Research progress of producing genetically modified pigs by CRISPR/Cas9 in the medical field_Journal of Biomedical Engineering

Authors：

GAO Mengyu ¹ , YANG Guang ² ,  BAO Ji ¹

1. Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
2. Experimental Animal Center, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;

Corresponding author：

Keywords：

clustered regularly interspersed short palindromic repeat/CRISPR-associated 9; large animal disease models; genetically modified pig; gene editing

DOI：

10.7507/1001-5515.201802046

Video：

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As pigs are similar to humans in anatomy, physiology and pathology, nutrition metabolism and disease characteristics, genetically modified pigs are already used for the studies of disease mechanism, pathology and toxicology and the evaluation of drugs. But the production of large modified animals is difficult, cumbersome, time-consuming and costly. With the breakthrough of gene editing technology, clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated 9( Cas9)(CRISPR/Cas9) technology has greatly improved the mutation efficiency, reduced the cost and simplified the steps, and promoted the widespread application of genetically modified pigs. In this paper, the production methods of genetically modified pigs and the research progress of genetically modified pigs by CRISPR/Cas9 in the medical field were reviewed.

Citation： GAO Mengyu, YANG Guang, BAO Ji. Research progress of producing genetically modified pigs by CRISPR/Cas9 in the medical field. Journal of Biomedical Engineering, 2018, 35(4): 637-642. doi: 10.7507/1001-5515.201802046 Copy

1.	Whyte J J, Prather R S. Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev, 2011, 78(10/11): 879-891.
2.	Niemann H, Petersen B. The production of multi-transgenic pigs: update and perspectives for xenotransplantation. Transgenic Res, 2016, 25(3): 361-374.
3.	Butler J R, Ladowski J M, Martens G R, et al. Recent advances in genome editing and creation of genetically modified pigs. Int J Surg, 2015, 23(Pt B): 217-222.
4.	Song Chanwoo, Lee J, Lee S Y. Genome engineering and gene expression control for bacterial strain development. Biotechnol J, 2015, 10(1): 56-68.
5.	Eid A, Mahfouz M M. Genome editing: the road of CRISPR/Cas9 from bench to clinic. Exp Mol Med, 2016, 48(10): e265.
6.	Ulain Q, Chung J Y, Kim Y H. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release, 2015, 205: 120-127.
7.	Samanta M K, Dey A, Gayen S. CRISPR/Cas9: an advanced tool for editing plant genomes. Transgenic Res, 2016, 25(5): 561-573.
8.	Lafountaine J S, Fathe K, Smyth H D. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm, 2015, 494(1): 180-194.
9.	Chen Fengjiao, Wang Ying, Yuan Yilin, et al. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting. Journal of Genetics and Genomics, 2015, 42(8): 437-444.
10.	Zhou Xiaoqing, Xin Jige, Fan Nana, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72(6): 1175-1184.
11.	Bi Yanzhen, Hua Zaidong, Liu Ximei, et al. Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP. Sci Rep, 2016, 6: 31729.
12.	Lai Sisi, Wei Shu, Zhao Bentian, et al. Generation of knock-in pigs carrying Oct4-tdTomato reporter through CRISPR/Cas9-mediated genome engineering. PLoS One, 2016, 11(1): e0146562.
13.	Wang Haoyi, Yang Hui, Shivalila C S, et al. One-Step Generation of mice carrying mutations in multiple genes by CRISPR/Cas-Mediated genome engineering. Cell, 2013, 153(4): 910-918.
14.	Peng Jin, Wang Yong, Jiang Junyi, et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Sci Rep, 2015, 5: 16705.
