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Despite some recent progress, the lack of well-established genetic manipulation methods for diverse research applications remains a challenge. We recently reported the establishment of targeted mutagenesis to disrupt genes in\n                      <jats:italic toggle=\"yes\">Ehrlichia<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Anaplasma<\/jats:italic>\n                      species. Many essential genes of the obligate pathogens, such as\n                      <jats:italic toggle=\"yes\">Ehrlichia chaffeensis<\/jats:italic>\n                      , are likely refractory to targeted mutagenesis; thus, we developed a novel targeted mutational approach leading to expression of an antisense RNA facilitating the knockdown of p28-Omp19 protein expression from\n                      <jats:italic toggle=\"yes\">ECH_1143<\/jats:italic>\n                      . This gene was selected as its encoded protein is among the highly immunogenic proteins of\n                      <jats:italic toggle=\"yes\">E. chaffeensis<\/jats:italic>\n                      , and our prior efforts to generate a loss-of-function mutation were unsuccessful. This method involved introducing a mutation at a distal genomic location within the\n                      <jats:italic toggle=\"yes\">E. chaffeensis<\/jats:italic>\n                      genome, allowing generating a 209-nucleotide-long antisense RNA that is complementary to\n                      <jats:italic toggle=\"yes\">ECH_1143<\/jats:italic>\n                      sense mRNA expressed from the same gene promoter, duplicated in the genome during mutagenesis. The mutational strategy was designed to retain the surrounding genomic regions unaltered. The antisense knockdown mutation resulted in a reduction of p28-Omp19 expression relative to wild-type\n                      <jats:italic toggle=\"yes\">E. chaffeensis<\/jats:italic>\n                      during its replication in a macrophage cell line, where the gene expression is known to occur, and the mutation caused a fitness defect. We anticipate that this novel mutational strategy will be broadly applicable to facilitate investigating essential genes of obligate intracellular bacterial pathogens.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      We developed an innovative targeted mutagenesis approach in the obligate intracellular bacterium\n                      <jats:italic toggle=\"yes\">Ehrlichia chaffeensis<\/jats:italic>\n                      to express specific antisense RNA (asRNA) to knockdown protein synthesis from a gene. The mutational strategy was carefully designed to retain the genomic sequences surrounding the mutational site unaltered. Our data demonstrate that this asRNA system effectively reduces protein synthesis by the unique mutational strategy and is ideal for investigating genes critical for the bacterial replication\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . This innovative molecular tool facilitates research in investigating essential gene research in\n                      <jats:italic toggle=\"yes\">E. chaffeensis<\/jats:italic>\n                      and holds strong potential to extend similar research on other bacterial pathogens having reduced genomes.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00334-26","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T18:13:28Z","timestamp":1785867208000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A targeted mutational strategy aiding in generating antisense RNA to knockdown the\n                    <i>Ehrlichia chaffeensis<\/i>\n                    P28-outer membrane protein 19 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