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研究揭示V型CRISPR转座子系统的靶标选择和重塑
2021-11-13 22:23

瑞士苏黎世大学Martin Jinek课题组揭示V型CRISPR转座子系统的靶标选择和重塑。2021年11月10日,国际知名学术期刊《自然》在线发表了这一成果。

研究人员报告了Cas12k指导的RNA-靶标DNA复合体和DNA结合的聚合TnsC丝的冷冻电镜结构,该系统来自光合蓝藻Scytonema hofmanni的CRISPR相关转座子系统。Cas12k复合物的结构揭示了复杂的向导RNA结构和向导RNA引导的靶标DNA识别的关键相互作用。TnsC螺旋丝的组装是依赖ATP的,并伴随着结合的DNA双链的结构重塑。体内转座实验证实了结构的关键特征,生化实验显示TniQ限制了TnsC的聚合,而TnsB直接与TnsC丝状物相互作用,在ATP水解时触发它们的解体。

这些结果表明,Cas12k的RNA定向靶标选择促进了TnsC的聚合和DNA重塑,为TnsB产生了一个招募平台,从而催化特定位点的转座子插入。这项工作的见解将为CRISPR相关转座子的发展提供信息,使其成为可编程的特定位点基因插入工具。

据了解,经典的CRISPR-Cas系统对移动遗传元件提供了适应性免疫。然而,I-F、I-B和V-K型系统已被Tn7类转座子采用,用于指导RNA引导的转座子插入。V-K型CRISPR相关转座子依赖于假核酸酶Cas12k、转座酶TnsB、AAA+ATP酶TnsC和锌指蛋白TniQ7,但RNA引导的DNA转座的分子机制仍然难以捉摸。

附:英文原文

Title: Target site selection and remodelling by type V CRISPR-transposon systems

Author: Querques, Irma, Schmitz, Michael, Oberli, Seraina, Chanez, Christelle, Jinek, Martin

Issue&Volume: 2021-11-10

Abstract: Canonical CRISPR–Cas systems provide adaptive immunity against mobile genetic elements1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2,3,4,5,6,7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA–target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.

DOI: 10.1038/s41586-021-04030-z

Source: https://www.nature.com/articles/s41586-021-04030-z

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:69.504
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

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