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  • 86
    New England Biolabs cas12a
    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to <t>Cas12a.</t> The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Cas12a, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    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, 2021-06
    86/100 stars
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    99
    New England Biolabs engen lba cas12a cpf1
    Optimization of TESTOR system. a , Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to the N0 gene at the indicated concentration. b , Reporters with A, T, G, or C nucleotide sequence was screened to identify the one with the best affinity to <t>Cas12a.</t> The same amount of RPA product of N0 gene was added to a Cas12a mixture with different reporter, and fluorescence was monitored by real-time or taken at 30 min after incubation at 37 °C. c , Primers modified with phosphorothioate on different phosphate backbones were compared for reaction efficiency by real-time (left panel) or endpoint (right panel) method. d , TESTOR approach for detection of ORF1ab gene of SARS-CoV-2. e , Fluorescence kinetics of Cas12a cleavage using product of RPA for ORF1ab gene as input. f , Quantification of the fluorescence intensity of TESTOR method or routine two-step method (from Fig. 2 d and 2e) after 30 min of incubation at 37 °C. g , h , Determination of LoDs for N0 ( g ) and ORF1ab ( h ) genes using the optimized conditions for TESTOR system. Representative plot of fluorescence intensity over time for N0 and ORF1ab genes of SARS-CoV-2 (left panel) or fluorescent signal was taken at 30 min after reaction (right panel). Error bars represent the mean ± s.d., where n = 3–6 replicates ( a , b, c, f, g, h ). ***, P
    Engen Lba Cas12a Cpf1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/engen lba cas12a cpf1/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    engen lba cas12a cpf1 - by Bioz Stars, 2021-06
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    97
    Integrated DNA Technologies cas12a
    Steric interference by a proximal nuclease enhances nucleosomal DNA cleavage. ( A ) Diagram depicting dCas9 (light blue arrows) and <t>Cas12a</t> (color-coded arrows) targets. Only targets discussed in the main text are included here; others are shown in fig. S3. ( B ) Proxy-CRISPR reaction scheme. ( C ) Proxy-CRISPR nucleosome cleavage rates for indicated dCas9 and Cas12a pairs. n.a., original nucleosome cleavage rate without dCas9 prebound. Data points are normalized to the DNA cleavage rate of each target. Each data point is the mean of three replicates; error bars: SEM. ( D ) Sp Cas9 [PDB: 4UN3 ( 81 )] modeled to bind target 5p, 13 bp away from the nucleosome [PDB: 3LZ0 ( 80 )]. Linker DNA was generated using sequence to structure modeling. Extension of the distal linker DNA shows a steric clash with the bound Cas9.
    Cas12a, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    cas12a - by Bioz Stars, 2021-06
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    93
    Addgene inc cas12a
    PAM preference for FnCas12a and genome editing at additional targets. ( A ) Yeast strains containing same gRNA targets but alternative PAMs with synonymous mutations were tested to measure the genome editing efficiency. ( B ) Transformants from parents strains with the PAM sequence CTTA compared to TTTA. ( C ) <t>Cas12a</t> editing efficiency targeting leu1 , his3 and lys9 , with mean values ± S.E.M. shown here. The original colony counts are listed in Supplementary Table S7 .
    Cas12a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas12a/product/Addgene inc
    Average 93 stars, based on 1 article reviews
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    cas12a - by Bioz Stars, 2021-06
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    Standard format Plasmid sent in bacteria as agar stab
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    Standard format Plasmid sent in bacteria as agar stab
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    Standard format Plasmid sent in bacteria as agar stab
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    Image Search Results


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

    Journal: Bioactive Materials

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

    doi: 10.1016/j.bioactmat.2021.05.005

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

    Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].

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

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

    Journal: Bioactive Materials

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

    doi: 10.1016/j.bioactmat.2021.05.005

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

    Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].

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

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

    Journal: Bioactive Materials

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

    doi: 10.1016/j.bioactmat.2021.05.005

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

    Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].

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

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

    Journal: Bioactive Materials

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

    doi: 10.1016/j.bioactmat.2021.05.005

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

    Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].

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

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

    Journal: Bioactive Materials

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

    doi: 10.1016/j.bioactmat.2021.05.005

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

    Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].

