CRISPR/Cas9技术在工业微生物中的应用

近年来，通过基因编辑技术对工业微生物底盘细胞改造从而获得的优良细胞工厂，促进了农业、医学、环境、能源等领域的可持续发展，提高了人民的生活水平。微生物底盘细胞的改造离不开基因编辑，作为现阶段主要的基因编辑技术，规律间隔成簇短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）/Cas9系统自被发现以来，依靠其低成本、高效率等编辑优点，被广泛用于工业微生物底盘细胞的改造。本文主要简述了以CRISPR/Cas9为基础而衍伸出的各种基因编辑技术，提出了常用的工业微生物对应底盘细胞的改造策略，以期为研究者在进行微生物底盘细胞改造时选择出合适的基因编辑方法。最后指出了CRISPR基因编辑技术面临的PAM位点的依赖性、脱靶效应和应用广泛性等问题。

Application of CRISPR/Cas9 Technology in Industrial Microorganisms

As a new interdisciplinary subject, microbial synthetic biology aims to transform existing microbial cells or create new microbial cells to provide them with specific physiological functions or the ability to produce target products. To achieve this process, efficient, rapid and accurate gene manipulation tools are important. Since its creation, CRISPR/Cas9 system has been widely used in synthetic biology in recent years due to its ability to accurately identify and cut specific DNA sequences, as well as its characteristics of simple operation, low cost, high efficiency and diverse functions. In this paper, various gene editing techniques derived from CRISPR/Cas9 and their specific applications in microbial synthetic biology are reviewed. At the same time, the commonly used industrial model microorganisms and the corresponding transformation methods of chassis cells are introduced. A variety of industrial microbial chassis cells exist in nature and have their own metabolic characteristics to produce suitable products. However, natural chassis cells also have problems such as intermediate metabolite toxicity, end-product feedback inhibition, and branch pathway, which lead to low target yield, or the host itself does not have the ability to produce the product. Therefore, it is necessary to modify chassis cells by gene editing method, so as to obtain more excellent strains. The advantages of the edited cells compared to the control in the synthesis of target products were listed, and the characteristics of each gene editing technology were summarized in order to select the appropriate gene editing methods for the researchers in the transformation of microbial chassis cells. Finally, the existing problems of CRISPR gene editing technology, such as PAM dependence, off-target effect and universality of application, are put forward.