B6;129-Gt(ROSA)26Sor^{tm1(CAG-cas9*,-EGFP)Fezh}/J

Stock number: 024857
Product name: Rosa26-LSL-Cas9 knockin
Strain synonyms: Rosa26-floxed STOP-Cas9 knockin
Research areas: Neurobiology Research, Research Tools

Description

A C57BL/6J congenic version of this strain is available as Stock No. 026175.

These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease, a 3X-FLAG epitope tag and EGFP directed by a CAG promoter. Expression of Cas9 and EGFP is prevented by an upstream Lox-Stop-Lox (LSL) sequence. When used in combination with single guide RNAs and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.

Detailed description

These Rosa26-LSL-Cas9 knock-in mice have a floxed-STOP cassette preventing expression of the downstream bicistronic sequences (Cas9 and EGFP). Although under control of a CAG promoter, widespread expression of cas9 and EGFP is prevented by the STOP cassette. After exposure to Cre recombinase, expression of cas9 and EGFP is observed. The donating investigator reports cas9 expression is tightly controlled in a Cre-dependent manner. Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these Rosa26-LSL-Cas9 mice only require one 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 Rosa26-LSL-Cas9 knock-in allele are viable and fertile.

The Rosa26-LSL-Cas9 knock-in mice are also available on a FVB/NJ congenic background (Stock No. 026481), a C57BL/6J congenic background (Stock No. 026175), a NOD/ShiLtJ congenic background (Stock No. 026431), as well as a C57BL/6NJ congenic background (Stock No. 026556).

Development

The Rosa26-LSL-Cas9 targeting vector used was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm. The construct was electroporated into 129S-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6N blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6N mice. The mice were then backcrossed for four generations onto C57BL/6N. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony. SNP (single nucleotide polymorphism) analysis performed as part of The Jackson Laboratory quality control effort indicates an incomplete backcross.

A 32 SNP panel analysis (27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains) was performed on the rederived live colony at The Jackson Laboratory Repository. Five of the 27 markers were segregating for 129. This suggests an incomplete backcross. All 5 markers that distinguish C57BL/6J from C57BL/6N were found to be segregating. This further suggests the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-cas9*,-EGFP)Fezh}
Name: targeted mutation 1, Feng Zhang
MGI accession ID: MGI:5583839
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Endonuclease
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,; cas9,CRISPR associated protein 9,
Site of expression: EGFP and Cas9 will be expressed in cells where promoters driving Cre recombinase are expressed.
Molecular note: The targeting vector inserted into the locus was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm.