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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,8,12]]},"abstract":"<jats:p>\n                    Compact programmable nucleases provide versatile genome editing tools with therapeutic potential, particularly when delivered via adeno-associated virus (AAV) vectors. However, their limited editing efficacy and stringent protospaceradjacent motif (PAM) requirements impose significant limitations in practical application. Here, we engineered MiniCasUltra, an optimized Un1Cas12f1 variant, through rational mutagenesis. MiniCasUltra exhibits sixfold higher editing activity than Un1Cas12f1, minimal off-target effects (on\/off-target ratio &gt; 10), and an expanded PAM preference (5\u2032-WBTR). Using a single AAV vector encoding MiniCasUltra and two single-guide RNAs, we achieved simultaneous editing of two disease-causing genes (\n                    <jats:italic toggle=\"yes\">Pten<\/jats:italic>\n                    and\n                    <jats:italic toggle=\"yes\">Fah<\/jats:italic>\n                    ) in mouse liver, with indel rates of 15.82% and 29.39%, respectively- significantly surpassing CasMINI V3.1 (3.45% and 10.98%). Furthermore, AAV delivery of MiniCasUltra targeting a noncanonical 5\u2032-TCTG PAM site in human vascularendothelial growth factor A reduced choroidal neovascularization (CNV) lesions in a laser-induced CNV mouse model of neovascular age-related macular degeneration, a leading global cause of blindness. The broad and effective targeting capabilities of MiniCasUltra, coupled with its compact size, highlight its potential for in vivo genome editing and therapeutic interventions.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2501292122","type":"journal-article","created":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T16:26:02Z","timestamp":1754411162000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":9,"title":["Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8978-0864","authenticated-orcid":false,"given":"Li","family":"Chen","sequence":"first","affiliation":[{"name":"College of Life Sciences and Oceanography, Shenzhen University","place":["Shenzhen, China"]},{"name":"College of Physics and Optoelectronic Engineering, Shenzhen University","place":["Shenzhen, China"]},{"name":"Department of Ophthalmology, Shanghai Eye, Ear, Nose and Throat Hospital, State Key Laboratory of Brain Function and Disorders, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xujiao","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Shanghai Eye, Ear, Nose and Throat Hospital, State Key Laboratory of Brain Function and Disorders, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengsi","family":"Huang","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Oceanography, Shenzhen University","place":["Shenzhen, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yirou","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Shanghai Eye, Ear, Nose and Throat Hospital, State Key Laboratory of Brain Function and Disorders, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changchang","family":"Xin","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Shanghai Eye, Ear, Nose and Throat Hospital, State Key Laboratory of Brain Function and Disorders, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxu","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Shanghai Eye, Ear, Nose and Throat Hospital, State Key Laboratory of Brain Function and Disorders, Fudan University","place":["Shanghai, China"]},{"name":"State Key Laboratory of Medical Neurobiology, Fudan University","place":["Shanghai, China"]},{"name":"National Health Commission Key laboratory of Myopia and Related Eye Diseases","place":["Shanghai, China"]},{"name":"Shanghai Key Laboratory of Rare Disease Gene Editing and Cell Therapy; Shanghai Engineering Research Center of Synthetic Immunology","place":["Shanghai, China"]},{"name":"Department of Ophthalmology, Children\u2019s Hospital of Fudan University, National Pediatric Medical Center of China","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5188-4622","authenticated-orcid":false,"given":"Yu","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Oceanography, Shenzhen University","place":["Shenzhen, China"]},{"name":"Shanghai Key Laboratory of Rare Disease Gene Editing and Cell Therapy; Shanghai Engineering Research Center of Synthetic Immunology","place":["Shanghai, China"]},{"name":"State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences","place":["Shenzhen, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,8,5]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.09.038"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature14299"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-019-0012-9"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0209-1"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav4294"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa208"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-023-01857-x"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05933-9"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05826-x"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-04058-1"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-01898-9"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.abj6856"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2021.08.008"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-01009-z"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-021-00868-6"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.08.031"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-023-01380-9"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-023-00995-4"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-37829-7"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-023-01420-4"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.11.035"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab179"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1002\/2211-5463.13198"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-33346-1"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu048"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3117"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2021.101088"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2009.246"},{"key":"e_1_3_4_31_2","first-page":"375","article-title":"Clinical chemistry reference intervals for C57BL\/6J, C57BL\/6N, and C3HeB\/FeJ mice (Mus musculus)","volume":"55","author":"Otto G. 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NCBI Sequence Read Archive. https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA1121205. Deposited 7 June 2024."}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2501292122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T16:26:07Z","timestamp":1754411167000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2501292122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,5]]},"references-count":42,"journal-issue":{"issue":"32","published-print":{"date-parts":[[2025,8,12]]}},"alternative-id":["10.1073\/pnas.2501292122"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2501292122","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,5]]},"assertion":[{"value":"2025-01-24","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-06-20","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-08-05","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2501292122"}}