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Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA.

Wang Y, Du Y, Shen B, Zhou X, Li J, Liu Y, Wang J, Zhou J, Hu B, Kang N, Gao J, Yu L, Huang X, Wei H - Sci Rep (2015)

Bottom Line: Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species.By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%.Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China.

ABSTRACT
Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

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Related in: MedlinePlus

Npc1l1 sgRNA:Cas9-mediated modifications in the ovaries of founder C2-5.(a) PCR products of the targeted region of Npc1l1 in the ovaries of founder C2-5. (b) Detection of Npc1l1 sgRNA:Cas9-mediated cleavage of Npc1l1 in ovaries. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in the ovaries of founder C2-5. PCR products of the targeted region of Npc1l1 were amplified from the ovaries and sequenced. The PAM sequences are highlighted in green; the targeting sequences in red; the mutations are shown in blue, lower case. Deletions, (−); and Insertions, (+). N/N indicates positive colonies out of total sequenced.
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f4: Npc1l1 sgRNA:Cas9-mediated modifications in the ovaries of founder C2-5.(a) PCR products of the targeted region of Npc1l1 in the ovaries of founder C2-5. (b) Detection of Npc1l1 sgRNA:Cas9-mediated cleavage of Npc1l1 in ovaries. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in the ovaries of founder C2-5. PCR products of the targeted region of Npc1l1 were amplified from the ovaries and sequenced. The PAM sequences are highlighted in green; the targeting sequences in red; the mutations are shown in blue, lower case. Deletions, (−); and Insertions, (+). N/N indicates positive colonies out of total sequenced.

Mentions: To establish genetically modified animals requires germline transmission of genetic mutations. Because the gene-modified Bama miniature pigs have not reached sexual maturity, we currently cannot perform breeding to determine germline transmission. But the highly efficient occurrences of the target mutations in different tissues strongly suggest it is very possible that the Cas9/sgRNA-mediated genome targeting will integrate into the pig gonads. To prove the possibility, the founder C2-5 was sacrificed and the ovaries were isolated. The examination of the target mutagenesis was performed as above. The PCR and T7EN1 assays revealed that the cleavage of Npc1l1 did occur in the ovaries (Figure 4a & b). This was confirmed by sequencing of the target mutations, which were identical to those detected in the somatic tissues, such as a 1-bp insertion, 9-, 13-, and 24-bp deletions (Figure 4c and Table 6), and absence of wild-type sequence, demonstrating the Cas9/sgRNA-mediated genome targeting successfully integrated into the gonads too. It is notifying, the gonads are composed of germ cells and somatic cells. The sequencing results of the ovaries showed that the efficiency of Cas9/sgRNA-induced targeting was 100% in the ovaries, strongly suggesting that the Cas9/sgRNA-mediated targeting was most likely transmitted into the pig germ cells. Nevertheless, breeding results will provide direct evidence of germline transmission in the future.


Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA.

Wang Y, Du Y, Shen B, Zhou X, Li J, Liu Y, Wang J, Zhou J, Hu B, Kang N, Gao J, Yu L, Huang X, Wei H - Sci Rep (2015)

Npc1l1 sgRNA:Cas9-mediated modifications in the ovaries of founder C2-5.(a) PCR products of the targeted region of Npc1l1 in the ovaries of founder C2-5. (b) Detection of Npc1l1 sgRNA:Cas9-mediated cleavage of Npc1l1 in ovaries. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in the ovaries of founder C2-5. PCR products of the targeted region of Npc1l1 were amplified from the ovaries and sequenced. The PAM sequences are highlighted in green; the targeting sequences in red; the mutations are shown in blue, lower case. Deletions, (−); and Insertions, (+). N/N indicates positive colonies out of total sequenced.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317696&req=5

f4: Npc1l1 sgRNA:Cas9-mediated modifications in the ovaries of founder C2-5.(a) PCR products of the targeted region of Npc1l1 in the ovaries of founder C2-5. (b) Detection of Npc1l1 sgRNA:Cas9-mediated cleavage of Npc1l1 in ovaries. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in the ovaries of founder C2-5. PCR products of the targeted region of Npc1l1 were amplified from the ovaries and sequenced. The PAM sequences are highlighted in green; the targeting sequences in red; the mutations are shown in blue, lower case. Deletions, (−); and Insertions, (+). N/N indicates positive colonies out of total sequenced.
Mentions: To establish genetically modified animals requires germline transmission of genetic mutations. Because the gene-modified Bama miniature pigs have not reached sexual maturity, we currently cannot perform breeding to determine germline transmission. But the highly efficient occurrences of the target mutations in different tissues strongly suggest it is very possible that the Cas9/sgRNA-mediated genome targeting will integrate into the pig gonads. To prove the possibility, the founder C2-5 was sacrificed and the ovaries were isolated. The examination of the target mutagenesis was performed as above. The PCR and T7EN1 assays revealed that the cleavage of Npc1l1 did occur in the ovaries (Figure 4a & b). This was confirmed by sequencing of the target mutations, which were identical to those detected in the somatic tissues, such as a 1-bp insertion, 9-, 13-, and 24-bp deletions (Figure 4c and Table 6), and absence of wild-type sequence, demonstrating the Cas9/sgRNA-mediated genome targeting successfully integrated into the gonads too. It is notifying, the gonads are composed of germ cells and somatic cells. The sequencing results of the ovaries showed that the efficiency of Cas9/sgRNA-induced targeting was 100% in the ovaries, strongly suggesting that the Cas9/sgRNA-mediated targeting was most likely transmitted into the pig germ cells. Nevertheless, breeding results will provide direct evidence of germline transmission in the future.

Bottom Line: Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species.By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%.Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China.

ABSTRACT
Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

Show MeSH
Related in: MedlinePlus