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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.
Liu et al. (Wed,) studied this question.