(326f) Genome-Scale CRISPR Interference Screening in Rhodococcus Ruber

Rhodococcus species are gram-positive bacteria known for their high tolerance to organic solvents and versatile metabolic capabilities, making them invaluable in biocatalysis, biosynthesis, and bioremediation. Understanding the genotype-phenotype relationships in these bacteria is crucial for optimizing their biocatalytic applications, yet high-throughput investigative methods have been scarce. In this study, we developed a genome-scale CRISPR interference (CRISPRi) screening technique combined with next-generation sequencing (NGS) in Rhodococcus ruber, an industrial strain for production of acrylamide from acrylonitrile. This approach allows for the rapid, high-throughput identification of novel genetic targets. Initially, we engineered a CRISPRi system in R. ruber by integrating the gene for dCas9 from Streptococcus pyogenes into the bacterial genome and expressing guide RNAs from a plasmid under a strong, constitutive promoter, which achieved up to 98.4% gene repression efficiency. We then constructed a comprehensive guide RNA library comprising 43,656 sequences that target 98.9% of all coding genes and 92.3% of noncoding genes in R. ruber, and introduced this library into strains containing the dCas9 system. Our CRISPRi screenings led to the discovery of a previously unidentified restriction enzyme in R. ruber, whose suppression increased transformation efficiency by 11 times. Additionally, we explored gene fitness under selective conditions with acrylamide and acrylonitrile, finding that inhibition of the genes guaB, encoding IMP dehydrogenase, and glpX, encoding fructose-1,6-bisphosphatase, significantly bolstered the resistance of R. ruber to these compounds. Overall, our CRISPRi screening method offers a powerful tool for uncovering unknown genetic targets to enhance the biotechnological utility of R. ruber.

Acknowledgements: This work was supported by the National Natural Science Foundation (No. 22208189; No. 22078173) of China and the National Key R&D Program of China (2018YFA0902200).