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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

Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb.(a) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. (b) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in (a). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. (c) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
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f1: Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb.(a) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. (b) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in (a). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. (c) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.

Mentions: As some homologous genes are located adjacent to one another to form a gene cluster, it is difficult to generate multiple-gene knockout animals by crossing due to the low homologous recombination efficiency within short distances in the genome. Therefore, we next tried to delete a genomic region which spans 95 kb in the mouse genome containing three receptors for formyl peptide to extend the deletion range through direct embryo injection (Fig. 1a). After injection of Cas9 protein:sgRNAs, 20 F0 pups were obtained. PCR and subsequent DNA sequencing confirmed that two pups were founders bearing the desired 95 kb genomic DNA deletion (Fig. 1b, Table 1). The deletion was germline transmissible as determined by genotyping of F1 progeny (Fig. 1c). We demonstrated that through a single injection of Cas9 proteins and two sgRNAs, large DNA fragment deleted animals could be easily generated.


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)

Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb.(a) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. (b) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in (a). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. (c) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb.(a) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. (b) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in (a). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. (c) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
Mentions: As some homologous genes are located adjacent to one another to form a gene cluster, it is difficult to generate multiple-gene knockout animals by crossing due to the low homologous recombination efficiency within short distances in the genome. Therefore, we next tried to delete a genomic region which spans 95 kb in the mouse genome containing three receptors for formyl peptide to extend the deletion range through direct embryo injection (Fig. 1a). After injection of Cas9 protein:sgRNAs, 20 F0 pups were obtained. PCR and subsequent DNA sequencing confirmed that two pups were founders bearing the desired 95 kb genomic DNA deletion (Fig. 1b, Table 1). The deletion was germline transmissible as determined by genotyping of F1 progeny (Fig. 1c). We demonstrated that through a single injection of Cas9 proteins and two sgRNAs, large DNA fragment deleted animals could be easily generated.

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