Gt(ROSA)26Sor^{tm1(CAG-cas9*/EP300*)Gers}

The targeting vector was 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 dCas9 sequence (dCas9-NLS-p300; see below), a 1xFLAG epitope tag, 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 used here is composed of a codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein (dCas9) DNA fused at its C-terminus to a nucleoplasmin bipartite nuclear localization signal and the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300 (EP300). (J:342286)