CRISPR-based programmable RNA editing
Inventors
Wiedenheft, Blake A. • NICHOLS, Joseph E. • NEMUDRAIA, Anna A. • NEMUDRYI, Artem A.
Assignees
Montana State University Bozeman • National Institutes of Health NIH
Publication Number
US-12139731-B2
Publication Date
2024-11-12
Expiration Date
2042-07-08
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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.
Core Innovation
The invention relates to programmable RNA editing using a non-naturally occurring type III CRISPR complex that enables sequence-specific modification of RNA molecules. This method includes the use of a programmable crRNA guide designed to target specific RNA sequences, combined with a crRNA-guided nuclease that cleaves RNA at defined positions. The cleavage is followed by ligation of the resulting RNA fragments, allowing for insertions, deletions, or replacements of specific RNA sequences.
A particular embodiment involves utilizing a type III-A Csm complex derived from Streptococcus thermophilus to specifically cleave regions of the SARS-CoV-2 RNA genome at increments of 6 nucleotides. After cleavage, a DNA splint is used to bridge the excision site, and RNA ligase repairs the RNA, resulting in edited RNA molecules. This workflow allows excision and/or replacement of 6, 12, 18, or 24 nucleotides at a targeted RNA location prior to cellular transfection, demonstrated on infectious SARS-CoV-2 RNA.
The primary problem addressed by this invention is the lack of efficient and reliable methods for targeted editing of RNA, particularly for RNA viruses such as SARS-CoV-2. Existing techniques for manipulating viral RNA are cumbersome, multi-step, error-prone, and not suited for precise deletion or replacement of target RNA sequences. The disclosed innovation provides a programmable, versatile system for sequence-specific editing of RNA in vitro, which is independent of protospacer adjacent motifs (PAMs), and generalizable to a wide range of RNA molecules, including for gene therapy.
Claims Coverage
There are two independent claims in this patent, each focusing on programmable RNA editing using a non-naturally occurring type III CRISPR complex with specific inventive features related to crRNA-guided sequence-specific RNA cleavage and repair.
Programmable RNA editing using a non-naturally occurring type III CRISPR complex
The method involves cleaving a ribonucleic acid (RNA) molecule with a non-naturally occurring type III Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex, which comprises: - A CRISPR RNA (crRNA) sequence designed to be complementary to a target sequence of the RNA molecule - A crRNA-guided nuclease that specifically cleaves the RNA molecule at the target portion when the complex is bound After cleavage, the method includes ligating cleaved ends of the RNA molecule that flank the excised target portion, resulting in a programmatically edited RNA molecule based on the excised region and ligated ends.
Editing RNA with six-nucleotide interval cleavage
This inventive feature centers on employing a non-naturally occurring type III CRISPR complex to cleave an RNA molecule at intervals of six nucleotides: - The complex includes a crRNA designed for complementarity to the target sequence and a crRNA-guided nuclease that cleaves the RNA at six-nucleotide intervals - Cleaved RNA ends flanking the excised target portion are ligated, programmatically editing the RNA molecule according to the excision and ligation.
The independent claims focus on programmable RNA editing through the use of specially designed, non-naturally occurring type III CRISPR complexes for sequence-specific cleavage and ligation of RNA molecules, enabling precise editing at user-defined positions and intervals, particularly at six-nucleotide increments.
Stated Advantages
The method enables efficient, sequence-specific editing of RNA molecules, including precise deletions and replacements, which were not possible using previous RNA editing systems.
Programmable crRNA guides allow targeting of any RNA sequence without reliance on protospacer adjacent motifs (PAMs), thereby simplifying guide design and expanding applicability.
The approach generalizes to any RNA molecule, making it broadly applicable to editing RNA viruses and to gene therapy.
Editing is performed in vitro, providing a straightforward and reproducible protocol that overcomes the cumbersome, error-prone nature of prior multi-step RNA manipulation processes.
Documented Applications
Sequence-specific editing of RNA viruses, including but not limited to the SARS-CoV-2 genome.
General gene therapy applications involving programmatic modification, deletion, or insertion within any RNA molecule.
Editing of viral RNA genomes to produce infectious, genetically-altered viral clones for research or therapeutic use.
Potential extension to any organism’s RNA, including use in mammalian, avian, and plant systems according to explicit document statements.
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