Joseph D Nichols

US Patent:

20230040061, Feb 9, 2023

Filed:

Jul 8, 2022

Appl. No.:

17/811391

Inventors:

- Bozeman MT, US
Joseph E. NICHOLS - Bozeman MT, US

International Classification:

C12N 9/22
C12N 15/66
C12N 7/00
C12N 15/11

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.