CRISPR/Cas9编辑系统在微生物天然产物研究中的应用

Applications of the CRISPR/Cas9 editing system in the study of microbial natural products

微生物作为天然产物的巨大宝库,一直以来都是研究人员挖掘和开发新的活性化合物的重要来源。目前,利用基因编辑工具发现、生物合成和代谢调控天然产物的研究方法受到该领域研究者的广泛关注。CRISPR/Cas9遗传编辑系统以其独特的灵活靶向优势克服了其他遗传编辑方法常见的对序列同源或位点限制,简化了实验步骤,提高了实验效率,促进了天然产物研究领域的发展。本文主要介绍CRISPR/Cas9系统在微生物天然产物发现、生物合成和工程改造方面的应用,分别从CRISPR/Cas9系统的发展、天然产物生物合成基因簇的克隆和遗传编辑、天然产物结构衍生化和代谢调节、沉默天然产物基因簇的激活这几个方面阐述CRISPR/Cas9系统在微生物天然产物研究领域的优势。最后,针对CRISPR/Cas9系统无法克服的重组效率和宿主适应性问题提供了可行的解决思路。相信随着合成生物学和信息技术的发展,越来越多的与CRISPR/Cas9系统相关的遗传操作工具和方法会被开发,将不断推动天然产物领域的发展进步。

Microorganisms have consistently been a crucial source for researchers to explore and develop new natural products.Currently,research methods involving gene editing tools for the discovery,biosynthesis,and metabolic engineering of natural products have garnered broad attention in this field.However,traditional methods for gene editing usually rely on the recombination ability of the host or introduced proteins.It’s difficult to establish a general platform for all bacteria mainly because of their complicated genetic background.This genetic diversity often causes laborious experimental operations with low efficiency.The CRISPR/Cas9 gene editing system,with its unique and flexible targeting advantages,overcomes common limitations such as sequence homology or site constraint in other gene editing methods and thus is more likely to function in diverse bacteria species.This simplifies experimental procedures,enhances work efficiency,and promotes the development of natural product research.This article introduces the applications of the CRISPR/Cas9 system for the discovery,biosynthesis,and metabolic engineering of natural products in microorganisms.It covers the development of the CRISPR/Cas9 system,cloning and genetic editing of natural product biosynthetic gene clusters,structural derivatization and metabolic engineering of natural products,and the activation of silenced natural product biosynthetic gene clusters.These aspects highlight the advantages of the CRISPR/Cas9 system in the research of natural products with microorganisms.Finally,solutions are proposed for addressing challenges that the CRISPR/Cas9 system currently faces in overcoming low recombination efficiency and host adaptability issues.Especially the CRISPR/Cas12a system which has broadened applications of the CRISRP/Cas9 system by preferring different PAM sites.In addition to functions that CRISPR/Cas9 system has realized,its potent multiple targeting ability further enhances the efficiency of target editing.It is believed that with the development of synthetic biology and information technology,an increasing number of genetic manipulation tools and methods related to the CRISPR/Cas9 system will be developed,continually driving progress in the research of natural products.