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EP-4737568-A1 - NOVEL CRISPR/CAS13 SYSTEM AND USE THEREOF

EP4737568A1EP 4737568 A1EP4737568 A1EP 4737568A1EP-4737568-A1

Abstract

The present specification discloses a novel CRISPR/Cas 13 system. The novel CRISPR/Cas13 system was identified from a public database. The novel /Cas 13 system has both target-specific nucleic acid cleavage activity and collateral cleavage activity. The inventors of the present specification have specified a novel CRISPR/Cas 13 system from a known database, and have demonstrated the function thereof through experimentation, thereby completing the invention.

Inventors

  • LEE, BONG HEE
  • BAYARSAIKHAN, Delger
  • BAYARSAIKHAN, Govigerel
  • GASIUNAS, Giedrius
  • STITILYTE, Migle
  • KOO, Okjae
  • KANG, HYUN A
  • LEE, JAE SUK

Assignees

  • Nsage Corp.

Dates

Publication Date
20260506
Application Date
20240624

Claims (14)

  1. A novel CRISPR/Cas 13 composition comprising: a Cas 13 protein comprising a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the Cas 13 protein; and a programmable guide RNA, or a nucleic acid encoding the programmable guide RNA, wherein the programmable guide RNA has a structure where a direct repeat and a guide domain are linked from a 5'-end to a 3'-end direction, wherein the direct repeat interacts with the Cas 13 protein to form a complex, wherein the direct repeat comprises an RNA sequence of SEQ ID NO: 2, and wherein the guide domain is capable of binding complementarily to a predetermined target nucleic acid.
  2. The novel CRISPR/Cas 13 composition of claim 1, wherein the nucleic acid encoding the Cas 13 protein is DNA or mRNA, and wherein the nucleic acid encoding the programmable guide RNA is DNA.
  3. The novel CRISPR/Cas 13 composition of claim 1 or claim 2, wherein the novel CRISPR/Cas 13 composition comprises the Cas 13 protein and the programmable guide RNA, wherein the Cas 13 protein and the programmable guide RNA form a protein-RNA complex.
  4. The novel CRISPR/Cas 13 composition of claim 1 or claim 2, wherein the novel CRISPR/Cas 13 composition comprises the nucleic acid encoding the Cas 13 protein and the nucleic acid encoding the programmable guide RNA.
  5. The novel CRISPR/Cas 13 composition of claim 4, wherein the nucleic acid encoding the Cas 13 protein and the nucleic acid encoding the programmable guide RNA are included in a single vector molecule.
  6. The novel CRISPR/Cas 13 composition of claim 4, wherein the nucleic acid encoding the Cas 13 protein and the nucleic acid encoding the programmable guide RNA are each included in two or more vector molecules.
  7. The novel CRISPR/Cas 13 composition of claim 5 or claim 6, wherein the vector is a viral vector, a non-viral vector, or a proper combination thereof.
  8. A method for cleaving non-target RNAs using a novel CRISPR/Cas 13 system, comprising: mixing the novel CRISPR/Cas 13 composition according to claim 3 with a sample, wherein the sample comprises a target RNA and the non-target RNA, and wherein the guide domain of the programmable guide RNA of the novel CRISPR/Cas 13 composition is capable of binding complementarily to the target RNA.
  9. A method for detecting a target RNA using a novel CRISPR/Cas 13 system, comprising: (a) mixing the novel CRISPR/Cas 13 composition according to claim 3, a sample, and a detecting agent, wherein the guide domain of the programmable guide RNA of the novel CRISPR/Cas 13 composition is capable of binding complementarily to the target RNA, wherein the detecting agent comprises a reporter, and the reporter comprises a probe nucleic acid, wherein when the probe nucleic acid is cleaved, the reporter generates a detectable signal, and wherein when the sample comprises the target RNA, the protein-RNA complex of the novel CRISPR/Cas 13 composition binds complementarily to the target RNA, thereby activating the collateral cleavage function of the protein-RNA complex, leading to the cleavage of the probe nucleic acid; and (b) measuring the detectable signal in the said mixture, wherein information on the target RNA can be obtained by measuring the detectable signal.
  10. The method for detecting the target RNA of claim 9, wherein the (b) is a process of measuring whether the detectable signal is generated; the intensity of the generated signal; the change in signal intensity over time; or any combination thereof.
  11. The method for detecting the target DNA of claim 9 or claim 10, wherein the information about the target RNA comprises whether the target RNA is present, how much of the target RNA is included in the sample, or both.
  12. The method for detecting the target DNA of any one of claims 9 to 11, wherein the detectable signal is a fluorescence signal.
  13. A method for providing information on diagnosing COVID-19, comprising: (a) mixing a novel CRISPR/Cas 13 complex, a specimen, and a detecting agent, wherein the specimen is collected from a patient suspected of having COVID-19 infection, wherein the novel CRISPR/Cas 13 complex comprises a Cas 13 protein of SEQ ID NO: 1; and guide RNA, wherein the guide RNA has a structure where a direct repeat of SEQ ID NO: 2 and a guide domain are linked sequentially from a 5' end to a 3' end, wherein the guide domain comprises an RNA sequence selected from a group consisting of SEQ ID NOS: 37 to 51, and SEQ ID NOS: 52 to 56, wherein the detecting agent comprises a reporter, and the reporter comprises a probe nucleic acid, wherein when the probe nucleic acid is cleaved, the reporter generates a fluorescence signal; (b) measuring the fluorescence signal in the mixture; and (c) obtaining information on whether the patient who provides the specimen is infected with COVID-19, by analyzing the fluorescence signal.
  14. The method for providing information on diagnosing COVID-19 of claim 13, wherein the (b) is a process of measuring whether the fluorescence signal is generated; an intensity of the fluorescence signal generated; a change in the intensity of the fluorescence signal over time; or any combination thereof.

