Igs2^{tm1(CAG-mCherry,-cas9/ZNF10*)Mtm}

The targeting construct contains (from 5' to 3') an attB site, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), the mCherry gene, a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), the dCas9/ZNF10* fusion gene followed by a nuclear localization signal (NLS), an SV40 polyA signal and a second attB site. The dCas9/ZFN10* fusion gene sequence (dK) is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA fused at its C-terminus to the repressive chromatin modifier domain mRNA from the Kruppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10). PhiC31 integrase-mediated transgenesis is used to insert the construct into the first and second attP sites of Igs2tm3.1Luo. The resulting knock-in allele is therefore H11::pCA::puro::T2A::mCherry::F2A::dCas9/KRAB-NLS-SV40pA::attP::frt5. The dCas9/KRAB binds to a specific DNA sequence/locus following introduction of a single guide RNA (sgRNA), and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain. (J:278806)