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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.


Rapidly selecting most effective sgRNAs by single blastocyst genotyping.(A) Overview and timeline of the experiment. Beginning with experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. (B) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
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f2: Rapidly selecting most effective sgRNAs by single blastocyst genotyping.(A) Overview and timeline of the experiment. Beginning with experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. (B) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

Mentions: The lack of a simple platform to unbiasedly evaluate the efficacies of sgRNA creates uncertainties and restricts the ability to modify the pig genome. Toward this end, we developed an experimental system to rapidly select the most favorable sgRNA for a specific gene locus based on single-blastocyst genotyping. An overview of the experimental process with approximate timings is shown in Fig. 2A.


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)

Rapidly selecting most effective sgRNAs by single blastocyst genotyping.(A) Overview and timeline of the experiment. Beginning with experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. (B) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Rapidly selecting most effective sgRNAs by single blastocyst genotyping.(A) Overview and timeline of the experiment. Beginning with experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. (B) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
Mentions: The lack of a simple platform to unbiasedly evaluate the efficacies of sgRNA creates uncertainties and restricts the ability to modify the pig genome. Toward this end, we developed an experimental system to rapidly select the most favorable sgRNA for a specific gene locus based on single-blastocyst genotyping. An overview of the experimental process with approximate timings is shown in Fig. 2A.

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.