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

    Steric interference by a proximal nuclease enhances nucleosomal DNA cleavage. ( A ) Diagram depicting dCas9 (light blue arrows) and Cas12a (color-coded arrows) targets. Only targets discussed in the main text are included here; others are shown in fig. S3. ( B ) Proxy-CRISPR reaction scheme. ( C ) Proxy-CRISPR nucleosome cleavage rates for indicated dCas9 and Cas12a pairs. n.a., original nucleosome cleavage rate without dCas9 prebound. Data points are normalized to the DNA cleavage rate of each target. Each data point is the mean of three replicates; error bars: SEM. ( D ) Sp Cas9 [PDB: 4UN3 ( 81 )] modeled to bind target 5p, 13 bp away from the nucleosome [PDB: 3LZ0 ( 80 )]. Linker DNA was generated using sequence to structure modeling. Extension of the distal linker DNA shows a steric clash with the bound Cas9.

    Journal: Science Advances

    Article Title: Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

    doi: 10.1126/sciadv.abd6030

    Figure Lengend Snippet: Steric interference by a proximal nuclease enhances nucleosomal DNA cleavage. ( A ) Diagram depicting dCas9 (light blue arrows) and Cas12a (color-coded arrows) targets. Only targets discussed in the main text are included here; others are shown in fig. S3. ( B ) Proxy-CRISPR reaction scheme. ( C ) Proxy-CRISPR nucleosome cleavage rates for indicated dCas9 and Cas12a pairs. n.a., original nucleosome cleavage rate without dCas9 prebound. Data points are normalized to the DNA cleavage rate of each target. Each data point is the mean of three replicates; error bars: SEM. ( D ) Sp Cas9 [PDB: 4UN3 ( 81 )] modeled to bind target 5p, 13 bp away from the nucleosome [PDB: 3LZ0 ( 80 )]. Linker DNA was generated using sequence to structure modeling. Extension of the distal linker DNA shows a steric clash with the bound Cas9.

    Article Snippet: Nucleic acids and proteins All DNA oligos and GeneBlocks were purchased from Integrated DNA Technologies (IDT) (table S1). crRNA for Cas12a and single guide RNA (sgRNA) for Cas9 were purchased from IDT or Synthego (table S1).

    Techniques: CRISPR, Generated, Sequencing

    Nucleosomes inhibit Cas12a’s two-step DNA binding. ( A ) Concentration-dependent Cas12a cleavage plots of DNA (dark triangles) and nucleosome substrates (light circles). Colored lines: Hyperbolic curve fitting. Second-order rate constants for each target are written below. The asterisk indicates that the target 5 DNA cleavage second-order rate constant is a lower limit due to lack of data points in the concentration-dependent phase. Each data point is the mean of at least three replicates; error bars: SEM. ( B ) Dissociation rates determined by electrophoretic mobility shift assay (fig. S2). Data points represent at least duplicates; error bars: SEM. ( C ) Diagrams showing the calculated nucleosomal inhibition on Cas12a PAM recognition and R-loop formation (fig. S2C). K PAM (Cas12a affinity for the targets’ PAM) increases and k R-loop (rate of R-loop formation) decreases due to the nucleosome.

    Journal: Science Advances

    Article Title: Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

    doi: 10.1126/sciadv.abd6030

    Figure Lengend Snippet: Nucleosomes inhibit Cas12a’s two-step DNA binding. ( A ) Concentration-dependent Cas12a cleavage plots of DNA (dark triangles) and nucleosome substrates (light circles). Colored lines: Hyperbolic curve fitting. Second-order rate constants for each target are written below. The asterisk indicates that the target 5 DNA cleavage second-order rate constant is a lower limit due to lack of data points in the concentration-dependent phase. Each data point is the mean of at least three replicates; error bars: SEM. ( B ) Dissociation rates determined by electrophoretic mobility shift assay (fig. S2). Data points represent at least duplicates; error bars: SEM. ( C ) Diagrams showing the calculated nucleosomal inhibition on Cas12a PAM recognition and R-loop formation (fig. S2C). K PAM (Cas12a affinity for the targets’ PAM) increases and k R-loop (rate of R-loop formation) decreases due to the nucleosome.

