The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic\nchallenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of\nhiPSC-derived NSCs (hiPS-NSCs) still hasmuch roomto improve.Here we attempted to promote the tumor tropismof hiPS-NSCs\nby manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration.\nWe first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the\npromoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9).We then applied this CRISPRi system\nto hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster.\nMeanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET,\nand MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster\nsignificantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of\nCRISPRi in NSC-based tumor-targeted gene therapy.
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