Lowering whole cost for sugarcane‐based ethanol production by engineered <i>Zymomonas mobilis</i>

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knock-out by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene was may attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence several strategies are proposed to enhance gene knockout and plasmid curing.

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Lowering whole cost for sugarcane‐based ethanol production by engineered Zymomonas mobilis

Abstract: This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 3 publications

References 54 publications

Expanding the horizons of levan: from microbial biosynthesis to applications and advanced detection methods

Expanding the horizons of levan: from microbial biosynthesis to applications and advanced detection methods

Sugar fermentation: C2 (ethanolic) platform

The escape of CRISPR-mediated gene editing inZymomonas mobilis

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