{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T00:03:43Z","timestamp":1773014623013,"version":"3.50.1"},"reference-count":28,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,11,6]],"date-time":"2021-11-06T00:00:00Z","timestamp":1636156800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["PTDC\/BIA-MIC\/28779\/2017"],"award-info":[{"award-number":["PTDC\/BIA-MIC\/28779\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological\/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve\/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness\/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.<\/jats:p>","DOI":"10.3390\/life11111198","type":"journal-article","created":{"date-parts":[[2021,11,7]],"date-time":"2021-11-07T20:41:14Z","timestamp":1636317674000},"page":"1198","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803"],"prefix":"10.3390","volume":"11","author":[{"given":"Marina","family":"Santos","sequence":"first","affiliation":[{"name":"i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4000-008 Porto, Portugal"},{"name":"IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4000-008 Porto, Portugal"},{"name":"Programa Doutoral em Biologia Molecular e Celular (MCbiology), Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar (ICBAS), Universidade do Porto, 4000-008 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0741-1529","authenticated-orcid":false,"given":"Catarina C.","family":"Pacheco","sequence":"additional","affiliation":[{"name":"i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4000-008 Porto, Portugal"},{"name":"IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4000-008 Porto, Portugal"}]},{"given":"Lun","family":"Yao","sequence":"additional","affiliation":[{"name":"Science for Life Laboratory, KTH Royal Institute of Technology, 10004 Stockholm, Sweden"},{"name":"Department of Protein Science, KTH Royal Institute of Technology, 10004 Stockholm, Sweden"}]},{"given":"Elton P.","family":"Hudson","sequence":"additional","affiliation":[{"name":"Science for Life Laboratory, KTH Royal Institute of Technology, 10004 Stockholm, Sweden"},{"name":"Department of Protein Science, KTH Royal Institute of Technology, 10004 Stockholm, Sweden"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4396-2122","authenticated-orcid":false,"given":"Paula","family":"Tamagnini","sequence":"additional","affiliation":[{"name":"i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4000-008 Porto, Portugal"},{"name":"IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4000-008 Porto, Portugal"},{"name":"Departamento de Biologia, Faculdade de Ci\u00eancias, Universidade do Porto, 4000-008 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1111\/j.1574-6976.2009.00183.x","article-title":"Complexity of cyanobacterial exopolysaccharides: Composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly","volume":"33","author":"Pereira","year":"2009","journal-title":"FEMS Microbiol. 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