15.	Wu Jun, Vilarino M, Suzuki K, et al. CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci Rep, 2017, 7(1): 10487.
16.	Lei Shaohua, Ryu J, Wen Ke, et al. Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency. Sci Rep, 2016, 6: 25222.
17.	Sato M, Koriyama M, Watanabe S, et al. Direct injection of CRISPR/Cas9-Related mRNA into cytoplasm of partheno genetically activated porcine oocytes causes frequent mosaicism for indel mutations. Int J Mol Sci, 2015, 16(8): 17838-17856.
18.	Wang Xianlong, Zhou Jinwei, Cao Chunwei, et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci Rep, 2015, 5: 13348.
19.	Wu Jinqing, Mei Gui, Liu Zhiguo, et al. Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system. Yi Chuan, 2015, 37(1): 55-62.
20.	Tao Li, Yang Mingyao, Wang Xiaodong, et al. Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system. Biochem Biophys Res Commun, 2016, 476(4): 225-229.
21.	Wang Kepin, Jin Qin, Ruan Degong, et al. Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing. Genome Res, 2017, 27(12): 2061-2071.
22.	Hai Tang, Teng Fei, Guo Runfa, et al. One-step Generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res, 2014, 24(3): 372-375.
23.	Lotem J, Levanon D, Negreanu V, et al. Runx3 at the interface of immunity, inflammation and cancer. Biochim Biophys Acta, 2015, 1855(2): 131-143.
24.	Kang J T, Ryu J, Cho B, et al. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting. Reproduction in Domestic Animals, 2016, 51(6): 970-978.
25.	Wang Kankan, Ouyang Hongsheng, Xie Zicong, et al. Efficient Generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep, 2015, 5: 16623.
26.	Yan Sen, Tu Zhuchi, Liu Zhaoming, et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington's disease. Cell, 2018, 173(4): 989-1002.
27.	Wang Xianlong, Cao Chunwei, Huang Jiaojiao, et al. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Sci Rep, 2016, 6: 20620.
28.	Yang Luhan, Gueell M, Niu Dong, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 2015, 350(6264): 1101-1104.
29.	Niu Dong, Wei Hongjiang, Lin Lin, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science, 2017, 357(6357): 1303-1307.
30.	Estrada J L, Martens G, Li Ping, et al. Evaluation of human and non-human Primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation, 2015, 22(3): 194-202.
31.	Butler J R, Paris L L, Blankenship R L, et al. Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers. Transplantation, 2016, 100(3): 571-576.
32.	Gao Hanchao, Zhao Chengjiang, Xiang Xi, et al. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning. J Reprod Dev, 2017, 63(1): 17-26.
33.	Petersen B, Frenzel A, Lucas-Hahn A, et al. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation, 2016, 23(5): 338-346.
34.	Kang J T, Cho B, Ryu J, et al. Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs. Reprod Biol Endocrinol, 2016, 14(1): 74.
35.	Wu Zhenfang, Li Zicong, Yang Jinzeng. Transient transgene transmission to piglets by intrauterine insemination of spermatozoa incubated with DNA fragments. Mol Reprod Dev, 2008, 75(1): 26-32.
36.	Lavitrano M, Giovannoni R, Cerrito M G. Methods for sperm-mediated gene transfer. Methods Mol Biol, 2013, 927: 519-529.
37.	Zhang Yongliang, Xi Qianyun, Ding Jinghua, et al. Production of transgenic pigs mediated by pseudotyped lentivirus and sperm. PLoS One, 2012, 7(4): e35335.
38.	Oddi S, Bernabò N, Di Tommaso M, et al. DNA uptake in swine sperm: effect of plasmid topology and methyl-beta-cyclodextrin-mediated cholesterol depletion. Mol Reprod Dev, 2012, 79(12): 853-860.

