Figure 4. Identification of mutations induced by nsd2 Cas9.

(A) A schematic diagram showing primers for detecting mutations in Cas9 target site of nsd2 gene. (B) The relative amplification efficiency of nsd2 Pf primers to Po primers. nsd2 Cas9 mRNA injection dramatically reduced the amplification efficiency of nsd2 Pf primers. 5 embryos from the wild-type or the nsd2-Cas9-injected group were pooled and analyzed by qPCR, and 3 pools of each group were analyzed in an independent experiment. Similar results were obtained in three independent experiments. (C) Identification of a mutation in a single allele with T-CIA. Upper panel was an agarose electrophoresis result of PCR products amplified with nsd2 Po and Pf primers. Lower panel listed the sequences of mutant colonies identified in upper panel.