Figure 4
Heritable, BOC-inducible editing of an endogenous eGFP transgene by cRNA-encoded Cas9K510B. (A) Offspring produced by serial injection of C57BL/6 (B6) mII oocytes with RNA (+) or not injected (−), and incubated in the presence (+) or absence (−) of 1 mM BOC for 4–5 h as indicated, followed by injection of an eGFP+ sperm. Two-cell embryos were transferred non-selectively to recipients and the resulting offspring imaged on postnatal day 3 under UV illumination. Injected RNA (RNA) comprised a mixture of PylRS cRNA (600 ng/μl), Cas9K510B cRNA (600 ng/μl), Pyl tRNA (1,200 ng/μl) and eGFP gRNA (200 ng/μl). (B) Developmental efficiency and green fluorescence in offspring following BOC-inducible editing by Cas9K510B of (A) in the presence (+) or absence of 1 mM BOC. The number of embryos (embryos) transferred to recipients (tf) is given relative to the number of recipients that became pregnant (preg). *Died perinatally. (C) Sequences of genomic PCR products encompassing the targeted region of eGFP, derived from non-fluorescing blastocysts in the experiment of (A). The sequence corresponding to the eGFP target (green) plus adjacent sequences are displayed on the top row and mutants beneath, with the proto-spacer adjacent motif (PAM) highlighted in green. Mutations are indicated in red type-face. All (n = 12) non-fluorescing blastocysts examined had undergone mutagenesis whereas no controls (n = 12) in which either RNA or BOC had been omitted harbored mutations.
