Successful establishment of CRISPR-based genome-edited clonal cell populations from primary cells of buffalo, goats, and sheep

Abstract

Genome editing technology has great potential for precise DNA modification in mammalian cells. The ability to precisely generate the clonal population of CRISPR-edited geno-type is of great importance in gene function/ pathway analysis, drug discovery, and production of genome-edited animals. In the present study, we demonstrated an efficient method to generate CRISPR-edited single-cell clonal populations of farm animals, including buffalo, goats, and sheep. To generate clonal cell populations, the primary fibroblasts were established through explant culture and then electroporated with CRISPR/Cas RNPs targeted for the disrupted MSTN gene. We used a single-cell pickup method in which one cell was picked up using an ultra-fined glass capillary and transferred into each well of a 96-well plate. For promoting the growth of single cells, we used growth factor-supplemented media. After seeding a single cell to each well, the plate was kept undisturbed for 5-7 days, and then cell attachment rates were noted. We reported that the cell attachment rates for buffalo, goat, and sheep cells were 40%, 77.08%, and 83.67%, respectively. The proliferation rates were 70.83%, 75.67%, and 78.05% for buffalo, goat, and sheep cells, respectively. We noticed that cell attachment and proliferation rates were better in the case of goat and sheep cells; also, these cells exhibited less vacuolation compared to buffalo cells. In the present study, we generated 11, 20, and 20 single-cell clones of MSTN-gene-edited buffalo, goat, and sheep cells. In conclusion, our method can be efficiently used to generate genome-edited single-cell clones to harness the potential of CRISPR technologies in farm animals.