Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, State Key Laboratory of Genetic Engineering at School of Life Sciences, Fudan University, Shanghai, 200438, China.
Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Fudan University, Shanghai, 200438, China.
Adv Sci (Weinh). 2024 Sep;11(36):e2400469. doi: 10.1002/advs.202400469. Epub 2024 Jul 30.
The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system represents the third family of CRISPR-Cas systems that are harnessed for genome editing. However, only a few nucleases have demonstrated activity in human cells, and their in vivo therapeutic potential remains uncertain. In this study, a green fluorescent protein (GFP)-activation assay is conducted to screen a panel of 15 Cas12b orthologs, and four of them exhibited editing activity in mammalian cells. Particularly noteworthy is the NiCas12b derived from Nitrospira sp., which recognizes a "TTN" protospacer adjacent motif (PAM) and facilitates efficient genome editing in various cell lines. Importantly, NiCas12b also exhibits a high degree of specificity, rendering it suitable for therapeutic applications. As proof of concept, the adeno-associated virus (AAV) is employed to introduce NiCas12b to target the cholesterol regulatory gene proprotein convertase subtilisin/ kexin type 9 (Pcsk9) in the mouse liver. After 4 weeks of injections, an impressive is observed over 16.0% insertion/deletion (indel) efficiency, resulting in a significant reduction in serum cholesterol levels. NiCas12b provides a novel option for both basic research and clinical applications.
RNA 指导的簇状规律间隔短回文重复序列 (CRISPR)/Cas12b 系统代表了被用于基因组编辑的第三代 CRISPR-Cas 系统。然而，只有少数几种核酸酶在人类细胞中表现出活性，其体内治疗潜力仍不确定。在这项研究中，通过绿色荧光蛋白 (GFP) 激活测定法筛选了一组 15 种 Cas12b 同源物，其中有 4 种在哺乳动物细胞中具有编辑活性。特别值得注意的是来源于 Nitrospira sp. 的 NiCas12b，它识别“TTN”原间隔基序 (PAM)，并促进各种细胞系中的高效基因组编辑。重要的是，NiCas12b 还具有高度的特异性，使其适用于治疗应用。作为概念验证，腺相关病毒 (AAV) 被用于将 NiCas12b 引入小鼠肝脏中靶向胆固醇调节基因前蛋白转化酶枯草杆菌蛋白酶/ kexin 9 型 (Pcsk9)。经过 4 周的注射后，观察到令人印象深刻的超过 16.0%的插入/缺失 (indel) 效率，导致血清胆固醇水平显著降低。NiCas12b 为基础研究和临床应用提供了一种新的选择。
