CRISPR-based programmable RNA editing

Abstract

CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

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