas12a-crRNA complex can bind complementary target dsDNA and cleave it. Some mismatched targets can be nicked rapidly by Cas12a but undergo slow linearization. After this cleavage event, Cas12a releases the PAM-distal cleavage product and remains bound to at least the PAM-proximal cleaved target strand, thus remaining in an activated confirmation. The active RuvC domain can then accept other nucleic acid substrates. Target-activated Cas12a can further nick, linearize, and degrade nonspecific dsDNA, ssDNA, and ssRNA substrates.

    Techniques Used:

    11) 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

    12) Product Images from "Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection"

    Article Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18615-1

    The trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see Supplementary Figs. 1 – 4 . a Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3′- or 5′-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. b A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. c , d , and e 3′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. f , g , and h 5′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signal of a sample with the activator to the corresponding sample without activator. For c – h , error bars represent mean ± SEM, where n = 6 replicates (three technical replicates examined over two independent experiments). Statistical analysis was performed using a two-way ANOVA test with Dunnett’s multiple comparison test, where ns = not significant with p > 0.05, and the asterisks (*, **, ***, ****) denote significant differences with p values listed above. Source data are available in the Source Data file.
    Figure Legend Snippet: The trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see Supplementary Figs. 1 – 4 . a Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3′- or 5′-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. b A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. c , d , and e 3′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. f , g , and h 5′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signal of a sample with the activator to the corresponding sample without activator. For c – h , error bars represent mean ± SEM, where n = 6 replicates (three technical replicates examined over two independent experiments). Statistical analysis was performed using a two-way ANOVA test with Dunnett’s multiple comparison test, where ns = not significant with p > 0.05, and the asterisks (*, **, ***, ****) denote significant differences with p values listed above. Source data are available in the Source Data file.

    Techniques Used: Activity Assay, Modification, Fluorescence, Reporter Assay

    Characterization of ENHANCE with various crRNA modifications and different Cas12a systems. a Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5′-end of the crRNA is cleaved by LbCas12a in the absence of the activator). b Comparison of trans-cleavage activity between AT-rich extensions and GC-rich 7-nt DNA 3′-end extensions on the crRNA + 3′DNA7. For a , b ), error bars denote mean ± SD, where n = 3 technical replicates. c Trans-cleavage activity of LbCas12a with non-fully phosphorothioate (PS) modified crRNA targeting GFP fragment. Sequence representation of 6 non-fully PS extension on the 3′-end of crGFP ranging from 1 to 6 PS. The asterisk symbol (*) signifies the phosphorothioated nucleotide. The graph below the sequence representation shows fold change of the LbCas12a fluorescence-based reporter assay with the activator normalized to the corresponding samples without the activator at t = 20 min. d kinetics of the LbCas12a fluorescence-based reporter assay in c , e Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. For c , d , and e , n = 6 replicates (three technical replicates examined over two independent experiments), where error bars in e represent mean ± SEM. Source data are available in the Source Data file.
    Figure Legend Snippet: Characterization of ENHANCE with various crRNA modifications and different Cas12a systems. a Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5′-end of the crRNA is cleaved by LbCas12a in the absence of the activator). b Comparison of trans-cleavage activity between AT-rich extensions and GC-rich 7-nt DNA 3′-end extensions on the crRNA + 3′DNA7. For a , b ), error bars denote mean ± SD, where n = 3 technical replicates. c Trans-cleavage activity of LbCas12a with non-fully phosphorothioate (PS) modified crRNA targeting GFP fragment. Sequence representation of 6 non-fully PS extension on the 3′-end of crGFP ranging from 1 to 6 PS. The asterisk symbol (*) signifies the phosphorothioated nucleotide. The graph below the sequence representation shows fold change of the LbCas12a fluorescence-based reporter assay with the activator normalized to the corresponding samples without the activator at t = 20 min. d kinetics of the LbCas12a fluorescence-based reporter assay in c , e Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. For c , d , and e , n = 6 replicates (three technical replicates examined over two independent experiments), where error bars in e represent mean ± SEM. Source data are available in the Source Data file.

