Developing a CRISPR‐assisted base‐editing system for genome engineering of Pseudomonas chlororaphis. Issue 9 (16th May 2022)

• Record Type: Journal Article
• Authors: Yue, Sheng‐Jie; Huang, Peng; Li, Song; Cai, Yu‐Yuan; Wang, Wei; Zhang, Xue‐Hong; Nikel, Pablo I.; Hu, Hong‐Bo
• Is Part Of: Microbial biotechnology. Volume 15:Issue 9(2022)
• Page Start: 2324
• Page End: 2336
• Publication Date: 2022-05-16
• Subjects: Microbial biotechnology -- Periodicals; Biotechnology; Microbiology; 660.62
• DOI: 10.1111/1751-7915.14075
• Languages: English
• ISSNs: 1751-7915

Abstract

Summary: Pseudomonas chlororaphis is a non‐pathogenic, plant growth‐promoting rhizobacterium that secretes phenazine compounds with broad‐spectrum antibiotic activity. Currently available genome‐editing methods for P. chlororaphis are based on homologous recombination (HR)‐dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ‐Red–like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time‐consuming, laborious and inefficient. Here, we established a CRISPR‐assisted base‐editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced‐specificity Cas9 nickase (eSpCas9pp D10A ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P . chlororaphis were successfully interrupted at high efficiency. The phzO ‐inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR‐based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non‐traditional bacterial hosts. Abstract : In this study, we established a CRISPR‐assisted base‐editing (CBE) system for Pseudomonas chlororaphis . This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted with high efficiency.