CRISPR/Cas9 Assisted Genome Editing Technique In Escherichia Coli

The CRISPR/Cas9 system is a powerful,revolutionary tool for genome editing.However,it is not without limitations.There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system,and off-target activity impedes its broader applications.To avoid these drawbacks,we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step(CAGO)genome editing technique which does not require the construction of a plasmid to express a specific gRNA.Instead,a universal N20 sequence with a very high targeting efficiency is inserted into the E.coli chromosome by homologous recombination,which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process.This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli.When applied to multi-locus editing,CAGO was able to modify one locus in two days with a near 100% editing efficiency.Further more,modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency.Finally,genome editing by CAGO only requiresa transformation procedure and the construction of a linear donor DNA cassette,which was further simplified by applying a modular design strategy.Although the technique was established in E.coli,it should be applicable to other organisms with only minor modifications.Genome editing for site-specific chromosome modification is one of the most significant techniques in biological research,although various techniques have been established,conventional techniques usually deal with one genomic locus at a time.Multiple genomic targets are often required to be modified to develop microbial cell factories.It is necessary to develop techniques able to edit multiple loci simultaneously with minimal labor andtime.However,transformation efficiency,recombination efficiency and construction of complex editing plasmid hindered multiplex editing techniques.In this work,we developed a CRISPR/Cas9 assisted multiplex genome editing(CMGE)technique in Escherichia coli.With this editing method,all functional parts were assembled into replicable plasmids,and stringent inducible expression systems were used to control Cas9 gene expression,which was to decouple transformation from editing process.A modular assembly strategy was designed to construct the complex multi-gRNA plasmid.Furthermore,the long presence period and multiple copies of plasmid based donor DNA increased editing efficiency.With this technique,two and three loci were able to be modified with 100% and 90% efficiency,while four loci could be edited with an average efficiency of 40%.These are the best results reported with multiplex genome editing technique in E.coli.Although developed in model organism,CMGE could be adapted to other prokaryotic hosts with some modification.This is a well-designed convenient technique with no special requirement,can be used by any biological lab easily.