    Techniques Used: Activity Assay, Modification, Sequencing, Fluorescence, Reporter Assay

    13) Product Images from "Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection"

    Article Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.036079

    Trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see supplementary figures 1-4. (a) Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3’- or 5’-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. (b) A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. (c) , (d) , and (e) 3’-end ssDNA, ssRNA and phosphorothioate ssDNA extensions of crRNA, respectively. (f) , (g) , and (h) 5’-end ssDNA, ssRNA and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signals of sample with activator to the corresponding sample without activator. Error bars represent ± SEM, where n = 6 replicates; two-way ANOVA test two-way ANOVA (n=3, N=2, ns P > 0.05, *P
    Figure Legend Snippet: Trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested. For other fluorophore-quencher systems, see supplementary figures 1-4. (a) Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3’- or 5’-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. (b) A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. (c) , (d) , and (e) 3’-end ssDNA, ssRNA and phosphorothioate ssDNA extensions of crRNA, respectively. (f) , (g) , and (h) 5’-end ssDNA, ssRNA and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signals of sample with activator to the corresponding sample without activator. Error bars represent ± SEM, where n = 6 replicates; two-way ANOVA test two-way ANOVA (n=3, N=2, ns P > 0.05, *P

    Techniques Used: Activity Assay, Modification, Fluorescence, Reporter Assay

    Mechanism and kinetics of LbCas12a trans-cleavage with modified crGFP. (a) Interactions of fluorescently labeled crRNAs with LbCas12a and dsDNA activator, characterized by PAGE analysis. In the absence of the activator, the modified crRNA (pre-crRNA) is trimmed by LbCas12a on its 5’-end (the first Uracil is cleaved, so-called truncated-crRNA or tru-crRNA). In the presence of the activator, the crRNA extensions are further trimmed, possibly leaving a 3’overhang. (b) Schematic diagram of putative processing of crRNA cleavage sites in the presence and absence of activator GFP. (c) schematic diagram of different cleavage sites in the presence and absence of activator GFP. (c) Enzyme kinetic data of LbCas12a with crGFP vs. crGFP+3’DNA7. (d) Michaelis-Menten kinetic study of the wild-type crGFP vs. crGFP+3’DNA7. For this assay, 100nM of LbCas12a, 100 nM of crRNA, and 7.4 nM of GFP fragment were used. (e) Time-dependent cis-cleavage of LbCas12a on GFP in the presence of nonspecific ssDNA M13mp18. The reaction mixture was taken out every five minutes and quenched with 6X SDS-containing loading dye. (f) Effect of different types of fluorophore-quencher systems on trans-cleavage activity with various modifications of crRNA. (g) Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5’-end of the crRNA is cleaved by LbCas12a in the absence of the activator). (h) Comparison of trans-cleavage activity between AT-rich extensions and GC-rich extensions of the crRNA. (i) Dissociation constants of crGFP vs. crGFP+3’DNA7. The Kd was determined by the biolayer interferometry binding kinetic assay with R 2 > 0.9. (j) Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. (k) Single-point mutations (m1-m20) on the target strand of a dsDNA GFP activator. The heat map displays relative fluorescence intensity normalized to wild-type (WT) activator after 3 hours. Error bars represent ± SEM, where n = 6 replicates. The experiments were repeated at least twice with n = 3 per experiment.
    Figure Legend Snippet: Mechanism and kinetics of LbCas12a trans-cleavage with modified crGFP. (a) Interactions of fluorescently labeled crRNAs with LbCas12a and dsDNA activator, characterized by PAGE analysis. In the absence of the activator, the modified crRNA (pre-crRNA) is trimmed by LbCas12a on its 5’-end (the first Uracil is cleaved, so-called truncated-crRNA or tru-crRNA). In the presence of the activator, the crRNA extensions are further trimmed, possibly leaving a 3’overhang. (b) Schematic diagram of putative processing of crRNA cleavage sites in the presence and absence of activator GFP. (c) schematic diagram of different cleavage sites in the presence and absence of activator GFP. (c) Enzyme kinetic data of LbCas12a with crGFP vs. crGFP+3’DNA7. (d) Michaelis-Menten kinetic study of the wild-type crGFP vs. crGFP+3’DNA7. For this assay, 100nM of LbCas12a, 100 nM of crRNA, and 7.4 nM of GFP fragment were used. (e) Time-dependent cis-cleavage of LbCas12a on GFP in the presence of nonspecific ssDNA M13mp18. The reaction mixture was taken out every five minutes and quenched with 6X SDS-containing loading dye. (f) Effect of different types of fluorophore-quencher systems on trans-cleavage activity with various modifications of crRNA. (g) Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5’-end of the crRNA is cleaved by LbCas12a in the absence of the activator). (h) Comparison of trans-cleavage activity between AT-rich extensions and GC-rich extensions of the crRNA. (i) Dissociation constants of crGFP vs. crGFP+3’DNA7. The Kd was determined by the biolayer interferometry binding kinetic assay with R 2 > 0.9. (j) Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. (k) Single-point mutations (m1-m20) on the target strand of a dsDNA GFP activator. The heat map displays relative fluorescence intensity normalized to wild-type (WT) activator after 3 hours. Error bars represent ± SEM, where n = 6 replicates. The experiments were repeated at least twice with n = 3 per experiment.

