Liu Hong, Dong Jiantong, Wu Renzhi, Dai Jun, Lou Xiaoding, Xia Fan, Willner Itamar, Huang Fujian
State Key Laboratory of Geomicrobiology and Environmental Changes, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan, 430074, China.
The Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
Angew Chem Int Ed Engl. 2025 Jul 7;64(28):e202502892. doi: 10.1002/anie.202502892. Epub 2025 May 19.
A photo-triggered CRISPR/Cas12a machinery for in vitro and in vivo gene editing is introduced. The system consists of a caged, inactive ortho-nitrobenzyl phosphate ester photo-responsive crRNA, which, upon light-induced deprotection, yields the active CRISPR/Cas12a gene editing machinery (LAC12aGE). The LAC12aGE system induces specific thymidine-rich (TTTN) protospacer-adjacent motif (PAM)-guided double-stranded breaks in genomic DNA, which upon non-homologous end-joining lead to gene repair. The LAC12aGE machinery is applied for gene editing of an exogenous dual fluorescent reporter gene in living cells, as well as the endogenous gene encoding DNA methyltransferase 1. In addition, the LAC12aGE is applied for in vitro gene editing and disruption of the hepatocyte growth factor (HGF) gene in HepG2 cells, where knockout of the HGF gene results in inhibited cell proliferation and migration, as well as enhanced apoptosis. Moreover, the in vivo knockout and disruption of the HGF gene in HepG2 tumors by the LAC12aGE machinery is demonstrated. The cyclic temporal development of the LAC12aGE system in tumors shows effective inhibition of tumor growth and enhanced apoptosis/necrosis of tumor tissues compared to control systems.
介绍了一种用于体外和体内基因编辑的光触发CRISPR/Cas12a机制。该系统由一种笼状、无活性的邻硝基苄基磷酸酯光响应性crRNA组成，在光诱导脱保护后，产生活性CRISPR/Cas12a基因编辑机制（LAC12aGE）。LAC12aGE系统在基因组DNA中诱导特定的富含胸腺嘧啶（TTTN）的原间隔序列相邻基序（PAM）引导的双链断裂，经非同源末端连接后导致基因修复。LAC12aGE机制用于活细胞中外源双荧光报告基因以及编码DNA甲基转移酶1的内源基因的基因编辑。此外，LAC12aGE用于体外基因编辑和破坏HepG2细胞中的肝细胞生长因子（HGF）基因，其中HGF基因的敲除导致细胞增殖和迁移受到抑制，以及凋亡增强。此外，还证明了LAC12aGE机制在体内对HepG2肿瘤中HGF基因的敲除和破坏。与对照系统相比，LAC12aGE系统在肿瘤中的循环时间发展显示出对肿瘤生长的有效抑制和肿瘤组织凋亡/坏死的增强。