    Article Snippet: Nucleic acids and proteins All DNA oligos and GeneBlocks were purchased from Integrated DNA Technologies (IDT) (table S1). crRNA for Cas12a and single guide RNA (sgRNA) for Cas9 were purchased from IDT or Synthego (table S1).

    Techniques: Binding Assay, Concentration Assay, Electrophoretic Mobility Shift Assay, Inhibition

    DNA unwrapping regulates Cas12a cleavage of nucleosomal targets. ( A ) The Widom 601 positioning sequence is divided into quartiles indicating the inner (II and III) and outer (I and IV) wrap and is flanked by DNA. “****”: four TA dinucleotide repeats that produce tight wrapping of the inner left quartile. Arrows: Cas12a targets, pointing in the direction of R-loop formation; for top arrows, R-loop forms with the Crick (bottom) strand, and for bottom arrows, R-loop forms with the Watson (top) strand. ( B ) Cleavage reaction setup. nuc, nucleosome substrate. ( C ) Representative gels of target 4 cleavage by Cas12a. ( D ) Cleavage rates for the six DNA and nucleosome Cas12a targets at 25°C. Downward arrows signify that the value is an upper limit due to the lack of detectable cleavage. ( E ) Top: Diagram depicting variant 601 constructs (fig. S1E). Bottom: Cas12a cleavage of variant nucleosome substrates normalized to original 601 nucleosome substrate. ( F ) Top: Crystal structure of the 601 nucleosome [Protein Data Bank (PDB): 3LZ0 ( 80 )] highlighting the amino acid modifications H3Y41E and H3K56Q, which mimic H3Y41ph and H3K56ac. Bottom: Cleavage rates of H3 mutant nucleosome normalized to the original wt nucleosome. n.d., no data, as no cleavage was observed for targets 3 and 4 for all nucleosome substrates. (D to F) Each data point is the mean of at least three replicates; error bars: SEM.

    Journal: Science Advances

    Article Title: Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

    doi: 10.1126/sciadv.abd6030

    Figure Lengend Snippet: DNA unwrapping regulates Cas12a cleavage of nucleosomal targets. ( A ) The Widom 601 positioning sequence is divided into quartiles indicating the inner (II and III) and outer (I and IV) wrap and is flanked by DNA. “****”: four TA dinucleotide repeats that produce tight wrapping of the inner left quartile. Arrows: Cas12a targets, pointing in the direction of R-loop formation; for top arrows, R-loop forms with the Crick (bottom) strand, and for bottom arrows, R-loop forms with the Watson (top) strand. ( B ) Cleavage reaction setup. nuc, nucleosome substrate. ( C ) Representative gels of target 4 cleavage by Cas12a. ( D ) Cleavage rates for the six DNA and nucleosome Cas12a targets at 25°C. Downward arrows signify that the value is an upper limit due to the lack of detectable cleavage. ( E ) Top: Diagram depicting variant 601 constructs (fig. S1E). Bottom: Cas12a cleavage of variant nucleosome substrates normalized to original 601 nucleosome substrate. ( F ) Top: Crystal structure of the 601 nucleosome [Protein Data Bank (PDB): 3LZ0 ( 80 )] highlighting the amino acid modifications H3Y41E and H3K56Q, which mimic H3Y41ph and H3K56ac. Bottom: Cleavage rates of H3 mutant nucleosome normalized to the original wt nucleosome. n.d., no data, as no cleavage was observed for targets 3 and 4 for all nucleosome substrates. (D to F) Each data point is the mean of at least three replicates; error bars: SEM.

    Article Snippet: Nucleic acids and proteins All DNA oligos and GeneBlocks were purchased from Integrated DNA Technologies (IDT) (table S1). crRNA for Cas12a and single guide RNA (sgRNA) for Cas9 were purchased from IDT or Synthego (table S1).

