Fig. 2

Efficient mutagenesis of EGFP reporter transgenes. (A-C) Mutagenesis of the EGFP open reading frame in transgenic embryos that harbor a single copy of the ubiquitously expressed ubi:EGFP transgene using Cas9 and EGFP sgRNA. (A) 5dpf uninjected heterozygous ubi:EGFP control sibling and (B) embryo following injection of Cas9 mRNA with EGFP sgRNA leads to mosaic EGFP expression, as compared with (C) complete loss of EGFP signal upon Cas9 protein (Cas9p)-sgRNA RNP injection. Insets are brightfield images of fluorescence views. (D) Relative fluorescence quantification of ubi:EGFP and wt1b:EGFP mutagenesis versus RNP-injected non-transgenic (n.t.) controls. Error bars represent mean with s.e.m. (E-J) EGFP mutagenesis in heterozygous myl7:EGFP transgenic reporters that express EGFP specifically in heart muscle cells. Left column depicts EGFP fluorescence, right column shows the brightfield view. (E,F) Control sibling, (G,H) Cas9 mRNA and EGFP sgRNA injection causing mosaic EGFP expression (arrowhead, G), and (I,J) reconstituted Cas9p-sgRNA complex injection results in complete loss of EGFP signal (asterisk, I). (K-M) EGFP mutagenesis in kidney expressing wt1b:EGFP transgenic reporters. Brightfield overlay with EGFP fluorescence. (K) Control sibling, (L) mosaic EGFP expression following Cas9 mRNA and EGFP sgRNA injection (arrowhead), and (M) loss of EGFP following injection of reconstituted Cas9p-sgRNA complex (asterisk). (N) Western blot of EGFP and Tubulin loading control from ubi:EGFP transgenic embryos (two examples are shown) injected with EGFP sgRNA-containing Cas9-mCherry RNPs and control embryos.