    Techniques Used: Modification, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Binding Assay, Kinetic Assay, Fluorescence

    14) Product Images from "Rapid detection of SARS-CoV-2 with CRISPR-Cas12a"

    Article Title: Rapid detection of SARS-CoV-2 with CRISPR-Cas12a

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000978

    Sensitivity and specificity analysis. ( a, b ) Serial dilutions of in vitro–transcribed ORF1ab ( a ) and N ( b ) for LoD determination. After amplification at 42°C for 20 minutes, 10 μL of each reaction was transferred to 40 μL of the Cas12a mixture for cleavage assays. Bar graphs represent fluorescent signal obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD ( n = 15). Unpaired 2-tailed t test was used to analyze the difference from NTC. *** P
    Figure Legend Snippet: Sensitivity and specificity analysis. ( a, b ) Serial dilutions of in vitro–transcribed ORF1ab ( a ) and N ( b ) for LoD determination. After amplification at 42°C for 20 minutes, 10 μL of each reaction was transferred to 40 μL of the Cas12a mixture for cleavage assays. Bar graphs represent fluorescent signal obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD ( n = 15). Unpaired 2-tailed t test was used to analyze the difference from NTC. *** P

    Techniques Used: In Vitro, Amplification

    Primer screening. Primers for ORF1ab ( a–d ) and N ( e–h ) were screened using a single forward or reverse primer against the corresponding reverse or forward primers, selecting the best performing primer and then using it to perform second round screening to determine the primer pairs with the best performance. Fluorescent signal was obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD ( n = 3; b , d , f , h ). Numerical source data underlying this figure can be found in S1 Data . Cas, CRISPR associated proteins; RFU, relative fluorescence unit; SD, standard deviation.
    Figure Legend Snippet: Primer screening. Primers for ORF1ab ( a–d ) and N ( e–h ) were screened using a single forward or reverse primer against the corresponding reverse or forward primers, selecting the best performing primer and then using it to perform second round screening to determine the primer pairs with the best performance. Fluorescent signal was obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD ( n = 3; b , d , f , h ). Numerical source data underlying this figure can be found in S1 Data . Cas, CRISPR associated proteins; RFU, relative fluorescence unit; SD, standard deviation.

    Techniques Used: CRISPR, Fluorescence, Standard Deviation

    Validation of the system with pseudovirus and clinical samples. (a–d) The LoD was determined using the lysed pseudovirus for ORF1ab ( a, b ) and N ( c, d ). Fluorescent signal was obtained at 10 minutes for the Cas12a reaction ( b, d ). Fifteen replicates were conducted for each test. The data are presented as the means ± SD. Unpaired 2-tailed t test was used to analyze the difference from NTC. *** P
    Figure Legend Snippet: Validation of the system with pseudovirus and clinical samples. (a–d) The LoD was determined using the lysed pseudovirus for ORF1ab ( a, b ) and N ( c, d ). Fluorescent signal was obtained at 10 minutes for the Cas12a reaction ( b, d ). Fifteen replicates were conducted for each test. The data are presented as the means ± SD. Unpaired 2-tailed t test was used to analyze the difference from NTC. *** P

    Techniques Used:

    Workflow of Cas12a-based detection for SARS-CoV-2. Extracted RNA or lysed samples can be used as an input for RPA-Cas12a-based detection. ( a ) The results can be achieved within 50 minutes using a fluorescent reader or with visual detection methods. ( b ) The minimum equipment needed to run the assay following RNA extraction or rapid sample lysis includes UV light imagers, heat blocks (42°C and 37°C), pipettes and tips, Eppendorf tubes, reagents, and lateral flow strips. Cas, CRISPR associated proteins; RPA, recombinase polymerase amplification; RT, room temperature; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; UV, ultraviolet.
    Figure Legend Snippet: Workflow of Cas12a-based detection for SARS-CoV-2. Extracted RNA or lysed samples can be used as an input for RPA-Cas12a-based detection. ( a ) The results can be achieved within 50 minutes using a fluorescent reader or with visual detection methods. ( b ) The minimum equipment needed to run the assay following RNA extraction or rapid sample lysis includes UV light imagers, heat blocks (42°C and 37°C), pipettes and tips, Eppendorf tubes, reagents, and lateral flow strips. Cas, CRISPR associated proteins; RPA, recombinase polymerase amplification; RT, room temperature; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; UV, ultraviolet.

