FVB.129-Igs2^{tm1(CAG-mCherry,-dcas9/KRAB)Mtm}/J

Stock number: 031300
Product name: dCas9-KRAB , H11^dCas9-KRAB , FVB.dCas9-KRAB
Strain synonyms: FVB.129-Igs2^{tm1(CAG-mCherry,-cas9/ZNF10*)Mtm}/J
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

dCas9-KRAB knock-in mice (also called H11^dCas9-KRAB) have widespread 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). dCas9/KRAB binds to a specific DNA sequence/locus following introduction of a single guide RNA (sgRNA), and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain. These mice are a transcriptional repression (CRISPRi) tool enabling rapid and efficient suppression of specific genes (site-specific and programmable CpG-methylation) throughout primary tissues.

dCas9-KRAB knock-in mice are also available on a C57BL/6J-congenic background as Stock No. 030000.

Detailed description

dCas9-KRAB knock-in mice (also called H11^dCas9-KRAB) have widespread 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). Following introduction of a single guide RNA (sgRNA) that directs dCas9/KRAB binding to a specific DNA sequence/locus, the KRAB domain subsequently induces silencing of that locus via heterochromatin formation and/or DNA methylation. These mice are a transcriptional repression (CRISPRi) tool enabling rapid and efficient suppression of specific genes (site-specific and programmable CpG-methylation) throughout primary tissues. More specific details are below.

The dCas9-KRAB (H11^dCas9-KRAB) knock-in allele has a CAG promoter followed by a multi-cistronic cassette encoding an mCherry fluorescent protein and a dCas9/KRAB fusion gene - all inserted into the Igs2 locus (Hipp11 or H11; an intergenic region on mouse chromosome 11). The H11 locus is reported to support high-level global gene expression in conjunction with the CAG promoter, and a higher rate of mitotic/interchromosomal recombination compared to Gt(ROSA)26Sor.

Mice heterozygous or homozygous for the dCas9-KRAB allele are viable and fertile with no gross physical, motor, or behavioral abnormalities.

In normal biological processes, most DNA methylation occurs at cytosine residues - forming 5-methylcytosine (5mC) residues that can profoundly alter the behavior of gene expression. Furthermore, KRAB containing zinc-finger proteins may function in the initiation of epigenetic silencing via establishing repressive 5mC marks in the genome, which are maintained throughout development, and are ultimately targeted by methyltransferase-induced DNA methylation - permanently locking the targeted locus into a repressive state.

The donating investigator reports that when they introduced sgRNA targeting the cell surface protein CD81 gene, this showed dCas9-KRAB was able to efficiently direct KRAB-mediated CpG island 5mC methylation throughout development and potently silence the target gene to nearly undetectable levels. Also, the dCas9-KRAB activity is optimal when the sgRNA is targeted downstream and proximal to the transcription start site of the gene of interest.

Regarding mCherry fluorescence, the donating investigator reports that mCherry expression is very low (considered as background) in all tissues tested to date (May 2018).

dCas9-KRAB knock-in mice are available on an FVB/N-congenic background (Stock No. 031300) and a C57BL/6J-congenic background (Stock No. 030000).

Development

The dCas9-KRAB knock-in allele (also called H11^dCas9-KRAB) has an mCherry gene and a dCas9/KRAB fusion gene inserted into the Igs2 locus (Hipp11 or H11; an intergenic region on mouse chromosome 11 [cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci]). Specific details below.

This dCas9-KRAB allele was created in the laboratory of Dr. Michael T. McManus (University of California, San Francisco) using site-specific PhiC31 integrase-mediated transgenesis. First, the attB-pCA-puro-T2A-mCherry-F2A-dCas9/KRAB-NLS-SV40pA-attB replacement vector was designed with (from 5' to 3') an attB site, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), the mCherry gene (a monomeric DsRed fluorescent protein variant), a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), the dCas9/KRAB fusion gene (dK; details below) followed by a nuclear localization signal (NLS), an SV40 polyA signal and a second attB site.

The dCas9-KRAB fusion gene sequence (dK) is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA fused at its C-terminus to the repressive chromatin modifier domain mRNA from the Krüppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10).

