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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 Npc1l1 sgRNA:Cas9-mediated targeting in different tissues.(a) PCR products of the targeted region of Npc1l1 from 7 different tissues. (b) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in ear, heart, and liver. 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.
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f3: Detection of Npc1l1 sgRNA:Cas9-mediated targeting in different tissues.(a) PCR products of the targeted region of Npc1l1 from 7 different tissues. (b) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in ear, heart, and liver. 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.

Mentions: All of the targeting results described above were from the noninvasively available ear tissues. The dead founder pig provided us an opportunity to evaluate the integration of the Cas9/sgRNA-mediated Npc1l1 targeting into the derivatives of three germ layers, which generated detailed information for CRISPR/Cas9-mediated genome targeting in pigs via injection of zygotes with Cas9/sgRNA mixture. By PCR amplification and T7EN1 cleavage assays, we first performed extensive analysis of the target mutagenesis in 7 different somatic tissues, including heart, liver, spleen, lung, kidney, skin, and muscle from the dead founder. While the PCR bands did not differ between experimental and control groups (Figure 3a), but the T7EN1 assays showed that the cleavage bands existed in every reactions (Figure 3b), indicating that the Cas9/sgRNA-mediated mutations occurred in all tissues examined. The target mutations were further confirmed by sequencing the samples from ear, heart, and liver, which are derived from ectoderm, mesoderm, and endoderm, respectively, and the results showed that identical genetic modifications extensively integrated into all three germ layers (Figure 3c and Table 5). Again, no wild type allele was detected, indicating that CRISPR/Cas9-mediated bi-allelic mutations existed in all tissues examined. These results further substantiate the high efficiency of CRISPR/Cas9 system in pigs.


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 Npc1l1 sgRNA:Cas9-mediated targeting in different tissues.(a) PCR products of the targeted region of Npc1l1 from 7 different tissues. (b) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in ear, heart, and liver. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Detection of Npc1l1 sgRNA:Cas9-mediated targeting in different tissues.(a) PCR products of the targeted region of Npc1l1 from 7 different tissues. (b) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1. All PCR products from (a) were subjected to T7EN1 cleavage assays. (c) Sequencing results of modified Npc1l1 alleles detected in ear, heart, and liver. 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.
Mentions: All of the targeting results described above were from the noninvasively available ear tissues. The dead founder pig provided us an opportunity to evaluate the integration of the Cas9/sgRNA-mediated Npc1l1 targeting into the derivatives of three germ layers, which generated detailed information for CRISPR/Cas9-mediated genome targeting in pigs via injection of zygotes with Cas9/sgRNA mixture. By PCR amplification and T7EN1 cleavage assays, we first performed extensive analysis of the target mutagenesis in 7 different somatic tissues, including heart, liver, spleen, lung, kidney, skin, and muscle from the dead founder. While the PCR bands did not differ between experimental and control groups (Figure 3a), but the T7EN1 assays showed that the cleavage bands existed in every reactions (Figure 3b), indicating that the Cas9/sgRNA-mediated mutations occurred in all tissues examined. The target mutations were further confirmed by sequencing the samples from ear, heart, and liver, which are derived from ectoderm, mesoderm, and endoderm, respectively, and the results showed that identical genetic modifications extensively integrated into all three germ layers (Figure 3c and Table 5). Again, no wild type allele was detected, indicating that CRISPR/Cas9-mediated bi-allelic mutations existed in all tissues examined. These results further substantiate the high efficiency of CRISPR/Cas9 system in pigs.

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