Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Frontiers in genome editing Pub Date : 2024-12-19 eCollection Date: 2024-01-01 DOI:10.3389/fgeed.2024.1495968
Yuvaraj Bhoobalan-Chitty, Mathieu Stouf, Marianne De Paepe
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引用次数: 0

Abstract

CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, α-gt and β-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.

利用CRISPR-Cas13b系统对T4噬菌体进行基因操作。
CRISPR-Cas II型和V型系统由于其DNA的过度修饰,在修饰T4噬菌体基因组方面效率低下。在这里,我们提出了一种使用VI型CRISPR-Cas系统对T4噬菌体进行基因组编辑的技术。利用BzCas13b靶向T4噬菌体,我们能够单独删除T4糖基转移酶基因α-gt和β-gt。此外,我们利用这种方法突变了T4 DNA聚合酶中的两个保守残基,并将黄色荧光蛋白(YFP)编码序列引入T4噬菌体基因组,使我们能够可视化噬菌体感染。该T4基因组编辑方案经过优化,可在6小时内生成重组噬菌体。最后,利用与多种T4基因同源的间隔子研究了Cas13b靶向的普遍性,揭示了靶向效率的重要变异性。总之,该方法是一种快速有效的产生特异性T4噬菌体突变体的方法,可推广到其他T4样噬菌体。
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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CiteScore
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审稿时长
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