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

Abstract:

[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 pTarget plasmid in traditional CRISPR method was modified to an RK2ts-type plasmid with better temperature sensitivity. Two plasmids with different resistance markers (pTW-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, RK2ts-type plasmid was eliminated more easily than pSC101ts-type plasmid, and could selectively eliminate pTW-A/S plasmid. Moreover, it eliminated pTW-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 BspanD and aspA 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 pTarget 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.