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.

细胞培养肉为传统肉类生产所面临的环境与伦理挑战提供了极具前景的解决方案，但在规模化生产与成本效益层面仍存在显著障碍。以牛间充质干细胞（bovine mesenchymal stem cells, bMSCs）为代表的细胞来源，其有限的增殖能力是核心瓶颈之一。本研究采用CRISPR基因敲除筛选技术，鉴定出若干影响牛间充质干细胞增殖的关键基因与通路。值得注意的是，TP53与PTEN基因敲除在混合池CRISPR筛选中位列顶级命中结果，相较于所有其他基因敲除组，其在30天内的细胞丰度提升幅度最为显著。后续通过单基因敲除实验验证证实，相较于非靶向sgRNA（single guide RNA）对照，TP53与PTEN的缺失均显著提升了牛间充质干细胞的增殖速率。本研究还在线粒体丙酮酸载体与SMAD信号通路中鉴定出可改善间充质干细胞增殖与分化能力的靶点。上述研究结果证实了CRISPR技术在增强干细胞生长方面的潜力，可为实现更高效、更具成本效益且可规模化的细胞培养肉生产提供支撑。
总体实验设计：为探究调控间充质干细胞增殖的基因，本研究构建了靶向600个基因的混合池CRISPR敲除文库，并将其应用于脂肪源牛间充质干细胞。通过将混合池细胞群体扩增30天或200天开展增殖筛选，并在不同时间点提取基因组DNA、通过聚合酶链式反应（PCR）扩增sgRNA序列。借助MAGeCK工具，我们鉴定并排序了随时间显著富集或耗竭的sgRNA，以此推断哪些基因敲除会改变脂肪源牛间充质干细胞的增殖能力。此外，我们开展了基因本体（Gene Ontology, GO）富集分析，以鉴定与牛间充质干细胞增殖增减相关的显著富集GO术语。