Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat [RNA-Seq]
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 the role of various genes in regulating MSC proliferation  , we established a pooled CRISPR knockout library population targeting 600 genes in adipose-derived bovine MSCs. We then performed gene expression profiling analysis using data obtained from RNA-seq of three MSCs populations at two time points. Comparative gene expression profiling analysis of RNA-seq data for CRISPR-Library-KO MSC cells at day 125 compared to early WT at day 25 or late WT at day 125 with three replicated for each.