Zhang Xin, Lv Sihan, Luo Zhenhuan, Hu Yongfei, Peng Xin, Lv Jie, Zhao Shanshan, Feng Jianqi, Huang Guanjie, Wan Qin-Li, Liu Jun, Huang Hongxin, Luan Bing, Wang Dong, Zhao Xiaoyang, Lin Ying, Zhou Qinghua, Zhang Zhen-Ning, Rong Zhili
Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.
Department of Endocrinology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200072, China.
Nucleic Acids Res. 2021 Apr 19;49(7):4171-4185. doi: 10.1093/nar/gkab174.
CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.
CRISPR介导的基因激活（CRISPRa）是一种很有前景的治疗性基因编辑策略，不会诱导DNA双链断裂（DSB）。然而，这些CRISPRa系统在体内的应用仍然是一个挑战。在此，我们报告了一种用于体内激活内源性靶基因的紧凑且强大的小型Cas9激活剂（称为miniCAFE）。该系统依赖于通过单链向导RNA将来自空肠弯曲菌的工程化最小无核酸酶Cas9和强效转录激活剂募集到靶位点。即使只有一个DNA拷贝，它也能在人类细胞中实现强大的基因激活，并且能够通过激活长寿调节基因来延长秀丽隐杆线虫的寿命。作为概念验证，在一体化腺相关病毒（AAV）中递送时，miniCAFE可以激活成年小鼠肝脏中的Fgf21表达并调节能量代谢。因此，miniCAFE在治疗人类疾病方面具有巨大的治疗潜力。
