Gt(ROSA)26Sor^{tm2(CAG-cas9*/ZNF10*)Gers}

The targeting vector is designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette, a 3xFLAG epitope tag, an SV40 nuclear localization signal, a dCas9-KRAB sequence (dCas9-NLS-KRAB; see below), a WPRE, a bGH polyA signal and an attB-PGK-frt-Neo-polyA-attP cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. The dCas9-KRAB is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA fused at its C-terminus to a nuclear localization signal and the repressive chromatin modifier domain mRNA from the Kruppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10). (J:342286)