    Techniques Used: Recombinase Polymerase Amplification, RNA Extraction, Lysis, CRISPR

    15) 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

    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 "Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria"

    Article Title: Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.2010196117

    Schematic of one-pot SHERLOCK assay. RT-RPA amplifies Plasmodium species target sequences and occurs in parallel with programmed Cas12a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore-labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout using Au-NP, gold nanoparticles.
    Figure Legend Snippet: Schematic of one-pot SHERLOCK assay. RT-RPA amplifies Plasmodium species target sequences and occurs in parallel with programmed Cas12a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore-labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout using Au-NP, gold nanoparticles.

    Techniques Used: Recombinase Polymerase Amplification, Labeling

    18) Product Images from "Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes"

    Article Title: Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1264

    Potential steric clashes between target nucleotide 5-ghmC modifications and CRISPR effector proteins. ( A ) Multiple clashes (indicated in red) are observed between the polypeptide chains of Cascade and 5-ghmC modifications of nucleotides in the target strand (TS, complementary to the crRNA) and non-target strand (NTS). ( B ) Clashes are mostly observed between the polypeptide chains of Cas9 and 5-ghmC modifications of nucleotides in the seed region. ( C ) No clashes are observed in the seed region for Cas12a.
    Figure Legend Snippet: Potential steric clashes between target nucleotide 5-ghmC modifications and CRISPR effector proteins. ( A ) Multiple clashes (indicated in red) are observed between the polypeptide chains of Cascade and 5-ghmC modifications of nucleotides in the target strand (TS, complementary to the crRNA) and non-target strand (NTS). ( B ) Clashes are mostly observed between the polypeptide chains of Cas9 and 5-ghmC modifications of nucleotides in the seed region. ( C ) No clashes are observed in the seed region for Cas12a.

    Techniques Used: CRISPR

    Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

    Techniques Used: CRISPR, Modification, Cleavage Assay, Marker, Electrophoretic Mobility Shift Assay

    19) 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

    20) 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

    Related Articles

    other:

    Article Title: Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA
    Article Snippet: For each target, two Cas9-crRNA were designed to flank the region of interest to be protected and two Cas12a-crRNA at least 100 bases away from the Cas9-crRNA position. (The complete list of crRNA is available in Supplementary Data ).

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    In Vitro:

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020
    Article Snippet: At the DNMT1-3 target, Lb2-KY activity was slightly less than that of As-Ultra. .. The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D). .. Note that it is not clear that this trans-cleavage activity is deleterious in physiological settings, because ssDNA in replication bubbles and homologous recombination complexes are protected by DNA-binding proteins, , , and because LbCas12a-mediated HDR with ssDNA repair templates is as efficient as that mediated by AsCas12a.

    Activity Assay:

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020
    Article Snippet: At the DNMT1-3 target, Lb2-KY activity was slightly less than that of As-Ultra. .. The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D). .. Note that it is not clear that this trans-cleavage activity is deleterious in physiological settings, because ssDNA in replication bubbles and homologous recombination complexes are protected by DNA-binding proteins, , , and because LbCas12a-mediated HDR with ssDNA repair templates is as efficient as that mediated by AsCas12a.

    Concentration Assay:

    Article Title: Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology
    Article Snippet: Cas12a-mediated transcleavage of the ssDNA reporter, for the purpose of fluorescence detection, was carried out at room temperature (approximately 23 °C) for 10 min. .. The EnGen Lba Cas12a enzyme (NEB) at 1 μM concentration was preincubated with 1.25 μM gRNA in 1× NEBuffer 2.1 (NEB) to form the ribonucleoprotein (RNP) complex. .. An aliquot of the RNP complex solution was placed inside the cap of a PCR tube.

    CRISPR:

    Article Title: Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens
    Article Snippet: Targeted genes of SARS-CoV-2 were amplified by incubating at 39 °C for 30 min, then the reaction was inactivated at 75 °C for 5 min. .. The CRISPR-Cas12a detection method consists of 1X NEBuffer 2.0 (New England Biolabs, USA), 30 nM crRNA, 330 nM EnGen® Lba Cas12a endonuclease (New England Biolabs, USA), 200 nM 5' 6-FAM / 3' BHQ-1®, Dual Labeled Fluorescent Probe and DEPC-treated water in a total reaction volume of 15 µl. .. The amplified products were added in a CRISPR-Cas12a reaction and then incubated at 39 °C for 15 min.

