Abstract
As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.
Highlights
The practicability of targeted gene editing is important for understanding the biological functions of genes
We isolated a RepA mutant (RepAA56V), namely pCasM (Figures 2A,B), which both supported the culture of bacteria at 37◦C and facilitated the rapid elimination of RepAA56V plasmids in the absence of antibiotics (Figure 2C)
This mutant was employed for genome editing in this study and may be used in other applications that require final elimination of the plasmids
Summary
The practicability of targeted gene editing is important for understanding the biological functions of genes. Several homology-directed repair (HDR)-based genetic modification technologies have been developed in the model microorganism, Escherichia coli, and two-step strategies including a negative selection step make it possible to perform markerless genome editing (Datsenko and Wanner, 2000; Tischer et al, 2006; Peters et al, 2013; Wang et al, 2014). A large number of toxic genes have been verified to work well as negative selection markers in E. coli, including sacB, ccdB and codAB (Yu et al, 2008; Kostner et al, 2013; Wang et al, 2014). A general and accessible method for the modification of genomic sequences of interest in bacteria would be of great value in multiple applications such as metabolic engineering
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