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

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 FGF20 and 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 HR genes in chickens and for FGF20 gene in quails using genetic constructs based on the pX458 vector.