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CRISPR RNA介导的整合酶可对细菌基因组实现高效多重操作
作者:小柯机器人 发布时间:2020/11/24 17:01:11

美国哥伦比亚大学Samuel H. Sternberg研究小组取得一项新突破。他们发现CRISPR RNA介导的整合酶可实现高效、多重细菌基因组操作。 该项研究成果发表在2020年11月23日出版的《自然-生物技术》上。

为了解决现有细菌基因组特异性整合的缺点,研究人员研发了基于先前报道的霍乱弧菌Tn7样转座子的显著改进版本,该转座子使用I-F CRISPR–Cas系统进行可编程的、RNA引导的转座。引导RNA辅助靶向(INTEGRATE)系统优化了转座因子的插入效率,在细菌中以约100%的效率实现了高达10 KB碱基高度准确且无标记的DNA整合。使用多间隔CRISPR阵列,研究人员通过结合正交整合和重组酶,在三个基因组位点中实现了同时多重插入和高效的多位点缺失。

最后,研究人员证明了其在生物医学和工业相关细菌中的强大功能,并在复杂细菌群落中实现了针对靶点和物种的整合。这项工作揭示了INTEGRATE可作为多用途、千碱基规模因组工程改造的多功能性工具。

据悉,现有特异性整合细菌中千碱基DNA序列的技术受到效率低下、依赖重组效率、对多种载体需求以及多重挑战的局限。

附:英文原文

Title: CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

Author: Phuc Leo H. Vo, Carlotta Ronda, Sanne E. Klompe, Ethan E. Chen, Christopher Acree, Harris H. Wang, Samuel H. Sternberg

Issue&Volume: 2020-11-23

Abstract: Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.

DOI: 10.1038/s41587-020-00745-y

Source: https://www.nature.com/articles/s41587-020-00745-y

期刊信息

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:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex