Approaches to genome editing in agricultural animals

Abstract

The results of research into genetic methods of breeding agricultural animals with improved characteristics are presented. By now a significant number of animals with an edited genome have been selected. Methods of genome modification in cattle are constantly improving. The approaches of gene editing of bovine embryos, delivery of edited constructs and improvement of embryo survival after introduction of editing systems have been studied. The studies were performed on cattle embryos. BLG and SD209 gene editing systems were developed and validated. Delivery options of the editing system into cattle cells were studied: microinjection into the zygote of plasmid DNA encoded sequence with CRISPR/Cas9 c sgRNA, the method of viral vectors (adeno-associated AAV viruses, serotypes AAV1, AAV2, AAV6, AAV9, AAVDJ), co-injection of plasmid DNA and sperm into the oocyte at the MII stage, and microinjection of Cas9 and guide RNAs. Different techniques for performing microinjections have been investigated and refined, and different preparation of Cas9 RNA and guide RNA mixtures have been tested. Based on these results, the protocol for performing microinjection of the editing system was optimized and a model experiment was performed on 160 oocytes, with 80 cells per each construction. The findings have shown that the efficiency of editing has generally improved. When injected with guide RNA against BLG gene and spCas9 mRNA, 84% of the surviving cells initiated cleavage, blastulation was 20%, and BLG knockout was 69.2%. When injected against the CD209 gene and spCas9 mRNA, 44.4% of the surviving embryos started cleavage, blastulation was 16.7%, with CD209 knockout at 44.4%. The novelty of the work lies in obtaining data on the development of editing systems with specific target genes, in improving the delivery system and cultivation of bovine embryos.