Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: Agarose gels and quantification of DNA cleavage with (A, B) Sa Cas9, (C, D) Fn Cas9, and (E, F) Nme Cas9. Time points for Sa Cas9 and Nme Cas9: 0, 1, 2, 5, 10 min. Time points for Fn Cas9: 0, 10, 20, 30, 60 min. Graphs represent the mean of three replicates. Error bars: S.E.M.

Article Snippet: Indel frequencies at the Sa Cas9 target site were assessed via a T7E1 assay with the EnGen Mutation Detection Kit (NEB), using the manufacturer’s recommendations.

Techniques:

Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: (A) HEK293T cells are transiently transfected with plasmids carrying Sa Cas9 + sgRNA. A second plasmid encoding either AcrIIA4 or AcrIIA11 is included, as indicated. (B, C) Representative agarose gel and quantification of indel percentage for the CACNA1D site. Error bars are the standard deviation of three replicates. P-values (not significant [ns], p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) were determined using a Student’s t-test. (D) Quantification of the editing efficiency when AcrIIA11 or AcrIIA4 are present relative to when no Acr is expressed.

Article Snippet: Indel frequencies at the Sa Cas9 target site were assessed via a T7E1 assay with the EnGen Mutation Detection Kit (NEB), using the manufacturer’s recommendations.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Standard Deviation

Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: (A) Schematic of the double-tethered DNA curtains assay. Buffer flow is turned off after both ends of the DNA are tethered between the chromium barriers and pedestals and the protein enters the flowcell. (B) Kymographs showing diffusing Sa Cas9 RNPs (top) and stationary AcrIIA11: Sa Cas9 RNP complexes (bottom). The white arrow indicates the time when Sa Cas9 binds, and the yellow arrow indicates when Sa Cas9 releases the DNA (C) Most Sa Cas9 RNPs diffuse without AcrIIA11 (N=89) but are stationary with AcrIIA11 (N=93). P-value was determined by a Chi-Squared test (p = 3 × 10 −9 ). (D) Lifetime of Sa Cas9 (N=30) and AcrIIA11: Sa Cas9 (N=49) on non-specific DNA. Half-lives are indicated for each curve. (E) Diffusion coefficients of Sa Cas9 with (N=33) and without (N=33) AcrIIA11. P-value was determined by a Mann-Whitney U-test (p = 9.6 × 10 −7 ). Red diamonds indicate the mean diffusion coefficient.

Article Snippet: Indel frequencies at the Sa Cas9 target site were assessed via a T7E1 assay with the EnGen Mutation Detection Kit (NEB), using the manufacturer’s recommendations.

Techniques: Diffusion-based Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: (A) Image of single-tethered DNA molecules that are cleaved by WT Sa Cas9 RNPs. The target site (T) is indicated with a yellow dotted line. DNA is stained with a fluorescent intercalating dye YOYO-1 after incubation with Sa Cas9. (B) The DNA remains full-length after AcrIIA11: Sa Cas9 complexes are injected into the flowcell. (C) Schematic and images of Sa Cas9 binding (top, left), cleaving/ releasing the DNA (top, middle), and the cleaved DNA (top, right). Schematic and images of AcrIIA11: Sa Cas9 binding at an off-target site on the DNA (bottom, left), dissociating from the DNA (bottom, middle), and the remaining full-length DNA (bottom, right). The target site is labeled with a yellow box. (D) Percentage of WT Sa Cas9 at the target with (N=108) and without AcrIIA11 (N=89). P-value was determined by a Chi-Squared test (p = 1 × 10 −6 ). (E) Representative EMSAs of the strong 5x PAM and weak 5x PAM DNA substrates with d Sa Cas9 and AcrIIA11. (F) Quantification of weak PAM, 1x, 2x, and 5x strong PAM EMSAs. Error bars: S.E.M. of three replicates.

Article Snippet: Indel frequencies at the Sa Cas9 target site were assessed via a T7E1 assay with the EnGen Mutation Detection Kit (NEB), using the manufacturer’s recommendations.

Techniques: Staining, Incubation, Injection, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: (A) Sa Cas9 diffuses along the DNA in search of strong PAMs (black squares) that are adjacent to the target sequence. Once a possible target is recognized, Sa Cas9 forms an R-loop between the sgRNA and the target DNA, followed by DNA cleavage. (B) We speculate that AcrIIA11 dimerizes Sa Cas9 on DNA. The AcrIIA11:Cas9 complex is sequestered at off-target PAM-rich sites, preventing Sa Cas9 from recognizing and cleaving the target site.

Article Snippet: Indel frequencies at the Sa Cas9 target site were assessed via a T7E1 assay with the EnGen Mutation Detection Kit (NEB), using the manufacturer’s recommendations.

Techniques: Sequencing