Hybridization‐Mediated Reversible sgRNA Caging Enables Tumor‐Selective Gene Editing for Potentiating Antitumor Immunity via cGAS Demethylation

Abstract

ABSTRACT Spatiotemporal precision remains a critical challenge against the gene editing technology used for disease treatment. We herein developed a tumor cell‐selective CRISPR/Cas9 gene editing system by introducing a complementary single‐stranded DNA (ssDNA) containing apurinic/apyrimidinic (AP) sites (AP‐ssDNA) along the target region of single‐guided RNA (sgRNA). The engineered hybrid (AP‐ssDNA/sgRNA) could tracelessly release the native sgRNA in tumor cells due to cleavage of AP‐ssDNA by the overexpressed apurinic/apyrimidinic endonuclease 1 (APE1), thus enabling tumor‐selective gene editing. Subsequently, a biodegradable lipid nanoparticle based on dynamic covalent bonding was developed to codeliver Cas9‐encoding mRNA (mCas9) with AP‐ssDNA/sgRNA targeting the SUV39H1 gene, which mediated silence of cyclic GMP‐AMP synthase (cGAS) methylation to activate stimulator of interferon genes (STING) and enhanced antitumor immunity. As thus, when coupled with radiotherapy, the gene editing platform provoked robust antitumor efficacy in B16F10 xenograft tumor‐bearing mice. This study provides a versatile paradigm for conditionally controlled gene editing and highlights the therapeutic potential of cGAS demethylation in reactivating cGAS‐STING pathway for antitumor immunotherapy.