{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T04:09:09Z","timestamp":1788581349021,"version":"build-2803163510"},"reference-count":69,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"17","funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["MCB # 2225776"],"award-info":[{"award-number":["MCB # 2225776"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["MCB CAREER award #2441696"],"award-info":[{"award-number":["MCB CAREER award #2441696"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"name":"BioDiscovery Institute","award":["Seed Funding"],"award-info":[{"award-number":["Seed Funding"]}]},{"name":"U.S. Department of Energy\/MSI STEM Research & Development Consortium","award":["#W911SR-14-2-0001"],"award-info":[{"award-number":["#W911SR-14-2-0001"]}]},{"name":"BioDiscovery Institute","award":["Seed Funding"],"award-info":[{"award-number":["Seed Funding"]}]}],"content-domain":{"domain":["www.science.org"],"crossmark-restriction":true},"short-container-title":["Sci. Adv."],"published-print":{"date-parts":[[2026,4,24]]},"abstract":"<jats:p>\n                    Methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH\n                    <jats:sub>4<\/jats:sub>\n                    ) and have potential to mitigate climate-active gases. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a CRISPR interference (CRISPRi) system to enable functional genomic screening in methanotrophic bacteria. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph,\n                    <jats:italic toggle=\"yes\">Methylococcus capsulatus<\/jats:italic>\n                    , consisting of ~45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH\n                    <jats:sub>4<\/jats:sub>\n                    identified genes associated with essential methanotrophic bacterial processes and previously unidentified essential gene candidates, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs. The CRISPRi screen also led to nitrate mineral salts medium optimization to improve methanotroph biomass productivity from CH\n                    <jats:sub>4<\/jats:sub>\n                    . Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify genetic determinants of methanotrophy and isolation of improved methanotroph biocatalysts.\n                  <\/jats:p>","DOI":"10.1126\/sciadv.aed4234","type":"journal-article","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T17:58:32Z","timestamp":1777053512000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":2,"title":["CRISPRi-based functional genomic screening identifies genes essential for CH\n                    <sub>4<\/sub>\n                    -dependent growth in a methanotrophic bacterium"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-6736-2083","authenticated-orcid":true,"given":"Jessica M.","family":"Henard","sequence":"first","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Spencer A.","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4227-3180","authenticated-orcid":true,"given":"Yao-Chuan","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1494-5434","authenticated-orcid":true,"given":"Danyang","family":"Shao","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rajeev K.","family":"Azad","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4574-587X","authenticated-orcid":true,"given":"Calvin A.","family":"Henard","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.678057"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a041671"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.3c00727"},{"key":"e_1_3_2_5_2","doi-asserted-by":"crossref","unstructured":"M. G. Kalyuzhnaya O. A. Gomez J. C. Murrell in Taxonomy Genomics and Ecophysiology of Hydrocarbon-Degrading Microbes T. J. McGenity Ed. 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