Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing^1,2,3,4. Several genetic interventions have been found to ameliorate old brain function^5,6,7,8, but systematic functional testing of genes in old NSCs—and more generally in old cells—has not been done. Here we develop in vitro and in vivo high-throughput CRISPR–Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR–Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

衰老会损害神经干细胞 （NSC） 在成年哺乳动物大脑中从静止过渡到增殖的能力。NSC 的功能下降导致衰老过程中受伤后新神经元的产生减少和再生缺陷^1,2,3,4。已经发现几种遗传干预可以改善老年大脑功能^5,6,7,8，但尚未对老年 NSC（更普遍地说是老细胞）中的基因进行系统的功能测试。在这里，我们开发了体外和体内高通量 CRISPR-Cas9 筛选平台，以系统地揭示促进老年小鼠 NSC 活化的基因敲除。我们在年轻和老年 NSC 的原代培养物中进行了全基因组筛选，发现了 300 多个基因敲除，这些基因敲除专门恢复了老年 NSC 的激活。排名靠前的基因敲除参与纤毛组织和葡萄糖输入。我们还建立了一个可扩展的体内 CRISPR-Cas9 筛选平台，该平台确定了 24 个基因敲除，这些基因敲除可促进 NSC 激活和旧大脑中新神经元的产生。值得注意的是，敲除编码 GLUT4 葡萄糖转运蛋白的 Slc2a4 是改善老年 NSC 功能的主要干预措施。衰老过程中 NSC 的葡萄糖摄取增加，短暂的葡萄糖饥饿恢复了老年 NSC 的激活能力。因此，葡萄糖摄取的增加可能导致 NSC 激活随年龄的增长而下降。我们的工作提供了可扩展的平台，以系统地识别增强老年 NSC 功能的遗传干预，包括体内，对对抗衰老过程中的再生衰退具有重要意义。