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Genetically modified (GM) animals are necessary to solve the global problems of humanity related to nutrition and health. Rabbits, as laboratory, domestic and farm animals, occupy a special niche in research. GM rabbits are promising as bioreactors for producing biologically active (BA) proteins with milk or blood, and are in demand in Biomedicine as biomodels of diseases. To date, many GM rabbits-biomodels, producers of recombinant proteins have been created in the world using CRISPR/Cas9 technology. All-Russian Research Institute of Animal Physiology, Biochemistry and Nutrition has experience in obtaining transgenic rabbitsproducers of human BA proteins with milk by microinjecting recombinant DNA into zygote pronuclei. The possibility of site-specific modification of the rabbit whey acidic protein (WAP) gene using CRISPR/Cas9 technology is discussed. A DNA matrix containing homology arms to the WAP rabbit gene and site-specific CRISPR/Cas9 components in plasmid form were obtained. Microinjections of rabbit zygotes were performed and embryo survival was evaluated in vitro. The efficiency of using the green fluorescent protein gene under the cytomegalovirus promoter in the DNA matrix as an indicator of homologically directed repair was evaluated. This work can be useful for obtaining rabbits that produce with milk BA protein instead of WAP.

Beta-lactoglobulin (BLG) is the main protein in milk serum in almost all mammals, with the exception of rodents and primates. Regulatory regions of the beta-lactoglobulin gene in ruminants (sheep, goats, and cattle) as part of genetic constructs provide tissue - specific expression of recombinant protein in the mammary gland and have been actively used in genetic engineering since the beginning of the era of creating transgenic animals. To work effectively with the CRISPR/Cas9 genomic editing method, it is necessary to know the exact DNA sequence of the target gene: this is necessary both for creating a DNA matrix for homologous recombination and for the targeted accuracy of guide RNAs. A polymorphic variant of the bovine BLG gene was identified, whose sperm was used to fertilize cow oocytes in vitro. The aim of this work was to create a plasmid containing 5’ - and 3’ - arms of homology (ha) to the bovine BLG gene. Based on ??TZ57R/T, the pTZhaBLG plasmid was obtained, which has a unique site for EagI restriction at the junction of the homology arms. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at this restriction site. We created the pBLGcmvEGFP plasmid containing the green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter: protein expression can serve as a reliable indicator of successful integration of the transgene into the genome. The resulting plasmids in circular or linearized form are intended for site-specific integration by homologous recombination repair into the BLG gene using CRISPR/Cas9 components.

Using on-line programs, sites were selected for obtaining double-stranded breaks in the BLG gene of cattle. The strategy for making double-stranded cuts in the BLG gene was developed taking into account the polymorphic variant of the gene (A-allele): DNA was isolated from bovine sperm used for fertilization of cow eggs in vitro. Four pX330 plasmids encoding Cas9 endonuclease and gRNAs specific to the selected BLG gene sequences were obtained. A strategy was developed for analyzing possible genetic modifications resulting from the operation of the CRISPR/Cas9 system components and the genetic construct microinjected into zygotes (NHEJ, HDR). The pBLGcmvEGFP plasmid containing the green fluorescent protein gene under the cytomegalovirus promoter was proposed as a model genetic construct for replacing the BLG gene. The use of a plasmid containing the reporter protein gene under its own regulatory sequences, flanked by homology arms to the beta-lactoglobulin gene, can be useful for evaluating the effectiveness of site-specific activity of the CRISPR/Cas9 system components in vitro.