转座子编辑技术与CRISPR编辑技术在乳酸菌定向改良中应用的研究进展

Research Progress of Application of Transposon Editing Technique and CRISPR Editing Technique in Directional Improvement of Lactic Acid Bacteria

乳酸菌具有降胆固醇、维持菌群平衡、增强免疫等多种益生特性,是一类重要的食品工业微生物,而菌种改良经常用于改善工业上重要的微生物性能和功能。概述了传统改良、转座子诱变和CRISPR编辑3种乳酸菌改良技术的特点与应用。传统的乳酸菌改良技术包括适应性进化、随机诱变和原生质体融合,这些技术虽能改良乳酸菌,但操作复杂且收效低;转座子诱变是基于阻断转录或翻译过程而导致基因失活的一项技术,常被用于乳酸菌突变体库的构建,该技术高效简便,但依赖DNA链断裂、安全性低、不适用于广泛的宿主特性;CRISPR编辑技术在乳酸菌改良中应用广泛,可实现多位点同时编辑,是基因沉默和转录调控的强大工具,但该技术依赖DNA链断裂和同源重组,脱靶率较高。阐述了目前新出现的CRISPR相关转座子编辑系统的构建进行。CRISPR相关转座子编辑系统可实现供体DNA大片段(>10 kbp)的高效、精准、无标记编辑,且不依赖DNA双链断裂和同源重组,具有极低的脱靶率,在精准靶向和高效整合技术中显示出了极大优势,为未来乳酸菌精准高效的基因编辑技术提供了参考。希望可以为后续的乳酸菌遗传改良与基因功能分析提供相关理论指导。

With a variety of probiotic properties such as lowering cholesterol,maintaining flora balance and enhancing immunity,lactic acid bacteria(LAB)is an important microorganism in the food industry.Strain development is frequently used to improve the performance and functionality of industrially important microbes.A brief overview of the characteristics and applications of three improved techniques of LAB,including traditional improvement,transposon mutagenesis and CRISPR was summaried.Traditional techniques for LAB improvement include adaptive evolution,random mutagenesis and protoplast fusion,which can improve LAB but are complex and ineffective.Transposon mutagenesis,a technique based on the inactivation of genes by blocking transcriptional or translational processes,is often used in the construction of LAB mutant libraries,which is efficient and simple,but is dependent on DNA strand breaks,with a low safety profile and being not applicable to a wide range of host characteristics.CRISPR editing technology is widely used in LAB,which enables simultaneous editing of multiple loci and is a powerful tool for gene silencing and transcriptional regulation,but the technique relies on DNA strand breaks and homologous recombination and has a high off-target rate.In addition,the construction and perspective of currently emerging CRISP R-associated transposon(CAST)editing systems are also discussed.The CAST enables efficient,accuracy,marker-free insertion of large fragments(>10 kbp)of donor DNA without relying on double strand break and homologous recombination,having an extremely low off-target rate,showing great advantages in high accuracy targeting and efficient integration techniques,providing a reference in high accuracy and efficient LAB gene editing in future.This review could provide relevant theoretical guidance for the subsequent genetic improvement and function analysis of LAB gene.