Abstract

CRISPR/Cas system has been successfully harnessed for programmable RNA-guided genome editing in many fields. But CRISPR-based genome engineering strategies have off-target cleaves, variable gRNA efficiency, protospacer-adjacent motif (PAM) specificity and other problems. These hinder us obtaining strains that are accurately edited, prolonging the time of strain construction and screening, costing more manpower and materials.

Here, we developed a CRISPR-based purification system “strainer”, which utilizes double stranded DNA breaks (DSBs) as a signal to start the transcription of gRNA targeting on the plasmid harboring inducible toxic gene SacB. Then, only the strain with DSBs and recombineering can survive in the media with sucrose, because SacB converts sucrose to levan that is toxic to E. coli, thereby improving the overall editing efficiency during the genome engineering process. We then applied “strainer” for an isopropanol high-producing strain construction efficiently. “Strainer” can be potentially used as an universal method for cell factory construction.

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