美国斯克利普斯研究所Xin Jin研究组发现，大规模并行体内Perturb-seq技术揭示大脑皮层发育过程中细胞类型特异性转录网络。这一研究成果于2024年5月20日在线发表在国际学术期刊《细胞》上。
利用AAV的多功能向性和标记能力，研究人员扩大了体内CRISPR筛选的规模，在胚胎到成人大脑和外周神经系统中进行了单细胞转录组表型分析。通过对AAV血清型中的86种载体结合转座子系统进行广泛测试，研究人员大大提高了标记效果，并将体内基因递送从数周加速到数天。子宫内的原理性验证筛选确定了Foxg1的多效应，强调了它对第6层皮层丘脑神经元细胞命运特化所必需的独特网络的严格调控。
值得注意的是，该平台可以标记大于6%的脑细胞，超过了目前慢病毒标记小于0.1%的最先进效率，从而在一次实验中分析超过30000个细胞，并实现大规模并行体内Perturb-seq。它与各种表型测量（单细胞或空间多组学）兼容，提供了一种灵活的方法，可用于体内跨细胞类型的基因功能检测，将基因变异转化为其因果功能。
附：英文原文
Title: Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development
Author: Xinhe Zheng, Boli Wu, Yuejia Liu, Sean K. Simmons, Kwanho Kim, Grace S. Clarke, Abdullah Ashiq, Joshua Park, Jiwen Li, Zhilin Wang, Liqi Tong, Qizhao Wang, Keerthi T. Rajamani, Rodrigo Muoz-Castaeda, Shang Mu, Tianbo Qi, Yunxiao Zhang, Zi Chao Ngiam, Naoto Ohte, Carina Hanashima, Zhuhao Wu, Xiangmin Xu, Joshua Z. Levin, Xin Jin
Issue&Volume: 2024-05-20
Abstract: 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.
DOI: 10.1016/j.cell.2024.04.050
Source: https://www.cell.com/cell/fulltext/S0092-8674(24)00476-8