改造的I型CRISPR-Cas系統可用於人類基因組編輯

改造的I型CRISPR-Cas系統可用於人類基因組編輯

作者：

小柯機器人

發布時間：2019/11/19 15:55:10

美國馴鹿生物科學公司Samuel H. Sternberg、Peter Cameron等研究人員合作利用I型CRISPR-Cas系統在人類細胞中實現基因組編輯。2019年11月18日，相關論文在線發表在《自然—生物技術》上。

研究人員將Cascade融合到依賴二聚化的非特異性FokI核酸酶結構域，並在多個人類細胞系中以高特異性和高達50％的效率實現RNA指導的基因編輯。FokI–Cascade可以通過優化的雙組分表達系統重建，該系統在單個多順反子載體上編碼CRISPR相關（Cas）蛋白，在單獨的質粒上編碼RNA（gRNA）。完整的Cascade–Cas3複合物在人細胞中的表達導致定向缺失的長度高達約200 kb。這些工作表明，可以利用高度豐富、尚未開發的I型CRISPR–Cas系統在真核細胞中進行基因組工程應用。

據了解，I型CRISPR–Cas系統是細菌和古細菌中最豐富的適應性免疫系統。靶標幹擾依賴於稱為Cascade的RNA引導的多亞基複合物，該複合物募集了反式解旋酶核酸酶Cas3用於靶標降解。由於多組分級聯複合物的異源表達相對困難，I型系統很少用於真核基因組工程應用。

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