A compact Cascade-Cas3 system for targeted genome engineering.
A compact Cascade-Cas3 system for targeted genome engineering.
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DOI：
10.1038/s41592-020-00980-w
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发表时间：
2020-12
期刊：
影响因子：
48
通讯作者：
Bondy-Denomy J
中科院分区：
文献类型：
--
作者：
Csörgő B;León LM;Chau-Ly IJ;Vasquez-Rifo A;Berry JD;Mahendra C;Crawford ED;Lewis JD;Bondy-Denomy J
CRISPR-Cas技术提供了可编程的基因编辑工具，彻底改变了研究。领先的CRISPR-Cas9和Cas12a酶是编程遗传操作的理想选择，然而，它们在基因组规模干预方面受到限制。在这里，我们利用cas3为基础的系统，具有一个基因组工程的过程核酸酶。这个最小的Cascade-Cas3系统（Type I-C），用单个crRNA编程，经过优化，以接近100%的效率产生缺失，并用于在铜绿假单胞菌中快速产生7 - 424 kb的大缺失。相比之下，Cas9产生了小的缺失和点突变。cas3产生的缺失边界是高度可变的，但通过同源定向修复（HDR）模板成功指定。与Cas9相比，当病变由Cas3产生时，HDR更有效。最小的I-C型系统也是便携式的；使用“all-in-one”载体，可以有效地在丁香假单胞菌、大肠杆菌和肺炎克雷伯菌中产生大量缺失。值得注意的是，Cas3产生了源自程序化切割位点的双向缺失，利用10种不同的crrna快速迭代地减少了铜绿假单胞菌基因组837 kb（13.5%）。我们还通过开发一种“anti-anti-CRISPR”策略来规避内源性CRISPR-Cas抑制剂蛋白，从而增强Cas3系统的效用。CRISPR-Cas3可以促进合成生物学和代谢工程目的的快速菌株操作，基因组最小化，移动遗传元件去除以及分析未知功能的大区域。
CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilize a Cas3-based system featuring a processive nuclease for genome engineering. This minimal Cascade-Cas3 system (Type I-C), programmed with a single crRNA, was optimized to generate deletions with near-100% efficiency, and used to rapidly generate large deletions ranging from 7 - 424 kb in Pseudomonas aeruginosa. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were highly variable, but successfully specified by a homology-directed repair (HDR) template. HDR was much more efficient when lesions were generated by Cas3, compared to Cas9. The minimal Type I-C system is also portable; using an “all-in-one” vector, large deletions could be efficiently generated in Pseudomonas syringae, Escherichia coli, and Klebsiella pneumoniae. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to rapidly and iteratively reduce a P. aeruginosa genome by 837 kb (13.5 %) using 10 distinct crRNAs. We also enhance the utility of Cas3 systems by developing an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biology and metabolic engineering purposes, genome minimization, mobile genetic element removal, and the analysis of large regions of unknown function.
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影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
DOI：
10.1073/pnas.1731982100
发表时间：
2003-09-02
影响因子：
11.1
作者：
Buell, CR;Joardar, V;Collmer, A
通讯作者：
Collmer, A
影响因子：
48
作者：
Choi, KH;Gaynor, JB;Schweizer, HP
通讯作者：
Schweizer, HP
影响因子：
14.9
作者：
Cui L;Bikard D
通讯作者：
Bikard D
DOI：
10.1126/science.1159689
发表时间：
2008-08-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Brouns SJ;Jore MM;Lundgren M;Westra ER;Slijkhuis RJ;Snijders AP;Dickman MJ;Makarova KS;Koonin EV;van der Oost J
通讯作者：
van der Oost J