{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T19:28:38Z","timestamp":1793388518693,"version":"4.5.0"},"reference-count":45,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T00:00:00Z","timestamp":1738627200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T00:00:00Z","timestamp":1738627200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100008119","name":"Norges Milj\u00f8- og Biovitenskapelige Universitet","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100008119","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,2,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:sec>\n            <jats:title\/>\n            <jats:p>\n              <jats:italic>Lactiplantibacillus plantarum,<\/jats:italic>\n              a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR\/Cas9-system for chromosomal gene insertion in\n              <jats:italic>L. plantarum<\/jats:italic>\n              . The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR\/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Genetic engineering of lactic acid bacteria, such as\n              <jats:italic>Lactiplantibacillus plantarum,<\/jats:italic>\n              has proven to be difficult. This study presents an inducible two-plasmid CRISPR\/Cas9-system for inserting genes into the chromosome of\n              <jats:italic>Lactiplantibacillus plantarum<\/jats:italic>\n              . Our system successfully knock-in four expression cassettes varying in length from ~800\u20131,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR\/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR\/Cas9 engineering of\n              <jats:italic>L. plantarum<\/jats:italic>\n              strains.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02025-24","type":"journal-article","created":{"date-parts":[[2025,1,16]],"date-time":"2025-01-16T14:03:48Z","timestamp":1737036228000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":13,"title":["CRISPR\/Cas9-mediated genomic insertion of functional genes into\n            <i>Lactiplantibacillus plantarum<\/i>\n            WCFS1"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-8566-5979","authenticated-orcid":false,"given":"Kamilla","family":"Wiull","sequence":"first","affiliation":[{"name":"Faculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences","place":["\u00c5s, Norway"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa K.","family":"Haugen","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences","place":["\u00c5s, Norway"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9220-8743","authenticated-orcid":true,"given":"Vincent G. 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