小柯机器人

美国加州大学旧金山分校Joseph Bondy-Denomy研究团队的一项最新研究提出了一个紧凑的Cascade-Cas3系统有针对性的基因工程。 该项研究成果发表在2020年10月19日的《自然—方法学》上。

研究人员使用进行性核酸酶Cas3以及最小的基于I-C类型Cascade酶系统在细菌中进行靶向基因组工程。单个CRISPR RNA引导下的DNA解理可以产生大的删除(7 - 424 个碱基)，在铜绿假单胞菌中能达到将近100%的效率，而Cas9只产生小的删除和点突变。Cas3产生开始于编程位点的双向删除，可以实现减少铜绿假单胞菌基因组的837 kb (13.5%)。大型删除的边界可以在Cascade-Cas3编辑过程中被同源进化修复模板有效识别，然而Cas9不行。

可转移的“多合一”载体可在大肠杆菌、丁香假单胞菌和肺炎克雷伯菌中发挥作用，并且内源性CRISPR-Cas通过“抗-抗-CRISPR”策略得到了增强。铜绿假单胞菌I-C类型Cascade-Cas3 (PaeCas3c)促成快速菌株操纵，在合成生物学、基因组最小化和消除大型基因组区域都有应用。

研究人员表示，CRISPR-Cas技术可对真核生物和原核生物的基因进行编程编辑。然而，前导Cas9和Cas12a酶对于大型删除的能力仍然有限。

附：英文原文

Title: A compact Cascade–Cas3 system for targeted genome engineering

Author: Blint Csrg, Lina M. Len, Ilea J. Chau-Ly, Alejandro Vasquez-Rifo, Joel D. Berry, Caroline Mahendra, Emily D. Crawford, Jennifer D. Lewis, Joseph Bondy-Denomy

Issue&Volume: 2020-10-19

Abstract: CRISPR–Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7–424kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade–Cas3, but not Cas9. A transferable ‘all-in-one’ vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR–Cas use was enhanced with an ‘anti-anti-CRISPR’ strategy. P. aeruginosa Type I-C Cascade–Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

DOI: 10.1038/s41592-020-00980-w

Source: https://www.nature.com/articles/s41592-020-00980-w

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex