基于 CRISPR 的高通量基因组学方法的兴起与未来。

IF 10.1 2区 生物学 Q1 MICROBIOLOGY
Silke Vercauteren, Simon Fiesack, Laetitia Maroc, Natalie Verstraeten, Liselot Dewachter, Jan Michiels, Sibylle C Vonesch
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引用次数: 0

摘要

有规律间隔短回文重复序列(CRISPR)给基因组编辑领域带来了革命性的变化。为了规避传统 CRISPR 技术造成的永久性修改,并促进对重要和非重要基因的研究,CRISPR 干扰(CRISPRi)应运而生。这种基因沉默技术利用失活的 Cas 效应蛋白和引导 RNA 来阻断转录的启动或延伸。对 CRISPRi 机制的不断改进和深入了解扩大了其应用范围,促进了全基因组高通量筛选,以研究表型的遗传基础。此外,新出现的基于 CRISPR 的替代方法也进一步扩大了基因筛选的可能性。本综述深入探讨了 CRISPRi 的机制,将其与其他高通量基因扰乱技术进行了比较,并强调了其在研究复杂微生物性状方面的卓越能力。我们还探讨了 CRISPRi 的演化过程,强调了它的增强功能,包括多重性、可诱导性、可滴定性、可预测的基因敲除效果以及对非模式微生物的适应性。除了 CRISPRi 之外,我们还讨论了 CRISPR 激活、RNA 靶向 CRISPR 系统和单核苷酸分辨率扰动技术在微生物全基因组高通量筛选中的潜力。总之,这篇综述全面概述了全基因组 CRISPRi 筛选的一般工作流程,并对优缺点、未来方向和潜在替代方案进行了广泛讨论。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The rise and future of CRISPR-based approaches for high-throughput genomics.

Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized the field of genome editing. To circumvent the permanent modifications made by traditional CRISPR techniques and facilitate the study of both essential and nonessential genes, CRISPR interference (CRISPRi) was developed. This gene-silencing technique employs a deactivated Cas effector protein and a guide RNA to block transcription initiation or elongation. Continuous improvements and a better understanding of the mechanism of CRISPRi have expanded its scope, facilitating genome-wide high-throughput screens to investigate the genetic basis of phenotypes. Additionally, emerging CRISPR-based alternatives have further expanded the possibilities for genetic screening. This review delves into the mechanism of CRISPRi, compares it with other high-throughput gene-perturbation techniques, and highlights its superior capacities for studying complex microbial traits. We also explore the evolution of CRISPRi, emphasizing enhancements that have increased its capabilities, including multiplexing, inducibility, titratability, predictable knockdown efficacy, and adaptability to nonmodel microorganisms. Beyond CRISPRi, we discuss CRISPR activation, RNA-targeting CRISPR systems, and single-nucleotide resolution perturbation techniques for their potential in genome-wide high-throughput screens in microorganisms. Collectively, this review gives a comprehensive overview of the general workflow of a genome-wide CRISPRi screen, with an extensive discussion of strengths and weaknesses, future directions, and potential alternatives.

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来源期刊
FEMS microbiology reviews
FEMS microbiology reviews 生物-微生物学
CiteScore
17.50
自引率
0.90%
发文量
45
审稿时长
6-12 weeks
期刊介绍: Title: FEMS Microbiology Reviews Journal Focus: Publishes reviews covering all aspects of microbiology not recently surveyed Reviews topics of current interest Provides comprehensive, critical, and authoritative coverage Offers new perspectives and critical, detailed discussions of significant trends May contain speculative and selective elements Aimed at both specialists and general readers Reviews should be framed within the context of general microbiology and biology Submission Criteria: Manuscripts should not be unevaluated compilations of literature Lectures delivered at symposia must review the related field to be acceptable
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