B6;129S6-Gt(ROSA)26Sor^{tm2(CAG-dcas9/KRAB)Gers}/J

Stock number: 033066
Product name: Rosa26-LSL-dCas9-KRAB , R26-LSL-dCas9-KRAB
Strain synonyms: B6;129S6-Gt(ROSA)26Sor^{tm2(CAG-cas9*/ZNF10*)Gers}/J
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Rosa26-LSL-dCas9-KRAB knock-in mice (also called R26-LSL-dCas9-KRAB) have a loxP-flanked STOP cassette preventing expression of the dCas9-KRAB fusion protein - a human codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to a Krüppel associated box domain (KRAB). Following Cre recombinase exposure and introduction of a single guide RNA (sgRNA), dCas9-KRAB binds to the target DNA sequence/locus, and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain. These mice allow Cre-inducible, RNA-guided transcriptional repression (CRISPRi) of target genes in a wide range of cellular processes.

Detailed description

Rosa26-LSL-dCas9-KRAB knock-in mice (also called R26-LSL-dCas9-KRAB) have a loxP-flanked STOP cassette preventing widespread expression of the RNA-guided DNA-binding fusion protein dCas9-KRAB - a human codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to a Krüppel associated box domain (KRAB). Following Cre recombinase exposure and introduction of a single guide RNA (sgRNA) that directs dCas9-KRAB binding to a specific DNA sequence/locus, the KRAB domain subsequently induces silencing of the target locus via heterochromatin formation and/or DNA methylation. These mice are a Cre-inducible, transcriptional repression (CRISPRi) tool enabling rapid and efficient suppression of specific genes (site-specific and programmable CpG-methylation) throughout a wide range of cellular processes. More specific details are below.

The Rosa26-LSL-dCas9-KRAB (R26-LSL-dCas9-KRAB) knock-in allele has a CAG promoter and loxP-flanked STOP cassette upstream of the dCas9-KRAB fusion gene - all inserted into the Gt(ROSA)26Sor locus.
Prior to introduction of Cre recombinase, mice heterozygous or homozygous for the Rosa26-LSL-dCas9-KRAB allele are viable and fertile with normal fertility, and no gross physical or behavioral abnormalities. The donating investigator reports little to no dCas9-KRAB expression is observed before Cre exposure (levels are barely detectable by Western blot).
Upon exposure to Cre recombinase, the STOP cassette is removed - resulting in dCas9-KRAB expression in the cells/tissues as determined by the Cre promoter. Specifically, the donating investigator reports when using Rosa26-LSL-dCas9-KRAB in conjunction with Cre recombinase expression directed to liver, T cells and neurons, the resulting mice all show the desired dCas9-KRAB expression levels, with appropriate target gene repression/methylation as determined by the sgRNA used.

Development

The Rosa26-LSL-dCas9-KRAB knock-in allele has a dCas9-KRAB fusion gene inserted into the Gt(ROSA)26Sor locus on mouse chromosome 6. Specific details are below.

This Rosa26-LSL-dCas9-KRAB allele (R26-LSL-dCas9-KRAB) was created in the laboratory of Dr. Charles A. Gersbach (Duke University). The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a frt site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a 3xFLAG epitope tag, an SV40 nuclear localization signal, a dCas9-KRAB sequence (dCas9-NLS-KRAB; see below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bGH polyA signal and an attB-PGK-frt-Neo-polyA-attP cassette. This entire construct (CAG::frt::LSL::3xFLAG::NLS::dCas9-NLS-KRAB::WPRE::bGHpA::attB-PGK-frt-Neo-pA-attP) was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Positive clones were expanded and injected into the 8-cell morulae of ICR mice. Chimeric animals were bred to C57BL/6J mice for germline transmission. The resulting Rosa26-LSL-dCas9-KRAB mice were backcrossed to C57BL/6J for at least one generation, and then bred together to be homozygous. In 2019, Rosa26-LSL-dCas9-KRAB males were sent to The Jackson Laboratory Repository as Stock No. 033066. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The dCas9-KRAB fusion gene sequence is based on the one described in Thakore et al. 2015 Nat Methods 12:1143 [PMID:26501517]. The dCas9-KRAB used here 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 Krüppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10). The dCas9 lacks endonuclease activity due to the D10A mutation in the RuvC-like domain and the H840A mutation in the HNH domain.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-dcas9/KRAB)Gers}
Name: targeted mutation 2, Charles Gersbach
MGI accession ID: MGI:6360904
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Epitope tag; Endonuclease
Synonyms: Rosa26-LSL-dCas9-KRAB; Gt(ROSA)26Sor^{tm2(CAG-cas9*/ZNF10*)Gers}
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: 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).