B6.Cg-Col1a1^{tm1(tetO-dcas9/KRAB)Jnxu}/J

Stock number: 036999
Product name: dCas9-KRAB
Strain synonyms: B6.Cg-Col1a1^{tm1(tetO-cas9*)Jnxu}/J
Research areas: Cell Biology Research, Neurobiology Research, Reproductive Biology Research, Research Tools

Description

dCas9-KRAB 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). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), dCas9-KRAB KI expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline. 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 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). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), dCas9-KRAB KI expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline. 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.

Homozygous mice 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.

Mice containing this dCas9-KRAB KI allele and the M2rtTA transactivator located in the Gt(ROSA)26Sor gene (Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}, see Stock No. 006965) are available as Stock No. 036998.

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 contained 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 generations by the donating laboratory (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony of double mutant dCas9-KRAB Rosa26-M2rtTA KI as Stock No. 036998, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes. The Rosa26-M2rtTA allele was bred out of this single allelic strain, Stock No. 036999.

In 2023, 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 rederived living colony at The Jackson Laboratory Repository. Two of the 43 markers throughout the genome were segregating with 129 on chromosomes 4 and 11. The Chromosome 11 marker is likely a passenger segment from Col1a1 mutation.

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)