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Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

Wang L, Shao Y, Guan Y, Li L, Wu L, Chen F, Liu M, Chen H, Ma Y, Ma X, Liu M, Li D - Sci Rep (2015)

Bottom Line: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously.Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency.In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

No MeSH data available.


Related in: MedlinePlus

Site-specific insertion of IRES-CreERT2-pA into the mouse Nfatc1 locus.(a) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. (b) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. (c) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2+/−:Rosa26-LacZ+/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.
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f4: Site-specific insertion of IRES-CreERT2-pA into the mouse Nfatc1 locus.(a) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. (b) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. (c) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2+/−:Rosa26-LacZ+/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.

Mentions: Site-specific insertion of functional cassettes containing relatively long DNA fragments into the mouse or rat genome through customized nucleases is challenging. As the CreERT2 cassette is widely used for inducible knockout and lineage tracing studies, we next tried to insert a 2.7 kb IRES-CreERT2 cassette into the mouse Nfatc1 locus (Fig. 4a). We constructed a donor template with about 600 bp homology arms flanking the IRES-CreERT2 cassette. Among 16 F0 pups 4 carried indels as shown by T7E1 assay (Fig. 4b upper) and 1 founder was identified with two sets of genotyping primers (Fig. 4b, Table 1). The insertion was also confirmed by sequencing the PCR products (data not shown). We observed highly efficient germline transmission in the F1 generation (Fig. 4b). To test if the inserted CreERT2 cassette functions in vivo, the Nfatc1-CreERT2 strain was crossed with the Rosa26-LSL-LacZ reporter strain. As Nfatc1 is expressed in CD34+ hair follicle stem cells33, we initiated cell lineage tracing by intraperitoneal injection of tamoxifen at the age of 4 weeks when the hair cycle is in the transition from first telogen to second anagen. 2 days after induction, LacZ positive cells were detected in the bulge area of the hair follicles (Fig. 4c mid), suggesting that Nfatc1 marks the bulge cells during anagen as reported33. 4 weeks after induction when the hair follicles were in the second telogen stage, LacZ positive cells occupied the entire hair follicle (Fig. 4c right), suggesting that Nfatc1-positive cells are follicle stem cells in the bulge. When induced at the 8th week for 2 days, only stem cells in the bulge area exhibited β-Gal staining (data not shown). To our knowledge, this is the first evidence to show that Nfatc1 marks the hair follicle stem cells through lineage-tracing studies.


Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

Wang L, Shao Y, Guan Y, Li L, Wu L, Chen F, Liu M, Chen H, Ma Y, Ma X, Liu M, Li D - Sci Rep (2015)

Site-specific insertion of IRES-CreERT2-pA into the mouse Nfatc1 locus.(a) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. (b) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. (c) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2+/−:Rosa26-LacZ+/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664917&req=5

f4: Site-specific insertion of IRES-CreERT2-pA into the mouse Nfatc1 locus.(a) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. (b) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. (c) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2+/−:Rosa26-LacZ+/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.
Mentions: Site-specific insertion of functional cassettes containing relatively long DNA fragments into the mouse or rat genome through customized nucleases is challenging. As the CreERT2 cassette is widely used for inducible knockout and lineage tracing studies, we next tried to insert a 2.7 kb IRES-CreERT2 cassette into the mouse Nfatc1 locus (Fig. 4a). We constructed a donor template with about 600 bp homology arms flanking the IRES-CreERT2 cassette. Among 16 F0 pups 4 carried indels as shown by T7E1 assay (Fig. 4b upper) and 1 founder was identified with two sets of genotyping primers (Fig. 4b, Table 1). The insertion was also confirmed by sequencing the PCR products (data not shown). We observed highly efficient germline transmission in the F1 generation (Fig. 4b). To test if the inserted CreERT2 cassette functions in vivo, the Nfatc1-CreERT2 strain was crossed with the Rosa26-LSL-LacZ reporter strain. As Nfatc1 is expressed in CD34+ hair follicle stem cells33, we initiated cell lineage tracing by intraperitoneal injection of tamoxifen at the age of 4 weeks when the hair cycle is in the transition from first telogen to second anagen. 2 days after induction, LacZ positive cells were detected in the bulge area of the hair follicles (Fig. 4c mid), suggesting that Nfatc1 marks the bulge cells during anagen as reported33. 4 weeks after induction when the hair follicles were in the second telogen stage, LacZ positive cells occupied the entire hair follicle (Fig. 4c right), suggesting that Nfatc1-positive cells are follicle stem cells in the bulge. When induced at the 8th week for 2 days, only stem cells in the bulge area exhibited β-Gal staining (data not shown). To our knowledge, this is the first evidence to show that Nfatc1 marks the hair follicle stem cells through lineage-tracing studies.

Bottom Line: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously.Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency.In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

No MeSH data available.


Related in: MedlinePlus