B6;129S6-Gt(ROSA)26Sor^{tm1(CAG-dcas9/EP300*)Gers}/J

Stock number: 033065
Product name: Rosa26-LSL-dCas9-p300 , R26-LSL-dCas9^{p300 Core}
Strain synonyms: B6;129S6-Gt(ROSA)26Sor^{tm1(CAG-cas9*/EP300*)Gers}/J
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Rosa26-LSL-dCas9-p300 knock-in mice (also called R26-LSL-dCas9^{p300 Core}) have a loxP-flanked STOP cassette preventing expression of the dCas9^{p300 Core} fusion protein - a codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to the catalytic core of the human acetyltransferase p300. Following Cre recombinase and introduction of a single guide RNA (sgRNA), dCas9^{p300 Core} binds to the target DNA sequence/locus, and that locus is subsequently activated by the p300 core domain. These mice allow Cre-inducible, RNA-guided transcriptional activation of target genes from promoters and both proximal and distal enhancers in a wide range of cellular processes.

Detailed description

Rosa26-LSL-dCas9-p300 knock-in mice (also called R26-LSL-dCas9^{p300 Core}) have a loxP-flanked STOP cassette preventing widespread expression of the RNA-guided DNA-binding fusion protein dCas9^{p300 Core} - a codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to the catalytic histone acetyltransferase (HAT) core domain of the human acetyltransferase p300. Following Cre recombinase exposure and introduction of a single guide RNA (sgRNA) that directs dCas9^{p300 Core} binding to a specific DNA sequence/locus, the p300 domain subsequently catalyzes acetylation of histone H3 lysine 27 at its target sites - leading to robust transcriptional activation of the target locus. These mice allow Cre-inducible, RNA-guided transcriptional activation of target genes from promoters and both proximal and distal enhancers in a wide range of cellular processes. More specific details are below.

The Rosa26-LSL-dCas9-p300 (R26-LSL-dCas9^{p300 Core}) knock-in allele has a CAG promoter and loxP-flanked STOP cassette upstream of the dCas9^{p300 Core} 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^{p300 Core} allele are viable and fertile with normal fertility, and no gross physical or behavioral abnormalities. The donating investigator reports little to no dCas9^{p300 Core} 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^{p300 Core} expression in the cells/tissues as determined by the Cre promoter. Specifically, the donating investigator reports when using Rosa26-LSL-dCas9^{p300 Core} in conjunction with Cre recombinase expression directed to liver, T cells and neurons, the resulting mice all show the desired dCas9^{p300 Core} expression levels, with appropriate target gene upregulation/transcription as determined by the sgRNA used.

Development

The Rosa26-LSL-dCas9-p300 knock-in allele has a dCas9^{p300 Core} fusion gene inserted into the Gt(ROSA)26Sor locus on mouse chromosome 6. Specific details are below.

This Rosa26-LSL-dCas9-p300 allele (R26-LSL-dCas9^{p300 Core}) 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 dCas9^{p300 Core} sequence (dCas9-NLS-p300; see below), a 1xFLAG epitope tag, 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::dCas9-NLS-p300::1xFLAG::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-p300 mice were backcrossed to C57BL/6J for at least one generation, and then bred together to be homozygous. In 2019, Rosa26-LSL-dCas9-p300 males were sent to The Jackson Laboratory Repository as Stock No. 033065. 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^{p300 Core} fusion gene sequence is based on the one described in Hilton et al. 2015 Nat Biotechnol 33:510 [PMID:25849900]. The dCas9^{p300 Core} 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). 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^{tm1(CAG-dcas9/EP300*)Gers}
Name: targeted mutation 1, Charles Gersbach
MGI accession ID: MGI:6360899
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Epitope tag; Endonuclease
Synonyms: Rosa26-LSL-dCas9-p300; Gt(ROSA)26Sor^{tm1(CAG-cas9*/EP300*)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 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).