Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Rsky}

The template construct used for for homology-directed repair (HDR) was designed with (from 5' to 3') a 5' homology arm, splice acceptor, polyA elements, a ubiquitously expressed CAG promoter, two copies of the bovine growth hormone poly(A) signal, loxP-flanked Neo/Stop cassette, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) linked to an IRES-GFP (internal ribosome entry site and Green Fluorescent Protein) reporter, and a 3' homology arm. The allele was generated by injecting the template construct, sgRosa26-1 guide RNA, and cas9 mRNA (Cas9-162A, containing plasmid coded polyadenine (polyA) tail). (J:229086)