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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 reverse oriented SA-GFP-pA into the rat Lgr5 locus.(a) Left, schematic of the strategy to insert the inverted GFP reporter cassette into intron1 of the rat lgr5 locus. The reporter cassette consists of a splice acceptor (SA), GFP and polyA (pA) sequences flanked by heterospecific Loxp twin-sites in opposite orientations and homologous arms each about 800 bp long. Right, Genotyping of the founder and the F1 heterozygous mice. Arrowheads indicate the detection of the GFP reporter cassette insertion into the Lgr5 locus in F0 or F1 rats. Asterisk, the PCR band amplified from the wild-type allele. Arrows represent the positions of genotyping primers. M, DNA Marker. (b) Left, schematic depiction of Cre mediated cassette inversion, Loxp site excision and lgr5 inactivation. The reporter cassette was first inverted by Cre recombinase between either inverted Loxp pairs. Thereafter, Cre mediated excision deletes the sequence in between the identically-oriented Loxp pairs, leaving one WT and one mutant Loxp site. Right, RT-PCR to detect endogenous Lgr5 mRNA by primer pair RT1&RT3 and Lgr5-GFP fusion mRNA by primer pair RT1&RT2. Arrows represent the positions of RT-PCR primers. Lower, sequencing result indicating the fusion of Lgr5 and SA-GFP-pA. M, DNA Marker. (c) Detection of GFP (red arrowhead) in rat small intestine tissue after Cre-mediated recombination through immunohistochemical staining. Scale bar, 100 μm.
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f5: Site-specific insertion of reverse oriented SA-GFP-pA into the rat Lgr5 locus.(a) Left, schematic of the strategy to insert the inverted GFP reporter cassette into intron1 of the rat lgr5 locus. The reporter cassette consists of a splice acceptor (SA), GFP and polyA (pA) sequences flanked by heterospecific Loxp twin-sites in opposite orientations and homologous arms each about 800 bp long. Right, Genotyping of the founder and the F1 heterozygous mice. Arrowheads indicate the detection of the GFP reporter cassette insertion into the Lgr5 locus in F0 or F1 rats. Asterisk, the PCR band amplified from the wild-type allele. Arrows represent the positions of genotyping primers. M, DNA Marker. (b) Left, schematic depiction of Cre mediated cassette inversion, Loxp site excision and lgr5 inactivation. The reporter cassette was first inverted by Cre recombinase between either inverted Loxp pairs. Thereafter, Cre mediated excision deletes the sequence in between the identically-oriented Loxp pairs, leaving one WT and one mutant Loxp site. Right, RT-PCR to detect endogenous Lgr5 mRNA by primer pair RT1&RT3 and Lgr5-GFP fusion mRNA by primer pair RT1&RT2. Arrows represent the positions of RT-PCR primers. Lower, sequencing result indicating the fusion of Lgr5 and SA-GFP-pA. M, DNA Marker. (c) Detection of GFP (red arrowhead) in rat small intestine tissue after Cre-mediated recombination through immunohistochemical staining. Scale bar, 100 μm.

