CRISPR screen reveals genes and pathways regulating proliferation in bovine stem cells 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. 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.

培养肉（cultured meat）为传统肉类生产所带来的环境与伦理难题提供了极具前景的解决方案，但在规模化生产与成本效益方面仍面临显著障碍。细胞来源（如牛源间充质干细胞（bovine mesenchymal stem cells, bMSCs））的增殖能力有限是核心瓶颈之一。本研究采用CRISPR敲除筛选（CRISPR knockout screening）技术，鉴定出若干影响牛源间充质干细胞增殖的关键基因与信号通路。值得注意的是，TP53与PTEN敲除在本次混合CRISPR筛选（pooled CRISPR screen）中位列顶尖候选靶点，相较于其他所有基因敲除组，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）对照组，TP53与PTEN的缺失均显著提升了牛源间充质干细胞的增殖速率。本研究还鉴定出线粒体丙酮酸载体（mitochondrial pyruvate carrier）与SMAD信号通路（SMAD signaling pathway）中的潜在靶点，有望改善间充质干细胞的增殖与分化能力。上述研究结果证实了CRISPR技术在增强干细胞增殖能力方面的应用潜力，可助力实现更高效、更具成本效益且可规模化的培养肉生产。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CRISPR敲除文库（pooled CRISPR knockout library），并转染至脂肪来源牛源间充质干细胞（adipose-derived bovine MSCs, AD-bMSCs）中。通过将混合细胞群体扩增30天或200天，并在不同时间点提取基因组DNA、PCR扩增sgRNA序列，开展增殖筛选实验。借助MAGeCK工具，我们对随时间显著富集或耗竭的sgRNA进行鉴定与排序，以此推断哪些基因敲除会改变脂肪来源牛源间充质干细胞的增殖能力。我们还开展了基因本体（Gene Ontology, GO）过度富集分析，以鉴定与牛源间充质干细胞增殖增减相关的富集GO术语。