Journal: bioRxiv

Article Title: Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

doi: 10.1101/350504

Figure Lengend Snippet: a,b, A native gel of the sgRNA visualized by SYBR gold staining ( a ) and of the FAM-labeled target DNA ( b ), both of which were added last to NmeCas9 + Acr [and in ( b ), + sgRNA] incubation. c , Live-cell fluorescence images of U2OS cells transiently transfected with plasmids encoding dNmeCas9-(sfGFP) 3 , dSpyCas9-(mCherry) 3 , their respective telomeric sgRNAs, and Acrs. The plasmid encoding the Acr is also marked with the blue fluorescent protein, mTagBFP2, which is overlaid on a differential interference contrast (DIC) image of each cell. 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, overlay. Second column: dNmeCas9-(sfGFP) 3 . Third column: dSpyCas9-(mCherry) 3 . Fourth column: dNmeCas9-(sfGFP) 3 and dSpyCas9-(mCherry) 3 , merged. Scale bars, 5 μm.

Article Snippet: pHAGE-TO-DEST dSpyCas9-(mCherry) 3 and dNmeCas9-(sfGFP) 3 plasmids were purchased from Addgene (#64108 and #64109, respectively) and used directly for no-sgRNA control experiments. dNmeCas9-(sfGFP) 3 and dSpyCas9-(mCherry) 3 all-in-one plasmids have been described previously (Pawluk et al., 2016).

Techniques: Staining, Labeling, Incubation, Fluorescence, Transfection, Plasmid Preparation, Imaging