B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-dcas9/KRAB)Jnxu}/J

Stock number: 036998
Product name: dCas9-KRAB Rosa26-M2rtTA KI
Strain synonyms: CRISPRi Rosa26-M2rtTA KI , B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-cas9*)Jnxu}/J
Research areas: Cell Biology Research, Neurobiology Research, Reproductive Biology Research, Research Tools

Description

dCas9-KRAB Rosa26-M2rtTA KI mice have a tetracycline-inducible promoter (TRE or tetO) driving expression of the dCas9-KRAB fusion protein - a human codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to a Krüppel associated box domain (KRAB). They also carry a M2rtTA transactivator located in the Gt(ROSA)26Sor gene (Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}, see Stock No. 006965). Following doxycycline-administration and introduction of a single guide RNA (sgRNA), dCas9-KRAB binds to the target DNA sequence/locus, and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain. These mice allow doxycycline-inducible, RNA-guided transcriptional repression (CRISPRi) of target genes in a wide range of cellular processes.

Detailed description

dCas9-KRAB Rosa26-M2rtTA KI mice have a tetracycline-inducible promoter (TRE or tetO) driving expression of the dCas9-KRAB fusion protein - a human codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to a Krüppel associated box domain (KRAB). They also carry a M2rtTA transactivator located in the Gt(ROSA)26Sor gene (Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}, see Stock No. 006965). Following doxycycline-administration and introduction of a single guide RNA (sgRNA), dCas9-KRAB binds to the target DNA sequence/locus, and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain.

Prior to doxycycline administration, mice homozygous for both alleles are viable and fertile with normal fertility, and no gross physical or behavioral abnormalities. These mice allow doxycycline-inducible, RNA-guided transcriptional repression (CRISPRi) of target genes in a wide range of cellular processes.

The dCas9-KRAB KI allele is available without the Rosa26-M2rtTA allele as Stock No. 036999.

Development

A targeting vector was designed to insert an FRT-flanked PGK-Neo-pA 500 bp downstream of the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cell clones was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a rabbit beta-globin intron/polyA signal and PGK-puromycin cassette, all downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the FRT-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a dCas9-KRAB (CRISPR associated protein 9 (KRAB)) gene under control of the tetracycline-inducible promoter (TRE or tetO) and flp recombinase. The dCas9-KRAB plasmid contains a splice acceptor-polyA sequence and the tetracycline responsive element (tetO) upstream of the dCas9-KRAB fusion gene.

The dCas9/KRAB fusion gene sequence (dK) is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA, an HA tag, and two copies of a nuclear localization sequence (NLS), and the repressive chromatin modifier domain mRNA from the Krüppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10). The dCas9-KRAB cassette was amplified from the pHR-TRE3B-KRAB-dCas9-P2A-mCherry vector provided by Dr. Luke Gilbert at UCSF.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and dCas9-KRAB in the Col1a1 locus (Col1a1-dCas9-KRAB), were injected into recipient blastocysts. The resulting dCas9-KRAB Rosa26-M2rtTA KI mice were bred with C57BL/6J mice (Stock No. 000664) for at least 6 generation by the donating laboratory (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

In 2021, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 males sent to The Jackson Laboratory Repository. Five of the 43 markers throughout the genome were segregating with 129, suggesting an incomplete backcross.

Allele

Symbol: Col1a1^{tm1(tetO-dcas9/KRAB)Jnxu}
Name: targeted mutation 1, Jian Xu
MGI accession ID: MGI:6835795
Generation method: Targeted
Attributes: Inducible; Epitope tag; Endonuclease
Synonyms: dCas9-KRAB; Col1a1^{tm1(tetO-cas9*)Jnxu}
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Marker synonyms: CAFYD; Col1a-1; Cola1; Cola-1; COLIA1; EDSARTH1; EDSC; Mov-13; OI1; OI2; OI3; OI4
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: The plasmid contains a splice acceptor-polyA sequence and the tetracycline responsive element (tetO) upstream of the dCas9-KRAB fusion gene replacing the FRT-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus. The dCas9/KRAB fusion gene sequence (dK) is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA, an HA tag, and two copies of a nuclear localization sequence (NLS), and the repressive chromatin modifier domain mRNA from the Kruppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10)

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}
Name: targeted mutation 1, Rudolf Jaenisch
MGI accession ID: MGI:3702294
Generation method: Targeted
Attributes: Transactivator
Synonyms: Gt(ROSA)26Sor^{tm1(M2rtTA)Jae}; M2; M2-rtTA; R26-M2rtTA; R26-rtTA; Rosa26-M2rtTA
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/6 x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Expresses an optimized rtTA protein (rtTA-M2). Inducible target gene expression is detected in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression is detected in the brain and testes.
Molecular note: An optimized form of reverse tetracycline controlled transactivator (rtTA-M2) was inserted downstream of the Gt(ROSA)26Sor promoter and was followed by a PGK-puro selection cassette. This mutant form of rtTA termed M2 has five amino acid substitutions in the tetR moiety of tTA: S12G, E19G, A56P, D148E and H179R. This mutated form of transactivator protein has increased doxycycline sensitivity. Mice have widespread expression of the rtTA-M2 protein.
General note: This mutation was created during the generation of mutant ES cell line KH2, which also contains a frt docking site downstream of the endogenous Col1a1 locus on Chr 11 (Col1a1). The two mutations have subsequently been bred apart.