Metagenome-Derived CRISPR-Cas12a Mining and Characterization.

IF 4 4区 生物学 Q2 GENETICS & HEREDITY
CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-05-21 DOI:10.1089/crispr.2024.0099
Kalani Gast, Sydney Baker, Adair L Borges, Stephanie Ward, Jillian F Banfield, Rodolphe Barrangou
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引用次数: 0

Abstract

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.

宏基因组衍生的CRISPR-Cas12a挖掘和表征。
基于集群规则间隔短回文重复序列(CRISPR)的技术的出现彻底改变了基因组编辑,人们对用多样化、高效和特异性效应物扩展CRISPR相关蛋白(Cas)工具箱的兴趣不断增加。CRISPR-Cas12a是一种有效的、可编程的rna引导双缺口酶,广泛用于基因组编辑。在这里,我们为CRISPR-Cas系统挖掘奶牛微生物宏基因组,揭示了新的Cas12a酶。使用硅管道,我们表征和预测了CRISPR-Cas12a活性的关键驱动因素,包括五个系统的引导和原间隔邻近基序。接下来,我们利用基于gfp的荧光读数在无细胞转录翻译试验中评估了它们的功能潜力。最后,我们通过产生1kb的敲除,确定了它们在大肠杆菌体内的基因组编辑潜力。出乎意料的是,我们观察到表现最好的候选基因的桥-螺旋结构域的自然序列变化,并使用诱变来改变Cas12a同源物的活性,从而提高了相对低效的候选基因的基因编辑能力。这项研究说明了未充分开发的宏基因组序列多样性对基因组编辑效应物的开发和改进的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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