A CRISPR Interference System for Inducible Gene Knockdown in soil bacterium Sinorhizobium meliloti

Abstract

Symbiotic relationships have an important role in most life forms, but the molecular and cellular processes that establish and maintain these harmonious interactions remain largely unknown. The relationship between leguminous plants and rhizobial bacteria is a classic example of symbiosis, where the bacterium converts atmospheric nitrogen to plant-usable ammonia in exchange for fixed carbon and nutrients. Some legumes such as Medicago truncatula has evolved a set of small peptides that exploit this relationship, turning its bacterial partner, Sinorhizobium meliloti, into a terminally differentiated bacterium that loses its capability to survive outside the host. However, the mechanisms of how this transformation happens remain elusive due to the absence of high-throughput tools for targeted gene knockdowns in the bacterium. To overcome these limitations in the plant-rhizobia field, we developed an inducible CRISPR-interference knockdown system which can reversibly block the transcription of a target gene through the combined action of a deactivated-Cas9 (dCas9) and single-guide RNAs (sgRNAs). We used a taurine-inducible promoter to achieve fine-tunable expression levels of dCas9 in free-living S. meliloti and demonstrated that this tool is suitable for the study of essential genes that could be involved in the symbiotic process, including hemH, dnaN and ctrA. Our cost-effective inducible CRISPRi strategy will contribute to understanding the molecular mechanisms underlying legume-rhizobia symbiosis, ultimately allowing soil improvement and reducing chemical fertilizers usage while meeting global food demands.