CRISPRi loss-of-function mutations revealed through experimental evolution in Burkholderia cenocepacia
Year: 2026
Type: preprint
Abstract: Essential genes encode proteins required for cell growth and survival, which makes them ideal targets for controlling bacterial growth during infection or in industrial applications. A gene silencing approach, CRISPR interference (CRISPRi), enables transcriptional control of essential genes, allowing phenotypic characterization of knockdown mutants. We previously developed a CRISPRi system for the Gram-negative bacteria Burkholderia, consisting of a chromosomally encoded rhamnose-inducible dCas9 and a plasmid encoding constitutively expressed synthetic guide RNAs (sgRNAs) targeting essential genes. Despite the system's utility, the CRISPRi mutants often reverted to wild-type growth phenotypes, hindering their use in gene-to-function studies that require many generations. To understand the reasons for the CRISPRi mutants’ phenotypic reversions, we performed experimental evolution of 15 CRISPRi mutants under dCas9-inducing and non-inducing conditions, followed by quantitative gene expression analysis and whole-genome sequencing (WGS). While the CRISPRi machinery remained genetically stable in the CRISPRi-evolved mutants, reverse transcription quantitative PCR revealed a loss of CRISPRi repression in several mutants. Immunoblotting analysis further demonstrated that dCas9 expression was abolished, suggesting a defect in rhamnose uptake. One evolved CRISPRi mutant showed single-nucleotide polymorphisms (SNPs) affecting putative ABC sugar transporters. This work highlights the strengths and limitations of CRISPRi-based downregulation of essential genes for extended control of bacterial growth.
Source: Research Square
Institutions: University of Manitoba, Université de Montréal
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7169542476