Figure 3. Use of Embryo Splitting to Assess Ubi-Cas9 Targeting Efficiency.

(A) Embryo splitting at the 4-cell stage to separate single cells (I, II, III, and IV), which were placed into individual empty zonae pellucidae to further develop to divided embryos that consist of 4 cells (1, 2, 3, and 4). Single cells (1, 2, 3, and 4) were then isolated and used for PCR analysis. (B) Photographs of development of divided monkey embryos: a single cell isolated from 4-cell embryos was transferred into an empty zona pellucida, and then developed to 2- or 4-cell or hatched blastocyst. (C) Hpy188I digestion of the targeted Pink1 gene showing that Ubi-Cas9 mRNA injection yielded a high rate of homogenous mutations in monkey embryos. PCR products of single cells (1, 2, 3, 4) from divided embryos were analyzed. For Hpy188I digestion, red arrows indicate biallelic mutations, whereas green arrows indicate a single allele mutation. Images of full-length gels are seen in supplemental information. (D) In cultured blastomeres that were isolated from embryos injected with Ubi-Cas9 or WT-Cas9, Ubi-Cas9 yielded biallelic mutations in 53% of blastomeres whereas WT-Cas9 generated bialleic mutations in 28% of balstomeres.