Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children's Medical Center, Children's Hospital, Fudan University, Shanghai, China.
Department of Pediatrics, Qilu Hospital of Shandong University, Ji'nan, 250012, China.
Nat Commun. 2023 Jan 26;14(1):413. doi: 10.1038/s41467-023-36004-2.
Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.
尽管最近开发了微型 CRISPR-Cas12f 系统，但衍生的微型胞嘧啶和腺嘌呤碱基编辑器（miniCBE 和 miniABEs）的编辑效果和靶向范围尚未得到全面解决。此外，功能性 miniCBE 尚未建立。在这里，我们生成了各种具有改进编辑活性和多样化靶向范围的 Cas12f 衍生的 miniCBE 和 miniABEs。我们揭示了使用传统胞嘧啶脱氨酶生成的 miniCBE 具有广泛的编辑窗口和高脱靶效应。为了改善经典 CBE 和衍生的 miniCBE 的编辑特征，我们通过诱变筛选工程化 TadA 脱氨酶，生成具有高精度和最小脱靶效应的强效 miniCBE。我们表明，新设计的 miniCBE 和 miniABEs 能够在细胞系中纠正致病突变，并通过腺相关病毒在体内大脑中有效引入遗传突变。总之，这项研究为 CBE 的开发提供了替代策略，扩展了 miniCBE 和 miniABEs 的工具包，并为临床应用提供了有前途的治疗工具。
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