110 Development competence of β-lactoglobulin gene editing bovine embryos producing by CRISPR/Cas9 and somatic cell nuclear transfer
E. N. Shedova A , G. N. Singina A , V. P. Sergiev B C , M. P. Rubtsova D , N. V. Ravin E , A. V. Lopukhov A , I. A. Polejaeva F , O. A. Dontsova C D and N. A. Zinovieva AA L.K. Ernst Federal Research Center for Animal Husbandry, Podolsk, Moscow region, Russia
B Institute of Functional Genomics, Lomonosov Moscow State University, Moscow, Russia
C Center of life sciences, Skolkovo Institute of Science and Technology, Moscow, Russia
D Department of chemistry, Lomonosov Moscow State University, Moscow, Russia
E Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia
F Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA
Reproduction, Fertility and Development 34(2) 292-292 https://doi.org/10.1071/RDv34n2Ab110
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The transfer of β-lactoglobulin (BLG) knockout embryos into surrogate animals would allow the production of gene-edited cows producing milk with reduced allergenic properties. To achieve this, we aimed to disrupt the BLG gene in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate BLG-edited embryos by somatic cell nuclear transfer (SCNT). Considering the fact that, in the bovine genome, the BLG gene is duplicated and represented by two closely related paralogs, BLG (PAEP) itself and BLG-like gene (LOC100848610), we inactivated both paralogous genes. We designed a single sgRNA specific for PAEP and a pair of sgRNA specific for LOC100848610. The targeting vectors were constructed by using the pX458 plasmid and transfected (by electroporation) into bovine BFFs. High-throughput sequencing of the amplicons of the target regions of the PAEP and LOC100848610 genes in population of BFFs showed the presence, respectively, of 12 and 7.5% of mutant sequences containing deletions and insertions of nucleotides at the sites of putative breaks. To obtain individual colonies of BLG-edited cell, the early-passage BFFs were electroporated with a mixture of plasmids encoding Cas9 and gRNA, aimed at inactivating the PAEP and LOC100848610 genes and then cultured in Dulbecco’s modified Eagle medium, supplemented with 5% fetal bovine serum. After 2 days, the total pool of cells expressing the genes for components of the CRISPR/Cas9 system was sorted and then the cells were seeded individually in 96-well plates and cultured until colonies appeared. The proportion of the formed colonies was 21.3% of the total number of seeded cells (90/389). To evaluate BLG editing, genomic DNA from individual BFF colonies was isolated, amplified by PCR, and the PCR product was Sanger sequenced. Biallelic knockouts of PAEP and LOC100848610 genes were established in four out of 90 individual BFF colonies, which corresponds to a gene editing efficiency of 4.4%. The BLG-edited BFFs were then used to generate embryos by SCNT. After IVM (20 h), the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.27 m mannitol fusion medium (0.1 mM calcium, 0.1 mM magnesium) by two pulses of 35 V for 20 µs. Reconstructed oocytes were activated by exposure to 5 mM ionomycin for 5 min, followed by 4 h of incubation in 2 mM 6-(dimethylamino)purine and 10 mg mL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. We found that 61.8% (183/296) of SCNT-oocytes formed 2-cell embryos and 18.9% (51/296) developed to the blastocyst stage. We conclude that BLG-BFFs edited by CRISPR/Cas9 can be used to produce SCNT bovine embryos.
This research was supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of the Russian Federation (theme No. 0445-2021-0004).
