Figure 4. Ubi-Cas9 Targeting of Pink1 Gene.

(A) A diagram showing single cell PCR using intact or divided monkey embryos. (B,C) PCR diagnosis of Pink1 exon 2 targeting via Ubi-Cas9 in single cells from intact embryos (B) or divided embryos (C). Gene mutations were diagnosed via digestion of PCR products by Hpy188I. Green arrows indicate one allele mutation, and red arrows indicate biallelic mutations. (D) Quantitative assessment of Ubi-Ca9-mediated homogenous mutations in single cells from intact embryos at the 4–8-cell stage or from divided embryos derived from 4-cell intact embryos. In this graph, 31 intact embryos were used to isolate single cells, and 13 4-cell-stage embryos were divided to single cells for further development to 37 single blastomere embryos; data are presented as mean ± SEM. **p < 0.01. (E) DNA sequencing verified the homogenous mutations in single cells from divided embryos injected with Ubi-Cas9 and gRNA for Pink1 exon 2.