The Application of CRISPR/Cas9 Technology for Farm Animals: A Review

DOI: 10.18805/ag.R-2163    | Article Id: R-2163 | Page : 54-61
Citation :- The Application of CRISPR/Cas9 Technology for Farm Animals: A Review.Agricultural Reviews.2022.(43):54-61
Vinay Kumar Mehra, Satish Kumar vinay28mehra@yahoo.com
Address : Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India.
Submitted Date : 18-01-2021
Accepted Date : 16-07-2021


Livestock animal are important for agriculture economy and biomedical research. They are sources of Milk, meat, carcass, organic manure and other products. The development of genome editing technologies, especially CRISPR-Cas have revolutionized the generation of gene edited farm animals. In this review, we briefly introduce the CRISPR-Cas9 technology and highlight its application on livestock such as human disease modeling, disease resistant animal, and generation of hornless cattle, animal welfare and other agricultural and biomedical related traits which enhance the livestock production in order to meet the increasing demand of food worldwide. The ability to transfer sperm-producing stem cells or spermatogonial stem cells (SSCs) from a donor animal into the testes of a recipient male could have multiple applications. Production of BLG free milk in cattle provides a promising way to those who have allergy to cow milk. The knockdown of myostatin gene in different species like Sheep, Goat, Cattle and pig is very helpful in the economy of meat industry. Besides the several benefits of CRISPR-Cas9 technology, the risk factors and ethics issues related to this technology should be reconsidered before they enter into CRISPR era.


