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Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.


Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis.Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.
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f4: Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis.Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.

Mentions: The dominant strategy for generating transgenic pigs is to first genetically modify fibroblasts and then conduct SCNT. To determine whether the selected sgRNA by mRNA injection into PA-derived blastocysts can induce highly efficient mutations in somatic cells, we investigated the mutagenesis efficiencies of different sgRNAs in porcine primary fibroblasts. The plasmids expressing Cas9 and R1 or R2 sgRNA were transfected into fibroblasts, and transfected single cells were sorted and cultured in 96-well plates. Of 17 colonies obtained by R1 sgRNA transfections, eight carried mutations in the target sequence and seven had biallelic mutations. However, of the 10 colonies obtained by R2 sgRNA transfection, only two colonies carried mutations in the target sequence, and none had biallelic mutations (Fig. 4). The results (summarized in Table 3) demonstrated that the mutagenesis efficiencies of sgRNAs in fibroblasts were comparative with those in single blastocyst assays, suggesting that the mutagenesis efficiencies of a given sgRNA were consistent in both embryo and somatic cells.


Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis.Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543986&req=5

f4: Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis.Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.
Mentions: The dominant strategy for generating transgenic pigs is to first genetically modify fibroblasts and then conduct SCNT. To determine whether the selected sgRNA by mRNA injection into PA-derived blastocysts can induce highly efficient mutations in somatic cells, we investigated the mutagenesis efficiencies of different sgRNAs in porcine primary fibroblasts. The plasmids expressing Cas9 and R1 or R2 sgRNA were transfected into fibroblasts, and transfected single cells were sorted and cultured in 96-well plates. Of 17 colonies obtained by R1 sgRNA transfections, eight carried mutations in the target sequence and seven had biallelic mutations. However, of the 10 colonies obtained by R2 sgRNA transfection, only two colonies carried mutations in the target sequence, and none had biallelic mutations (Fig. 4). The results (summarized in Table 3) demonstrated that the mutagenesis efficiencies of sgRNAs in fibroblasts were comparative with those in single blastocyst assays, suggesting that the mutagenesis efficiencies of a given sgRNA were consistent in both embryo and somatic cells.

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.