Journal: Nucleic Acids Research

Article Title: Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18

doi: 10.1093/nar/gkab1197

Figure Lengend Snippet: Biochemical and functional characterizations of AcrIIA17 and AcrIIA18. (A, B) In vitro DNA cleavage assay of Cas9 in the presence of AcrIIA17 ( A ) or AcrIIA18 ( B ). The assay was performed with different orders of addition as described in the schematic (lower inset). The pre-incubated components are shown in parentheses. These gels are representative of three replicate experiments. (C, D) A TBE-urea denaturing gel showing AcrIIA17- ( C ) or AcrIIA18-mediated ( D ) cleavage of sgRNA. The assay was performed with different orders of addition. dCas9: dead mutant Cas9. These gels are representative of three replicate experiments. (E, F) The gel electrophoresis mobility shift assay (EMSA) on sgRNA in the presence of AcrIIA17 ( E ) or AcrIIA18 ( F ). The assay was performed with different orders of addition. These gels are representative of three replicate experiments.

Article Snippet: The cleavage products were separated and visualized by 0.5% TBE agarose gel pertained with StarStain Red Nucleic Acid Dye (GenStar).

Techniques: Functional Assay, In Vitro, DNA Cleavage Assay, Incubation, Mutagenesis, Nucleic Acid Electrophoresis, Mobility Shift

Journal: Nucleic Acids Research

Article Title: Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18

doi: 10.1093/nar/gkab1197

Figure Lengend Snippet: The N-terminal β-hairpin in NTD is critical for AcrIIA18 function. ( A ) The charged residues of AcrIIA18 selected for mutagenesis are shown in pink sphere representations ( B ) In vitro DNA cleavage assay in the presence of the wild-type or mutated AcrIIA18. This gel is representative of three replicate experiments. ( C ) Representative TBE-urea denaturing gels showing the sgRNA cleavage by wild-type and mutated AcrIIA18 in the presence of Cas9. This gel is representative of three replicate experiments. ( D ) The V-shaped groove of AcrIIA18. Charged residues Lys15, Arg19 and Arg52 are located in the V-shaped groove. Tyr17 stacks with Lys15 and Arg19. ( E ) Y17A mutant almost failed to inhibit Cas9-mediated DNA cleavage. This gel is representative of three replicate experiments. ( F ) Y17A mutant significantly impaired the AcrIIA18-induced sgRNA cleavage. This gel is representative of three replicate experiments.

Article Snippet: The cleavage products were separated and visualized by 0.5% TBE agarose gel pertained with StarStain Red Nucleic Acid Dye (GenStar).

Techniques: Mutagenesis, In Vitro, DNA Cleavage Assay

Journal: Nucleic Acids Research

Article Title: Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18

doi: 10.1093/nar/gkab1197

Figure Lengend Snippet: AcrIIA18 triggers sgRNA truncation, and the product is incapable of inducing Cas9-mediated DNA cleavage. ( A ) EMSA was performed using the carboxyfluorescein (FAM)-labeled target DNA as a probe. EMSA showing Cas9 binding to DNA in the presence of either AcrIIA18 or 15 nt sgRNA. 15 nt sgRNA: sgRNA with 15 nt spacer. This gel is representative of three replicate experiments. ( B ) Schematic diagrams of sgRNA. The proposed cleavage site of spacer is indicated by a scissors. ( C ) AcrIIA18 induces the truncation of sgRNA to 15 nt spacer. 15 nt sgRNA and 14 nt sgRNA were used as marker. This gel is representative of three replicate experiments. ( D ) Representative TBE–urea denaturing gels showing sgRNAs of 30, 20 and 15-nt, respectively, cleavage by AcrIIA18. This gel is representative of three replicate experiments. ( E ) In vitro DNA cleavage assay for Cas9 complexed with full-length (20 nt) or 15 nt sgRNA. This gel is representative of three replicate experiments.

Article Snippet: The cleavage products were separated and visualized by 0.5% TBE agarose gel pertained with StarStain Red Nucleic Acid Dye (GenStar).

Techniques: Labeling, Binding Assay, Marker, In Vitro, DNA Cleavage Assay