Skip to main content
Log in

Innovative Approaches to Genome Editing in Chickens

  • Published:
Cytology and Genetics Aims and scope Submit manuscript

Abstract

Obtaining productive animals, including chickens, with specified characteristics is a promising area of modern animal husbandry. The most relevant traits for chickens are increased meat and egg productivity, resistance to infectious diseases, products with reduced allergenicity, and the production of integrated transgenic proteins. The rapidly developing methods of molecular genetics, such as genome editing, allow for solving these problems. In birds, unlike mammals, access to the unicellular zygote—when editing the genome is most effective—is difficult due to the special structure of the reproductive system. As a result, innovative methods have been developed for genetic engineering of birds, the most common of which is the use of primordial germ cells (PGC), precursors of poultry reproductive cells. This review provides a brief description and discussion of modern methods of editing the chicken genome using endonucleases, such as transcription activator-like effector nucleases (TALEN) and the system of clustered short palindromic repeats CRISPR/Cas9. Particular attention is paid to methods of gene editing in birds using primordial germ cells (PGC). Various strategies for the delivery of guide RNA (gRNA) and Cas9 protein into poultry cells based on the use of plasmid vectors, an alternative method of delivery of genetic constructs using spermatozoa (STAGE), the RNP method, which consists in direct delivery of gRNA and Cas9 protein in the form of a complex ribonucleoproteins, and the RNP method based on the use of nanobubbles were considered.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1.

Similar content being viewed by others

REFERENCES

  1. Abu-Bonsrah, K.D., Zhang, D., and Newgreen, D.F., CRISPR/Cas9 targets chicken embryonic somatic cells in vitro and in vivo and generates phenotypic abnormalities, Sci. Rep., 2016, vol. 6, art. ID 34524.https://doi.org/10.1038/srep34524

  2. Ball, B.A., Sabeur, K., and Allen, W.R., Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer, Cytology and Genetics, 2022, Vol. 56 No. 2 Innovative approaches to genome editing. 205 Equine Vet. J., 2008, vol. 40, no. 1, pp. 76–82.https://doi.org/10.2746/042516407X235786

  3. Barrangou, R., Cas9 targeting and the CRISPR revolution, Science, 2014, vol. 344, no. 6185, pp. 707–708.https://doi.org/10.1126/science.1252964

  4. Bernardo, A.D.M., Sprenkels, K., and Rodrigues, G., Chicken primordial germ cells use the anterior vitelline veins to enter the embryonic circulation, Biol. Open, 2012, vol. 1, no. 11, pp. 1146–1152.https://doi.org/10.1242/bio.20122592

  5. Bibikova, M., Carroll, D., and Segal, D.J., Stimulation of homologous recombination through targeted cleavage by chimeric nucleases, Mol. Cell Biol., 2001, vol. 21, no. 1, pp. 289–297.https://doi.org/10.1128/mcb.21.1.289-297.2001

  6. Bibikova, M., Golic, M., and Golic, K.G., Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases, Genetics, 2002, vol. 161, no. 3, pp. 1169–1175. https://doi.org/10.1093/genetics/161.3.1169

  7. Challagulla, A., Jenkins, K.A., and O’Neil, T.E., Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs, Anim. Biotechnol., 2020, vol. 24, pp. 1–10.https://doi.org/10.1080/10495398.2020.1789869

  8. Chojnacka-Puchta, L. and Sawicka, D., CRISPR/Cas9 gene editing in a chicken model: current approaches and applications, J. Appl. Genet., 2020, vol. 61, pp. 221–229.https://doi.org/10.1007/s13353-020-00537-9

  9. Cho, S.W., Kim, S., Kim, Y., Kweon, J., Kim, H.S., Bae, S., and Kim, J.-S., Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases, Genome Res., 2014, vol. 24, no. 1, pp. 132–141.https://doi.org/10.1101/gr.162339.113

  10. Collares, T., Campos, V.F., and De Leon, P.M., Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide, J. Biosci., 2011, vol. 36, pp. 613–620.https://doi.org/10.1007/s12038-011-9098-x

  11. Cong, L., Ran, F.A., Cox, D., et al., Multiplex genome engineering using CRISPR/Cas systems, Science, 2013, vol. 339, no. 6121, pp. 819–823.https://doi.org/10.1126/science.1231143

