{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T19:01:09Z","timestamp":1790103669797,"version":"4.0.1"},"reference-count":34,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Project of the Health Commission of Xi\u2019an City","award":["2025qn19"],"award-info":[{"award-number":["2025qn19"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31900121"],"award-info":[{"award-number":["31900121"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["31900121"],"award-info":[{"award-number":["31900121"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017596","name":"Natural Science Basic Research Program of Shaanxi Province","doi-asserted-by":"publisher","award":["2024JC-YBQN-0196"],"award-info":[{"award-number":["2024JC-YBQN-0196"]}],"id":[{"id":"10.13039\/501100017596","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Species of Cronobacter are emerging foodborne pathogens that pose a significant threat to neonates. Functional genomic studies in Cronobacter have been hindered by the lack of efficient genetic manipulation tools. Here, we established a CRISPR\/Cas9-based genome editing platform for Cronobacter. We developed a dual-plasmid platform, pAmpCRISPR\/pCasCm, by integrating CRISPR\/Cas9 with the \u03bb-Red recombination machinery. This platform enables scarless genome editing in Cronobacter malonaticus and proved effective for gene knockout and large-fragment integration under the tested conditions. Furthermore, we validated this platform in another clinically relevant species, Cronobacter sakazakii. The development of this genome editing toolkit provides a useful approach for fundamental research into Cronobacter pathogenesis, such as bacterial physiology studies, drug target exploration, and metabolic engineering.<\/jats:p>","DOI":"10.3390\/pathogens15090990","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T17:56:41Z","timestamp":1790099801000},"page":"990","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Dual-Plasmid CRISPR\/Cas9-Based Genome Editing System for Efficient Gene Knockout and Large DNA Integration in Cronobacter malonaticus"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7653-8810","authenticated-orcid":false,"given":"Shuwei","family":"Yan","sequence":"first","affiliation":[{"name":"Department of Laboratory, Xi\u2019an No.3 Hospital, the Affiliated Hospital of Northwest University, Xi\u2019an 710018, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"conceptualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6827-8532","authenticated-orcid":false,"given":"Jing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"given":"Yufei","family":"Han","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"},{"name":"School of Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6084-5133","authenticated-orcid":false,"given":"Jia","family":"Jia","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"},{"name":"School of Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Ge","family":"Yun","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"},{"name":"School of Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"supervision","vocabulary":"credit"}]},{"given":"Dan","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"},{"name":"School of Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Yue","family":"Ma","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"},{"name":"School of Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"}]},{"given":"Zhimeng","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Faculty of Life Science and Medicine, Northwest University, Xi\u2019an 710069, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"given":"Yuanyuan","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Medical Technology, Shaanxi University of Chinese Medicine, Xi Xian New Area, Xi\u2019an 712046, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"}]},{"given":"Haifeng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Laboratory, Xi\u2019an No.3 Hospital, the Affiliated Hospital of Northwest University, Xi\u2019an 710018, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Joseph, S., Desai, P., Ji, Y., Cummings, C.A., Shih, R., Degoricija, L., Rico, A., Brzoska, P., Hamby, S.E., and Masood, N. 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