“Usage a CRISPR/Cas9-based for obtaining knockouts of economically significant cattle genes”

¹ Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences, 119334, 34/5, Vavilova str., Moscow, Russia
² Federal State Budgetary Scientific Institution Ural Federal Agrarian Scientific Research Centre, Ural Branch of the Russian Academy of Sciences (FSBSI UrFASRC, UrB of RAS), 112 А, Belinskogo St., 620000 Еkaterinburg, Russia

^* Corresponding author: isaeva.05@bk.ru

Abstract

An experimental work dealing with the gene modification using the Cas9 RNA-based editing system was performed. Point site-specific breakpoints in gDNA were introduced at the zygote stage by microinjection of spCas9 mRNA protein and guide RNAs into the zygote cytoplasm. Oocytes that extruded the first and second polar bodies were used for the injection. 2 series of microinjections of gene editing designs for early bovine embryos were made. The degeneration ranged from 10% to 56% in different groups. A total of 100 injections were performed. Cleavage was started by 78% of the surviving oocytes; 5 embryos reached the blastocyst stage, which was 16% of the number of dividing embryos. All the resulting embryos were analyzed to evaluate the efficiency of editing. gDNA was isolated from all embryos that had reached the blastocyst stage. Using Sanger sequencing of genes of interest in pre-implantation bovine embryos and biopsies from them, it was shown that in 5 out of 17 embryos resulting from microinjections of guide RNA against the BLG gene and SpCas9 mRNA, and in 2 out of 9 embryos after microinjections of guide RNA against CD209 gene and SpCas9 mRNA, the required genome modifications were found. This is indicative of the high efficiency of this delivery method of the editing system.