B6(C)-Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Rsky}/J

Stock number: 028555
Product name: Rosa26-Cas9 on B6 , Rosa26^Cas9 knock-in
Research areas: Neurobiology Research, Research Tools

Description

These CRISPR/Cas9 knockin mice constitutively express CRISPR associated protein 9 (cas9) endonuclease and EGFP in a widespread fashion under the direction of a CAG promoter. The mice were generated in C57BL/6 zygotes using a CRISPR/cas9 protocol and as such retain C57BL/6 sequence flanking the mutation.

Detailed description

CRISPR/cas9 endonuclease mediated genome editing of the Gt(ROSA)26Sor, gene was used to introduce a Cas9 cassette into C57BL/6 zygotes. These Rosa26 ^Cas9 knock-in mice have a CAG promoter directing the expected widespread expression (Cas9 and EGFP). Since the knock-in allele was introduced into C57BL/6 zygotes, CRISPR/Cas9 mediated mutants generated from this strain will be on the inbred C57BL/6 genetic background. Mice homozygous for the Rosa26-Cas9 knockin allele are viable and fertile.

This knock-out strain was generated from the floxed-STOP Cre recombinase-dependent strain, Stock No. 028551.

Development

The pRosa-Cas9 targeting vector, the template for homology-directed repair (HDR), was designed with (from 5' to 3') a 5' homology arm, splice acceptor, polyA elements, a ubiquitously expressed CAG promoter, two copies of the bovine growth hormone poly(A) signal, loxP-flanked Neo/Stop cassette, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) linked to an IRES-GFP (internal ribosome entry site and Green Fluorescent Protein) reporter, and a 3' homology arm. This template construct, sgRosa26-1 guide RNA, cas9 mRNA (Cas9-162A, containing plasmid coded polyadenine (polyA) tail) were microinjected into C57BL/6NCrl (Charles River, Sulzbach, Germany) zygotes, which had just been supplemented with Cas9 protein. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and bred to C57BL/6J (Stock No. 000664) once, generating the floxed stop ROSA^LSL-Cas9 line (see Stock No. 028551). The ROSA^LSL-Cas9 mice were then crossed to a human cytomegalovirus minimal promoter driven Cre recombinase strain (Stock No. 006054) to excise the floxed STOP cassette. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

A 32 SNP (single nucleotide polymorphism) panel analysis, with 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 living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Rsky}
Name: endonuclease-mediated mutation 1.1, Klaus Rajewsky
MGI accession ID: MGI:5750729
Generation method: Endonuclease-mediated
Attributes: Reporter; Endonuclease
Synonyms: Rosa26-Cas9
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6NCrl
Expressed genes: cas9,CRISPR associated protein 9,; GFP,Green Fluorescent Protein,
Site of expression: Widespread
Molecular note: The template construct used for for homology-directed repair (HDR) was designed with (from 5' to 3') a 5' homology arm, splice acceptor, polyA elements, a ubiquitously expressed CAG promoter, two copies of the bovine growth hormone poly(A) signal, loxP-flanked Neo/Stop cassette, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) linked to an IRES-GFP (internal ribosome entry site and Green Fluorescent Protein) reporter, and a 3' homology arm. The allele was generated by injecting the template construct, sgRosa26-1 guide RNA, and cas9 mRNA (Cas9-162A, containing plasmid coded polyadenine (polyA) tail). Cre-mediated recombination removed the floxed Neo/Stop cassette.