Wang Xinyi, Dong Kaili, Kong Deqiang, Zhou Yang, Yin Jianli, Cai Fengfeng, Wang Meiyan, Ye Haifeng
Synthetic Biology and Biomedical Engineering Laboratory, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Dongchuan Road 500, Shanghai 200241, China.
Department of Breast Surgery, Yangpu Hospital, School of Medicine, Tongji University, 450 Tengyue Road, Shanghai 200090, China.
Sci Adv. 2021 Dec 10;7(50):eabh2358. doi: 10.1126/sciadv.abh2358.
The CRISPR-Cas12a has been harnessed as a powerful tool for manipulating targeted gene expression. The possibility to manipulate the activity of CRISPR-Cas12a with a more precise spatiotemporal resolution and deep tissue permeability will enable targeted genome engineering and deepen our understanding of the gene functions underlying complex cellular behaviors. However, currently available inducible CRISPR-Cas12a systems are limited by diffusion, cytotoxicity, and poor tissue permeability. Here, we developed a far-red light (FRL)–inducible CRISPR-Cas12a (FICA) system that can robustly induce gene editing in mammalian cells, and an FRL-inducible CRISPR-dCas12a (FIdCA) system based on the protein-tagging system SUperNova (SunTag) that can be used for gene activation under light-emitting diode–based FRL. Moreover, we show that the FIdCA system can be deployed to activate target genes in mouse livers. These results demonstrate that these systems developed here provide robust and efficient platforms for programmable genome manipulation in a noninvasive and spatiotemporal fashion.
CRISPR-Cas12a已成为用于操纵靶向基因表达的强大工具。以更精确的时空分辨率和深部组织渗透性来操纵CRISPR-Cas12a活性的可能性，将实现靶向基因组工程，并加深我们对复杂细胞行为背后基因功能的理解。然而，目前可用的可诱导CRISPR-Cas12a系统受到扩散、细胞毒性和组织渗透性差的限制。在此，我们开发了一种远红光（FRL）诱导的CRISPR-Cas12a（FICA）系统，其可在哺乳动物细胞中强力诱导基因编辑，以及一种基于蛋白质标记系统超新星（SunTag）的FRL诱导的CRISPR-dCas12a（FIdCA）系统，该系统可用于在基于发光二极管的FRL下激活基因。此外，我们表明FIdCA系统可用于激活小鼠肝脏中的靶基因。这些结果表明，我们在此开发的这些系统为以无创和时空方式进行可编程基因组操纵提供了强大且高效的平台。
