Heritable, BOC-induced editing of an endogenous paternal genomic eGFP transgene by endogenous maternal transgene-expressed Cas9K510B. (A) Structures of maternally-derived endogenous transgene (tg) encoding Cas9K510B (rightmost) or control Cas9 (centre), showing their corresponding mRNA expression levels ± s.e.m. (determined by qPCR; n = 3) relative to levels of H3f3 mRNA in immature germinal vesicle (G) and mature metaphase II (mII) oocytes. The activity relative to that in germinal vesicle oocytes (set at 100%) of the native pZP3 promoter is indicated by similarly determining ZP3 transcript levels (n = 4) in wt oocytes and embryos (leftmost; B, E4.0 blastocyst). nd, not detected. (B) Editing of a paternally-derived transgenic eGFP allele following eGFP+ sperm injection (ICSI) as determined by the percentage of blastocysts ( ±s.e.m.) that fluoresced on E4.5. In controls, wt C57BL/6 (B6) mII oocytes (leftmost) or mII oocytes from pZP3-Cas9 transgenic females [centre, corresponding to the tg structure of (A), above] were injected with eGFP+ sperm and a mixture of Cas9 cRNA (3,300 ng/μl) and eGFP gRNA (800 ng/μl). Also (rightmost), mII oocytes from pZP3-Cas9K510B transgenic females (tg structure above) were injected with eGFP+ sperm and a mixture of PylRS cRNA (600 ng/μl), Pyl tRNA (1,200 ng/μl) and eGFP gRNA (800 ng/μl) (Supplementary Table S1) but no cRNA encoding Cas9K510B. Incubation either omitted BOC (−) or included 1 mM BOC for 5 h (+).