Chen Li, Zhou Xujiao, Huang Chengsi, Zhang Yirou, Xin Changchang, Hong Jiaxu, Wang Yu
College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China.
Proc Natl Acad Sci U S A. 2025 Aug 12;122(32):e2501292122. doi: 10.1073/pnas.2501292122. Epub 2025 Aug 5.
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 > 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 ( and ) 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.
紧凑型可编程核酸酶提供了具有治疗潜力的多功能基因组编辑工具，尤其是通过腺相关病毒（AAV）载体递送时。然而，它们有限的编辑效率和对原间隔相邻基序（PAM）的严格要求在实际应用中造成了重大限制。在此，我们通过合理诱变设计了MiniCasUltra，这是一种优化的Un1Cas12f1变体。MiniCasUltra的编辑活性比Un1Cas12f1高六倍，脱靶效应最小（on/off靶比>10），并且PAM偏好性有所扩展（5'-WBTR）。使用单个编码MiniCasUltra和两个单向导RNA的AAV载体，我们在小鼠肝脏中实现了对两个致病基因（和）的同时编辑，插入缺失率分别为15.82%和29.39%，显著超过了CasMINI V3.1（3.45%和10.98%）。此外，在新生血管性年龄相关性黄斑变性（全球主要致盲原因）的激光诱导脉络膜新生血管（CNV）小鼠模型中，以人血管内皮生长因子A中的非经典5'-TCTG PAM位点为靶点的MiniCasUltra的AAV递送减少了脉络膜新生血管（CNV）病变。MiniCasUltra广泛而有效的靶向能力，加上其紧凑的尺寸，突出了其在体内基因组编辑和治疗干预方面的潜力。
