The escape of CRISPR-mediated gene editing inZymomonas mobilis

Abstract

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.

Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation

“…Consequently, we successfully constructed deleting strains Δ zur and Δ zur/fur in ZM532 ( Supplementary Figure S6B ). However, it is worth noting that our attempts to obtain a pure Δ fur deletion strain were unsuccessful ( 54 ). Editing of the fur gene in the wild-type strain resulted in the transfer of the target gene to the editing plasmid, the editing plasmid could not be cured ( 54 ).…”

“…However, it is worth noting that our attempts to obtain a pure Δ fur deletion strain were unsuccessful ( 54 ). Editing of the fur gene in the wild-type strain resulted in the transfer of the target gene to the editing plasmid, the editing plasmid could not be cured ( 54 ). The escape gene in the editing plasmid remained capable of expression ( Supplementary Figure S6C ).…”