E3S Web Conf.
Volume 395, 2023International Scientific and Practical Conference “Ensuring the Technological Sovereignty of the Agro-Industrial Complex: Approaches, Problems, Solutions” (ETSAIC2023)
|Number of page(s)||8|
|Section||Creation of Safe and High Quality, Including Functional Food Products Based on Modern Genetic and Selection Methods in Crop and Livestock Production|
|Published online||06 June 2023|
“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: email@example.com
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.
© The Authors, published by EDP Sciences, 2023
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.
Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.
Initial download of the metrics may take a while.