Figure 1.

CRISPR/Cas9-mediated indel mutations in mouse embryos delivered by electroporation. (A) Genotyping of mouse embryos targeted at the Tet1 locus. Mouse embryos were electroporated with Cas9 mRNA (100 ng/μl) and sgRNA targeting the Tet1 locus (50 ng/μl), cultured to blastocyst stage of development, and RFLP analysis performed as shown in the top panel. PCR products are sequenced and those showing overlapping sequencing traces were cloned and individual clones sequenced. In the bottom half, mutant alleles are shown for embryos 1 and 10 (indicated by red star) from the group treated with AT for 10 sec. The SacI restriction site at the target region, used for RFLP analysis, is bold and underlined. The protospacer adjacent motif (PAM) sequence is colored in green. Mutated bases are labeled in red. (B) Genotyping of mouse embryos targeted at theTet2 locus. Mutant alleles identified from embryos 4, 6, and 8 (indicated by red star) are shown. Only one mutant allele was recovered from embryos 4 and 8. The EcoRV site located within the target sequence is bold and underlined and PAM sequence colored in green.