CRISPR/Cas9技术在益生菌编辑中的应用与进展

doi:10.13560/j.cnki.biotech.bull.1985.2025-0146

摘要/Abstract

摘要：

基因编辑技术是一种通过分子工具对生物基因组DNA序列进行高效精准修饰，从而实现基因功能调控或性状改良的前沿生物技术。近年来，基因编辑技术的革新为益生菌功能优化开辟了新路径，借助基因编辑技术对益生菌进行工程化改造已广泛应用于农业、医学、生产及科学研究等领域，其中CRISPR/Cas9作为细菌适应性免疫的一部分，相较于传统的锌指核酸酶和类转录激活因子效应物核酸酶技术，具有更高的编辑效率和更低的使用成本，迅速发展成为生命科学领域的革命性基因编辑工具。CRISPR/Cas9技术已成功实现了对多种细菌和真菌的精确基因修饰，随着研究的不断深入，通过优化改进单链向导RNA、选择新型核酸酶、使用双CRISPR切割系统及结合碱基编辑技术等方式，CRISPR/Cas9技术为益生菌基因编辑提供了更多高效且精确的修饰策略。本文首先对CRISPR/Cas9技术进行介绍，阐述了其结构组成及作用机制，接着探讨了目前通过CRISPR/Cas9技术编辑益生菌的必要性和潜在价值，再结合具体实例详细介绍了CRISPR/Cas9技术在益生菌领域的实际应用现状，同时指出了CRISPR/Cas9技术目前存在的脱靶率高、染色体异常、编辑后的细胞毒性等技术问题，最后对CRISPR/Cas9技术的应用前景进行了展望，指出其健康有序发展离不开政策法规的监管，旨在为CRISPR/Cas9技术在益生菌编辑应用方面提供参考。

关键词: CRISPR/Cas9, 基因编辑, 益生菌, 菌株功能定制, 基因功能分析, 疾病治疗

Abstract:

Gene editing is an advanced biotechnology that allows for the efficient and precise modification of the DNA sequence of an organism's genome, enabling the regulation gene functions or the improvement of traits. In recent years, the revolution of gene editing technology has opened up new avenues for the optimization of probiotic functions. The engineering of prob through gene editing has been widely applied in agriculture, medicine, production, and scientific research. Among them, CRISPR/Cas9, as a part of the bacterial adaptive immune system, has a higher editing efficiency and lower cost of use compared with traditional zine finger endonuclease and transcription activator-like effector nuclease technology. CRISPR/Cas9 has rapidly become a revolutionary gene editing tool in the field of life sciences. At present, the probiotic industry is developing rapidly, and CRISPR/Cas9 technology has successfully achieved precise gene modification in bacteria, cells, animals and various plants. With the continuous deepening of research, CRISPR/Cas9 technology provides more efficient and precise modification strategies for probiotic gene editing by optimizing and sgRNA, selecting new nucleases, using dual CRISPR cutting systems combining base editing technology and other ways. This article first gives a brief introduction to CRISPR/Cas9 technology, elucidates its structural composition and action mechanism, then explores the necessity and potential value of editing probiotics through CRISPR/Cas9 technology, and combines specific examples to show the current practical application status of this technology in the field of probiotics in detail. Then, the article p lists the issues of high off-target rate, chromosomal abnormalities, and edited cytotoxicity. Finally, the article prospects the application trends of CRISPR/Cas9 technology, and points out that its healthy and orderly development can not be without the supervision of policies and laws, aiming to provide references for the application of CRISPR/Cas9 technology in probiotic editing.

Key words: CRISPR/Cas9, gene editing, probiotics, strain function customization, gene function analysis, disease treatment

刘梓琦, 钟沛, 李琴, 郭成, 张艳梅, 张乃锋, 屠焰, 刁其玉, 毕研亮. CRISPR/Cas9技术在益生菌编辑中的应用与进展[J]. 生物技术通报, 2025, 41(11): 89-99.

LIU Zi-qi, ZHONG Pei, LI Qin, GUO Cheng, ZHANG Yan-mei, ZHANG Nai-feng, TU Yan, DIAO Qi-yu, BI Yan-liang. Application and Progress of CRISPR/Cas9 Technology in Probiotic Editing[J]. Biotechnology Bulletin, 2025, 41(11): 89-99.

