Figure 3

Screening of inactive sgRNAs with scaffold mutations in mammalian cells. (A) Schematic representation of wild-type and mutant (ATG to GTG) EGFP with PAM sequence insertion. (B) Experimental scheme used in (C,E–F) and Fig. 4A. 293 T cells were transduced with the EGFP mutant. The mutant EGFP-expressing 293 T cells were then transfected with nCas9-CDA and sgRNAs targeting the T > C mutation. (C) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (D) Schematic representation of wild-type (left) and PAM-inserted sgRNAs (original version: middle, optimized version: right). Note that the G-U interaction at the 7th base is lost in the original PAM-inserted sgRNA. The optimized version of the PAM-inserted sgRNA contains additional sequences in the stem-loop region to preserve the G-U interaction at the seventh base. (E) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (F) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with different sgRNAs. The indicated T > C mutations were introduced into the control sgRNA. mut; mutation.