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Its genetic intractability, due to a rigid cell wall and lack of natural competence, has limited its development as a microbial chassis. To facilitate its genetic transformation, a hyperosmotic electroporation protocol was optimised using cell wall weakening agents, achieving efficiencies comparable to other recalcitrant bacilli. A comprehensive and reusable genetic tool was developed centred on a temperature\u2010sensitive\n                    <jats:italic>\n                      <jats:styled-content style=\"fixed-case\">E. coli<\/jats:styled-content>\n                      \u2013\n                      <jats:styled-content style=\"fixed-case\">S. clausii<\/jats:styled-content>\n                    <\/jats:italic>\n                    shuttle vector (pM4B522) specifically engineered for compatibility with Golden Gate assembly. The plasmid backbone includes a spectinomycin resistance marker and an integrated red fluorescent protein reporter for transformants selection. A removable AmilCP chromoprotein cassette streamlines the assembly process by enabling blue\/white screening in\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>E. coli<\/jats:italic>\n                    <\/jats:styled-content>\n                    . This plasmid, employing a two\u2010step pop\u2010in\/pop\u2010out integration strategy, has demonstrated high versatility for genome editing in both\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>S. clausii<\/jats:italic>\n                    <\/jats:styled-content>\n                    and\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>Bacillus subtilis<\/jats:italic>\n                    <\/jats:styled-content>\n                    as evidenced by its successful use in multiple cases: (i) sequential, markerless deletions of the non\u2010essential catabolic genes\n                    <jats:italic>xylA<\/jats:italic>\n                    and\n                    <jats:italic>lacA<\/jats:italic>\n                    in\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>S. clausii<\/jats:italic>\n                    <\/jats:styled-content>\n                    DSM 8716, with a success rate exceeding 60%; (ii) replacement of the\n                    <jats:italic>lacA<\/jats:italic>\n                    coding sequence with a\n                    <jats:italic>gfp<\/jats:italic>\n                    coding sequence, resulting in fluorescence induction in lactose\u2010supplemented medium; (iii) introduction of a single\u2010base substitution generating a premature stop codon in\n                    <jats:italic>lacA<\/jats:italic>\n                    , showcasing scar\u2010free point mutagenesis; and (iv) transfer of the system to\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>B. subtilis<\/jats:italic>\n                    <\/jats:styled-content>\n                    168, highlighting its broader applicability across Gram\u2010positive bacteria. Given the precision and scarless nature of these genetic modifications, this system holds strong potential for the development of next\u2010generation probiotics and synthetic biology applications.\n                  <\/jats:p>","DOI":"10.1111\/1751-7915.70287","type":"journal-article","created":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T17:40:26Z","timestamp":1770658826000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Tool for High\u2010Efficiency, Markerless and Iterative Genome Editing in\n                    <i>Shouchella clausii<\/i>"],"prefix":"10.1111","volume":"19","author":[{"given":"Claudia","family":"Cappella","sequence":"first","affiliation":[{"name":"Department of Biosciences, Biotechnologies and Environment University of Bari \u201cAldo Moro\u201d  Bari 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