    Techniques: Sequencing, Variant Assay, Construct, Mutagenesis

    Phase-separated nucleosome arrays minimally inhibit DNA accessibility. ( A ) Diagram of a 12-mer 601 array. Gray boxes: 601 positioning sequences; black lines: unique linker DNAs. Cas12a targets are highlighted (L4/5, magenta; Ex5, green; L8/9, cyan), and the associated arrows represent direction of R-loop formation. ( B ) Phase-separated nucleosome array droplets colocalize with dCas12a-L4/5. Histone H2B is labeled with AF594; L4/5 crRNA is labeled with AF488. Scale bar, 10 μm. ( C ) Reaction scheme depicting Cas12a cleavage of DNA arrays or phase-separated nucleosome arrays. ( D ) Representative agarose gel showing Cas12a cleavage of phase-separated nucleosome arrays, detected by ethidium bromide. All reactions are from the same gel; the black line represents cropping of the gel. ( E ) Cas12a cleavage plot of DNA and nucleosome arrays. Data points are color-coordinated with (A). Rates are included in table S5. ( F ) Concentration-dependent Cas12a cleavage of L4/5, targeting DNA, and nucleosome arrays. (E and F) Each data point represents the mean of at least three replicates (exception: DNA cleavage at 30 and 200 nM Cas12a was done in duplicate). Error bars: SEM. ( G ) Model of Cas12a cleavage inhibition by the nucleosome and chromatin. Chromatin has a small impact on Cas12a cleavage efficiency, which can be attributed to neighboring nucleosomes. The nucleosome inhibits Cas12a binding to wrapped DNA.

    Journal: Science Advances

    Article Title: Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

    doi: 10.1126/sciadv.abd6030

    Figure Lengend Snippet: Phase-separated nucleosome arrays minimally inhibit DNA accessibility. ( A ) Diagram of a 12-mer 601 array. Gray boxes: 601 positioning sequences; black lines: unique linker DNAs. Cas12a targets are highlighted (L4/5, magenta; Ex5, green; L8/9, cyan), and the associated arrows represent direction of R-loop formation. ( B ) Phase-separated nucleosome array droplets colocalize with dCas12a-L4/5. Histone H2B is labeled with AF594; L4/5 crRNA is labeled with AF488. Scale bar, 10 μm. ( C ) Reaction scheme depicting Cas12a cleavage of DNA arrays or phase-separated nucleosome arrays. ( D ) Representative agarose gel showing Cas12a cleavage of phase-separated nucleosome arrays, detected by ethidium bromide. All reactions are from the same gel; the black line represents cropping of the gel. ( E ) Cas12a cleavage plot of DNA and nucleosome arrays. Data points are color-coordinated with (A). Rates are included in table S5. ( F ) Concentration-dependent Cas12a cleavage of L4/5, targeting DNA, and nucleosome arrays. (E and F) Each data point represents the mean of at least three replicates (exception: DNA cleavage at 30 and 200 nM Cas12a was done in duplicate). Error bars: SEM. ( G ) Model of Cas12a cleavage inhibition by the nucleosome and chromatin. Chromatin has a small impact on Cas12a cleavage efficiency, which can be attributed to neighboring nucleosomes. The nucleosome inhibits Cas12a binding to wrapped DNA.

    Article Snippet: Nucleic acids and proteins All DNA oligos and GeneBlocks were purchased from Integrated DNA Technologies (IDT) (table S1). crRNA for Cas12a and single guide RNA (sgRNA) for Cas9 were purchased from IDT or Synthego (table S1).

    Techniques: Labeling, Agarose Gel Electrophoresis, Concentration Assay, Inhibition, Binding Assay

    PAM preference for FnCas12a and genome editing at additional targets. ( A ) Yeast strains containing same gRNA targets but alternative PAMs with synonymous mutations were tested to measure the genome editing efficiency. ( B ) Transformants from parents strains with the PAM sequence CTTA compared to TTTA. ( C ) Cas12a editing efficiency targeting leu1 , his3 and lys9 , with mean values ± S.E.M. shown here. The original colony counts are listed in Supplementary Table S7 .

    Journal: Nucleic Acids Research

    Article Title: CRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference

    doi: 10.1093/nar/gkaa329

    Figure Lengend Snippet: PAM preference for FnCas12a and genome editing at additional targets. ( A ) Yeast strains containing same gRNA targets but alternative PAMs with synonymous mutations were tested to measure the genome editing efficiency. ( B ) Transformants from parents strains with the PAM sequence CTTA compared to TTTA. ( C ) Cas12a editing efficiency targeting leu1 , his3 and lys9 , with mean values ± S.E.M. shown here. The original colony counts are listed in Supplementary Table S7 .

