{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T12:55:19Z","timestamp":1791204919180,"version":"4.1.0"},"reference-count":28,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35 GM118061"],"award-info":[{"award-number":["R35 GM118061"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2026,9,29]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Targeted gene repression using CRISPRi has become a foundational technique for microbiology. The effector protein in most CRISPRi systems is a catalytically inactivated version of Cas9, an RNA-guided DNA endonuclease from\n                      <jats:italic toggle=\"yes\">Streptococcus pyogenes<\/jats:italic>\n                      (SpCas9). The application of SpCas9-based CRISPRi systems in increasingly ambitious experiments in diverse bacteria raises important questions about the effects of growth temperature on CRISPRi efficacy, particularly because SpCas9 loses cutting efficacy at elevated temperatures. Here, we test the effect of temperature on SpCas9\n                      <jats:italic toggle=\"yes\">-<\/jats:italic>\n                      based CRISPRi knockdown efficacy by performing a series of genome-wide screens using a well-characterized CRISPRi system in the model firmicute\n                      <jats:italic toggle=\"yes\">Bacillus subtilis<\/jats:italic>\n                      . We observe very little repression at high (45\u00b0C) growth temperatures and confirm this observation using single cell measurements of GFP fluorescence in CRISPRi strains grown at different temperatures. Our data suggest that\n                      <jats:italic toggle=\"yes\">Streptococcus pyogenes-<\/jats:italic>\n                      based CRISPRi systems are ill-suited for thermophiles and should not be used for elevated-temperature or heat shock experiments in mesophilic bacteria.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      CRISPRi is a common technique for inducible gene depletion in bacteria and has been used for many different types of experiments in increasingly diverse bacteria. We identify increased temperatures as an important limitation of the most common CRISPRi effector (\n                      <jats:italic toggle=\"yes\">Streptococcus pyogenes<\/jats:italic>\n                      dCas9). This finding both affects experimental design and informs the kinds of bacterial species amenable to manipulation by\n                      <jats:italic toggle=\"yes\">S. pyogenes<\/jats:italic>\n                      -based CRISPRi systems.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00379-26","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T13:00:38Z","timestamp":1787144438000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Elevated temperature undermines the efficacy of\n                    <i>Streptococcus pyogenes<\/i>\n                    dCas9-based CRISPRi systems"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3875-2542","authenticated-orcid":false,"given":"Lili M.","family":"Kim","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, University of California, San Francisco","place":["San Francisco, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Byoung-Mo","family":"Koo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, University of California, San Francisco","place":["San Francisco, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5595-9732","authenticated-orcid":true,"given":"Carol A.","family":"Gross","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, University of California, San Francisco","place":["San Francisco, USA"]},{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cell and Tissue Biology, University of California, San Francisco","place":["San Francisco, USA"]},{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"California Institute of Quantitative Biology, University of California, San Francisco","place":["San Francisco, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5556-6085","authenticated-orcid":true,"given":"Horia","family":"Todor","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043mz5j54","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, University of California, San Francisco","place":["San Francisco, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2020.11.005"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/mmbr.00170-22"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.02.022"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt520"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1159689"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.12.041"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2021.100521"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-04209-5"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa294"},{"key":"e_1_3_4_12_2","volume-title":"Bergey\u2019s manual of systematic bacteriology. 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R package version 2.24.0. https:\/\/doi.org\/10.18129\/B9.bioc.flowCore."}],"container-title":["mSphere"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00379-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00379-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T12:37:10Z","timestamp":1791203830000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/msphere.00379-26"}},"subtitle":[],"editor":[{"given":"Paul D.","family":"Fey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,29]]},"references-count":28,"journal-issue":{"issue":"9","published-print":{"date-parts":[[2026,9,29]]}},"alternative-id":["10.1128\/msphere.00379-26"],"URL":"https:\/\/doi.org\/10.1128\/msphere.00379-26","relation":{},"ISSN":["2379-5042"],"issn-type":[{"value":"2379-5042","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,29]]},"assertion":[{"value":"2026-05-26","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-28","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-19","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00379-26"}}