{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T05:18:07Z","timestamp":1762233487089,"version":"build-2065373602"},"reference-count":45,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,11,2]],"date-time":"2025-11-02T00:00:00Z","timestamp":1762041600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2023YFD2400300"],"award-info":[{"award-number":["2023YFD2400300"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012428","name":"Central Public Interest Scientific Institution Basal Research Fund, CAFS","doi-asserted-by":"crossref","award":["2024XT01","2023TD45"],"award-info":[{"award-number":["2024XT01","2023TD45"]}],"id":[{"id":"10.13039\/501100012428","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Heilongjiang Province Seed Industry Innovation and Development Fund Project"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5\u2032-SNAAAG-3\u2032, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 \u00b0C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 \u00b0C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.<\/jats:p>","DOI":"10.3390\/ijms262110681","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:15:14Z","timestamp":1762175714000},"page":"10681","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum"],"prefix":"10.3390","volume":"26","author":[{"given":"Ran","family":"Zhao","sequence":"first","affiliation":[{"name":"Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianqiang","family":"Zhu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Di","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2797-6859","authenticated-orcid":false,"given":"Xinting","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Bioinformatics, School of Science, Xi\u2019an Jiaotong-Liverpool University, Suzhou 215123, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-8050-1882","authenticated-orcid":false,"given":"Lanlan","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Bioinformatics, School of Science, Xi\u2019an Jiaotong-Liverpool University, Suzhou 215123, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1682-7830","authenticated-orcid":false,"given":"Shaowu","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"},{"name":"National and Local Joint Engineering Laboratory for Freshwater Fish Breeding, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1843","DOI":"10.1126\/science.1165771","article-title":"CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA","volume":"322","author":"Marraffini","year":"2008","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1038\/nature15386","article-title":"CRISPR-Cas immunity in prokaryotes","volume":"526","author":"Marraffini","year":"2015","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1089\/hum.2015.091","article-title":"The Bacterial Origins of the CRISPR Genome-Editing Revolution","volume":"26","author":"Sontheimer","year":"2015","journal-title":"Hum. 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