    Article Title: Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus
    Article Snippet: Finally, crRNAs were quantified using the Nanodrop 2000C (Thermo Fisher Scientific) and stored at − 80 °C. .. CRISPR-Cas12a assay The detection of ASFV by probe-based qPCR has been described previously [ – ]. .. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay.

    Labeling:

    Article Title: Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens
    Article Snippet: Targeted genes of SARS-CoV-2 were amplified by incubating at 39 °C for 30 min, then the reaction was inactivated at 75 °C for 5 min. .. The CRISPR-Cas12a detection method consists of 1X NEBuffer 2.0 (New England Biolabs, USA), 30 nM crRNA, 330 nM EnGen® Lba Cas12a endonuclease (New England Biolabs, USA), 200 nM 5' 6-FAM / 3' BHQ-1®, Dual Labeled Fluorescent Probe and DEPC-treated water in a total reaction volume of 15 µl. .. The amplified products were added in a CRISPR-Cas12a reaction and then incubated at 39 °C for 15 min.

    Expressing:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Plasmid Preparation:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Polymerase Chain Reaction:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Amplification:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Real-time Polymerase Chain Reaction:

    Article Title: Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus
    Article Snippet: Finally, crRNAs were quantified using the Nanodrop 2000C (Thermo Fisher Scientific) and stored at − 80 °C. .. CRISPR-Cas12a assay The detection of ASFV by probe-based qPCR has been described previously [ – ]. .. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay.

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    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.
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    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

    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.

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2021.02.012

    Figure Lengend 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.

    Article Snippet: The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D).

    Techniques: 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

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2021.02.012

    Figure Lengend 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

    Article Snippet: The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D).

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

    Time courses of fluorescence detection with different concentrations of LbaCas12a: crRNA: ssDNA-FQ reporter to target M ( A ), N ( B ), and S ( C ) of SARS-CoV-2, respectively. The condition of 640 nM LbaCas12a: 640 nM crRNA: 800 nM FAM quencher reporter had significantly increased fluorescence signals for quickly detecting the genes compared with the other conditions.

    Journal: Biomedicines

    Article Title: Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

    doi: 10.3390/biomedicines9030239

    Figure Lengend Snippet: Time courses of fluorescence detection with different concentrations of LbaCas12a: crRNA: ssDNA-FQ reporter to target M ( A ), N ( B ), and S ( C ) of SARS-CoV-2, respectively. The condition of 640 nM LbaCas12a: 640 nM crRNA: 800 nM FAM quencher reporter had significantly increased fluorescence signals for quickly detecting the genes compared with the other conditions.

    Article Snippet: EnGen® LbaCas12a (New England Biolabs, Ipswich, MA, USA) was pre-assembled with each crRNA, respectively, for 30 min and mixed with custom ssDNA-FQ reporter (IDT) ( ).

    Techniques: Fluorescence

    Overview of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a assay to detect SARS-CoV-2. RNA or raw sample was subjected to RT-recombinase polymerase amplification reaction (RT-RPA). The RT-RPA product was mixed with LbaCas12a, CRISPR RNAs (crRNA), and single stranded DNA-FAM-quencher (ssDNA-FQ) reporter. Reaction was performed at 42 °C. Results are read by a fluorescence plate reader, a UV light illuminator, or a paper strip.

    Journal: Biomedicines

    Article Title: Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

    doi: 10.3390/biomedicines9030239

    Figure Lengend Snippet: Overview of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a assay to detect SARS-CoV-2. RNA or raw sample was subjected to RT-recombinase polymerase amplification reaction (RT-RPA). The RT-RPA product was mixed with LbaCas12a, CRISPR RNAs (crRNA), and single stranded DNA-FAM-quencher (ssDNA-FQ) reporter. Reaction was performed at 42 °C. Results are read by a fluorescence plate reader, a UV light illuminator, or a paper strip.

    Article Snippet: EnGen® LbaCas12a (New England Biolabs, Ipswich, MA, USA) was pre-assembled with each crRNA, respectively, for 30 min and mixed with custom ssDNA-FQ reporter (IDT) ( ).

    Techniques: CRISPR, Recombinase Polymerase Amplification, Fluorescence, Stripping Membranes

    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

    Journal: bioRxiv

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

    doi: 10.1101/2020.05.22.110494

    Figure Lengend 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

    Article Snippet: For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ).

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.05.22.110494

    Figure Lengend 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.

    Article Snippet: For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ).

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.05.22.110494

    Figure Lengend 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.

    Article Snippet: For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ).

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.05.22.110494

    Figure Lengend 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

    Article Snippet: For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ).

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.05.22.110494

    Figure Lengend 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.

    Article Snippet: For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ).

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