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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

Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA.(a) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. (b) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.
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f3: Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA.(a) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. (b) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.

Mentions: Our above results suggest that delivery of Cas9 protein:sgRNA:ssODN is capable of introducing mutations through an HDR mechanism. Since HDR is cell cycle dependent and Cas9 protein should function faster than Cas9 mRNA once injected into the pronucleus, we assumed that the HDR efficiency following Cas9 protein injection was different from that of Cas9 mRNA injection. To compare the HDR efficiency between injection of Cas9 protein and Cas9 mRNA, we designed an sgRNA targeting the mouse Sirt3 locus and synthesized the corresponding ssODN template which contains 4 substitutions in the target sequence and 1 substitution near the PAM on the opposite side (Fig. 3a). Of 21 newborn pups in the Cas9 mRNA group, 12 pups carried indels. Only 2 of them were founders bearing the desired knock-in mutation. In the Cas9 protein injection group 7 of 9 pups carried indels with 2 of them bearing the desired knock-in mutation. These results suggested that injection of Cas9 protein could give a slightly higher indel (7/9 = 78% vs 12/21 = 63%) and increased HDR (2/9 = 22% vs 2/21 = 9.5%) rate than Cas9 mRNA injection (Fig. 3b and Table 1); however more experiments should be carried out to confirm this phenomenon on different genomic loci. In addition, the group that received Cas9 mRNA injection exhibited higher chimerism in some pups compared to the Cas9 protein injection group (Fig. 3b).


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)

Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA.(a) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. (b) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA.(a) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. (b) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.
Mentions: Our above results suggest that delivery of Cas9 protein:sgRNA:ssODN is capable of introducing mutations through an HDR mechanism. Since HDR is cell cycle dependent and Cas9 protein should function faster than Cas9 mRNA once injected into the pronucleus, we assumed that the HDR efficiency following Cas9 protein injection was different from that of Cas9 mRNA injection. To compare the HDR efficiency between injection of Cas9 protein and Cas9 mRNA, we designed an sgRNA targeting the mouse Sirt3 locus and synthesized the corresponding ssODN template which contains 4 substitutions in the target sequence and 1 substitution near the PAM on the opposite side (Fig. 3a). Of 21 newborn pups in the Cas9 mRNA group, 12 pups carried indels. Only 2 of them were founders bearing the desired knock-in mutation. In the Cas9 protein injection group 7 of 9 pups carried indels with 2 of them bearing the desired knock-in mutation. These results suggested that injection of Cas9 protein could give a slightly higher indel (7/9 = 78% vs 12/21 = 63%) and increased HDR (2/9 = 22% vs 2/21 = 9.5%) rate than Cas9 mRNA injection (Fig. 3b and Table 1); however more experiments should be carried out to confirm this phenomenon on different genomic loci. In addition, the group that received Cas9 mRNA injection exhibited higher chimerism in some pups compared to the Cas9 protein injection group (Fig. 3b).

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