一种大肠杆菌快速连续CRISPR基因组编辑的新策略

Novel efficient strategy for continual CRISPR-mediated genome editing in Escherichia coli

【背景】通过CRISPR结合λ-Red同源重组技术进行染色体编辑是大肠杆菌遗传改造的重要手段。虽然目前已经有多个以CRISPR为基础的大肠杆菌基因组编辑策略,但这些方法往往涉及单独质粒消除、多片段组装等过程,存在效率低、操作繁琐、耗时长等问题。【目的】建立一种基于不同温度敏感程度的多种质粒协同使用的大肠杆菌快速、连续、高效的CRISPR基因组编辑方法,提高CRISPR在大肠杆菌基因编辑过程中的效率。【方法】将传统CRISPR方法中使用的pTarget质粒改造为RK2ts型温敏型质粒,并采用pTW-A/S两种抗性标记质粒交替使用的方法消除假阳性,实现质粒消除与下一步基因组编辑同步进行。【结果】在相同温度下RK2ts型质粒先于pSC101ts型质粒发生丢失,从而能够选择性缺失RK2ts型质粒pTW-A/S。同时实现pTW-A/S质粒的消除和下一轮基因整合过程中质粒与打靶片段的转入。利用该方法基因敲除/整合效率高达100%。通过对菌株BP01基因Bspan D与asp A进行高效连续整合,构建得到菌株BP03,成功提升产物β-丙氨酸的产量。【结论】建立起一种新的高效、方便、灵活的CRISPR/Cas9介导的大肠杆菌基因组连续编辑策略。通过这种不同温度敏感程度的多种质粒协同使用方法,一方面解决了传统方法中质粒消除繁琐等问题,另一方面也避免了大质粒多片段连接等步骤,并极大地缩短了实验周期,为代谢工程菌株改造提供有力工具。

[Background] Genome editing method coupling the CRISPR（Clustered regularly interspaced short palindromic repeats） system with λ-Red recombination technology has become an important access to Escherichia coli genomic editing. Recently, there have been several genomic editing strategies in E. coli based on CRISPR system. However, these methods often require many processes, such as the elimination of single plasmid or multi-fragment assembly, and are still inefficient, tedious and time-consuming. [Objective] Establish a fast, continuous and efficient CRISPR genome editing method based on several different temperature-sensitive plasmids in E. coli, and improve the editing efficiency of CRISPR in E. coli. [Methods] The p Target plasmid in traditional CRISPR method was modified to an RK2 ts-type plasmid with better temperature sensitivity. Two plasmids with different resistance markers（p TW-A/S） were constructed and used alternately to eliminate false positives. And this realized the elimination of plasmids and next round of gene integration simultaneously. [Results] At the same temperature, RK2 ts-type plasmid was eliminated more easily than p SC101 ts-type plasmid, and could selectively eliminate p TW-A/S plasmid. Moreover, it eliminated p TW-A/S plasmid and transformed other plasmids and target fragments during next round of gene integration simultaneously. The efficiency of gene knock-out/integration reached 100%. Then, we used this method to construct BP03 by modifying gene Bspan D and asp A on strain BP01 continuously, leading to increased production of β-alanine successfully. [Conclusion] A novel, efficient, convenient, continuous and flexible CRISPR/Cas9-mediated genome editing strategy has been established in E. coli. This coordinate use of multiple temperature-sensitive plasmids method solves the problem of incomplete elimination of p Target plasmids in traditional CRISPR systems, and it also avoids the low connection efficiency in process of large plasmids construction. Therefore, it shortens the experimental hours and provides a powerful tool for the construction of metabolic engineering strains.