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s muelleri cas9 121541 expression constructs  (Addgene inc)


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    Structured Review

    Addgene inc s muelleri cas9 121541 expression constructs
    AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the <t>Cas9</t> sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.
    S Muelleri Cas9 121541 Expression Constructs, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s muelleri cas9 121541 expression constructs/product/Addgene inc
    Average 93 stars, based on 2 article reviews
    s muelleri cas9 121541 expression constructs - by Bioz Stars, 2026-03
    93/100 stars

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    1) Product Images from "AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain"

    Article Title: AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae357

    AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the Cas9 sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.
    Figure Legend Snippet: AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the Cas9 sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.

    Techniques Used: SDS Page, Produced, Migration, Marker, Clear Native PAGE, Titration, In Vitro, CRISPR, Activity Assay, Staining

    Biding mode analysis indicated that the AcrIIA28 residues K7, W45 and S81 are critical for AcrIIA28 binding to Cas9. ( A ) The docking model of AcrIIA28 docked onto SpyCas9. Cas9 and docked AcrIIA28 (orange color) are presented by surface and cartoon models, respectively. ( B ) Analysis of PPI detail from the docking model of AcrIIA28/SpyCas9 complex. Four PPI regions are independently magnified. The residues involved in the interaction are labeled. ( C ) Interaction analysis between SpyCas9 and various tentative PPI-disrupting mutants of AcrIIA28 by SEC followed by SDS-PAGE analysis. SDS-PAGE gels produced by loading one of the main fractions from the SpyCas9 sample with wild-type or various mutants are shown. A black arrow indicates acrIIA28 mutants that co-migrated with SpyCas9. ( D ) The bar chart shows the quantified intensity of the co-eluted AcrII28 mutants. Data are presented as the mean ± standard deviation from three independent experiments. ( E ) Interaction analysis between SpyREC3 and three PPI-disrupting mutants (K7D, W45I and S81W) of AcrIIA28 by SEC followed by SDS-PAGE analysis.
    Figure Legend Snippet: Biding mode analysis indicated that the AcrIIA28 residues K7, W45 and S81 are critical for AcrIIA28 binding to Cas9. ( A ) The docking model of AcrIIA28 docked onto SpyCas9. Cas9 and docked AcrIIA28 (orange color) are presented by surface and cartoon models, respectively. ( B ) Analysis of PPI detail from the docking model of AcrIIA28/SpyCas9 complex. Four PPI regions are independently magnified. The residues involved in the interaction are labeled. ( C ) Interaction analysis between SpyCas9 and various tentative PPI-disrupting mutants of AcrIIA28 by SEC followed by SDS-PAGE analysis. SDS-PAGE gels produced by loading one of the main fractions from the SpyCas9 sample with wild-type or various mutants are shown. A black arrow indicates acrIIA28 mutants that co-migrated with SpyCas9. ( D ) The bar chart shows the quantified intensity of the co-eluted AcrII28 mutants. Data are presented as the mean ± standard deviation from three independent experiments. ( E ) Interaction analysis between SpyREC3 and three PPI-disrupting mutants (K7D, W45I and S81W) of AcrIIA28 by SEC followed by SDS-PAGE analysis.

    Techniques Used: Binding Assay, Labeling, SDS Page, Produced, Standard Deviation

    Direct interaction of AcrIIA28 is critical for Cas9 inhibition. ( A ) In vitro target DNA cleavage assay using wild-type Cas9 and various AcrIIA28 mutants. In vitro anti-CRISPR activity assay using various tentative PPI-disrupting mutants of AcrIIA28. In the enzyme reaction, + and – indicate added and not added, respectively. ( B ) Quantitative histogram of anti-Cas9 activity of AcrIIA28 according to (A). The inhibitory activity of wild-type AcrIIA28 was considered to be 100%. Data are presented as the mean ± standard deviation from three independent experiments.
    Figure Legend Snippet: Direct interaction of AcrIIA28 is critical for Cas9 inhibition. ( A ) In vitro target DNA cleavage assay using wild-type Cas9 and various AcrIIA28 mutants. In vitro anti-CRISPR activity assay using various tentative PPI-disrupting mutants of AcrIIA28. In the enzyme reaction, + and – indicate added and not added, respectively. ( B ) Quantitative histogram of anti-Cas9 activity of AcrIIA28 according to (A). The inhibitory activity of wild-type AcrIIA28 was considered to be 100%. Data are presented as the mean ± standard deviation from three independent experiments.

