Search

EP-4208544-B1 - SYSTEMS, METHODS, AND COMPOSITIONS FOR RNA-GUIDED RNA-TARGETING CRISPR EFFECTORS

EP4208544B1EP 4208544 B1EP4208544 B1EP 4208544B1EP-4208544-B1

Inventors

  • Abudayyeh, Omar
  • Gootenberg, Jonathan

Dates

Publication Date
20260513
Application Date
20210701

Claims (14)

  1. A composition comprising a guide RNA that specifically hybridizes to an RNA target, and a polypeptide comprising an amino acid sequence 85% identical to amino acid sequence SEQ ID NO: 1, wherein the polypeptide comprises aspartate at a position corresponding to position 429 of SEQ ID NO:1; and/or an aspartate at a position corresponding to position 654 of SEQ ID NO:1; and wherein the composition cleaves the RNA target.
  2. The composition of claim 1, wherein the guide RNA has a sequence with a length of from about 20 to about 53 nucleotides (nt), preferably from about 25 to about 53 nt, more preferably from about 29 to about 53 nt or from about 40 to about 50 nt.
  3. The composition of claim 1, wherein the guide RNA is a pre-crRNA or a mature crRNA.
  4. The composition of claim 1, wherein the RNA target is a single-strand RNA (ssRNA) and/or wherein the RNA target is in a prokaryotic cell or a eukaryotic cell.
  5. The composition of claim 5, wherein the eukaryotic cell is a mammalian cell, for example a human cell.
  6. The composition of claim 1, wherein the polypeptide further comprises a deaminase domain for programmable RNA base editing, optionally wherein the deaminase is selected from the group consisting of an adenosine deaminase, a cytidine deaminase, and a catalytic domain thereof.
  7. The composition of claim 1, wherein the deaminase is an adenosine deaminase, for example wherein the deaminase domain comprises an amino acid sequence 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42.
  8. The composition of claim 1, wherein the guide RNA comprises a mismatch that is about 20 to about 30 nucleotides from the non-pairing C of the guide RNA.
  9. A nucleic acid molecule encoding the polypeptide of any of the preceding claims.
  10. The nucleic acid molecule of claim 9, wherein the nucleic acid molecule further comprises a nucleic acid that encodes the guide RNA.
  11. A vector comprising the nucleic acid molecule of claims 9-10.
  12. A cell comprising the composition of claims 1-8, the nucleic acid molecule of claims 9-10 or the vector of claim 11, optionally wherein the cell is a prokaryotic cell or a eukaryotic cell.
  13. The composition of any one of claims 1 to 8, for use in medicine.
  14. The nucleic acid molecule of claim 9 or claim 10, or the vector of claim 11, for use in medicine.

Description

TECHNOLOGY FIELD The subject matter disclosed herein is generally related to systems, methods, and compositions for RNA-guided RNA-targeting CRISPR effectors for the treatment of diseases and diagnostics. BACKGROUND RNA targeting tools for genetic engineering are important to study RNA biology and develop RNA therapeutics. These tools can regulate intracellular and intercellular target-gene functions and expressions and manipulate specific target-genomic information. In contrast to DNA targeting tools, only a few RNA targeting tools for CRISPR applications have been developed, and these RNA specific tools present challenges. For instance, protein complexes for CRISPR RNA editing can have a weak activity in mammalian cells and present collateral effects which can be toxic in some cell types. Also, the size of the RNA editing tools can be a significant barrier to their use, as technologies such as programmable activation cannot be delivered in mouse models using common methods such as adeno-associated vectors (AAV). Development of effective gene and cell therapies requires genome editing tools and delivery technologies that can meet the demands for cell type specificity, large payload sizes, and efficient integration of diverse and large sequences. Many applications, including single-vector homology directed repair (HDR), CRISPR base editing, gene activation, and large gene delivery, are limited by AAV size restrictions. Thus, there remains a need for more effective tools for gene correction and delivery. WO2019222555A1 discusses systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion, including non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of DNA, RNA, and protein substrates. Each system includes one or more protein components and one or more nucleic acid components that together target DNA, RNA, or protein substrates. SUMMARY The present invention is defined in and by the appended claims, and is directed to systems, methods, and compositions for RNA-guided RNA-targeting CRISPR effectors for the treatment of diseases and diagnostics. Also disclosed are nucleotide deaminase functionalized CRISPR systems for RNA editing RNA knockdown, viral resistance, splicing modulation, RNA tracking, translation modulation, and epi-transcriptomic modifications. In one aspect of the invention according to claim 1, a composition is discussed, which comprises a guide RNA that specifically hybridizes to an RNA target, and a polypeptide comprising an amino acid sequence 85% identical to amino acid sequence SEQ ID NO: 1, wherein the polypeptide comprises aspartate at a position corresponding to position 429 of SEQ ID NO: 1; and/or an aspartate at a position corresponding to position 654 of SEQ ID NO: 1; and wherein the composition cleaves an RNA target. The polypeptide can comprise a glutamate at a position corresponding to position 70 of SEQ ID NO:1 and/or an aspartate at a position corresponding to position 487 of SEQ ID NO: 1. If desired, the polypeptide can comprise an amino acid sequence 90% identical to the amino acid sequence of SEQ ID NO: 1. If desired, the polypeptide can comprise an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO: 1. If desired, the polypeptide can comprise an amino acid sequence 99% identical to the amino acid sequence of SEQ ID NO: 1. If desired, the polypeptide can be the amino acid sequence of SEQ ID NO: 1. The guide RNA can comprise a mismatch distance that is 20-65% of the length of the guide. If desired, the guide RNA can comprise a mismatch that is about 20 to about 30 nucleotides from the non-pairing C of the guide RNA. If desired, the guide RNA can have a sequence with a length of from about 20 to about 53 nucleotides (nt), preferably from about 25 to about 53 nt, more preferably from about 29 to about 53 nt or from about 40 to about 50 nt. If desired, the guide RNA can be a pre-crRNA. If desired, the guide RNA can be a mature crRNA. The RNA target can be a single-strand RNA (ssRNA). If desired, the RNA target can be in a cell. The cell can be a prokaryotic cell. The cell can be a eukaryotic cell. The eukaryotic cell can be a mammalian cell. The mammalian cell can be a human cell. The polypeptide can comprise a deaminase domain. The deaminase can be selected from the group consisting of an adenosine deaminase, a cytidine deaminase, and a catalytic domain thereof. If desired, the deaminase can be an adenosine deaminase. If desired, the deaminase domain can comprise an amino acid sequence 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-31. If desired, the deaminase domain can comprise an amino acid sequence 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-42. Yet in another aspect according to claim 9, a nucleic acid molecule encoding the polypeptide is discussed. The nucleic acid molecule