B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}/J

Stock number: 038749
Product name: Cas9-TdT-gRNAs-M2; Col1a1^{tetO-Cas9-Tdt/gRNA-Array}:Rosa26^M2-rtTA

Strain synonyms: Cas9-TdT-gRNAs-M2
Research areas: Research Tools

Description

Inducible Cas9-TdT-gRNAs-M2 mice carry a tetO-driven cas9/Dntt (Tdt) fusion with ten tandem gRNAs driven by separate U6 promoters and a reverse tetracycline controlled transactivator (rtTA*M2).

The DARLIN model is created when Cas9-TdT-gRNAs-M2 mice are combined with CA/TA/RA triple target-array mice (Stock No. 038750). DARLIN is a lineage tracing mouse line with a 10¹⁸ lineage barcoding capacity and single cell lineage coverage with a 60% efficiency that can be induced at any time and across a wide range of tissues.

Detailed description

Cas9-TdT-gRNAs-M2 mice carry two targeted mutations: (1) a cas9 endonuclease fused to a mutant d138 terminal deoxynucleotidetransferase (Dntt, also known as Tdt) under the control of the a tetracycline responsive element (tetO or TRE) followed by ten tandem gRNAs driven by separate U6 promoters and (2) an optimized form of reverse tetracycline controlled transactivator (rtTA*M2) widely expressed from the Gt(ROSA)26Sor promoter. In this strain, administration of doxycycline induces widespread expression of cas9/Dntt which introduces site-directed double strand breaks in DNA through association with guide RNA.

The Cas9-TdT-gRNAs-M2 allele represents an improvement over the KH2-iCas9 allele (Stock No. 029415) by incorporating a template-independent DNA polymerase (Dntt) that can insert random nucleotides at both overhang and blunt 3' ends. Co-expression of Cas9/Dntt generates a higher fraction of rare alleles and higher barcode diversity as compared to cas9 alone.

To create the DARLIN model, Cas9-TdT-gRNAs-M2 mice are combined with CA/TA/RA triple target-array mice (B6;129S4-Gt(ROSA)26Sor^{tm1(UBC-GFP)Fcam} Igs7^{em1(CAG-mCherry)Fcam} Col1a1^{tm4(CAG-EGFP)Fcam}/J, Stock No. 038750). Administration of doxycycline to DARLIN mice activates expression of Cas9/Dntt allowing cut and repair of the target sequence creating a unique and inheritable "barcode". In comparison to the original CARLIN model (see Stock No. 035434), DARLIN can generate an estimated 10¹⁸ unique lineage barcodes and has 90% barcoding efficiency (compared to 30% in CARLIN). In single cell assays, 60% of captured cells have edited and rare lineage barcodes (10% in CARLIN).

Development

RMCE (recombinase-mediated cassette exchange) was utilized to insert the Cas9-Tdt (Dntt)-gRNA) construct into the Col1a1 locus on chromosome 11. KH2 embryonic stem cells (C57BL/6 x 129S4/SvJae), carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele (see Stock No. 006965) on chromosome 6, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The Cas9-Tdt (Dntt) fusion is composed of the SpCas9 (derived from S. pyogenes) coding sequence (that includes an N-terminal 3xFLAG peptide, the SV40-NLS [nulcear localization sequence], and a C-terminal NLS), and the coding sequence of the mutant d138 Tdt (formal designation Dntt) containing an N-terminal 6xHis tag. The fusion was created by replacing the Cas9 stop codon with a linker sequence followed by the entire d138 Tdt coding sequence. The 3’ UTR includes a WPRE and polyA sequence. Ten tandem gRNAs driven by separate U6 promoters were inserted downstream (in reverse orientation). The Cas9-Tdt-gRNA construct was cloned into the pBS31 targeting vector containing an FRT site and a TetO promoter. The construct was then co-electroporated with a FLPe recombinase-expressing vector (pCAGGS-FLPe-puro) into the RMCE-ready KH2 ES cells. The resulting correctly targeted ES cells, carrying rtTA*M2 in the Gt(ROSA)26Sor locus and Cas9-Tdt-gRNA in the Col1a1 locus, were injected into C57BL/6 embryos. The resulting chimeric animals were maintained on a C57BL/6 and 129S4 mixed background and then bred to homozygosity. Upon arrival, mice were bred to C57BL/6J for at least 1 generation to establish the colony.

Allele

Symbol: Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}
Name: targeted mutation 5, Fernando Camargo
MGI accession ID: MGI:7526271
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence
Synonyms: Cas9-TdT-gRNAs
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: RMCE (recombinase-mediated cassette exchange) was utilized to insert the Cas9-Tdt (Dntt)-gRNA) construct into the Col1a1 locus. KH2 embryonic stem cells, carrying the Gt(ROSA)26Sor(tm1(rtTA*M2)Jae) allele, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The Cas9-Dntt fusion is composed of the SpCas9 (derived from S. pyogenes) coding sequence (that includes an N-terminal 3xFLAG peptide, the SV40-NLS [nulcear localization sequence], and a C-terminal NLS), and the coding sequence of the mutant d138 Tdt (formal designation Dntt) containing an N-terminal 6xHis tag. The fusion was created by replacing the Cas9 stop codon with a linker sequence followed by the entire d138 Tdt coding sequence. The 3' UTR includes a WPRE and polyA sequence. Ten tandem gRNAs driven by separate U6 promoters were inserted downstream (in reverse orientation). The Cas9-Tdt-gRNA construct was cloned into the pBS31 targeting vector containing an FRT site and a TetO promoter. The construct was then co-electroporated with a FLPe recombinase-expressing vector (pCAGGS-FLPe-puro) into the RMCE-ready KH2 ES cells.
General note: KH2 embryonic stem cells carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus.

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.