For site-specific PhiC31 integrase-mediated transgenesis, the replacement vector and PhiC31 integrase mRNA were microinjected into FVB/N-congenic H11^{attPx3/attPx3} zygotes (the H11^attPx3 allele [Igs2^tm3.1Luo or H11P3] was originally targeted in 129-derived ES cells to have an unique sequence from the promoter of the yeast his3 gene followed by three shortened tandem attP sites and one frt5 site). Founder mice were identified carrying the correctly targeted insertion of replacement sequences into the first and second attP sites of the H11^attPx3 locus. The resulting dCas9-KRAB knock-in allele is therefore H11::pCA::puro::T2A::mCherry::F2A::dCas9/KRAB-NLS-SV40pA::attP::frt5. Of note, as a result of the attP/attB recombination, there remain two inactive/irreversible att recombined sites present (an att^P/B just upstream of pCA, and att^B/P just downstream of the SV40 polyA).

The dCas9-KRAB mice were bred with FVB/N wildtype mice for 1-2 generations, and then maintained as homozygotes prior to sending males with white coat color were sent to The Jackson Laboratory Repository in 2018. Upon arrival, males are used to cryopreserve sperm. To establish our live dCas9-KRAB mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from FVB/NJ inbred females (Stock No. 001800).

In 2018, a 48 SNP (single nucleotide polymorphism) panel analysis, with 48 markers covering all 19 chromosomes and the X chromosome, was performed on the males sent to The Jackson Laboratory Repository and our first generation rederived living colony. All markers tested were homozygous for FVB/N allele-type with the following exception - 1 marker on chromosome 10 [~60Mbp] was found to be segregating in one male. Importantly, this marker was FVB/N allele-type in our first generation rederived animals. Of note, the chromosome 11 marker closest to the targeted Igs locus was found to be heterozygous (FVB;129) in all our first generation dCas9-KRAB heterozygous mice - supporting that the original H11^attPx3 allele was created on 129 genetic background (before backcrossing onto FVB/N).

Allele

Symbol: Igs2^{tm1(CAG-mCherry,-dcas9/KRAB)Mtm}
Name: targeted mutation 1, Micheal McManus
MGI accession ID: MGI:6441034
Generation method: Targeted
Attributes: Reporter; Endonuclease
Synonyms: H11^dCas9-KRAB; Igs2^{tm1(CAG-mCherry,-cas9/ZNF10*)Mtm}
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
Chromosome: 11
Strain of origin: STOCK Igs2^tm3.1Luo/Luo
Site of expression: Widespread 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). Regarding mCherry fluorescence, the donating investigator reports that mCherry expression is very low (considered as background) in all tissues tested to date (May 2018).
Molecular note: The targeting construct contains (from 5' to 3') an attB site, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), the mCherry gene, a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), the dCas9/ZNF10* fusion gene followed by a nuclear localization signal (NLS), an SV40 polyA signal and a second attB site. The dCas9/ZFN10* fusion gene sequence (dK) is composed of a human codon-optimized, nuclease-deficient Streptococcus pyogenes Cas9 protein synthetic DNA fused at its C-terminus to the repressive chromatin modifier domain mRNA from the Kruppel associated box (KRAB) domain of the human zinc-finger protein 10 gene (ZNF10). PhiC31 integrase-mediated transgenesis is used to insert the construct into the first and second attP sites of Igs2^tm3.1Luo. The resulting knock-in allele is therefore H11::pCA::puro::T2A::mCherry::F2A::dCas9/KRAB-NLS-SV40pA::attP::frt5. The dCas9/KRAB binds to a specific DNA sequence/locus following introduction of a single guide RNA (sgRNA), and that locus is subsequently silenced via heterochromatin formation and/or DNA methylation induced by the KRAB domain.
General note: This allele was produced in zygotes collected from mice carrying the Igs2^tm3.1Luo allele, which was generated in (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 ES cells on a mixed background with 129S1/SvJ, 129X1/SvJ, C57BL/6J, DBA/2J, and FVB/N.