  12. Cooper, C.A., Challagulla, A., Jenkins, K.A., et al., Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE), 2017, Transgenic Res., vol. 26, no. 3, pp. 331–347. https://doi.org/10.1007/s11248-016-0003-0

    Article  CAS  PubMed  Google Scholar 

  13. Dimitrov, L., Pedersen, D., Ching, K.H., et al., Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells, PLoS One, 2016, vol. 11, no. 4, p. e0154303. https://doi.org/10.1371/journal.pone.0154303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fagerlund, R., Staals, R.H., Fineran, P.C., The Cpf1 CRISPR-Cas protein expands genome-editing tools, Genome Biol., 2015, vol. 16, art. ID 251. https://doi.org/10.1186/s13059-015-0824-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fu, Y., Sander J.D., Reyon, D., Cascio, V.M., and Joung, J.K., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs, Nature Biotechnology, 2014, vol. 32, no. 3, pp. 279–284.https://doi.org/10.1038/nbt.2808

  16. Gaj, T., Gersbach, C.A., and Barbas, C.F., ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering, Trends Biotechnol., 2013, vol. 31, pp. 397–405.https://doi.org/10.1016/j.tibtech.2013.04.004

  17. Hamburger, V. and Hamilton, H.L., A series of normal stages in the development of the chick embryo, J. Morphol., 1951, vol. 88, pp. 49–92.

  18. Hsu, P.D., Scott, D.A., and Weinstein, J.A., DNA targeting specificity of RNA-guided Cas9 nucleases, Nat. Biotechnol., 2013, vol. 31, no. 9, рр. 827–832.https://doi.org/10.1038/nbt.2647

  19. Hsu, P.D., Lander, E.S., and Zhang, F., Development and applications of CRISPR-Cas9 for genome engineering, Cell., 2014, vol. 157, рр. 1262–1278.https://doi.org/10.1016/j.cell.2014.05.010

  20. Hung, M.E. and Leonard, J.N., A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery, J. Extracell Vesicles, 2016, vol. 5, pp 310–327.https://doi.org/10.3402/jev.v5.3102

  21. Ishino, Y., Shinagawa H., and Makino, K., Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product, J. Bacteriol., 1987, vol. 169, pp.5429-5433.

  22. Jeong, D.K., Hong, Y.H., and Han, J.Y., Simple separation of chicken gonadal primordial germ cells with and without foreign genes, Cell Biol. Int., 2002, vol. 26, pp. 647–651.https://doi.org/10.1006/cbir.2002.0928

  23. Jiang, F. and Doudna, J.A., CRISPR–Cas9 structures and mechanisms, Annu Rev. Biophys., 2017, vol. 46, pp. 505–529.https://doi.org/10.1146/annurev-biophys-062215-010822

  24. Jinek, M., Chylinski, K., and Fonfara, I., A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity, Science, 2012, vol. 337(6096), pp. 816–821.https://doi.org/10.1126/science.1225829

  25. Johnson, R.D. and Jasin, M., Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells, EMBO J., 2000, vol. 19, pp. 3398–3407. https://doi.org/10.1093/emboj/19.13.3398

  26. Jung, K.M., Kim, Y.M., and Ono, T., Sizedependent isolation of primordial germ cells from avian species, Mol. Reprod. Dev., 2017, vol. 84. pp. 508–516.https://doi.org/10.1002/mrd.22802

  27. Khan, S.H., Genome-editing technologies: concept, pros, and cons of various genome-editing techniques and bioethical concerns for clinical application, Mol. Ther Nucleic Acids., 2019, vol. 16, pp. 326–334.https://doi.org/10.1016/j.omtn.2019.02.027

  28. Kadam, U.S., Shelake, R.M., Chavhan, R.L., and Suprasanna, P., “Concerns regarding ‘off-target’ activity of genome editing endonucleases,” Physiology and Biochemistry, 2018, vol. 131, pp. 22–30.https://doi.org/10.1016/j.plaphy.2018.03.027

