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Lipopolysaccharide (LPS) is an immunodominant and protective antigen for\n                    <jats:italic>Leptospira<\/jats:italic>\n                    , but its biosynthesis remains poorly understood. In this study, we employed CRISPR\/Cas9-non-homologous end-joining and CRISPR-Prime Editing to mutate key genes within the\n                    <jats:italic>rfb<\/jats:italic>\n                    locus of\n                    <jats:italic>L. interrogans<\/jats:italic>\n                    , including those involved in core oligosaccharide assembly, and the biogenesis, polymerization, and ligation of O-antigen. Mutants were successfully generated in LIC11312 (\n                    <jats:italic>waaF<\/jats:italic>\n                    , heptosyltransferase II) and LIC12137 (\n                    <jats:italic>wcaJ<\/jats:italic>\n                    , undecaprenyl-phosphate glycosyltransferase) but yielded only in-frame deletions suggesting their essentiality. Mutants were also successfully generated in LIC12143, a putative O-antigen polymerase, which exhibited truncated LPS that failed to induce acute leptospirosis in hamsters but retained the ability to colonize kidneys. Mutation of LIC_RS09320, an O-antigen ligase, did not display a change in LPS phenotype. Bacterins prepared with either control wild-type or LIC12143 mutant cells conferred complete homologous protection with sterile immunity, though failed to protect against heterologous challenge. These findings confirm LIC12143 as a functional O-antigen polymerase and underscore the challenges in generating knockout mutants to understand LPS biosynthesis in leptospires.\n                  <\/jats:p>","DOI":"10.1038\/s41598-026-43869-y","type":"journal-article","created":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T13:17:25Z","timestamp":1773407845000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase"],"prefix":"10.1038","volume":"16","author":[{"given":"Luis G. 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