Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage

• 成果类型: Article
• 署名作者: Chen, Li; Zhou, Xujiao; Huang, Chengsi; Zhang, Yirou; Xin, Changchang; Hong, Jiaxu; Wang, Yu
• 署名单位: Shenzhen University; Shenzhen University; Fudan University; Fudan University; Fudan University; Chinese Academy of Sciences; Shenzhen Institute of Advanced Technology, CAS
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8703
• DOI: 10.1073/pnas.2501292122
• 发表日期: 2025-08-05
• 关键词: endonuclease; cleavage; dna

摘要

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 minimal off-target effects (on/off-target ratio > 10), and an expanded PAM preference (5 '-WBTR). Using a single AAV vector encoding MiniCasUltra and two single-guide RNAs, we achieved simultaneous editing of two disease-causing genes (Pten and Fah) 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 '-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.