    Techniques Used: Inhibition, In Vitro, DNA Cleavage Assay, CRISPR, Activity Assay, Standard Deviation

    Proposed model of Cas9 inhibition by AcrIIA28. ( A and B ) EMSA analysis of the effect of AcrIIA28 on the binding of target DNA into Cas9. The number next to each protein indicates the order in which the proteins were added to form the complex. The red star indicates the band newly produced by the RNP/DNA complex. The experiments were performed three times with similar results. ( C and D ) An in vitro anti-CRISPR activity assay was used to compare the inhibitory effect of AcrIIA28 when added before (C) or after (D) the target DNA. Polyacrylamide gels (4%) were stained with SYBR GOLD. The amount of AcrIIA28 added to the reaction is indicated. The numbers next to the agents used in the experiment indicate the order in which agents were added to the reaction: one represents the sample added first, and three represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. ( E ) Proposed model of Cas9 inhibition by AcrIIA28.
    Figure Legend Snippet: Proposed model of Cas9 inhibition by AcrIIA28. ( A and B ) EMSA analysis of the effect of AcrIIA28 on the binding of target DNA into Cas9. The number next to each protein indicates the order in which the proteins were added to form the complex. The red star indicates the band newly produced by the RNP/DNA complex. The experiments were performed three times with similar results. ( C and D ) An in vitro anti-CRISPR activity assay was used to compare the inhibitory effect of AcrIIA28 when added before (C) or after (D) the target DNA. Polyacrylamide gels (4%) were stained with SYBR GOLD. The amount of AcrIIA28 added to the reaction is indicated. The numbers next to the agents used in the experiment indicate the order in which agents were added to the reaction: one represents the sample added first, and three represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. ( E ) Proposed model of Cas9 inhibition by AcrIIA28.

    Techniques Used: Inhibition, Binding Assay, Produced, In Vitro, CRISPR, Activity Assay, Staining



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    s muelleri cas9 121541 expression constructs  (Addgene inc)
    93
    Addgene inc s muelleri cas9 121541 expression constructs
    AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the <t>Cas9</t> sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.
    S Muelleri Cas9 121541 Expression Constructs, 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/s muelleri cas9 121541 expression constructs/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    s muelleri cas9 121541 expression constructs - by Bioz Stars, 2026-03
    93/100 stars
    		  Buy from Supplier

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    AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the Cas9 sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.

    Journal: Nucleic Acids Research

    Article Title: AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

    doi: 10.1093/nar/gkae357

    Figure Lengend Snippet: AcrIIA28 directly binds to only SpyCas9 via the REC3 domain. ( A ) Interaction analysis between AcrIIA28 and various Cas9s by SEC followed by SDS-PAGE analysis. SDS-PAGE gels are produced by loading one of the main fractions from the Cas9 sample with (+AcrIIA28) or without (-AcrIIA28) AcrIIA28. The black arrow indicates the co-migration of AcrIIA28 with Cas9. ( B ) Interaction analysis between AcrIIA28 and SpyCas9 by SEC followed by SDS-PAGE analysis. SEC profiles produced by AcrIIA28 (black line), SpyCas9 (blue line), and the mixture of AcrIIA28 and SpyCas9 (red line) are shown. SDS-PAGE gels produced by SpyCas9 alone and the mixture of AcrIIA28 and SpyCas9 are provided under the SEC profile. The red dot box on the SDS-PAGE gel indicates the co-migrated AcrIIA28 bands. Black lines indicate loaded fractions of SpyCas9/AcrIIA28 mixture for SDS-PAGE. M indicates protein size marker. ( C ) Native PAGE of SpyCas9, AcrIIA28 and SpyCas9 + AcrIIA28 mixture. The red arrow indicates a newly produced band that a complex might form. ( D ) Domain organization of SpyCas9. ( E ) Interaction analysis between AcrIIA28 and SpyREC3 domain by SEC. SEC profiles produced by AcrIIA28 (black line), SpyREC3 domain (blue line), and the mixture of AcrIIA28 and SpyREC3 domain (red line) are shown. SDS-PAGE gel produced by the AcrIIA28 and SpyREC3 domain mixture is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by the black bar. The black arrow indicated peak movement on the SEC profile. ( F ) ITC experiment showing titration of AcrIIA28 into a SpyREC3 solution. The raw calorimetric titration data are shown in the upper panel, and experimental fitting of the data to a single site interaction model is shown in the lower panel. ( G ) An in vitro anti-CRISPR activity assay of AcrIIA28 against SauCas9. Polyacrylamide gels (4%) were stained with SYBR GOLD. The numbers indicate the order in which the agents were added to the reaction: one represents the sample added first, and four represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. All the experiments for figure were performed three times with similar results.

