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

IF 3.2 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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引用次数: 0

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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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.