  29. Komor, A.C., Badran, A.H., and Liu, D.R., CRISPR-based technologies for the manipulation of eukaryotic genomes, Cell, 2017, vol. 168, nos. 1–2, pp. 20–36.https://doi.org/10.1016/j.cell.2016.10.044

  30. Lee, H.C., Lim, S., and Han, J.Y., Wnt/beta-catenin signalling pathway activation is required for proliferation of chicken primordial germ cells in vitro, Sci Rep., 2016, vol. 6, pp. 34510. https://doi.org/10.1038/srep34510

  31. Lee, H.J., Yoon, J.W., and Jung, K.M., Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development, FASEB J., 2019, vol. 33, no. 7, pp. 8519–8529.https://doi.org/10.1096/fj.201802671R

  32. Lee, J., Kim D.H., and Lee, K., Current approaches and applications in avian genome editing, Int. J. Mol. Sci., 2020, vol. 21, no. 11, p. 3937.https://doi.org/10.3390/ijms21113937

  33. Li, B.C., Chen, G.H., and Qin, J., Suitable stages for isolation and culture PGCs from chicken embryos, Int. J. Poult Sci., 2005, vol. 4, pp. 885–890.https://doi.org/10.3923/IJPS.2005.885.890

  34. Li, T., Huang S., and Jiang, W.Z., TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain, Nucleic Acids Res., 2011, vol. 39, no. 1, pp. 359–372.https://doi.org/10.1093/nar/gkq704

  35. Li, L., Natarajan, P., and Allen C., CGMP-compliant, clinical scale, non-viral platform for efficient gene editing using CRISPR/Cas9, Cytotherapy, 2014, vol. 16, no. 4, p. 37. https://doi.org/10.1016/j.jcyt.2014.01.125

  36. Lin, S., Staahl, B.T., and Alla, R.K., Enhanced homology directed human genome engineering by controlled timing of CRISPR/Cas9 delivery, Life, 2014, vol. 3, p. e04766.https://doi.org/10.7554/eLife.04766.001

  37. Love, J., Gribbin C., and Mather, C., Transgenic birds by DNA microinjection, Nat. Biotechnol., 1994, vol. 12, no. 1, pp. 60–63.https://doi.org/10.1038/nbt0194-60

  38. Luther, D.C., Lee, Y.W., Nagaraj, H., et al., Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges, Expert Opin Drug Deliv., 2018, vol. 15, no. 9, pp. 905–913.https://doi.org/10.1080/17425247.2018.1517746

  39. Macdonald, J., Glover, J.D., and Taylor, L., Characterisation and germline transmission of cultured avian primordial germ cells, PLoS One, 2010, vol. 5, p. 15518.https://doi.org/10.1371/journal.pone.0015518

  40. Malewska, A. and Olszańska, B., Accumulation and localisation of maternal RNA in oocytes of Japanese quail, Zygote, 1999, vol. 7, pp. 51–59.https://doi.org/10.1017/S0967199499000398

  41. Mojica, F.J., Díez-Villasenor, C., and Soria, E., Biological significance of a family of regularly spaced repeats in the genomes of Archaea, bacteria and mitochondria. Mol. Microbiol., 2000, vol. 36, pp. 244–246.https://doi.org/10.1046/j.1365-2958.2000.01838.x

  42. Motono, M., Ohashi, T., and Nishijima, K., Analysis of chicken primordial germ cells, Cytotechnology, vol. 2008, no. 57, pp. 199– 205.https://doi.org/10.1007/s10616-008-9156-x

  43. Mozdziak, P.E., Angerman-Stewart, J., and Rushton, B., Isolation of chicken primordial germ cells using fluorescence-activated cell sorting, Poult Sci., 2005, vol. 84, pp. 594–600. https://doi.org/10.1093/ps/84.4.594

  44. Naito, M., Embryo manipulation in chickens, J. Poultry Sci., 2015, vol. 52, pp. 7–14. https://doi.org/10.2141/jpsa.0140107

  45. Nakamura, Y., Yamamoto, Y., and Usui, F., Migration and proliferation of primordial germ cells in the early chicken embryo, Poult Sci., 2007, vol. 86, pp. 2182–2193.https://doi.org/10.1093/ps/86.10.2182