    Article Snippet: The S. pyogenes Cas9 (#62731), N. meningitides Cas9 (#71474), H. parainfluenzae Cas9 (#121540) and S. muelleri Cas9 (#121541) expression constructs were purchased from Addgene.

    Techniques: SDS Page, Produced, Migration, Marker, Clear Native PAGE, Titration, In Vitro, CRISPR, Activity Assay, Staining

    Biding mode analysis indicated that the AcrIIA28 residues K7, W45 and S81 are critical for AcrIIA28 binding to Cas9. ( A ) The docking model of AcrIIA28 docked onto SpyCas9. Cas9 and docked AcrIIA28 (orange color) are presented by surface and cartoon models, respectively. ( B ) Analysis of PPI detail from the docking model of AcrIIA28/SpyCas9 complex. Four PPI regions are independently magnified. The residues involved in the interaction are labeled. ( C ) Interaction analysis between SpyCas9 and various tentative PPI-disrupting mutants of AcrIIA28 by SEC followed by SDS-PAGE analysis. SDS-PAGE gels produced by loading one of the main fractions from the SpyCas9 sample with wild-type or various mutants are shown. A black arrow indicates acrIIA28 mutants that co-migrated with SpyCas9. ( D ) The bar chart shows the quantified intensity of the co-eluted AcrII28 mutants. Data are presented as the mean ± standard deviation from three independent experiments. ( E ) Interaction analysis between SpyREC3 and three PPI-disrupting mutants (K7D, W45I and S81W) of AcrIIA28 by SEC followed by SDS-PAGE analysis.

    Journal: Nucleic Acids Research

    Article Title: AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

    doi: 10.1093/nar/gkae357

    Figure Lengend Snippet: Biding mode analysis indicated that the AcrIIA28 residues K7, W45 and S81 are critical for AcrIIA28 binding to Cas9. ( A ) The docking model of AcrIIA28 docked onto SpyCas9. Cas9 and docked AcrIIA28 (orange color) are presented by surface and cartoon models, respectively. ( B ) Analysis of PPI detail from the docking model of AcrIIA28/SpyCas9 complex. Four PPI regions are independently magnified. The residues involved in the interaction are labeled. ( C ) Interaction analysis between SpyCas9 and various tentative PPI-disrupting mutants of AcrIIA28 by SEC followed by SDS-PAGE analysis. SDS-PAGE gels produced by loading one of the main fractions from the SpyCas9 sample with wild-type or various mutants are shown. A black arrow indicates acrIIA28 mutants that co-migrated with SpyCas9. ( D ) The bar chart shows the quantified intensity of the co-eluted AcrII28 mutants. Data are presented as the mean ± standard deviation from three independent experiments. ( E ) Interaction analysis between SpyREC3 and three PPI-disrupting mutants (K7D, W45I and S81W) of AcrIIA28 by SEC followed by SDS-PAGE analysis.