图/表 4

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菌株 Strain	改造后效果 Effect after transformation	参考文献 Reference
酿酒酵母	建立了莽草酸合成途径	［33］
多种梭菌属微生物	引入特定代谢路径，使菌株能够利用CO、CO₂及废弃甘油等生产生物燃料	［34］
保加利亚乳酸乳杆菌	编辑了保加利亚乳杆菌的ldh基因，并观察到其对酸奶特性的显著影响	［35］
双歧杆菌	编辑abfa基因簇，探索其影响宿主利用阿拉伯糖及肠道运动的机制	［36］
酵母菌	进行基因突变，成功构建乙醇耐受型酵母菌株	［37］
枯草芽胞杆菌	利用多轮基因编辑并结合发酵条件优化，赋予菌株利用木糖新功能并实现丰原素产量提高	［38］
副干酪乳杆菌	建立了高效编辑工具，验证尿嘧啶磷酸核糖基转移酶相关基因的功能	［39］
解淀粉芽胞杆菌	过表达bac基因簇，增强其对大肠杆菌和二叶乳杆菌的抗菌活性	［40］
共轭益生菌	构建COP系统高效消除小鼠肠道微生物群中>99.9%的靶向抗生素耐药大肠杆菌	［41］

菌株 Strain	改造后效果 Effect after transformation	参考文献 Reference
酿酒酵母	建立了莽草酸合成途径	［33］
多种梭菌属微生物	引入特定代谢路径，使菌株能够利用CO、CO₂及废弃甘油等生产生物燃料	［34］
保加利亚乳酸乳杆菌	编辑了保加利亚乳杆菌的ldh基因，并观察到其对酸奶特性的显著影响	［35］
双歧杆菌	编辑abfa基因簇，探索其影响宿主利用阿拉伯糖及肠道运动的机制	［36］
酵母菌	进行基因突变，成功构建乙醇耐受型酵母菌株	［37］
枯草芽胞杆菌	利用多轮基因编辑并结合发酵条件优化，赋予菌株利用木糖新功能并实现丰原素产量提高	［38］
副干酪乳杆菌	建立了高效编辑工具，验证尿嘧啶磷酸核糖基转移酶相关基因的功能	［39］
解淀粉芽胞杆菌	过表达bac基因簇，增强其对大肠杆菌和二叶乳杆菌的抗菌活性	［40］
共轭益生菌	构建COP系统高效消除小鼠肠道微生物群中>99.9%的靶向抗生素耐药大肠杆菌	［41］

菌株 Strain	改造后效果 Effect after transformation	参考文献 Reference
双歧杆菌	恢复菌株对四环素的敏感性	［42］
乳酸乳球菌	探究噬菌体侵染与细菌蛋白质组分间的关联	［43］
干酪乳杆菌	探究干酪乳杆菌中PyrR基因对嘧啶生物合成的调控作用及其对菌株抗菌能力等特性的影响	［45］
乳酸乳球菌	开发了双质粒转录抑制系统，鉴定llnz_07335基因编码的胆盐水解酶为乳酸乳球菌胆盐耐受的关键因子	［46］
鼠李糖乳杆菌	Muc2基因沉默可降低益生菌抑制大肠杆菌黏附和侵袭的能力	［31］
鼠李糖乳杆菌	丰富了基因表达工具	［47］
谷氨酸棒杆菌	证明柠檬酸合成酶基因glt A与L-赖氨酸产量提升有关	［48］
解脂耶氏酵母菌	激活β-葡萄糖苷酶基因表达	［49］
副干酪乳杆菌	利用内源性CRISPR-Cas9编辑技术研究发酵方式改变副干酪乳杆菌氧化应激耐受性的机制	［50］

菌株 Strain	改造后效果 Effect after transformation	参考文献 Reference
双歧杆菌	恢复菌株对四环素的敏感性	［42］
乳酸乳球菌	探究噬菌体侵染与细菌蛋白质组分间的关联	［43］
干酪乳杆菌	探究干酪乳杆菌中PyrR基因对嘧啶生物合成的调控作用及其对菌株抗菌能力等特性的影响	［45］
乳酸乳球菌	开发了双质粒转录抑制系统，鉴定llnz_07335基因编码的胆盐水解酶为乳酸乳球菌胆盐耐受的关键因子	［46］
鼠李糖乳杆菌	Muc2基因沉默可降低益生菌抑制大肠杆菌黏附和侵袭的能力	［31］
鼠李糖乳杆菌	丰富了基因表达工具	［47］
谷氨酸棒杆菌	证明柠檬酸合成酶基因glt A与L-赖氨酸产量提升有关	［48］
解脂耶氏酵母菌	激活β-葡萄糖苷酶基因表达	［49］
副干酪乳杆菌	利用内源性CRISPR-Cas9编辑技术研究发酵方式改变副干酪乳杆菌氧化应激耐受性的机制	［50］

菌株 Strain	改造后效果 Effect after transformation	参考文献 Reference
鼠李糖乳杆菌	利用益生菌作为载体，利用CRISPR/Cas9编辑工具在超声激活下实现对肿瘤免疫抑制相关基因的精确编辑	［51］
干酪乳杆菌	构建了重组干酪乳杆菌作为抗CRC疫苗	［52］
共轭益生菌	通过CRISPR/Cas9靶向切割耐药菌的耐药基因	［41］
干酪乳杆菌	成功表达猪IFN-λ，发挥抗病毒感染的作用	［53］
干酪乳杆菌	表达红色荧光蛋白	［54］
乳酸乳球菌	表达绿色荧光蛋白	［55］
产马乳酒乳杆菌	表达绿色荧光蛋白	［56］