  46. Nakamura, Y., Usui, F., and Ono, T., Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the Chicken, Biol. Reprod, 2010, vol. 83, pp. 130–137. https://doi.org/10.1095/biolreprod.110.083923

  47. Nakamura, Y., Kagami H., and Tagami T., Development, differentiation and manipulation of chicken germ cells, Dev. Growth Differ., 2013, vol. 55, pp. 20–40.https://doi.org/10.1111/dgd.12026

  48. Nakamura, Y., Avian biotechnology. Adv. Exp. Med. Biol., 2017, vol. 1001, pp. 187–214.https://doi.org/10.1007/978-981-10-3975-1_12

  49. Oishi I., Yoshii K., and Miyahara D., Targeted mutagenesis in chicken using CRISPR/Cas9 system, Sci Rep., 2016, vol. 6, p. 23980.https://doi.org/10.1038/srep23980

  50. Oishi, I., Yoshii, K., and Miyahara, D., Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens, Sci Rep., 2018, vol. 8, no. 1, pp. 102–103.https://doi.org/10.1038/s41598-018-28438-2

  51. Park, T.S., Lee, H.J., and Kim, K.H., Targeted gene knockout in chickens mediated by TALENs, Proc. Natl. Acad. Sci U.S.A., 2014, vol. 111, pp. 12716–12721.https://doi.org/10.1073/pnas.1410555111

  52. Pennisi, E., The CRISPR craze, Science, 2013, vol. 341, pp. 833–836.https://doi.org/10.1126/science.341.6148.833

  53. Pokhrel, N., Ben-Tal, Cohen E., Cellular and morphological characterization of blastoderms from freshly laid broiler eggs, Poult Sci., 2017, vol. 96, pp. 4399–4408.https://doi.org/10.3382/ps/pex242

  54. Porteus, M. and Carroll, D., Gene targeting using zinc finger nucleases, Nat Biotechnol., 2005, vol. 23, no. 8, pp. 967–973.https://doi.org/10.1038/nbt1125

  55. Qin, X., Xiao, N., and Xu, Y., Efficient knock-in at the chicken ovalbumin locus using adenovirus as a CRISPR/Cas9 delivery system, Biotech., 2019, vol. 9, no. 12, p. 454.https://doi.org/10.1007/s13205-019-1966-3

  56. Qomi, S.B., Asghari, A., and Mojarrad, M., An overview of the CRISPR-based genomic-and epigenome-editing system: function, applications, and challenges, Adv. Biomed. Res., 2019, vol. 8, p. 49.https://doi.org/10.4103/abr.abr_41_19

  57. Riordan, S.M., Heruth, D.P., Zhang, L.Q., et al., Application of CRISPR/Cas9 for biomedical discoveries, Cell Biosci., 2015, vol. 5, p. 33.https://doi.org/10.1186/s13578-015-0027-9

  58. Rouet P., Smih F., and Jasin M., Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease, Mol. Cell Biol., 1994, vol. 14, pp. 8096–8106.https://doi.org/10.1128/MCB.14.12.8096

  59. Sang, H., Prospects for transgenesis in the chick. Mech. Dev., 2004, vol. 121, no. 9, pp. 1179–1186.https://doi.org/10.1016/j.mod.2004.05.012

  60. Schusser, B., Collarini, E.J., and Yi, H. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells, Proc. Natl. Acad. Sci U.S.A., 2013, vol. 110, no. 50, pp. 20170–20175.https://doi.org/10.1073/pnas.1317106110

  61. Seydoux, G., Braun, R.E., Pathway to totipotency: lessons from germ cells, Cell, 2006, vol. 127, no. 5, pp. 891–904.https://doi.org/10.1016/j.cell.2006.11.016

  62. Shimada, K., Ono, T., and Mizushima, S., Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds, General and Comparative Endocrinology, 2014, vol. 196, pp. 100–105.https://doi.org/10.1016/j.ygcen.2013.11.001

  63. Song, Y., Duraisamy, S., and Ali, J., Characteristics of long-term cultures of avian primordial germ cells and gonocytes, Biol. Reprod., 2014, vol. 90, no. 1, p. 15. https://doi.org/10.1095/biolreprod.113.113381

