Engineering circular guide RNA and CRISPR-Cas13d-encoding mRNA for the RNA editing of Adar1 in triple-negative breast cancer immunotherapy.

Clustered regularly interspaced short palindromic repeat Cas endonuclease (CRISPR-Cas) systems, such as RNA-editing CRISPR-Cas13d, are poised to advance the gene therapy of various diseases. However, their clinical development has been challenged by 1) the limited biostability of linear guide RNAs (lgRNAs) susceptible to degradation, 2) the immunogenicity of prokaryotic microorganism-derived Cas proteins in human that restrains their long-term therapeutic efficacy, and 3) off-targeting gene editing caused by the prolonged Cas expression from DNA vectors. Here, we report the development of highly stable circular gRNAs (cgRNAs) and transiently-expressing Cas13d-encoding mRNA for efficient CRISPR-Cas13d editing of target mRNA. We first optimized cgRNA for CRISPR-Cas13d editing of adenosine deaminase acting on RNA type I (Adar1) transcript for the combination immunotherapy of triple negative breast cancer (TNBC). cgRNAs were synthesized by enzymatic ligation of lgRNA precursors. cgRNAs enhanced biostability with comparable Cas13d-binding affinity relative to lgRNA. Next, using ionizable lipid nanoparticles (LNPs), we co-delivered the resulting Adar1-targeting cgRNA with an mRNA encoding RfxCas13d (mRNA-RfxCas13d), a widely used Cas13d variant, to TNBC cells. As a result, relative to lgRNA, cgRNA significantly enhanced the efficiency of Adar1 knockdown with minimal collateral activity, which sensitized the cancer cells for cytokine-mediated cell apoptosis. In a 4T1 murine TNBC tumor model in syngeneic mice, Adar1-targeting cgRNA outperformed lgRNA for tumor immunotherapy in combination with immune checkpoint blockade (ICB). Collectively, these results demonstrate the great potential of cgRNA and mRNA-RfxCas13d for RNA-targeted gene editing.