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Figure 5 AcrIIC3 Nme Prevents DNA Binding by NmeCas9 in Human Cells Show full caption (A) Schematic representation of plasmids used for expression of dNmeCas9-(sfGFP) 3 , dSpyCas9-(mCherry) 3 , their respective telomeric sgRNAs, and anti-CRISPR protein. The plasmid encoding the anti-CRISPR protein is also marked with the blue fluorescent protein mTagBFP2. (B–F) Fluorescence images of U2OS cells transiently transfected with plasmids depicted in (A). The specific version of each plasmid set (with or without sgRNAs, with or without anti-CRISPRs) is given to the right of each row. First column: differential interference contrast (DIC) and mTagBFP2 imaging, merged. Second column: dNmeCas9-(sfGFP) 3 . Third column: dSpyCas9-(mCherry) 3 . Fourth column: dNmeCas9-(sfGFP) 3 and dSpyCas9-(mCherry) 3 , merged. (B) No sgRNAs for either dNmeCas9-(sfGFP) 3 or dSpyCas9-(mCherry) 3 . (C) No sgRNA for dNmeCas9-(sfGFP) 3 . (D) No Acr protein. (E) With AcrE2 (negative control anti-CRISPR). (F) With AcrIIC3 Nme . Scale bars, 5 μm. 3 telomeric foci, as judged by co-localization with dSpyCas9-(mCherry) 3 telomeric foci, in cells that express no anti-CRISPR, negative control anti-CRISPR (AcrE2), or AcrIIC3 Nme . Foci were scored blind, i.e., without the experimenter knowing the sample identities (see (G) Quantitation of dNmeCas9-(sfGFP)telomeric foci, as judged by co-localization with dSpyCas9-(mCherry)telomeric foci, in cells that express no anti-CRISPR, negative control anti-CRISPR (AcrE2), or AcrIIC3. Foci were scored blind, i.e., without the experimenter knowing the sample identities (see STAR Methods ). n = number of cells that were scored in each condition.

“Dead” Cas9 (dCas9) orthologs, including dNmeCas9 (), have proven to be exceptionally useful for RNA-guided DNA binding (without Cas9-catalyzed DNA cleavage), because a wide range of domains and functionalities can be fused or tethered to the DNA-bound dCas9/sgRNA complex (). In principle, anti-CRISPR inhibition of sgRNA-guided NmeCas9 DNA cleavage ( Figure 3 ) and genome editing ( Figure 4 ) could reflect either inhibition upstream of stable R-loop formation, or inhibition of NmeCas9 catalytic activation after stable R-loop formation. In the former case, the anti-CRISPR could be used as an off-switch not only for genome editing, but also for dNmeCas9 DNA binding applications such as CRISPRi and CRISPRa (). To determine whether our most potent genome editing inhibitor (AcrIIC3) can prevent stable DNA binding by dNmeCas9 in mammalian cells, we used a previously developed system in which superfolder (sf) GFP-labeled dNmeCas9 and mCherry-labeled dSpyCas9 are simultaneously colocalized to telomeric loci by cognate sgRNAs upon co-transfection of their expression plasmids in U2OS cells () ( Figure 5 A). We readily observed colocalizing telomeric dNmeCas9-(sfGFP)and dSpyCas9-(mCherry)foci as long as both of the telomere-directed sgRNAs were included for the two dCas9 orthologs ( Figures 5 B–5D), as reported previously (). We then repeated the experiment with the co-transfected, mTagBFP2-marked plasmid ( Figure 5 A, bottom) also carrying an anti-CRISPR expression cassette. AcrE2 had no effect on telomeric co-localization of dNmeCas9-(sfGFP)and dSpyCas9-(mCherry), as expected ( Figure 5 E). In contrast, co-expression of AcrIIC3prevented the co-localization of dNmeCas9-(sfGFP)with the dSpyCas9-(mCherry)telomeric foci ( Figure 5 F). We then repeated this experiment in a blinded fashion, with unidentified samples that had been coded by a separate experimenter. Only cells that exhibited mTagBFP2 and sfGFP fluorescence as well as dSpyCas9-(mCherry)telomeric foci were assessed for the presence or absence of co-localizing dNmeCas9-(sfGFP)telomeric foci, and all such imaged cells were included in our quantitations. The results were tabulated, decoded, and plotted as a bar graph ( Figure 5 G). Telomeric dNmeCas9-(sfGFP)foci were observed in 94% (31 out of 33) of cells in the absence of any Acr protein and 88% (31 out of 37) of cells in the presence of the negative control AcrE2 protein. By contrast, 0% of cells (0 out of 46) exhibited dNmeCas9-(sfGFP)telomeric foci when AcrIIC3was coexpressed. These results confirm the robust inhibitory effect of AcrIIC3on stable, sgRNA-programmed DNA binding by dNmeCas9 and indicate that it can be used as a potent off-switch not only for NmeCas9 genome editing, but also for dNmeCas9-based applications in mammalian cells.