Cas9 gene knockout using single or dual gRNAs.

<p><b>A.</b> mESC cell line L10a-GFP, in which one Rosa26 locus expresses GFP. <b>B.</b> Experimental outline and gRNAs. gRNAs #9 and #5 generate 5′ overhangs for dual nickase strategy. <b>C.</b> Single gRNA-directed GFP inactivation. <b>D.</b> Comparison of gRNA alone or gRNA+HDR donor containing a translational stop signal. n = 3. Bar = s.e.m. Red circles indicate individual data points. <b>E.</b> Gene inactivation frequency of single compared to paired gRNAs. <b>F–G.</b> Assessment of GFP expression in mESCs in the GFP-FACS fraction by fluorescent microscopy and western blotting. <b>H.</b> Effective gene knockout using dual gRNAs and the hCas9-D10A nickase mutant.