Mentions: Mosaic mutant analysis is a powerful strategy to study individual mutant cells in a wild-type cellular environment. The challenge of this technique is to label the truly knockout cells while avoiding mis-labeling wild-type cells when the reporter activation is independent of conditional knockout of the target gene34. We attempted to generate a conditional knockout rat strain whose cells can be labeled after disruption of the target gene by Cre recombinase. We took advantage of the gene trap strategy together with Cre-Loxp-mediated DNA inversion35 to inactivate the target gene while simultaneously labeling cells with GFP (Fig. 5a,b). Lgr5 is a biomarker of adult stem cells in certain tissues, including the intestine, stomach, hair follicle36. However, the study of adult stem cells is hampered in rats due to the lack of reporter strains. We intended to generate an Lgr5 conditional knockout/reporter line by using the above strategy to facilitate monitoring adult stem cells in their native context. Using the Cas9 protein/sgRNA system, we inserted a DNA cassette (composed of a splice acceptor (SA) sequence, the green fluorescent protein (GFP) coding sequence and a polyadenylation (PA) sequence flanked by two pairs of lox66 and lox71 sites) in the reverse orientation into intron 1 of the rat Lgr5 locus (Fig. 5a left). After PCR and subsequent sequencing, 2 founders were confirmed from 3 F0 pups (Fig. 5a right, Table 1). Interestingly, founder #3 was highly likely to carry homozygous mutation identified from tail DNA. However, after crossing with a wild-type rat, only half of the F1 generation from founder #3 was heterozygous suggesting that the founder was a mosaic in germ cells (Fig. 5a right). In principle, upon Cre-mediated recombination, the reverse-oriented SA-GFP-pA cassette will be inverted, then produce a truncated Lgr5 protein fused with GFP. Thus the GFP fusion proteins will label the cells having a spontaneous disruption of the endogenous Lgr5 gene (Fig. 5b left). To test whether the cassette is functional in vivo, we injected Cre mRNA into heterozygous one-cell embryos due to the unavailability of the appropriate rat Cre line. As shown in Fig. 5b, the reporter cassette was inverted in the genome, and the Lgr5-GFP fusion mRNA was detected in the rat intestine and confirmed by sequencing. Furthermore, the GFP+ cells were detected in the bottom of the intestinal crypts where Lgr5+ stem cells reside (Fig. 5c and Supplementary Fig. S3). To our knowledge, this is the first report of marking stem cells in the rat intestine. Intercrossing F1 heterozygotes of Lgr5gfp/+ rats yielded 35 F2 progenies including 13 wild types and 22 heterozygotes (WT: HZ≈1:1.7 ). The lack of homozygous rats in the F2 progenies indicates prenatal lethality of Lgr5 knockout in rats which is a more severe phenotype than that observed in Lgr5 knockout mice which exhibited neonatal lethality37.


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 reverse oriented SA-GFP-pA into the rat Lgr5 locus.(a) Left, schematic of the strategy to insert the inverted GFP reporter cassette into intron1 of the rat lgr5 locus. The reporter cassette consists of a splice acceptor (SA), GFP and polyA (pA) sequences flanked by heterospecific Loxp twin-sites in opposite orientations and homologous arms each about 800 bp long. Right, Genotyping of the founder and the F1 heterozygous mice. Arrowheads indicate the detection of the GFP reporter cassette insertion into the Lgr5 locus in F0 or F1 rats. Asterisk, the PCR band amplified from the wild-type allele. Arrows represent the positions of genotyping primers. M, DNA Marker. (b) Left, schematic depiction of Cre mediated cassette inversion, Loxp site excision and lgr5 inactivation. The reporter cassette was first inverted by Cre recombinase between either inverted Loxp pairs. Thereafter, Cre mediated excision deletes the sequence in between the identically-oriented Loxp pairs, leaving one WT and one mutant Loxp site. Right, RT-PCR to detect endogenous Lgr5 mRNA by primer pair RT1&RT3 and Lgr5-GFP fusion mRNA by primer pair RT1&RT2. Arrows represent the positions of RT-PCR primers. Lower, sequencing result indicating the fusion of Lgr5 and SA-GFP-pA. M, DNA Marker. (c) Detection of GFP (red arrowhead) in rat small intestine tissue after Cre-mediated recombination through immunohistochemical staining. Scale bar, 100 μm.
