B6J.129(Cg)-Igs2^{tm1.1(CAG-cas9*)Mmw}/J

Stock number: 028239
Product name: H11^Cas9 on B6J
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

These 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.



In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

H11^Cas9 knock-in mice are also available on a STOCK genetic background (Stock No. 027650).

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. These 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 as Stock No. 027650. Upon arrival, some mice were then backcrossed to C57BL/6J inbred mice (Stock No. 000664) using a marker assisted protocol - the resulting C57BL/6J-congenic H11^Cas9 strain is Stock No. 028239.

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.