Cas13a has already been successfully applied to virus detection. However, as a new gene interference tool, its potential in cancer treatment was not fully explored until now. This study constructed a new Cas13a expression vector, decoy minimal promoter-Cas13a-U6-guide RNA (DMP-Cas13a-U6-gRNA [DCUg]), by controlling the Cas13a and gRNA expression with a nuclear factor κB (NF-κB)-specific promoter and U6 promoter, respectively. DCUg could specifically and effectively knock down the expression of reporter genes in the 293T and HepG2 cells. DCUg could also similarly knock down the expression of endogenous oncogenes (TERT, EZH2, and RelA) at both mRNA and protein levels in a human hepatoma cell HepG2, which led to significant apoptosis and growth inhibition. In contrast, the same transfection did not affect the target gene expression, cell apoptosis, and growth of a human normal liver cell HL7702. Finally, DCUg targeting these oncogenes was packaged into adeno-associated virus (AAV) and treated four cells (HepG2, HL7702, WEHI-3, and Hepa1-6) and tumor-bearing mice. As results, the recombinant AAV significantly inhibited the growth of three cancer cells (HepG2, Hepa1-6, and WEHI-3) in vitro and the xenografted Hepa1-6 and WEHI-3 tumors in mice. This study therefore developed a new tool for the CRISPR-Cas13a-based cancer gene therapy.

Cas13a 已经成功应用于病毒检测。然而，作为一种新的基因干扰工具，其在癌症治疗中的潜力直到现在还没有得到充分的探索。本研究构建了一个新的Cas13a表达载体，通过用核因子κB（NF-κB）控制Cas13a和gRNA的表达，诱骗最小启动子-Cas13a-U6-guide RNA（DMP-Cas13a-U6-gRNA [DCUg]）——分别为特异性启动子和 U6 启动子。DCUg 可以特异性有效地抑制 293T 和 HepG2 细胞中报告基因的表达。DCUg 还可以类似地在人肝癌细胞 HepG2 中在 mRNA 和蛋白质水平上敲低内源性癌基因（TERT、EZH2 和 RelA）的表达，从而导致显着的细胞凋亡和生长抑制。相比之下，同样的转染不影响目的基因表达、细胞凋亡、人类正常肝细胞 HL7702 的生长和生长。最后，靶向这些癌基因的 DCUg 被包装到腺相关病毒 (AAV) 中并处理四种细胞（HepG2、HL7702、WEHI-3 和 Hepa1-6）和荷瘤小鼠。结果，重组 AAV 显着抑制了三种癌细胞（HepG2、Hepa1-6 和 WEHI-3）的生长体外和小鼠异种移植的 Hepa1-6 和 WEHI-3 肿瘤。因此，这项研究为基于 CRISPR-Cas13a 的癌症基因治疗开发了一种新工具。