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Vetokh A.N., Sergiev P.V., Rubtsova M.P., Volkova N.A.,. Tomgorova E.K, Volkova L.A., Zinovieva N.A. Creation of genome editing systems based on CRISPR-CAS9 for knockout in FGF20 and HR genes of embryonic and generative cells from chicken and quails. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2021, Vol. 56, № 6, p. 1099-1110.
(how to cite this article)

doi: 10.15389/agrobiology.2021.6.1099eng

UDC: 636.5:573.6.086.83:577.21

Acknowledgements:
Supported financially by Russian Foundation for Basic Research, grant No. 18-29-07079

 

CREATION OF GENOME EDITING SYSTEMS BASED ON CRISPR-CAS9 FOR KNOCKOUT IN FGF20 AND HR GENES OF EMBRYONIC AND GENERATIVE CELLS FROM CHICKEN AND QUAILS

A.N. Vetokh1 ✉, P.V. Sergiev2, 3, 4, M.P. Rubtsova2, 5, N.A. Volkova1,
E.K. Tomgorova1, L.A. Volkova1, N.A. Zinovieva1

1Ernst Federal Research Center for Animal Husbandry,60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail anastezuya@mail.ru (✉ corresponding author), natavolkova@inbox.ru, tomgorova@rambler.ru, ludavolkova@inbox.ru, n_zinovieva@mail.ru;
2Faculty of Chemistry, Lomonosov Moscow State University, GSP-1, 1-3, Leninskie Gory, Moscow, 119991 Russia, e-mail petya@genebee.msu.ru, mprubtsova@gmail.com;
3Center of Life Sciences, Skolkovo Institute of Science and Technology, 30-1, Bolshoi bulvar, Skolkovo, Moscow Province, 143026 Russia, e-mail petya@genebee.msu.ru;
4Institute of Functional Genomics, Lomonosov Moscow State University, 1, Leninskie Gory, Moscow, 119991 Russia, e-mail petya@genebee.msu.ru;
5Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, ul. Mikluho-Maklaya, Moscow, 117997 Russia, e-mail mprubtsova@gmail.com

ORCID:
Vetokh A.N. orcid.org/0000-0002-2865-5960
Tomgorova E.K. orcid.org/0000-0001-5398-8815
Sergiev P.V. orcid.org/0000-0001-8866-1863
Volkova L.A. orcid.org/0000-0002-9407-3686
Rubtsova M.P. orcid.org/0000-0002-4808-5416
Zinovieva N.A. orcid.org/0000-0003-4017-6863
Volkova N.A. orcid.org/0000-0001-7191-3550

Received September 30, 2021

 

Genome editing technologies using site-specific nucleases (ZNF, TALEN, CRISPR/Cas9) are used more and more in animal husbandry, including poultry farming. With the use of these technologies, scientists hope not only to speed up the process of creating breeds with improved economically useful traits, high resistance to infectious diseases, but also to create individuals carrying phenotypes, the introduction of which into animal and bird populations by traditional breeding methods is impossible or difficult. The creation of individuals devoid of plumage in order to improve the commercial qualities of poultry product is of interest for industrial poultry farming. For this, we selected the FGF20 and HR genes associated with the development and growth of hair in mammals (F. Benavides et al., 2009) and feathers in birds (K.L. Wells et al., 2012). The aim of the study was to create a system for knocking out the FGF20and HR genes in chickens and FGF20 in quails by genome editing techniques. We inactivated FGF20 and HR genes in the region of the third exons based on the analysis of their structure. The optimal cutting regions of these genes and guide RNAs and primers for amplifying the FGF20 and HR DNA fragments were selected bioinformatically and using internet resources (https://zlab.bio/guide-design-resources, https://www.ncbi.nlm.nih.gov/). To create genetic constructs for cutting in the regions encoding FGF20 and HR, the vector pX458 was selected (F.A. Ran et al., 2013). The hybridized oligonucleotides 5´-CACCGAAAGATGGTACTCCCAGAGA-3´ and 3´-CT-TTCTACCATGAGGGTCTCTCAAA-5´ (for FGF20 gene in chicken), 5´-CACCGTCCATGTTTGTACACGTTGG-3´ and 3´-CAGGTACAAACATGTGCAACCCAAA-5´ (for FGF20 gene in chicken and in quails); 5´-CACCGACGTGGCTGACGCGGCACT-3´ and 3´-CTGCACCGACTGCGC-CGTGACAAA-5´ (for gene HR) were used for ligation. The effectiveness of cloning constructs was confirmed by sequencing. The plasmids that were obtained were used for edit the genome of embryonic (fibroblasts) and generative (primordial germ cells — PGCs, spermatogonia) chicken and quail cells in in vitro experiments. Target cells were transfected by electroporation. Efficiency of electroporation was evaluated on a high-performance fluorescent cell sorter BD FacsAria III («BD Biosciences», USA) by expression of the eGFP marker gene. The proportion of in vitro transfected embryonic fibroblasts, PGCs and spermatogonia from chickens with a knockout of the FGF20 gene reached 5.7, 0.9, and 1.2 %, with a knockout of the HR gene — 7.4, 0.8, and 1.0 %, respectively. The percentage of embryonic fibroblasts, PGCs and spermatogonia from quails with a knockout of the FGF20 gene was 6.3, 0.9, and 1.1 %, respectively. Genomic DNA was isolated from transformed chicken and quail cells and used for amplification and sequencing of the regions of the FGF20 and HR genes in which deletions were introduced. The presence of multiple mutations in the amplified DNA regions was shown. The data obtained indicate the success of the knockout system creation for FGF20 and HRgenes in chickens and for FGF20 gene in quails using genetic constructs based on the pX458 vector.

Keywords: genome editing, CRISPR/Cas9, chicken, quail, primordial germ cells (PGCs), spermatogonia cells, FGF20, HR.

 

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