1. Whyte J J, Prather R S. Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev, 2011, 78(10/11): 879-891.
2. Niemann H, Petersen B. The production of multi-transgenic pigs: update and perspectives for xenotransplantation. Transgenic Res, 2016, 25(3): 361-374.
3. Butler J R, Ladowski J M, Martens G R, et al. Recent advances in genome editing and creation of genetically modified pigs. Int J Surg, 2015, 23(Pt B): 217-222.
4. Song Chanwoo, Lee J, Lee S Y. Genome engineering and gene expression control for bacterial strain development. Biotechnol J, 2015, 10(1): 56-68.
5. Eid A, Mahfouz M M. Genome editing: the road of CRISPR/Cas9 from bench to clinic. Exp Mol Med, 2016, 48(10): e265.
6. Ulain Q, Chung J Y, Kim Y H. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release, 2015, 205: 120-127.
7. Samanta M K, Dey A, Gayen S. CRISPR/Cas9: an advanced tool for editing plant genomes. Transgenic Res, 2016, 25(5): 561-573.
8. Lafountaine J S, Fathe K, Smyth H D. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm, 2015, 494(1): 180-194.
9. Chen Fengjiao, Wang Ying, Yuan Yilin, et al. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting. Journal of Genetics and Genomics, 2015, 42(8): 437-444.
10. Zhou Xiaoqing, Xin Jige, Fan Nana, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72(6): 1175-1184.
11. Bi Yanzhen, Hua Zaidong, Liu Ximei, et al. Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP. Sci Rep, 2016, 6: 31729.
12. Lai Sisi, Wei Shu, Zhao Bentian, et al. Generation of knock-in pigs carrying Oct4-tdTomato reporter through CRISPR/Cas9-mediated genome engineering. PLoS One, 2016, 11(1): e0146562.
13. Wang Haoyi, Yang Hui, Shivalila C S, et al. One-Step Generation of mice carrying mutations in multiple genes by CRISPR/Cas-Mediated genome engineering. Cell, 2013, 153(4): 910-918.
14. Peng Jin, Wang Yong, Jiang Junyi, et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Sci Rep, 2015, 5: 16705.
15. Wu Jun, Vilarino M, Suzuki K, et al. CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci Rep, 2017, 7(1): 10487.
16. Lei Shaohua, Ryu J, Wen Ke, et al. Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency. Sci Rep, 2016, 6: 25222.
17. Sato M, Koriyama M, Watanabe S, et al. Direct injection of CRISPR/Cas9-Related mRNA into cytoplasm of partheno genetically activated porcine oocytes causes frequent mosaicism for indel mutations. Int J Mol Sci, 2015, 16(8): 17838-17856.
18. Wang Xianlong, Zhou Jinwei, Cao Chunwei, et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci Rep, 2015, 5: 13348.
19. Wu Jinqing, Mei Gui, Liu Zhiguo, et al. Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system. Yi Chuan, 2015, 37(1): 55-62.
20. Tao Li, Yang Mingyao, Wang Xiaodong, et al. Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system. Biochem Biophys Res Commun, 2016, 476(4): 225-229.
21. Wang Kepin, Jin Qin, Ruan Degong, et al. Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing. Genome Res, 2017, 27(12): 2061-2071.
22. Hai Tang, Teng Fei, Guo Runfa, et al. One-step Generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res, 2014, 24(3): 372-375.
23. Lotem J, Levanon D, Negreanu V, et al. Runx3 at the interface of immunity, inflammation and cancer. Biochim Biophys Acta, 2015, 1855(2): 131-143.
24. Kang J T, Ryu J, Cho B, et al. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting. Reproduction in Domestic Animals, 2016, 51(6): 970-978.
25. Wang Kankan, Ouyang Hongsheng, Xie Zicong, et al. Efficient Generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep, 2015, 5: 16623.
26. Yan Sen, Tu Zhuchi, Liu Zhaoming, et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington's disease. Cell, 2018, 173(4): 989-1002.
27. Wang Xianlong, Cao Chunwei, Huang Jiaojiao, et al. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Sci Rep, 2016, 6: 20620.
28. Yang Luhan, Gueell M, Niu Dong, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 2015, 350(6264): 1101-1104.
29. Niu Dong, Wei Hongjiang, Lin Lin, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science, 2017, 357(6357): 1303-1307.
30. Estrada J L, Martens G, Li Ping, et al. Evaluation of human and non-human Primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation, 2015, 22(3): 194-202.
31. Butler J R, Paris L L, Blankenship R L, et al. Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers. Transplantation, 2016, 100(3): 571-576.
32. Gao Hanchao, Zhao Chengjiang, Xiang Xi, et al. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning. J Reprod Dev, 2017, 63(1): 17-26.
33. Petersen B, Frenzel A, Lucas-Hahn A, et al. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation, 2016, 23(5): 338-346.
34. Kang J T, Cho B, Ryu J, et al. Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs. Reprod Biol Endocrinol, 2016, 14(1): 74.
35. Wu Zhenfang, Li Zicong, Yang Jinzeng. Transient transgene transmission to piglets by intrauterine insemination of spermatozoa incubated with DNA fragments. Mol Reprod Dev, 2008, 75(1): 26-32.
36. Lavitrano M, Giovannoni R, Cerrito M G. Methods for sperm-mediated gene transfer. Methods Mol Biol, 2013, 927: 519-529.
37. Zhang Yongliang, Xi Qianyun, Ding Jinghua, et al. Production of transgenic pigs mediated by pseudotyped lentivirus and sperm. PLoS One, 2012, 7(4): e35335.
38. Oddi S, Bernabò N, Di Tommaso M, et al. DNA uptake in swine sperm: effect of plasmid topology and methyl-beta-cyclodextrin-mediated cholesterol depletion. Mol Reprod Dev, 2012, 79(12): 853-860.

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Research progress of producing genetically modified pigs by CRISPR/Cas9 in the medical field

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