Genetic code expansion allows BOC-inducible, Cas9-mediated genome editing in mouse preimplantation embryos. (A) Schematic diagram depicting the experimental strategy: serial injection first of a mouse metaphase II (mII) oocyte with the indicated RNAs and 4–5 h later by an eGFP+ sperm carrying a ubiquitously-expressed eGFP transgene which fertilizes the oocyte. BOC (1 mM) is either omitted from the culture or included for the durations indicated after RNA injection. Oocytes were injected with a mixture of Cas9K510B cRNA (600 ng/μl), Pyl tRNA (1,200 ng/μl) and PylRS cRNA (600 ng/μl) and 5 h later by eGFP gRNA (200 ng/μl) together with the eGFP+ sperm. (B) Vertically paired bright field (BF, upper) and fluorescence images of blastocysts produced by the method of (A) on the fourth day of culture (E4.0). Oocytes had been injected (+) with RNA, or RNA injection omitted (-), followed by exposure to BOC for 5 h (+) or not (-) as indicated. Bar, 100 μm. (C) Histograms showing the percentage (±s.e.m.) of 1-cell embryos of (B) developing to the blastocyst stage at E4.0 (open columns) and the percentages of blastocysts that fluoresced green (green columns), with embryo numbers (n) given above columns. (D) Histograms showing the percentage (±s.e.m.) of 1-cell embryo development to 2-cell (2 C), 4-cell (4 C), morula (mor) and blastocyst (Bla) stages following serial injection first of mII oocytes with a mixture of Cas9K510B cRNA (600 ng/μl), Pyl tRNA (1,200 ng/μl) and PylRS cRNA (600 ng/μl) and 5 h later by eGFP gRNA (200 ng/μl) together with an eGFP+ sperm. Incubation was in media containing BOC at the concentrations indicated for 5 or 24 h after the first injection (ie respectively 5 h before ICSI and 19 h after). Percentages of blastocysts that were green fluorescent are represented by green columns. (E) Histograms (left) showing the percentage (±s.e.m.) of 1-cell embryos as per (D) except that Cas9K742B cRNA was injected instead of Cas9K510B cRNA and incubation was in the presence (+) or absence (−) of 1 mM BOC for 5 h after the first injection. Vertically paired images of E4.0 embryos are to the right, with culture in the presence (+) or absence (−) of 1 mM BOC for 5 h after the first injection. Bright field (BF) is shown (upper) with eGFP (fluorescence). Scale bar, 100 μm. (F) As per (E) except that cRNA encoding the Cas9K510B,K742B double mutant was injected instead of Cas9K742B cRNA.