小柯机器人

美国驯鹿生物科学公司Samuel H. Sternberg、Peter Cameron等研究人员合作利用I型CRISPR-Cas系统在人类细胞中实现基因组编辑。2019年11月18日，相关论文在线发表在《自然—生物技术》上。

研究人员将Cascade融合到依赖二聚化的非特异性FokI核酸酶结构域，并在多个人类细胞系中以高特异性和高达50％的效率实现RNA指导的基因编辑。FokI–Cascade可以通过优化的双组分表达系统重建，该系统在单个多顺反子载体上编码CRISPR相关（Cas）蛋白，在单独的质粒上编码RNA（gRNA）。完整的Cascade–Cas3复合物在人细胞中的表达导致定向缺失的长度高达约200 kb。这些工作表明，可以利用高度丰富、尚未开发的I型CRISPR–Cas系统在真核细胞中进行基因组工程应用。

据了解，I型CRISPR–Cas系统是细菌和古细菌中最丰富的适应性免疫系统。靶标干扰依赖于称为Cascade的RNA引导的多亚基复合物，该复合物募集了反式解旋酶核酸酶Cas3用于靶标降解。由于多组分级联复合物的异源表达相对困难，I型系统很少用于真核基因组工程应用。

附：英文原文

Title: Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Author: Peter Cameron, Mary M. Coons, Sanne E. Klompe, Alexandra M. Lied, Stephen C. Smith, Bastien Vidal, Paul D. Donohoue, Tomer Rotstein, Bryan W. Kohrs, David B. Nyer, Rachel Kennedy, Lynda M. Banh, Carolyn Williams, Mckenzi S. Toh, Matthew J. Irby, Leslie S. Edwards, Chun-Han Lin, Arthur L. G. Owen, Tim Knne, John van der Oost, Stan J. J. Brouns, Euan M. Slorach, Chris K. Fuller, Scott Gradia, Steven B. Kanner, Andrew P. May, Samuel H. Sternberg

Issue&Volume: 2019-11-18

Abstract: Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5,6,7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8,9,10,11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

DOI: 10.1038/s41587-019-0310-0

Source: https://www.nature.com/articles/s41587-019-0310-0

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex