B6(C)-Igs2^{em1.1(CAG-cas12a*)Mmw}/J

Stock number: 038389
Product name: H11^Cas12a
Research areas: Research Tools

Description

H11^Cas12a knock-in mice have constitutive expression of an enhanced CRISPR associated protein 12a (cas12a) endonuclease directed by a CAG promoter. When used in with guide RNAs, they allow editing of single or multiple mouse genes in vivo or ex vivo.

Note that the donating investigator has also made a Cre-inducible H11^LSL-Cas12a knock-in mouse line available (Stock No. 038388).

Detailed description

These H11^Cas12a knock-in (KI) mice constitutively express an enhanced CRISPR-associated protein 12a (cas12a previously known as cpf1) sequence, that was inserted in to 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).

This H11^Cas12a KI strain was generated by crossing a Cre-inducible H11^LSL-Cas12a knock-in mouse line (Stock No. 038388) to a CMV-Cre mouse line (Stock No. 006054), resulting in ubiquitous expression of an optimized Acidaminococcus cas12a variant (enAsCas12). Cas12a has several feature that distinguish it from cas9, including the ability to more easily target several genes simultaneously. Whereas gene editing via viral delivery of cas12a is burdened by packaging size limits and potential immunogenicity, these H11^Cas12a mice only require delivery of a CRISPR RNA array that can contain multiple guides for generating single or multiple simultaneous mutations. Mice homozygous for the H11^Cas12a knock-in allele are viable and fertile. The donating laboratory has confirmed presence of Cas12a protein across multiple tissues by western blot and immunohistochemical staining.

Development

The H11^Cas12a knock-in (KI) allele was generated by insertion of an enhanced AsCas12a variant (enAsCas12a) 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 targeting vector contains a CMV enhancer/chicken beta-actin core promoter (CAGGS), a loxP-STOP(6xSV40 polyA)-loxP cassette (LSL), an enhanced Acidaminococcus cas12a variant, including several substitutions (E174R/N282A/S542R/K548R; enAsCas12a), a nucleoplasmin nuclear localization signal (NLS), a 3xHA epitope sequence, an SV40 NLS and a rabbit β-globin polyadenylation signal (RBG pA). An sgRNA to mouse Hipp11 (H11) locus, the donor vector cassette, and cas9 mRNA were co-injected into fertilized C57BL/6J zygotes. F0 founder animals were identified by PCR followed by sequence analysis. The resulting founder mice were bred with C57BL/6J to establish germline transmission. The resulting heterozygous H11^LSL-Cas12a KI mice were then crossed to a CMV-Cre deleter strain (Stock No. 006054) to generate the constitutive-expressing H11^Cas12a KI mice. The CMV-Cre was removed through selective breeding by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Igs2^{em1.1(CAG-cas12a*)Mmw}
Name: endonuclease-mediated mutation 1.1, Monte Winslow
MGI accession ID: MGI:7574256
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Epitope tag
Synonyms: H11^Cas12a
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
Chromosome: 11
Strain of origin: C57BL/6J
Molecular note: The targeting vector contains a CMV enhancer/chicken beta-actin core promoter (CAGGS), a loxP-STOP(6xSV40 polyA)-loxP cassette (LSL), an enhanced Acidaminococcus Cas12a variant, including several substitutions (E174R/N282A/S542R/K548R; enAsCas12a), a nucleoplasmin nuclear localization signal (NLS), a 3xHA epitope sequence, an SV40 NLS and a rabbit beta-globin polyadenylation signal (RBG pA). Cre-mediated recombination removed the STOP cassette.