Increasing the efficiency of gene editing with CRISPR-Cas9 via concurrent expression of the Beta protein

Abstract

Escherichia coli has emerged as an important host for the production of biopharmaceuticals or other industrially relevant molecules. An efficient gene editing tool is indispensable for ensuring high production levels and optimal release of target products. However, in Escherichia coli, the CRISPR-Cas9 system has been shown to achieve gene modifications with relatively low frequency. Large-scale PCR screening is required, hindering the identification of positive clones. The beta protein, which weakly binds to single-stranded DNA but tightly associates with complementary strand annealing products, offers a promising solution to this issue. In the present study, we describe a targeted and continuous gene editing strategy for the Escherichia coli genome. This strategy involves the coexpression of the beta protein alongside the CRISPR-Cas9 system, enabling a variety of genome modifications such as gene deletion and insertion with an efficiency exceeding 80 %. The integrity of beta proteins is essential for the CRISPR-Cas9/Beta-based gene editing system. In this work, the deletion of either the N- or C-terminal domain significantly impaired system efficiency. Overall, our findings established the CRISPR-Cas9/Beta system as a suitable gene editing tool for various applications in Escherichia coli.