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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 Npc1l1sgRNA:Cas9-mediated modifications of NPC1L1 in founder pigs.(a) A photo showing 38-day-old pigs carrying Npc1l1 mutations. (b) PCR products of the targeted region of Npc1l1 from founder pigs. The first litter of five live pigs were named C1-1 to C1-5. The second litter of six live pigs were named from C2-1 to C2-6. Con denotes wild-type pig as a control. Notably, the PCR products of C1-3, C1-4, C2-1, C2-4 and C2-6 differ from the control in size, suggesting a large fragment deletion or insertion. (c) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1 by T7EN1 cleavage assays. All PCR products from (b) were subjected to T7EN1 cleavage assays. All the samples were digested by T7EN1, suggesting that all founders carry Npc1l1 mutations. (d) Sequencing results of modified Npc1l1 alleles detected in founder pigs. At least 12 TA clones of the PCR products were analyzed. Sequences complementary to sgRNA are labeled in red, and PAM sequences in green. Mutations, blue, lower case; deletions, (−); insertions, (+). N/N indicates positive colonies out of total sequenced samples. See also Figure S1.
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f2: Detection of Npc1l1sgRNA:Cas9-mediated modifications of NPC1L1 in founder pigs.(a) A photo showing 38-day-old pigs carrying Npc1l1 mutations. (b) PCR products of the targeted region of Npc1l1 from founder pigs. The first litter of five live pigs were named C1-1 to C1-5. The second litter of six live pigs were named from C2-1 to C2-6. Con denotes wild-type pig as a control. Notably, the PCR products of C1-3, C1-4, C2-1, C2-4 and C2-6 differ from the control in size, suggesting a large fragment deletion or insertion. (c) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1 by T7EN1 cleavage assays. All PCR products from (b) were subjected to T7EN1 cleavage assays. All the samples were digested by T7EN1, suggesting that all founders carry Npc1l1 mutations. (d) Sequencing results of modified Npc1l1 alleles detected in founder pigs. At least 12 TA clones of the PCR products were analyzed. Sequences complementary to sgRNA are labeled in red, and PAM sequences in green. Mutations, blue, lower case; deletions, (−); insertions, (+). N/N indicates positive colonies out of total sequenced samples. See also Figure S1.

Mentions: With the success in pig parthenogenetic embryos, we set out to generate knockout Chinese Bama miniature pigs. A total of 110 porcine early embryos (1-cell stage) were surgically collected from 20 mated sows. The Cas9 mRNA and Npc1l1 sgRNA mixtures were injected into cytoplasm of embryo cells as described above. A total of 105 out of 110 injected embryos were transferred into 4 surrogate females. Of the 4 recipient mothers, 2 pregnancies were established (50%; 2 out of 4). After full-term (~114 days) pregnancy, 2 litters (Litter 1: C1-1 ~ C1-5; Litter 2: C2-1 ~ C2-6) of 12 piglets were successfully delivered alive, but one died immediately after birth (Figure 2a and Table 3).


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 Npc1l1sgRNA:Cas9-mediated modifications of NPC1L1 in founder pigs.(a) A photo showing 38-day-old pigs carrying Npc1l1 mutations. (b) PCR products of the targeted region of Npc1l1 from founder pigs. The first litter of five live pigs were named C1-1 to C1-5. The second litter of six live pigs were named from C2-1 to C2-6. Con denotes wild-type pig as a control. Notably, the PCR products of C1-3, C1-4, C2-1, C2-4 and C2-6 differ from the control in size, suggesting a large fragment deletion or insertion. (c) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1 by T7EN1 cleavage assays. All PCR products from (b) were subjected to T7EN1 cleavage assays. All the samples were digested by T7EN1, suggesting that all founders carry Npc1l1 mutations. (d) Sequencing results of modified Npc1l1 alleles detected in founder pigs. At least 12 TA clones of the PCR products were analyzed. Sequences complementary to sgRNA are labeled in red, and PAM sequences in green. Mutations, blue, lower case; deletions, (−); insertions, (+). N/N indicates positive colonies out of total sequenced samples. See also Figure S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Detection of Npc1l1sgRNA:Cas9-mediated modifications of NPC1L1 in founder pigs.(a) A photo showing 38-day-old pigs carrying Npc1l1 mutations. (b) PCR products of the targeted region of Npc1l1 from founder pigs. The first litter of five live pigs were named C1-1 to C1-5. The second litter of six live pigs were named from C2-1 to C2-6. Con denotes wild-type pig as a control. Notably, the PCR products of C1-3, C1-4, C2-1, C2-4 and C2-6 differ from the control in size, suggesting a large fragment deletion or insertion. (c) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1 by T7EN1 cleavage assays. All PCR products from (b) were subjected to T7EN1 cleavage assays. All the samples were digested by T7EN1, suggesting that all founders carry Npc1l1 mutations. (d) Sequencing results of modified Npc1l1 alleles detected in founder pigs. At least 12 TA clones of the PCR products were analyzed. Sequences complementary to sgRNA are labeled in red, and PAM sequences in green. Mutations, blue, lower case; deletions, (−); insertions, (+). N/N indicates positive colonies out of total sequenced samples. See also Figure S1.
Mentions: With the success in pig parthenogenetic embryos, we set out to generate knockout Chinese Bama miniature pigs. A total of 110 porcine early embryos (1-cell stage) were surgically collected from 20 mated sows. The Cas9 mRNA and Npc1l1 sgRNA mixtures were injected into cytoplasm of embryo cells as described above. A total of 105 out of 110 injected embryos were transferred into 4 surrogate females. Of the 4 recipient mothers, 2 pregnancies were established (50%; 2 out of 4). After full-term (~114 days) pregnancy, 2 litters (Litter 1: C1-1 ~ C1-5; Litter 2: C2-1 ~ C2-6) of 12 piglets were successfully delivered alive, but one died immediately after birth (Figure 2a and Table 3).

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