Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}

RMCE (recombinase-mediated cassette exchange) was utilized to insert the Cas9-Tdt (Dntt)-gRNA) construct into the Col1a1 locus. KH2 embryonic stem cells, carrying the Gt(ROSA)26Sor(tm1(rtTA*M2)Jae) allele, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The Cas9-Dntt fusion is composed of the SpCas9 (derived from S. pyogenes) coding sequence (that includes an N-terminal 3xFLAG peptide, the SV40-NLS [nulcear localization sequence], and a C-terminal NLS), and the coding sequence of the mutant d138 Tdt (formal designation Dntt) containing an N-terminal 6xHis tag. The fusion was created by replacing the Cas9 stop codon with a linker sequence followed by the entire d138 Tdt coding sequence. The 3' UTR includes a WPRE and polyA sequence. Ten tandem gRNAs driven by separate U6 promoters were inserted downstream (in reverse orientation). The Cas9-Tdt-gRNA construct was cloned into the pBS31 targeting vector containing an FRT site and a TetO promoter. The construct was then co-electroporated with a FLPe recombinase-expressing vector (pCAGGS-FLPe-puro) into the RMCE-ready KH2 ES cells. (J:341616)