RNA editing with CRISPR-Cas13

TLDR

RNA editing at the RNA level can rescue disease‑relevant sequences, and Type VI CRISPR‑Cas systems provide a programmable RNA‑guided ribonuclease, Cas13. The authors aimed to engineer a Cas13 ortholog for robust knockdown and develop a programmable A‑to‑I RNA editing system, REPAIR, capable of editing full‑length transcripts with pathogenic mutations. REPAIR uses catalytically inactive Cas13 fused to ADAR2 to direct A‑to‑I editing without strict sequence constraints, and the authors engineered a high‑specificity, minimized variant suitable for viral delivery. REPAIR presents a promising RNA‑editing platform with broad applicability for research, therapeutics, and biotechnology.

Abstract

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.