  64. Taylor, L., Carlson D.F., and Nandi S., Efficient TALEN-mediated gene targeting of chicken primordial germ cells, Development, 2017, vol. 144, pp. 928–34.https://doi.org/10.1242/dev.145367

  65. Tyack, S.G., Jenkins, K.A., and O’Neil, T.E., A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells, Transgenic Res., 2013, vol. 22, pp. 1257–1264.https://doi.org/10.1007/s11248-013-9727-2

  66. Van de Lavoir, M.C., Diamond, J.H., and Leighton, P.A., Germline transmission of genetically modified primordial germ cells, Nature, 2006, vol. 441, pp. 766–769.https://doi.org/10.1038/nature04831

  67. Van de Lavoir, M.C., Collarini, E.J., and Leighton, P.A., Interspecific germline transmission of cultured primordial germ cells, PLoS One, 2012, vol. 7, p. e35664. https://doi.org/10.1371/journal.pone.0035664

  68. Veron, N., Qu, Z., and Kipen, P.A., CRISPR mediated somatic cell genome engineering in the chicken, Dev. Biol., 2015, vol. 407, no. 1, pp. 68–74.https://doi.org/10.1016/j.ydbio.2015.08.007

  69. Vick, L., Li, Y., and Simkiss, K., Transgenic birds from transformed primordial germ cells, Proc. Biol. Sci., 1993, vol. 251, pp. 179–182.https://doi.org/10.1098/rspb.1993.0026

  70. Whyte, J., Glover, J.D., and Woodcock, M., FGF, insulin, and SMAD signaling cooperate for avian primordial germ cell self-renewal, Stem Cell Rep., 2015, vol. 5, no. 6, pp. 1171–1182. https://doi.org/10.1016/j.stemcr.2015.10.008

  71. Woodcock, M.E., Gheyas, A.A., and Mason, A.S., Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds, Proc. Natl. Acad. Sci U.S.A., 2019, vol. 116, pp. 20930–20937. https://doi.org/10.1073/pnas.1906316116

  72. Wu, X., Scott, D.A., and Kriz, A.J., Genomewide binding of the CRISPR endonuclease Cas9 in mammalian cells, Nat. Biotechnol., 2014, vol. 32, no. 7, pp. 670–676.https://doi.org/10.1038/nbt.2889

  73. Yamamoto Y., Usui F., and Nakamura Y., A novel method to isolate primordial germ cells and its use for the generation of Germline chimeras in chicken, Biol. Reprod., 2007, vol. 77, pp. 115–119. https://doi.org/10.1095/biolreprod.107.061200

  74. Yeh, C.D., Richardson, C.D., and Corn, J.E., Advances in genome editing through control of DNA repair pathways, Nat. Cell Biol., 2019, vol. 21, pp. 1468–1478.https://doi.org/10.1038/s41556-019-0425-z

  75. Zhang, Y., Wang, Y., and Zuo, Q., CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation, PLoS One, 2017, vol. 12, no. 2, p. e0172207. https://doi.org/10.1371/journal.pone.0172207

  76. Zhao, X., Wei, C., and Li, J., Cell cycle-dependent control of homologous recombination, Acta Biochim. Biophys. Sin., 2017, vol. 49, pp. 655–668.https://doi.org/10.1093/abbs/gmx055

  77. Zuo, Q., Wang, Y., Cheng, S. et al., Site-directed genome knockout in chicken cell line and embryos can use CRISPR/Cas gene editing technology, G3: Genes, Genomes, Genetics, 2016, vol. 6, no. 6, pp. 1787–1792.https://doi.org/10.1534/g3.116.028803

Download references

Funding

The study was supported by a grant from the Russian Science Foundation (project no. 20-76-10006).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to O. Yu. Barkova.

Ethics declarations

The authors declare that they have no conflict of interests. This article does not contain any studies involving animals or human participants performed by any of the authors.

Additional information

Translated by P. Kuchina

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Barkova, O.Y., Larkina, T.A., Krutikova, A.A. et al. Innovative Approaches to Genome Editing in Chickens. Cytol. Genet. 56, 196–207 (2022). https://doi.org/10.3103/S0095452722020037

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.3103/S0095452722020037

Keywords:

Navigation