Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle

G N Singina; P V Sergiev; A V Lopukhov; M P Rubtsova; N P Taradajnic; N V Ravin; E N Shedova; T E Taradajnic; I A Polejaeva; A V Dozev; G Brem; O A Dontsova; N A Zinovieva

doi:10.1134/S1607672921010099

Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle

Dokl Biochem Biophys. 2021 May;496(1):48-51. doi: 10.1134/S1607672921010099. Epub 2021 Mar 10.

Authors

G N Singina¹, P V Sergiev^{2

3

4}, A V Lopukhov⁵, M P Rubtsova⁴, N P Taradajnic⁵, N V Ravin⁶, E N Shedova⁵, T E Taradajnic⁵, I A Polejaeva⁷, A V Dozev⁵, G Brem⁸, O A Dontsova^{3

4

9

10}, N A Zinovieva⁵

Affiliations

¹ Ernst Federal Science Center for Animal Husbandry, Podolsk, Russia. g_singina@mail.ru.
² Institute of Functional Genomics, Moscow State University, Moscow, Russia.
³ Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, Russia.
⁴ Faculty of Chemistry, Moscow State University, Moscow, Russia.
⁵ Ernst Federal Science Center for Animal Husbandry, Podolsk, Russia.
⁶ Research Center of Biotechnology, Moscow, Russia.
⁷ Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA.
⁸ Department of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.
⁹ Belozersky Research Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
¹⁰ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Abstract

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.

Keywords: Bos taurus; beta-lactoglobulin knock-out; gene editing; somatic cloning.

MeSH terms

Animals
Animals, Genetically Modified / embryology
Animals, Genetically Modified / genetics*
CRISPR-Cas Systems*
Cattle / embryology
Cattle / genetics*
Cloning, Organism / methods*
Embryo, Mammalian / cytology*
Embryo, Mammalian / metabolism
Fibroblasts / cytology*
Fibroblasts / metabolism
Gene Editing / methods*
Gene Knockout Techniques / methods
Nuclear Transfer Techniques