CRISPRi-based functional genomic screening identifies genes essential for CH ₄ -dependent growth in the methanotroph Methylococcus capsulatus Bath

Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH ₄ ) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a CRISPR interference (CRISPRi) system to enable functional genomic screening in methanotrophic bacteria. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus , consisting of ∼45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH ₄ identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs. The CRISPRi screen uncovered that 50 ppb Co ²⁺ in methanotroph Nitrate Mineral Salts medium is growth inhibitory and decreasing Co ²⁺ to < 10 ppb improved biomass productivity from CH ₄ . Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy and isolation of improved methanotroph biocatalysts.