Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, La Jolla, CA 92037, USA.
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Cell. 2024 Jun 20;187(13):3236-3248.e21. doi: 10.1016/j.cell.2024.04.050. Epub 2024 May 20.
Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.
利用 AAV 的多功能嗜性和标记能力，我们通过单细胞转录组表型分析，在胚胎到成年大脑和外周神经系统中扩大了体内 CRISPR 筛选的规模。通过对 86 种 AAV 血清型结合转座子系统的广泛测试，我们大大提高了标记效率，并将体内基因传递速度从数周缩短到数天。我们在子宫内的初步筛选确定了 Foxg1 的多效性作用，突出了其对层 6 皮质丘脑神经元细胞命运特化所必需的不同网络的紧密调控。值得注意的是，我们的平台可以标记超过 6%的脑细胞，超过了当前最先进的慢病毒<0.1%的效率，从而在一次实验中实现对超过 30000 个细胞的分析，并支持大规模的体内 Perturb-seq。与各种表型测量方法（单细胞或空间多组学）兼容，它为在体内研究不同细胞类型中的基因功能提供了一种灵活的方法，将基因变异转化为其因果功能。
