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 methanotroph CRISPR interference (CRISPRi) system to enable functional genomic screening. 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, including the identification of novel essential genes. M. capsulatus growth was inhibited when the CRISPRi system was used to individually target genes identified in the screen, validating their essentiality for methanotrophic growth. 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. These CRISPRi screening methodologies can also be applied to high-throughput engineering approaches for isolation of improved methanotroph biocatalysts.