Cow milk allergy CRISPR Disease model Genome editing Livestock


  1. Adli M (2018). The CRISPR tool kit for genome editing and beyond. Nat Commun. 9(1): 1911.
  2. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes Science. 3: 1709-1712.
  3. Bjoern Petersen (2017). Basics of genome editing technology and its application in livestock species. Reprod Domest Anim. 52(3): 4-13. 
  4. Carlson DF, Lancto CA, Zang B, et al. (2016). Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology. 34(5): 479-481. 
  5. Chekani azar, Saeid and Mombeni, Ehsan and Birhan, Mastewal and Yousefi, Mahshad. (2020). CRISPR/Cas9 gene editing technology and its application to the coronavirus disease (COVID-19), A review. Journal of World’s Poultry Research. 10: 1-09. 
  6. Chen F, Wang Y, Yuan Y, et al. (2015). Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting. Journal of Genetics and Genomics. 42(8): 437- 444. 
  7. Cho, S., Kim, S., Kim, J. et al. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 31: 230-232.
  8. Ciccarelli M, Giassetti MI, Miao D, Oatley MJ, Robbins C, Lopez- Biladeau B, Waqas MS, Tibary A, Whitelaw B, Lillico S, Park CH, Park KE, Telugu B, Fan Z, Liu Y, Regouski M, Polejaeva IA, Oatley JM. (2020). Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males. Proc. Natl. Acad. Sci. U S A. 117(39): 24195-24204. 
  9. Cong L, Ran FA, Cox D, et al (2013). Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121): 819- 823. 
  10. Cui, C., Song, Y., Liu, J. et al. (2015). Gene targeting by TALEN-induced homologous recombination in goats directs production of â-lactoglobulin-free, high-human lactoferrin milk. Sci. Rep. 5: 10482. 
  11. Datsomor, A.K., Zic, N., Li, K. et al. (2019). CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes. Sci Rep. 9, 7533. 
  12. Davis P.J., C.M. Smales, D.C. James (2001). How can thermal processing modify the antigenicity of proteins? Allergy. 56: Suppl. 67: 56-60.
  13. Eaton, S.L., Proudfoot, C., Lillico, S.G. et al. CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease). Sci Rep. 9, 9891 (2019). 
  14. Ehn Britt Marie, Toomas Allmere, Esbjo Rn Telemo, Ulf Bengtsson and Bo Ekstrand (2005).Modification Of Ige Binding To Â-Lactoglobulin By Fermentation And Proteolysis of Cow’s Milk. Journal of Agricultural and Food Chemistry. 53(9): 3743-3748.
  15. Fiocchi A, Brozek J, Schünemann, H., Bahna, S.L., von Berg, A., Beyer, K., Bozzola, M., et al. (2010). World Allergy Organization (WAO) Special Committee on Food Allergy. World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow’s Milk Allergy (DRACMA) Guidelines. Pediatr Allergy Immunol. 21 Suppl. 21: 1-125. 
  16. Gao, Y., Wu, H., Wang, Y. et al. (2017). Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off- target effects. Genome Biol. 18, 13 (2017). 
  17. Gratacap, R.L., Regan, T., Dehler, C.E. et al. (2020). Efficient CRISPR /Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system. BMC Biotechnol. 20, 35 (2020).
  18. Grissa, I., Vergnaud, G. and Pourcel, C. (2007) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 8: 172 (2007). 
  19. Hsu PD, Lander ES, Zhang F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell. 157(6): 1262-1278. https://info.abmgood.com/crispr-crash-course -week-2?utm_campaign=CRISPR. Design and Validation.
  20. Ishino Y, H Shinagawa, K Makino, M Amemura, A Nakata (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli and identification of the gene product. Journal of Bacteriology. 169 (12): 5429-5433.
  21. Jiang, F. and J.A. Doudna (2017). CRISPR–Cas9 Structures and Mechanisms. Annual Review of Biophysics.  46: 1, 505- 529.
  22. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096): 816-821.
  23. Khalil, K., Elayat, M., Khalifa, E. et al. (2017). Generation of Myostatin Gene-Edited Channel Catfish (Ictalurus punctatus) via Zygote Injection of CRISPR/Cas9 System. Sci. Rep. 7: 7301 (2017). 
  24. Kurtz, Stefanie and Petersen, Björn. (2019). Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing. Theriogenology. 137. 10.1016/j. Theriogenology. 05.039. 
  25. Lino CA, Harper JC, Carney JP, Timlin JA (2018). Delivering CRISPR: A review of the challenges and approaches. Drug Deliv. 25(1): 1234-1257.
  26. Mali Prashant, Luhan Yang, Kevin M. Esvelt, John Aach, Marc Guell, James E. Dicarlo, Julie E. Norville, George M. Church (2013). RNA-Guided Human Genome Engineering via Cas9. Science. 15; 339(6121): 823-826.
  27. Menchaca A, I. Anegon, C.B.A. Whitelaw, H. Baldassarre, M. Crispo (2016). New insights and current tools for genetically engineered (GE) sheep and goats. Theriogenology. 2016 Jul 1; 86(1): 160-9. 
  28. Menchaca A, P.C. dos Santos Neto, A.P. Mulet , M. Crispo (2020). CRISPR in livestock: From editing to printing. Theriogenology. 01.06: 3.
  29. Moradpour Mahdi and Siti Nor Akmar Abdulah (2020).CRISPR/ dCas9 platforms in plants: strategies and applications beyond genome editing. Plant Biotechnology Journal 18: 32-44.
  30. Ni W, Qiao J, Hu S, et al. (2014). Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One. 9(9): e106718.
  31. Niu D, Wei H-J, Lin L, George H, Wang T, Lee I-H, et al. (2017). Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 357: 1303-7.
  32. Park, K., Kaucher, A., Powell, A. et al. (2017). Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Sci. Rep 7, 40176. 
  33. Peng, J., Wang, Y., Jiang, J. et al. (2015). Production of Human Albumin in Pigs Through CRISPR/Cas9-Mediated Knockin of Human cDNA into Swine Albumin Locus in the Zygotes. Sci. Rep. 5: 16705. 
  34. Qi LS, Larson MH, Gilbert LA, et al. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 152(5):1173-1183.
  35. Qian L., Tang M., Yang J. et al. (2015). Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Sci. Rep. 5: 14435.
  36. Sheets TP, Park CH, Park KE, Powell A, Donovan DM, Telugu BP (2016). Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs. Int. J .Mol. Sci. 17(12): 2031. 
  37. Sun, Z., Wang, M., Han, S. et al. (2018). Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA. Sci   Rep. 8: 15430. 
  38. Sung YH, Kim JM, Kim HT, et al. (2014). Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24(1): 125 131. 
  39. Thomas Mark, David Parry Smith and Vivek Iyer (2019). Best practice for CRISPR design using current tools and resources. Methods. (15): 164-165:3-17. 
  40. Wang K, Tang X, Xie Z, Zou X, Li M, Yuan H, et al. (2017).  CRISPR/ Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res. 26: 799-805.
  41. Wang, Y., Du, Y., Shen, B. et al. (2015). Efficient generation of gene- modified pigs via injection of zygote with Cas9 sgRNA. Sci. Rep. 5: 8256. 
  42. Wargelius A, Leininger S, Skaftnesmo KO, et al. (2016). Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon. Sci. Rep. 6: 21284. 
  43. Whitworth K., Rowland R., Ewen C. et al. (2016). Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat Biotechnol. 34: 20-22. 
  44. Wu, J., Vilarino, M., Suzuki, K. et al. (2017). CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci. Rep. 7, 10487. 
  45. Xue HY, Ji LJ, Gao AM, Liu P, He JD, Lu XJ (2015). CRISPR-Cas9 for medical genetic screens: applications and future perspectives. J. Med Genet. 53(2): 91-7. 
  46. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 154(6): 1370 1379. 
  47. Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, et al. (2015). Genomewide inactivation of porcine endogenous retroviruses (PERVs). Science. 350: 1101-4.
  48. Zhang Jiquan, Fengge Song, Yuying Sun, Kuijie Yu, Jianhai Xiang. (2018). CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda. Fish Shellfish Immunol. 77: 244-251. 
  49. Zhang Ju, Meng-Lan Cui, Yong-Wei Nie, Bai-Dai, Fei-Ran Li, Dong- Jun Liu, Hao Liang, Ming Cang. (2018). CRISPR/Cas9- mediated specific integration of fat-1 at the goat MSTN locus. FEBS J. 285(15): 2828-2839. 
  50. Zhao Jianguo, Liangxue Lai, Weizhi Ji, Qi Zhou (2019). Genome editing in large animals: current status and future prospects. National Science Review. 6(3): 402-420.
  51. Zheng, Q., Lin, J., Huang, J., Zhang, H., Zhang, R., Zhang, X., et al. (2017). Reconstitution of UCP1 using CRISPR/ Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. PNAS, 114(45): 9474-9482. 
  52. Zhengyi He, Ting Zhang, Lei Jiang, Minya Zhou, Daijin Wu, Junyan Mei and Yong Cheng (2018). Use of CRISPR/Cas9 technology efficiently targetted goat myostatin through zygotes microinjection resulting in double-muscled phenotype in goats. Biosci. Rep. 38(6).
  53. Zhou W, Wan Y, Guo R, et al. (2017). Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. PLoS One. 12(10).

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