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Notably,\n                      <jats:italic>Synechocystis<\/jats:italic>\n                      sp. strain PCC 6803 (\n                      <jats:italic>Synechocystis<\/jats:italic>\n                      ) has been engineered to produce isobutanol (IB) and 3-methyl-1-butanol (3M1B) via heterologous expression of \u03b1-ketoisovalerate decarboxylase (Kivd). Despite these advances, the achieved IB\/3M1B titers remain low. CRISPR interference (CRISPRi), an emerging tool for targeted gene repression, has demonstrated success in various cellular systems to enhance biochemical productivity.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      In this study, we developed a dCas12a-mediated CRISPRi system (CRISPRi-dCas12a) that effectively blocked the transcriptional initiation\/elongation of essential gene(s), resulting in up to 60% gene repression in\n                      <jats:italic>Synechocystis<\/jats:italic>\n                      . Subsequently, the CRISPRi-dCas12a system was successfully integrated into an IB\/3M1B producer strain, where it exhibited target gene repression under optimal cultivation conditions. To identify gene targets involved in metabolic pathways potentially limiting IB\/3M1B biosynthesis, we initially designed a CRISPR RNA (crRNA) library targeting fifteen individual gene(s), where repression of ten genes significantly increased IB\/3M1B production per cell. Moreover, a synergetic effect was observed on IB\/3M1B production by designing a single crRNA targeting multiple genes for simultaneous repression. A final strain HX106, featuring dual repression of\n                      <jats:italic>ppc<\/jats:italic>\n                      and\n                      <jats:italic>gltA<\/jats:italic>\n                      , both involved in the TCA cycle\n                      <jats:italic>,<\/jats:italic>\n                      resulted in 2.6-fold and 14.8-fold improvement in IB and 3M1B production per cell, respectively.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      Our findings underscore the effectiveness of the CRISPRi-dCas12a system in\n                      <jats:italic>Synechocystis<\/jats:italic>\n                      for identifying competing pathways and redirecting carbon flux to enhance IB\/3M1B production. Furthermore, this study established a solid groundwork for utilizing an expanded CRISPRi-crRNA library to undertake genome-wide exploration of potential competing pathways not only for IB\/3M1B biosynthesis but also for other diverse biofuels and biochemical production processes.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12934-025-02727-8","type":"journal-article","created":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T07:40:01Z","timestamp":1747122001000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["dCas12a-mediated CRISPR interference for multiplex gene repression in cyanobacteria for enhanced isobutanol and 3-methyl-1-butanol production"],"prefix":"10.1186","volume":"24","author":[{"given":"Hao","family":"Xie","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barbara","family":"Bourgade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karin","family":"Stensj\u00f6","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Lindblad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,5,13]]},"reference":[{"issue":"12","key":"2727_CR1","doi-asserted-by":"publisher","first-page":"1177","DOI":"10.1038\/nbt.1586","volume":"27","author":"S Atsumi","year":"2009","unstructured":"Atsumi S, Higashide W, Liao JC. 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