Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat [Short-Term screen]
Cultured meat offers a promising solution to the environmental and ethical challenges of conventional meat production, but faces significant hurdles in scalability and cost-effectiveness. A major bottleneck is the limited proliferation capacity of cell sources such as bovine mesenchymal stem cells (bMSCs). Here, we employ CRISPR knockout screening to identify several key genes and pathways influencing bMSC proliferation. Notably, TP53 and PTEN knockouts emerged as top hits in our pooled CRISPR screen, exhibiting the highest increase in cell abundance over 30 days compared to all other gene knockouts. Subsequent validation using individual knockouts confirmed that both TP53 and PTEN deletion significantly enhanced bMSC proliferation rates relative to non-targeting sgRNA controls. We also identified targets in mitochondrial pyruvate carrier and SMAD signaling pathways that may improve MSC proliferation and differentiation. These findings demonstrate CRISPR technology's potential to enhance stem cell growth for more efficient, cost-effective, and scalable cultured meat production. Overall design: To investigate genes regulating MSC proliferation, we established a pooled CRISPR knockout library targeting 600 genes in adipose-derived bovine MSCs. We conducted proliferation screens by expanding the pooled cell population for 30 or 200 days, extracting DNA and PCR-amplifying sgRNA sequences at various time points. Using MAGeCK, we identified and ranked significantly enriched or depleted sgRNAs over time, inferring which gene knockouts alter proliferation in AD-bMSCs. We performed Gene Ontology over-representation analysis to identify enriched GO terms associated with increased or decreased proliferation in bMSCs.