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Related In: Results  -  Collection

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f5: Site-specific insertion of reverse oriented SA-GFP-pA into the rat Lgr5 locus.(a) Left, schematic of the strategy to insert the inverted GFP reporter cassette into intron1 of the rat lgr5 locus. The reporter cassette consists of a splice acceptor (SA), GFP and polyA (pA) sequences flanked by heterospecific Loxp twin-sites in opposite orientations and homologous arms each about 800 bp long. Right, Genotyping of the founder and the F1 heterozygous mice. Arrowheads indicate the detection of the GFP reporter cassette insertion into the Lgr5 locus in F0 or F1 rats. Asterisk, the PCR band amplified from the wild-type allele. Arrows represent the positions of genotyping primers. M, DNA Marker. (b) Left, schematic depiction of Cre mediated cassette inversion, Loxp site excision and lgr5 inactivation. The reporter cassette was first inverted by Cre recombinase between either inverted Loxp pairs. Thereafter, Cre mediated excision deletes the sequence in between the identically-oriented Loxp pairs, leaving one WT and one mutant Loxp site. Right, RT-PCR to detect endogenous Lgr5 mRNA by primer pair RT1&RT3 and Lgr5-GFP fusion mRNA by primer pair RT1&RT2. Arrows represent the positions of RT-PCR primers. Lower, sequencing result indicating the fusion of Lgr5 and SA-GFP-pA. M, DNA Marker. (c) Detection of GFP (red arrowhead) in rat small intestine tissue after Cre-mediated recombination through immunohistochemical staining. Scale bar, 100 μm.
Mentions: Mosaic mutant analysis is a powerful strategy to study individual mutant cells in a wild-type cellular environment. The challenge of this technique is to label the truly knockout cells while avoiding mis-labeling wild-type cells when the reporter activation is independent of conditional knockout of the target gene34. We attempted to generate a conditional knockout rat strain whose cells can be labeled after disruption of the target gene by Cre recombinase. We took advantage of the gene trap strategy together with Cre-Loxp-mediated DNA inversion35 to inactivate the target gene while simultaneously labeling cells with GFP (Fig. 5a,b). Lgr5 is a biomarker of adult stem cells in certain tissues, including the intestine, stomach, hair follicle36. However, the study of adult stem cells is hampered in rats due to the lack of reporter strains. We intended to generate an Lgr5 conditional knockout/reporter line by using the above strategy to facilitate monitoring adult stem cells in their native context. Using the Cas9 protein/sgRNA system, we inserted a DNA cassette (composed of a splice acceptor (SA) sequence, the green fluorescent protein (GFP) coding sequence and a polyadenylation (PA) sequence flanked by two pairs of lox66 and lox71 sites) in the reverse orientation into intron 1 of the rat Lgr5 locus (Fig. 5a left). After PCR and subsequent sequencing, 2 founders were confirmed from 3 F0 pups (Fig. 5a right, Table 1). Interestingly, founder #3 was highly likely to carry homozygous mutation identified from tail DNA. However, after crossing with a wild-type rat, only half of the F1 generation from founder #3 was heterozygous suggesting that the founder was a mosaic in germ cells (Fig. 5a right). In principle, upon Cre-mediated recombination, the reverse-oriented SA-GFP-pA cassette will be inverted, then produce a truncated Lgr5 protein fused with GFP. Thus the GFP fusion proteins will label the cells having a spontaneous disruption of the endogenous Lgr5 gene (Fig. 5b left). To test whether the cassette is functional in vivo, we injected Cre mRNA into heterozygous one-cell embryos due to the unavailability of the appropriate rat Cre line. As shown in Fig. 5b, the reporter cassette was inverted in the genome, and the Lgr5-GFP fusion mRNA was detected in the rat intestine and confirmed by sequencing. Furthermore, the GFP+ cells were detected in the bottom of the intestinal crypts where Lgr5+ stem cells reside (Fig. 5c and Supplementary Fig. S3). To our knowledge, this is the first report of marking stem cells in the rat intestine. Intercrossing F1 heterozygotes of Lgr5gfp/+ rats yielded 35 F2 progenies including 13 wild types and 22 heterozygotes (WT: HZ≈1:1.7 ). The lack of homozygous rats in the F2 progenies indicates prenatal lethality of Lgr5 knockout in rats which is a more severe phenotype than that observed in Lgr5 knockout mice which exhibited neonatal lethality37.

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