    Article Snippet: The entry vectors for CRISPR genome editing with Cas12a were deposited to Addgene as accession number #132952 for pYZ221 and #132953 for pYZ673.

    Techniques: Sequencing

    Cas12a genome editing system. ( A ) FnCas12a and crRNA were expressed from a single plasmid with ura4 marker and ars1 replicating origin in fission yeast. As the first construct, FnCas12a and gRNA were expressed with adh1 and rrk1 promoter, respectively. ( B ) To test editing efficiency, we picked four gRNAs targeting the endogenous ade6 + CDS. One linear donor DNA was co-transformed to delete ade6 by homologous recombination. ( C ) The gRNA sequences used and their editing efficiency, with mean values ± S.E.M. The original colony counts are listed in Supplementary Table S6 . ( D ) Colony PCR to check the ade6 deletion, in red colonies and white colonies from plates with PMG5–Ura w/ low adenine media. The 1 kb Plus DNA ladder from NEB (Catalog# N3200L) was used as the molecular weight standard. ( E ) Spot assay on plates to check adenine auxotroph.

    Journal: Nucleic Acids Research

    Article Title: CRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference

    doi: 10.1093/nar/gkaa329

    Figure Lengend Snippet: Cas12a genome editing system. ( A ) FnCas12a and crRNA were expressed from a single plasmid with ura4 marker and ars1 replicating origin in fission yeast. As the first construct, FnCas12a and gRNA were expressed with adh1 and rrk1 promoter, respectively. ( B ) To test editing efficiency, we picked four gRNAs targeting the endogenous ade6 + CDS. One linear donor DNA was co-transformed to delete ade6 by homologous recombination. ( C ) The gRNA sequences used and their editing efficiency, with mean values ± S.E.M. The original colony counts are listed in Supplementary Table S6 . ( D ) Colony PCR to check the ade6 deletion, in red colonies and white colonies from plates with PMG5–Ura w/ low adenine media. The 1 kb Plus DNA ladder from NEB (Catalog# N3200L) was used as the molecular weight standard. ( E ) Spot assay on plates to check adenine auxotroph.

    Article Snippet: The entry vectors for CRISPR genome editing with Cas12a were deposited to Addgene as accession number #132952 for pYZ221 and #132953 for pYZ673.

    Techniques: Plasmid Preparation, Marker, Construct, Transformation Assay, Homologous Recombination, Polymerase Chain Reaction, Molecular Weight, Spot Test

    Expression of Cas12a crRNA using pol II promoters. ( A ) New constructs were designed using pol II promoters and terminators to express crRNA. ( B ) crRNA release from primary RNA transcripts. ( C ) New endogenous and exogenous pol II promoters tested and their editing efficiency targeting ade6 + . The same gRNA sequence (gRNA-3) was used here. Error bars represent mean ± S.E.M. using three technical replicates. An unpaired t -test was used to assess the significance of higher efficiency using nmt1 or fba1 promoter compared to the rrk1 promoter (** P

    Journal: Nucleic Acids Research

    Article Title: CRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference

    doi: 10.1093/nar/gkaa329

    Figure Lengend Snippet: Expression of Cas12a crRNA using pol II promoters. ( A ) New constructs were designed using pol II promoters and terminators to express crRNA. ( B ) crRNA release from primary RNA transcripts. ( C ) New endogenous and exogenous pol II promoters tested and their editing efficiency targeting ade6 + . The same gRNA sequence (gRNA-3) was used here. Error bars represent mean ± S.E.M. using three technical replicates. An unpaired t -test was used to assess the significance of higher efficiency using nmt1 or fba1 promoter compared to the rrk1 promoter (** P

    Article Snippet: The entry vectors for CRISPR genome editing with Cas12a were deposited to Addgene as accession number #132952 for pYZ221 and #132953 for pYZ673.

    Techniques: Expressing, Construct, Sequencing