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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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Detection of the Npc1l1 sgRNA:Cas9-mediated off-target cleavages in vivo.(a) PCR products of the potential off-target sites 2 and 4 (OTs 2 and 4) of Npc1l1 sgRNA:Cas9 from founder pigs. (b) Detection of Npc1l1 sgRNA:Cas9-mediated off-target cleavage at the OTs 2 and 4. All PCR products from (a) were subjected to T7EN1 cleavage assays. *, different cleavage band patterns compared to the control. The size of the digested bands does not match that expected. (c) Sequencing results of PCR products. No off-target indel mutation was observed in the T7EN7 cleavage bands. The two red arrows indicate potential off-target sites of OT2 and OT4, respectively. Con denotes the corresponding sequence of each retrieved from Ensembl.
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f5: Detection of the Npc1l1 sgRNA:Cas9-mediated off-target cleavages in vivo.(a) PCR products of the potential off-target sites 2 and 4 (OTs 2 and 4) of Npc1l1 sgRNA:Cas9 from founder pigs. (b) Detection of Npc1l1 sgRNA:Cas9-mediated off-target cleavage at the OTs 2 and 4. All PCR products from (a) were subjected to T7EN1 cleavage assays. *, different cleavage band patterns compared to the control. The size of the digested bands does not match that expected. (c) Sequencing results of PCR products. No off-target indel mutation was observed in the T7EN7 cleavage bands. The two red arrows indicate potential off-target sites of OT2 and OT4, respectively. Con denotes the corresponding sequence of each retrieved from Ensembl.

Mentions: Off-target mutagenesis is of a major concern for CRISPR/Cas9 system333435. The CRISPR/Cas9-induced off-target mutation is heritable both in mice and rats2324. We thus ask whether off-target mutation occurred in our genetic modified pigs. To this end, we screened the pig genome using the open SeqMap tool, and predicted a total of 8 most potential off-target sites (OT 1 ~ 8) (Supplementary Table 1). Using the primers listed in Supplementary Table 4, all the selected OTs were subjected to PCR amplification and T7EN1 cleavage assay. Results showed that except the OTs 2 and 4, no genetic modification was detected at the selected OTs (Figure 5a & b, Supplementary Figure 2). At the OTs 2 and 4, distinguishable cleavage bands of unexpected size were detected in 7 reactions of founders C1-1, C1-2, C1-5, C2-4, C2-6, including 2 (Founders C2-4 and C2-6) for OT2, and 5 (Founders C1-1, C1-2, C1-5, C2-4 and C2-6) for OT 4 (Figure 5b), indicating that the Npc1l1 sgRNA:Cas9 might have induced non-specific mutations at these two sites.


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)

Detection of the Npc1l1 sgRNA:Cas9-mediated off-target cleavages in vivo.(a) PCR products of the potential off-target sites 2 and 4 (OTs 2 and 4) of Npc1l1 sgRNA:Cas9 from founder pigs. (b) Detection of Npc1l1 sgRNA:Cas9-mediated off-target cleavage at the OTs 2 and 4. All PCR products from (a) were subjected to T7EN1 cleavage assays. *, different cleavage band patterns compared to the control. The size of the digested bands does not match that expected. (c) Sequencing results of PCR products. No off-target indel mutation was observed in the T7EN7 cleavage bands. The two red arrows indicate potential off-target sites of OT2 and OT4, respectively. Con denotes the corresponding sequence of each retrieved from Ensembl.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Detection of the Npc1l1 sgRNA:Cas9-mediated off-target cleavages in vivo.(a) PCR products of the potential off-target sites 2 and 4 (OTs 2 and 4) of Npc1l1 sgRNA:Cas9 from founder pigs. (b) Detection of Npc1l1 sgRNA:Cas9-mediated off-target cleavage at the OTs 2 and 4. All PCR products from (a) were subjected to T7EN1 cleavage assays. *, different cleavage band patterns compared to the control. The size of the digested bands does not match that expected. (c) Sequencing results of PCR products. No off-target indel mutation was observed in the T7EN7 cleavage bands. The two red arrows indicate potential off-target sites of OT2 and OT4, respectively. Con denotes the corresponding sequence of each retrieved from Ensembl.
Mentions: Off-target mutagenesis is of a major concern for CRISPR/Cas9 system333435. The CRISPR/Cas9-induced off-target mutation is heritable both in mice and rats2324. We thus ask whether off-target mutation occurred in our genetic modified pigs. To this end, we screened the pig genome using the open SeqMap tool, and predicted a total of 8 most potential off-target sites (OT 1 ~ 8) (Supplementary Table 1). Using the primers listed in Supplementary Table 4, all the selected OTs were subjected to PCR amplification and T7EN1 cleavage assay. Results showed that except the OTs 2 and 4, no genetic modification was detected at the selected OTs (Figure 5a & b, Supplementary Figure 2). At the OTs 2 and 4, distinguishable cleavage bands of unexpected size were detected in 7 reactions of founders C1-1, C1-2, C1-5, C2-4, C2-6, including 2 (Founders C2-4 and C2-6) for OT2, and 5 (Founders C1-1, C1-2, C1-5, C2-4 and C2-6) for OT 4 (Figure 5b), indicating that the Npc1l1 sgRNA:Cas9 might have induced non-specific mutations at these two sites.

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