Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli

IF 3.1 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS Biotechnology Journal Pub Date : 2025-03-20 DOI:10.1002/biot.70005

Xiaojie Zhou, Siqi Yang, Bingbing Sun, Feng Dong, Mingyu Yin, Yu Jiang, Zhiwei Huang, Sheng Yang

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Abstract

The preferred method for Escherichia coli genome editing relies on Cas9 from Streptococcus pyogenes (SpCas9) and λ-Red recombinase. Although SpCas9 is currently the most active RNA-guided DNA endonuclease, a significant number of escapers are often observed, making it inefficient across different sites, particularly when inserting large fragments. In this study, we identified two RAGATH RNA-associated DNA endonucleases (RADs) derived from IS607 transposons. Both of them exhibited high cleavage activity in E. coli. When combined with λ-Red recombinase, they achieved editing efficiencies approaching 100%. Even at target sites where SpCas9 exhibited low editing efficiency, RADs maintained efficiencies ranging from 57% to 94%. Moreover, RADs exhibited higher efficiencies in inserting large fragments in certain cases compared to SpCas9. Taken together, these RAD-based genome editing tools provide viable alternatives to SpCas9, particularly for challenging targets and/or large fragment insertions.

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RAGATH rna相关DNA内切酶作为基因组编辑工具在大肠杆菌中的应用

大肠杆菌基因组编辑的首选方法依赖于来自化脓性链球菌的Cas9 （SpCas9）和λ-Red重组酶。尽管SpCas9是目前最活跃的rna引导DNA内切酶，但经常观察到大量逃逸子，使其在不同位点上效率低下，特别是在插入大片段时。在这项研究中，我们鉴定了两个来自IS607转座子的RAGATH rna相关DNA内切酶（RADs）。两者在大肠杆菌中均表现出较高的裂解活性。当与λ-Red重组酶结合时，它们的编辑效率接近100%。即使在SpCas9表现出较低编辑效率的靶位点上，RADs的效率也保持在57%到94%之间。此外，与SpCas9相比，在某些情况下，RADs在插入大片段方面表现出更高的效率。总之，这些基于rad的基因组编辑工具为SpCas9提供了可行的替代方案，特别是对于具有挑战性的靶标和/或大片段插入。

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来源期刊

Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine

CiteScore

8.90

自引率

2.10%

发文量

123

审稿时长

1.5 months

期刊介绍： Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.