Description

Technical Field The present invention relates to a CRISPR/Cas 13 system, which belongs to a Class 2, Type VI CRISPR/Cas system. Background Art CRISPR/Cas 13 system Outline of CRISPR/Cas 13 system This specification discloses a novel CRISPR/Cas 13 system, which is a type of CRISPR/Cas system. The CRISPR/Cas 13 system includes an effector protein (Cas 13 protein) and a guide RNA. The CRISPR/Cas 13 system recognizes sequences complementary to a spacer sequence of the guide RNA, and a single-stranded RNA that has Protospacer Flanking Sequence (PFS), and cleaves them. In some CRISPR/Cas 13 systems, when the single-stranded RNA is cleaved, a collateral cleavage activity is triggered, and then the single-stranded RNA neighboring the system is randomly cleaved. The following provides a more detailed explanation of a structure and a function of the CRISPR/Cas 13 system. Classification of CRISPR/Cas 13 system A CRISPR/Cas 13 system belongs to Class 2, Type VI CRISPR/Cas systems. The CRISPR/Cas 13 system is further divided into several subtypes, including Cas 13a, Cas 13b, Cas 13c, Cas 13d, Cas 13x, and Cas 13y. These subtypes are also referred to as IV-a, IV-b, IV-c, IV-d, IV-x, and IVy, respectively. The CRISPR/Cas 13 systems within each subtype exhibit slight differences in a size of a Cas 13 protein and a structure of a guide RNA, etc. Composition and function of CRISPR/Cas 13 system A CRISPR/Cas 13 system includes a Cas 13 protein and a guide RNA. These Cas 13 protein and guide RNA function as a complex. This protein-RNA complex can bind to a target nucleic acid (recognize the target nucleic acid) and cleave it. Additionally, a certain protein-RNA complex has a collateral cleavage activity, wherein the complex randomly cleaves nucleic acids neighboring the complex after cleaving the target nucleic acid. This Cas 13 protein-guide RNA complex is also referred to as a Ribonucleoprotein (RNP). The Cas 13 protein can recognize a Protospacer Flanking Sequence (PFS) and cleave nucleic acids. The guide RNA can direct the Cas 13 protein-guide RNA complex to the target nucleic acid by binding to the target nucleic acid that has the target sequence. The CRISPR/Cas 13 system is known to function by targeting single-stranded RNA as a target nucleic acid. Structure of Cas 13 protein The Cas 13 protein can be broadly divided into a RECognition (REC) lobe and a NUClease (NUC) lobe. The NUC lobe contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains. The REC lobe plays a crucial part when the Cas 13 protein recognizes a guide RNA and forms a complex. The NUC lobe cleaves nucleic acids. A structure of the Cas 13 protein has been reported by various researchers. For instance, it is explained in detail by Xue et al.(Engineering CRISPR/Cas 13 System against RNA Viruses: From Diagnostics to Therapeutics. Bioengineering (Basel), July 2022; 9(7): 291). Structure of guide RNA A guide RNA of a CRISPR/Cas 13 system, unlike that of the CRISPR/Cas 9 system, is composed of single RNA molecule, referred to as a crRNA. The guide RNA includes a direct repeat and a spacer. A connection structure between the direct repeat and the spacer varies depending on a subtype of the CRISPR/Cas 13 system. For instance, in guide RNAs of Cas 13a and Cas 13d subtypes, the direct repeat and the spacer are connected sequentially from a 5'-end to a 3'-end direction. In contrast, in a guide RNA of a Cas 13b subtype, a spacer and a direct repeat are connected sequentially from a 5'-end to a 3'-end direction. Target-specific nucleic acid cleavage activity of CRISPR/Cas 13 system, and Protospacer Flanking Sequence (PFS) The CRISPR/Cas 13 system can cleave nucleic acids with target specificity. Two conditions have to be met for target-specific nucleic acid cleavage activity to exhibit. First, a nucleic acid has to contain a sequence of bases having certain length, which can be recognized by a Cas 13 protein. Second, there has to be a sequence that can bind complementarily to a spacer included in a guide RNA neighboring the sequence of bases having certain length. When 1) the Cas 13 protein recognizes the sequence of bases having certain length, and 2) the spacer binds complementarily to a region neighboring the sequence of bases having certain length, the CRISPR/Cas 13 system cleaves nucleic acids. Here, the sequence of bases having certain length recognized by the Cas 13 protein is referred to as a Protospacer Flanking Sequence (PFS), which has a concept corresponding to the Protospacer Adjacent Motif (PAM) of CRISPR/Cas 9 systems. The PFS is determined by a type of the Cas 13 protein. Knowing the PFS of the Cas 13 protein allows for designing CRISPR/Cas 13 systems that target nucleic acids neighboring the PFS by using the knowledge. Among Cas 13 subtypes, the CRISPR/Cas 13d system is known as not requiring the PFS. The CRISPR/Cas 13d system can cleave target nucleic acids without additional conditions (irrespective of a presence of the PFS s