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It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H\n                      <jats:sub>2<\/jats:sub>\n                      ) and carbon dioxide (CO\n                      <jats:sub>2<\/jats:sub>\n                      ) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for\n                      <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                      would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the\n                      <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                      genome. Gene knockout assays were conducted on the\n                      <jats:italic toggle=\"yes\">adh<\/jats:italic>\n                      gene, responsible for encoding alcohol dehydrogenase, and the\n                      <jats:italic toggle=\"yes\">ldh<\/jats:italic>\n                      gene, encoding lactate dehydrogenase. Furthermore, the\n                      <jats:italic toggle=\"yes\">adhE<\/jats:italic>\n                      gene from\n                      <jats:italic toggle=\"yes\">Thermoanaerobacter ethanolicus<\/jats:italic>\n                      , which encodes a bifunctional aldehyde\/alcohol dehydrogenase enzyme, was successfully integrated into the\n                      <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                      genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in\n                      <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                      for gene editing.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Thermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium\n                        <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                        . The system is an important expansion to the genetic toolbox of\n                        <jats:italic toggle=\"yes\">T. kivui<\/jats:italic>\n                        , enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01170-25","type":"journal-article","created":{"date-parts":[[2025,9,8]],"date-time":"2025-09-08T13:00:10Z","timestamp":1757336410000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium\n                    <i>Thermoanaerobacter 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