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.
Klaus Rajewsky, Max Delbruck Centre for Molecular Medicine
Ralf Kuehn, Max-Delbrück-Centrum for Molecular Medicine
Genetic Background | Generation |
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?+pN3F9
|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Endonuclease-mediated (Reporter, Endonuclease) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
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.
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 ROSALSL-Cas9 line (see Stock No. 028551). The ROSALSL-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.
Expressed Gene | GFP, Green Fluorescent Protein, |
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Expressed Gene | cas9, CRISPR associated protein 9, |
Site of Expression | Widespread |
Allele Name | endonuclease-mediated mutation 1.1, Klaus Rajewsky |
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Allele Type | Endonuclease-mediated (Reporter, Endonuclease) |
Allele Synonym(s) | Rosa26-Cas9 |
Gene Symbol and Name | Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano |
Gene Synonym(s) | |
Promoter | CAG, CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, |
Expressed Gene | GFP, Green Fluorescent Protein, |
Expressed Gene | cas9, CRISPR associated protein 9, |
Site of Expression | Widespread |
Strain of Origin | C57BL/6NCrl |
Chromosome | 6 |
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. |
When maintaining a live colony, these mice can be bred as homozygotes.
When using the Rosa26-Cas9 on B6 , Rosa26Cas9 knock-in mouse strain in a publication, please cite the originating article(s) and include JAX stock #028555 in your Materials and Methods section.
Service/Product | Description | Price |
---|---|---|
Heterozygous or wildtype for Gt(ROSA)26Sor<em1.1(CAG-cas9*,-EGFP)Rsky> |
Frozen Mouse Embryo | B6(C)-Gt(ROSA)26Sor<em1.1(CAG-cas9* -EGFP)Rsky>/J Frozen Emb | $2595.00 |
Frozen Mouse Embryo | B6(C)-Gt(ROSA)26Sor<em1.1(CAG-cas9* -EGFP)Rsky>/J Frozen Emb | $2595.00 |
Frozen Mouse Embryo | B6(C)-Gt(ROSA)26Sor<em1.1(CAG-cas9* -EGFP)Rsky>/J Frozen Emb | $3373.50 |
Frozen Mouse Embryo | B6(C)-Gt(ROSA)26Sor<em1.1(CAG-cas9* -EGFP)Rsky>/J Frozen Emb | $3373.50 |
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