Targeted nuclear RNA cleavage and polyadenylation with CRISPR-Cas
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Publication Number
US-12371698-B2
Publication Date
2025-07-29
Expiration Date
Abstract
The present invention provides proteins, nucleic acids, systems and methods for visualizing or modulating RNA.
Core Innovation
The invention relates to fusion proteins comprising a CRISPR-associated (Cas) protein and a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21. The content describes Postscriptr as a Cas protein fused to NUDT21, and the fusion includes nuclear localization signals such as Ty1/Ty1-like NLS.
The system achieves crRNA-guided, site-specific RNA cleavage and polyadenylation in mammalian cells, and it enables alternative cleavage/polyadenylation of endogenous transcripts. Cleavage/poly(A) sites are mapped, including by 3′ RACE mapping, and transcript outcomes are evaluated using reporter readouts and endogenous targets such as sfGFPapa reporter, human SREBP1, and DMPK.
Additional configurations include EraseR and HilightR as Cas13-based nuclear RNA targeting fusion systems. EraseR is described as dCas13 fused to an NLS for nuclear RNA cleavage/degradation, and HilightR is described as dCas13 fused to a fluorescent protein for nuclear RNA visualization.
Claims Coverage
The consolidated claim coverage includes one independent fusion-protein claim centered on a CRISPR-associated Cas protein fused to NUDT21, with dependent refinements that specify dCas13, defined NUDT21 and NLS sequence selections, and a method of modulating RNA transcript cleavage and/or polyadenylation in a subject.
CRISPR-associated Cas protein fused to NUDT21 cleavage or polyadenylation protein
A fusion protein comprising a CRISPR-associated (Cas) protein and a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21.
Catalytically dead Cas13 fusion configuration
The fusion protein uses catalytically dead Cas13 (dCas13) as the Cas protein.
NUDT21 sequence selection for the fusion
NUDT21 contains a sequence selected from SEQ ID NOs: 51-58, or a variant thereof.
Fusion protein includes a nuclear localization signal
The fusion protein further includes a nuclear localization signal (NLS).
Ty1-like NLS sequence selection
The NLS consists of a sequence selected from SEQ ID NOs: 75-695, or a variant thereof.
Modulation of RNA transcript cleavage and/or polyadenylation in a subject
A method for modulating cleavage and/or polyadenylation of an RNA transcript in a subject by administering a fusion protein or encoding nucleic acid together with a guide nucleic acid complementary to a target RNA sequence.
The claim coverage centers on a Cas-NUDT21 fusion protein for RNA cleavage/polyadenylation modulation, with refinements for dCas13, specified NUDT21 and NLS sequence selections, and a subject-based method using a complementary guide nucleic acid.
Stated Advantages
Enables crRNA-guided, site-specific RNA cleavage and polyadenylation in mammalian cells.
Allows alternative cleavage/polyadenylation of endogenous transcripts.
Manipulation of RNA transcripts without genomic DNA editing is described as a justification/advantage in the provided content.
Avoidance of germline DNA editing risk is described in the provided content.
Supports therapeutic targeting for myotonic dystrophy (DM1) by promoting premature termination upstream of toxic CUG expansions.
Enables degradation/visualization of toxic nuclear RNA foci using CRISPR-Cas13 with Ty1 NLS (hilightR/eraseR).
Documented Applications
crRNA-guided RNA cleavage/polyadenylation in mammalian cells using reporter and endogenous transcript targets, including sfGFPapa reporter and human SREBP1.
Targeting DMPK RNAs in myotonic dystrophy (DM1) to promote premature termination upstream of toxic CUG expansions.
Degradation/visualization of toxic nuclear RNA foci using CRISPR-Cas13 with Ty1 NLS (hilightR/eraseR).
Alternative cleavage/polyadenylation of endogenous transcripts, including DMPK and other endogenous targets described in the document.
Nuclear RNA targeting and editing using CRISPR-Cas13-based fusion-protein systems including Postscriptr, EraseR, and HilightR is described.
Modulating cleavage and/or polyadenylation of an RNA transcript in a subject using the claimed fusion protein or its encoding nucleic acid together with a complementary guide nucleic acid is described.
Nuclear RNA visualization is described for HilightR as a dCas13 fused to a fluorescent protein.
Systems for modulating RNA cleavage/polyadenylation and related RNA processing disease/disorder targets are described.
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