The second domestication of cattle:
Evolving bovine mesenchymal stem cells for cultured meat production by genome-wide CRISPR screen
2Department of Animal Science, Agricultural Research Organization - Volcani Institute, Israel
Can we cultivate meat better by adapting bovine-derived mesenchymal stem cells (bMSCs) to proliferate and differentiate more efficiently? Cultured meat is a rapidly growing alternative to traditional meat, aiming to mitigate adverse effects associated with live cattle; it is more efficient, sustainable, and humane, and has a smaller ecological footprint. However, several aspects must be improved to achieve efficient and economical production of cultured meat at large scale, including cell lines adapted for cellular agriculture. Two important traits for such cell lines are faster proliferation rate, and improved differentiation efficiency. To identify genes and pathways associated with proliferation and differentiation of bMSC, we chose to employ a genome-wide CRISPR knockout screen targeting Bos taurus genome, in conjunction with directed evolution. First, we isolated MSC from bovine adipose tissues. Following multiple passages, the MSCs were shown to retain their proliferation potential, multipotency, and the ability to incorporate foreign DNA by lentiviral transduction. Concurrently, CRISPR KO sgRNA library was curated by filtering whole-genome bovine sgRNA library (btCRISPRko.v1), to 86,000 unique sgRNAs targeting 21,027 bovine genes. Using lentiviral transduction, each sgRNA is transduced into a single bMSC cell. With the appropriate selective pressure, each cell`s fitness depends on the contained sgRNA, changing the composition of sgRNAs in the population, which is quantified using NGS. This will allow the discovery of important genes and pathways governing proliferation and differentiation. The identification, and ultimately harnessing of said genes and pathways in bMSC will lead to a more economical and efficient cultured meat production.