STOCK Igs2^{tm1.1(CAG-cas9*)Mmw}/J

Stock number: 027650
Product name: H11^Cas9
Research areas: Research Tools

Description

H11^Cas9 CRISPR/Cas9 knock-in mice have constitutive expression of CRISPR associated protein 9 (cas9) endonuclease directed by a CAG promoter. When used in with single guide RNAs, they allow editing of single or multiple mouse genes in vivo or ex vivo.

Detailed description

H11^Cas9 mice express the human codon-optimized S. pyogenes cas9 gene (hSpCas9) under control of a CAG promoter from the endogenous intergenic site 2, Igs2 locus (Hipp11 or H11; an intergenic region on chromosome 11 [cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci] reported to support high-level global gene expression in conjunction with the CAG (CAGGS) promoter. Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these H11^Cas9 mice only require delivery of a specific single guide RNA (sgRNA) for generating single or multiple simultaneous mutations. Mice homozygous for the H11^Cas9 knock-in allele are expected to be viable and fertile.

H11^Cas9 knock-in mice are also available on a C57BL/6J congenic genetic background (Stock No. 028239).

Development

A pattB-LSL-Cas9 plasmid and ΦC31 integrase mRNA were microinjected into the pronucleus and cytoplasm of H11^{attPx3/attPx3} zygotes on a 129 background. One founder mouse carried the correctly targeted transgenic insertion of pattB-LSL-Cas9 into the first and second attP sites of the H11^attPx3 locus. The mouse was bred to 2 other mutant lines with B6;129 backgrounds. The H11^LSL-Cas9 mice were bred to B6.C-Tg(CMV-cre)1Cgn/J (Stock No. 006054) mice to remove the floxed STOP cassette. The mice were then bred to remove the Cre recombinase transgene. The resulting H11^Cas9 knock-in allele is therefore attL-CAGGS-loxP-3xFLAG-NLS-hSpCas9-NLS-polyA-attR-attP-FRT5. Male mice were sent to The Jackson Laboratory Repository in 2015. Upon arrival, sperm was cryopreserved. To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Igs2^{tm1.1(CAG-cas9*)Mmw}
Name: targeted mutation 1.1, Monte Winslow
MGI accession ID: MGI:5645518
Generation method: Targeted
Attributes: Endonuclease
Marker symbol: Igs2
Marker name: intergenic site 2
Chromosome: 11
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: cas9,CRISPR associated protein 9,
Molecular note: The targeting construct contains a full-length attB site, the CMV enhancer/chicken beta-actin core promoter (CAGGS), a loxP-STOP(3xSV40 polyA)- loxP cassette (LSL), a 3xFLAG sequence, a nuclear localization signal (NLS), a human codon-optimized S. pyogenes cas9 gene (hSpCas9), a second NLS and the SV40 polyA signal. The embryonic stem cells used were previously targeted with the H11 allele ( Igs2^tm3.1Luo) or H11P3; a unique sequence from the promoter of the yeast his3 gene followed by three shortened tandem attP sites and one FRT5 site. Cre-mediated recombination removed the LSL cassette.