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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.


Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA.(A) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. (C) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. 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. Deleted bases are marked with colons, and inserted bases are gray. (D) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.
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f5: Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA.(A) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. (C) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. 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. Deleted bases are marked with colons, and inserted bases are gray. (D) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.

Mentions: The ultimate aim of this study was to efficiently generate genetically modified pigs through the direct cytoplasmic injections of Cas9 mRNA and sgRNA into zygotes. Thus, we next transferred the Cas9 mRNA and R1 sgRNA-injected zygotes into surrogate pigs to produce piglets. A total of 40 embryos were delivered to 3 surrogates, and one pregnancy was established (Table 4). Two live-born piglets were obtained and showed the white coat-color phenotype over its entire body (Fig. 5A); the wild-type pigs exhibited pigment deposition at the two ends of the body (Fig. 5A). RFLP (Fig. 5B) and sequence analysis (Fig. 5C) assays showed that the piglets were all genotyped as bi-allelic mutations, suggesting that R1 sgRNA could efficiently facilitate the CRISPR/Cas9 system to generate Mitf knockout pigs. The skin tissues of the tail were dissected from the mutant piglets, and Western blot analysis was performed to confirm the disruption of MITF in these pigs. Compared with wild-type piglet, MITF was completely absent in the two mutant piglets (Fig. 5D). Moreover, the genomic DNA isolated from the two mutant piglets were used to perform off-target analyses. The fragments around the potential off-target loci were amplified and sequenced. No unwanted mutations occurred at these genomic sites of the two mutant piglets (Supplemental Table S2 and Figure S1).


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)

Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA.(A) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. (C) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. 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. Deleted bases are marked with colons, and inserted bases are gray. (D) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA.(A) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. (C) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. 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. Deleted bases are marked with colons, and inserted bases are gray. (D) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.
Mentions: The ultimate aim of this study was to efficiently generate genetically modified pigs through the direct cytoplasmic injections of Cas9 mRNA and sgRNA into zygotes. Thus, we next transferred the Cas9 mRNA and R1 sgRNA-injected zygotes into surrogate pigs to produce piglets. A total of 40 embryos were delivered to 3 surrogates, and one pregnancy was established (Table 4). Two live-born piglets were obtained and showed the white coat-color phenotype over its entire body (Fig. 5A); the wild-type pigs exhibited pigment deposition at the two ends of the body (Fig. 5A). RFLP (Fig. 5B) and sequence analysis (Fig. 5C) assays showed that the piglets were all genotyped as bi-allelic mutations, suggesting that R1 sgRNA could efficiently facilitate the CRISPR/Cas9 system to generate Mitf knockout pigs. The skin tissues of the tail were dissected from the mutant piglets, and Western blot analysis was performed to confirm the disruption of MITF in these pigs. Compared with wild-type piglet, MITF was completely absent in the two mutant piglets (Fig. 5D). Moreover, the genomic DNA isolated from the two mutant piglets were used to perform off-target analyses. The fragments around the potential off-target loci were amplified and sequenced. No unwanted mutations occurred at these genomic sites of the two mutant piglets (Supplemental Table S2 and Figure S1).

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.