Strategy for modification of the bovine beta-lactoglobulin geneusing components of the CRISPR/Cas9 system in plasmid form

Abstract

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.