    Article Snippet: The S. pyogenes Cas9 (#62731), N. meningitides Cas9 (#71474), H. parainfluenzae Cas9 (#121540) and S. muelleri Cas9 (#121541) expression constructs were purchased from Addgene.

    Techniques: Binding Assay, Labeling, SDS Page, Produced, Standard Deviation

    Direct interaction of AcrIIA28 is critical for Cas9 inhibition. ( A ) In vitro target DNA cleavage assay using wild-type Cas9 and various AcrIIA28 mutants. In vitro anti-CRISPR activity assay using various tentative PPI-disrupting mutants of AcrIIA28. In the enzyme reaction, + and – indicate added and not added, respectively. ( B ) Quantitative histogram of anti-Cas9 activity of AcrIIA28 according to (A). The inhibitory activity of wild-type AcrIIA28 was considered to be 100%. Data are presented as the mean ± standard deviation from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

    doi: 10.1093/nar/gkae357

    Figure Lengend Snippet: Direct interaction of AcrIIA28 is critical for Cas9 inhibition. ( A ) In vitro target DNA cleavage assay using wild-type Cas9 and various AcrIIA28 mutants. In vitro anti-CRISPR activity assay using various tentative PPI-disrupting mutants of AcrIIA28. In the enzyme reaction, + and – indicate added and not added, respectively. ( B ) Quantitative histogram of anti-Cas9 activity of AcrIIA28 according to (A). The inhibitory activity of wild-type AcrIIA28 was considered to be 100%. Data are presented as the mean ± standard deviation from three independent experiments.

    Article Snippet: The S. pyogenes Cas9 (#62731), N. meningitides Cas9 (#71474), H. parainfluenzae Cas9 (#121540) and S. muelleri Cas9 (#121541) expression constructs were purchased from Addgene.

    Techniques: Inhibition, In Vitro, DNA Cleavage Assay, CRISPR, Activity Assay, Standard Deviation

    Proposed model of Cas9 inhibition by AcrIIA28. ( A and B ) EMSA analysis of the effect of AcrIIA28 on the binding of target DNA into Cas9. The number next to each protein indicates the order in which the proteins were added to form the complex. The red star indicates the band newly produced by the RNP/DNA complex. The experiments were performed three times with similar results. ( C and D ) An in vitro anti-CRISPR activity assay was used to compare the inhibitory effect of AcrIIA28 when added before (C) or after (D) the target DNA. Polyacrylamide gels (4%) were stained with SYBR GOLD. The amount of AcrIIA28 added to the reaction is indicated. The numbers next to the agents used in the experiment indicate the order in which agents were added to the reaction: one represents the sample added first, and three represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. ( E ) Proposed model of Cas9 inhibition by AcrIIA28.

    Journal: Nucleic Acids Research

    Article Title: AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

    doi: 10.1093/nar/gkae357

    Figure Lengend Snippet: Proposed model of Cas9 inhibition by AcrIIA28. ( A and B ) EMSA analysis of the effect of AcrIIA28 on the binding of target DNA into Cas9. The number next to each protein indicates the order in which the proteins were added to form the complex. The red star indicates the band newly produced by the RNP/DNA complex. The experiments were performed three times with similar results. ( C and D ) An in vitro anti-CRISPR activity assay was used to compare the inhibitory effect of AcrIIA28 when added before (C) or after (D) the target DNA. Polyacrylamide gels (4%) were stained with SYBR GOLD. The amount of AcrIIA28 added to the reaction is indicated. The numbers next to the agents used in the experiment indicate the order in which agents were added to the reaction: one represents the sample added first, and three represents the sample added last. In the enzyme reaction, + and – indicate added and not added, respectively. ( E ) Proposed model of Cas9 inhibition by AcrIIA28.

    Article Snippet: The S. pyogenes Cas9 (#62731), N. meningitides Cas9 (#71474), H. parainfluenzae Cas9 (#121540) and S. muelleri Cas9 (#121541) expression constructs were purchased from Addgene.

    Techniques: Inhibition, Binding Assay, Produced, In Vitro, CRISPR, Activity Assay, Staining