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Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3–4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.

Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli