Highly efficient genome editing in Bacillus subtilis via miniature DNA nucleases IscB

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology Pub Date : 2025-07-02 DOI:10.1016/j.synbio.2025.06.012

Jie Gao, Hengyi Wang, Jingtao Sun, Hongjie Tang, Yuhan Yang, Qi Li

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引用次数: 0

Abstract

Existing CRISPR-based genome editing techniques for Bacillus subtilis (B. subtilis) are limited due to the large size of the cas gene. IscB, a recently reported DNA nuclease, is one-third the size of Cas9, making it a potential tool for genome editing; however, its application in B. subtilis remains unexplored. In this study, two IscB and enhanced IscB (enIscB)-based genome editing systems, named pBsuIscB and pBsuenIscB were established in B. subtilis SCK6, and their deletion efficiencies ranging from 13.3 % to 100 %. Compared to the pBsuIscB system, the pBsuenIscB system showed higher deletion efficiency, inducing the deletion of a large genomic fragment with a single ωRNA. Additionally, the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B. subtilis SCK6 genome. Lastly, the pBsuenIscB system could efficiently conduct a second round of genome editing in B. subtilis SCK6. This study indicates that IscB can be used for genome editing in B. subtilis, enabling the efficient construction of engineered B. subtilis strains for large-scale biomolecule production.

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利用微型DNA核酸酶IscB对枯草芽孢杆菌进行高效基因组编辑

现有的基于crispr的枯草芽孢杆菌（B. subtilis）基因组编辑技术由于cas基因的大尺寸而受到限制。IscB是最近报道的一种DNA核酸酶，其大小是Cas9的三分之一，使其成为基因组编辑的潜在工具；然而，其在枯草芽孢杆菌中的应用尚未探索。本研究在枯草芽孢杆菌SCK6中建立了两个基于IscB和增强型IscB （enIscB）的基因组编辑系统，分别命名为pBsuIscB和pBsuenIscB，其缺失效率在13.3% ~ 100%之间。与pBsuIscB系统相比，pBsuenIscB系统显示出更高的缺失效率，可以用单个ωRNA诱导大的基因组片段缺失。此外，pBsuenIscB系统可以整合枯草芽孢杆菌SCK6基因组中的单拷贝和多拷贝mCherry基因。最后，pBsuenIscB系统可以有效地在枯草芽孢杆菌SCK6中进行第二轮基因组编辑。本研究表明，IscB可用于枯草芽孢杆菌的基因组编辑，从而高效构建工程枯草芽孢杆菌菌株，用于大规模生物分子生产。

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

Synthetic and Systems Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-

CiteScore

6.90

自引率

12.50%

发文量

审稿时长

67 days

期刊介绍： Synthetic and Systems Biotechnology aims to promote the communication of original research in synthetic and systems biology, with strong emphasis on applications towards biotechnology. This journal is a quarterly peer-reviewed journal led by Editor-in-Chief Lixin Zhang. The journal publishes high-quality research; focusing on integrative approaches to enable the understanding and design of biological systems, and research to develop the application of systems and synthetic biology to natural systems. This journal will publish Articles, Short notes, Methods, Mini Reviews, Commentary and Conference reviews.