Figure 2. Zygote RNA injection, blastocyst screening, and embryo transfer to derive genome-edited pigs.

(A) Strategy for in vivo genome editing of pig zygotes with injection of CRISPR sgRNAs and Cas9 mRNA into fertilized zygotes, and growth for 5–7 days in vitro, followed by DNA analysis of blastocysts or embryo transfer to a surrogate. RNP,. ribonucleoprotein. (B) Deletion PCR confirmation of gene editing of PAH exon 6 in a representative set of 15 pig blastocysts. PCR genotyping (as for Figure 1C) detects the WT band in 10 of these individual blastocysts while the expected deletion breakpoint (brkpt) band (1398 bp) is also detected in blastocysts 2, 7, and 8. Variant bands representing different deletion sizes are detected in blastocysts 9 and 11. Of a total of 57 blastocysts analyzed from 2 embryo transfers, 40 produced a PCR band(s), and of these 19 showed deletion alleles.