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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

Evaluation of Npc1l1 sgRNA:Cas9-mediated modifications of Npc1l1 in pig parthenogenetic embryos.(a) Schematic diagram of pig Npc1l1 partial protein coding region and the targeting locus of Npc1l1 sgRNA:Cas9. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9. (b) Sequencing results of the modified Npc1l1 alleles detected in injected parthenogenetic embryos. Sequences complementary to sgRNA are labeled in red, and PAM sequences are in green; mutations, blue, lower case; deletions, (−); insertions, (+). (c) The chromatographs of sequencing modified Npc1/1 alleles in parthenogenetic embryos in which overlapped peaks were observed. Con denotes wild-type sequence. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9.
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f1: Evaluation of Npc1l1 sgRNA:Cas9-mediated modifications of Npc1l1 in pig parthenogenetic embryos.(a) Schematic diagram of pig Npc1l1 partial protein coding region and the targeting locus of Npc1l1 sgRNA:Cas9. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9. (b) Sequencing results of the modified Npc1l1 alleles detected in injected parthenogenetic embryos. Sequences complementary to sgRNA are labeled in red, and PAM sequences are in green; mutations, blue, lower case; deletions, (−); insertions, (+). (c) The chromatographs of sequencing modified Npc1/1 alleles in parthenogenetic embryos in which overlapped peaks were observed. Con denotes wild-type sequence. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9.

Mentions: NPC1L1 is highly expressed in small intestine and plays a critical role in both dietary cholesterol absorption and biliary cholesterol reabsorption303132. In mice, NPC1L1 is exclusively expressed in small intestine and gallbladder, but human NPC1L1 is highly expressed in liver, intestine and perhaps other tissues. The tissue distribution of NPC1L1 expression in pigs was unknown. Considering similarities in physiology and metabolism between pigs and humans, we generated NPC1L1-deficient pig models for future definition of how NPC1L1 influences cardiovascular and metabolic diseases. One sgRNA targeting the exon 2 of Npc1l1 (Figure 1a) was designed as described previously22.


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)

Evaluation of Npc1l1 sgRNA:Cas9-mediated modifications of Npc1l1 in pig parthenogenetic embryos.(a) Schematic diagram of pig Npc1l1 partial protein coding region and the targeting locus of Npc1l1 sgRNA:Cas9. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9. (b) Sequencing results of the modified Npc1l1 alleles detected in injected parthenogenetic embryos. Sequences complementary to sgRNA are labeled in red, and PAM sequences are in green; mutations, blue, lower case; deletions, (−); insertions, (+). (c) The chromatographs of sequencing modified Npc1/1 alleles in parthenogenetic embryos in which overlapped peaks were observed. Con denotes wild-type sequence. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Evaluation of Npc1l1 sgRNA:Cas9-mediated modifications of Npc1l1 in pig parthenogenetic embryos.(a) Schematic diagram of pig Npc1l1 partial protein coding region and the targeting locus of Npc1l1 sgRNA:Cas9. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9. (b) Sequencing results of the modified Npc1l1 alleles detected in injected parthenogenetic embryos. Sequences complementary to sgRNA are labeled in red, and PAM sequences are in green; mutations, blue, lower case; deletions, (−); insertions, (+). (c) The chromatographs of sequencing modified Npc1/1 alleles in parthenogenetic embryos in which overlapped peaks were observed. Con denotes wild-type sequence. Red arrow indicates the targeting site of Npc1l1 sgRNA:Cas9.
Mentions: NPC1L1 is highly expressed in small intestine and plays a critical role in both dietary cholesterol absorption and biliary cholesterol reabsorption303132. In mice, NPC1L1 is exclusively expressed in small intestine and gallbladder, but human NPC1L1 is highly expressed in liver, intestine and perhaps other tissues. The tissue distribution of NPC1L1 expression in pigs was unknown. Considering similarities in physiology and metabolism between pigs and humans, we generated NPC1L1-deficient pig models for future definition of how NPC1L1 influences cardiovascular and metabolic diseases. One sgRNA targeting the exon 2 of Npc1l1 (Figure 1a) was designed as described previously22.

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