NOD/ShiLtJ-Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs}/DvsJ

Stock number: 027891
Product name: Rosa26^{CAG-LSL-Cas9-P2A-EGFP} (RCL-Cas9-EGFP)
Research areas: Cancer Research, Diabetes and Obesity Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The Rosa26^{CAG-LSL-Cas9-P2A-EGFP} knock-in allele (RCL-Cas9-EGFP) allows Cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease and EGFP. When these mice are used in combination with guide sequences and a Cre recombinase source, they allow editing of single or multiple mouse genes both in vivo and ex vivo.

Detailed description

The Gt(ROSA)26Sor^em1Dvs knock-in allele (Rosa26^{CAG-LSL-Cas9-P2A-EGFP} or RCL-Cas9-EGFP), although under control of the Gt(ROSA)26Sor and CAG promoters, has a floxed-STOP cassette preventing widespread expression of the individual genes cas9 and EGFP. After exposure to Cre recombinase, expression of cas9 and EGFP are observed.

Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these Rosa26^{CAG-LSL-Cas9-P2A-EGFP} mice only require the user to select a Cre recombinase driven by the promoter of their choosing and a specific guide sequence for generating single or multiple simultaneous mutations.

Mice homozygous for the Rosa26^{CAG-LSL-Cas9-P2A-EGFP} knock-in allele are viable and fertile. Mice on the NOD/ShiLtJ inbred background develop spontaneous autoimmune type 1 diabetes.

Development

The Gt(ROSA)26Sor^em1Dvs knock-in allele (Rosa26^{CAG-LSL-Cas9-P2A-EGFP} or RCL-Cas9-EGFP) was created by the Type 1 Diabetes Repository at The Jackson Laboratory using CRISPR/Cas9 genome engineering in NOD/ShiLtJ mouse zygotes (Stock No. 001976).

First, the CAG-LSL-Cas9-P2A-EGFP-WPRE-BGHpA sequences were obtained from Feng Zhang (Massachusetts Institute of Technology). Specifically, these sequences include CMV enhancer/chicken beta-actin promoter (CAG), a loxP-STOP(3xSV40 polyA)-loxP cassette (LSL), a 3xFLAG sequence, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]), a P2A ribosomal skip cleavage peptide sequence, an enhanced green fluorescent protein gene (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a bovine growth hormone polyA sequence (BGHpA).

To facilitate insertion into the Gt(ROSA)26Sor locus on chromosome 6 via homology directed repair (HDR), the LSL-Cas9-P2A-EGFP sequences were co-injected with a large piece of Gt(ROSA)26Sor locus homologous DNA.

Founder mice were bred to NOD/ShiLtJ inbred mice (Stock No. 001976), and then heterozygous mice are bred to NOD/ShiLtJ for one additional generations while characterizing the mutation. Rosa26^{CAG-LSL-Cas9-P2A-EGFP} heterozygous mice were then bred together to produce homozygous mice.

Allele

Symbol: Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs}
Name: endonuclease-mediated mutation 1, David V Serreze
MGI accession ID: MGI:6111351
Generation method: Endonuclease-mediated
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: NOD/ShiLtJ
Expressed genes: cas9,CRISPR associated protein 9,; GFP,Green Fluorescent Protein,
Site of expression: Expression of cas9 and EGFP is obtained in tissues expressing Cre recombinase driven by a desired promoter.
Molecular note: Sequences include CMV enhancer/chicken beta-actin promoter (CAG), a loxP-STOP(3xSV40 polyA)-loxP cassette (LSL), a 3xFLAG sequence, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]), a P2A ribosomal skip cleavage peptide sequence, an enhanced green fluorescent protein gene (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a bovine growth hormone polyA sequence (BGHpA). To facilitate insertion into the Gt(ROSA)26Sor locus on chromosome 6 via homology directed repair (HDR), the LSL-Cas9-P2A-EGFP sequences were co-injected with a large piece of Gt(ROSA)26Sor locus homologous DNA.