Zhang Xiaoqi, Tao Chen, Li Miaomiao, Zhang Sufang, Liang Puping, Huang Yan, Liu Huihui, Wang Yongming
Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, China.
Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing 100091, China.
Mol Ther Nucleic Acids. 2025 Jan 17;36(1):102455. doi: 10.1016/j.omtn.2025.102455. eCollection 2025 Mar 11.
SauriCas9 is a compact Cas9 nuclease showing promise for therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.
SauriCas9是一种紧凑的Cas9核酸酶，在治疗应用方面显示出前景。然而，对脱靶效应的担忧使得有必要提高其特异性。我们通过引入突变来消除Cas9与靶DNA之间的极性接触来解决这一问题，从而产生了具有更高特异性的SauriCas9-R253A变体。为了验证其功效，我们使用SauriCas9-R253A破坏三个基因（B2M、TRAC和PDCD1），这是开发同种异体嵌合抗原受体T细胞（CAR-T）疗法不可或缺的策略。我们的结果表明，SauriCas9-R253A最有效的单向导RNA表现出与SpCas9相当的活性，并且在这些基因的破坏中未检测到脱靶效应，突出了其治疗潜力。
