Development of a miniaturized CRISPR/Cas gene editing tool for human gut <i>Bacteroides</i>.

Yiwen ZHAN; Linggang ZHENG; Juntao SHEN; Yucan HU; Xuegang LUO; Lei DAI

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Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides.

Author: Yiwen ZHAN ¹ ; Linggang ZHENG ² ; Juntao SHEN ² ; Yucan HU ² ; Xuegang LUO ¹ ; Lei DAI ²
Author Information

1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
2. CAS Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China.
Publication Type:Journal Article
Keywords: Bacteroides; CRISPR/AsCas12f; asparaginase; gene editing
MeSH: CRISPR-Cas Systems/genetics*; Gene Editing/methods*; Bacteroides/genetics*; Humans; Gastrointestinal Microbiome/genetics*; Bacteroides fragilis/genetics*
From: Chinese Journal of Biotechnology 2025;41(6):2360-2372
CountryChina
Language:Chinese
Abstract: : Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.