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is an oncobacterium associated with colorectal cancer (CRC) development. Despite that () dominates the CRC niche in humans, studies on the pathogenesis of have been limited by the paucity of genetically tractable strains. Here, we constructed a conjugative DNA transfer system for genetic manipulation of clinical isolates. A mobilizable shuttle plasmid can be transferred by conjugation from to various strains, including 7_1, which has been considered genetically intractable using electroporation methods. The conjugation efficiency of 7_1 was further improved by ethanol treatment. Next, we manipulated the donor strain to express methyltransferases, which can protect plasmids from degradation by restriction systems in recipient strains. With this donor strain, we could isolate transconjugants of harboring chromosomally integrated plasmid. As a test case, we disrupted which is involved in cysteine metabolism and hydrogen sulfide (HS) production. The plasmid integrated into the locus was stably maintained without antibiotic selective pressure. The disruption in 7_1 significantly decreased the level of HS production in the presence of cysteine and resulted in lower cecal HS level in a mouse colonization model. The same conjugation-based system was successfully applied to another strain CTI-1, although this strain showed 10-fold lower conjugation efficiency compared to 7_1. Altogether, this work suggests that conjugation is an efficient method for genetic manipulation of strains, allowing us to mechanistically study their pathogenicity in CRC at the molecular level.IMPORTANCEAmong the species associated with colorectal cancer (CRC), () is the most clinically relevant species showing distinct genetic features. However, due to its genetic intractability, little is known experimentally about the molecular factors of contributing to CRC development. Here, we showed that foreign DNA can be transferred from to via conjugation. The conjugation system was developed by expressing -derived methyltransferases in the donor strain to transfer methylated plasmid to . Finally, the gene, encoding an enzyme responsible for hydrogen sulfide (HS) production, was successfully disrupted in strains. The effects of the disruption in on HS production were verified both and . This conjugation-based approach would be applied to not only but also a broader range of species, expanding our understanding of their virulence traits.