B6;129-Igs2^{tm1(CAG-cas9*)Mmw}/J

Stock number: 026816
Product name: H11^LSL-Cas9
Strain synonyms: H11
Research areas: Research Tools

Description

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

Detailed description

These H11^LSL-Cas9 knock-in mice have a loxP-flanked STOP cassette preventing expression of the downstream sequence (cas9), inserted in the 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 and a higher rate of mitotic/interchromosomal recombination compared to Gt(ROSA)26Sor). Although under control of a CAG promoter, widespread expression of cas9 is prevented by the STOP cassette. After exposure to Cre recombinase, expression of cas9 is observed. Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these H11^LSL-Cas9 mice only require the user to select a Cre recombinase driven by the promoter of their choosing and a specific single guide RNA (sgRNA) for generating single or multiple simultaneous mutations. Mice homozygous for the H11^LSL-Cas9 knock-in allele are viable and fertile.



H11^LSL-Cas9 knock-in mice are also available on a C57BL/6J congenic background (Stock No. 027632).

Development

The H11^LSL-Cas9 knock-in allele has attL-pCA-LSL-3xFLAG-NLS-hSpCas9-NLS-polyA-attR-attP-FRT5 inserted into the 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 and a higher rate of mitotic/interchromosomal recombination compared to Gt(ROSA)26Sor). Specific details below.
The pattB-LSL-Cas9 targeting vector was created with a full-length attB site, the CMV enhancer/chicken beta-actin core promoter (CAGGS), a loxP-STOP(3xSV40 polyA, CAGGS-NEO)- 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. (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells were previously targeted with the H11^attPx3 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). H11^attPx3 mice were backcrossed onto C57BL/6, and then made homozygous (H11^{attPx3/attPx3}). For site-specific ΦC31 integrase-mediated transgenesis, the pattB-LSL-Cas9 construct and ΦC31 integrase mRNA were microinjected into the pronucleus and cytoplasm of H11^{attPx3/attPx3} zygotes. One founder mouse carried the correctly targeted insertion of pattB-LSL-Cas9 into the first and second attP sites of the H11^attPx3 locus. The resulting H11^LSL-Cas9 knock-in allele is therefore attL-CAGGS-LSL-3xFLAG-NLS-hSpCas9-NLS-polyA-attR-attP-FRT5. The H11^LSL-Cas9LSL-Cas9 colony was bred to 2 other mutant lines with B6;129 backgrounds prior to sending agouti males 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(CAG-cas9*)Mmw}
Name: targeted mutation 1, Monte Winslow
MGI accession ID: MGI:5640626
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change; 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. One founder mouse carried the correctly targeted insertion of pattB-LSL-Cas9 into the first and second attP sites of the H11 locus. The resulting H11 knock-in allele is therefore attL-CAGGS-LSL-3xFLAG-NLS-hSpCas9-NLS-polyA-attR-attP-FRT5.