Modification of the specificity of non-coding RNA molecules for silencing gene expression in eukaryotic cells

Abstract

This invention provides a method for modifying genes that encode or are processed by non-coding RNA molecules that do not possess RNA silencing activity in eukaryotic cells. [Solution] The method is not for eukaryotic cells but for plant cells, and the method includes the step of introducing a DNA editing agent that confers silencing specificity of a non-coding RNA molecule to a target RNA of interest into the eukaryotic cells, and provides a method for modifying a gene that codes for an RNA silencing molecule against a target RNA or is processed by it in eukaryotic cells.

Claims

1. A method for modifying a gene that codes for or processes a non-coding RNA molecule that does not have RNA silencing activity in a eukaryotic cell, wherein the eukaryotic cell is not a plant cell, and the method comprises the steps of introducing a DNA editing agent into the eukaryotic cell that confers silencing specificity of the non-coding RNA molecule to a target RNA of interest, thereby modifying the gene that codes for or processes the non-coding RNA molecule.

2. The method according to claim 1, wherein the gene encoding or being processed by the non-coding RNA molecule is endogenous to the eukaryotic cell.

3. The method according to claim 1 or 2, wherein modifying the gene that encodes or processes the non-coding RNA molecule confers at least 45% complementarity to the target RNA of the object of the object to the non-coding RNA molecule.

4. The method according to any one of claims 1 to 3, wherein the silencing specificity of the non-coding RNA molecule is determined by measuring the RNA level or protein level of the target RNA of the purpose.

5. The method according to any one of claims 1 to 4, wherein, (a) The silencing specificity of the non-coding RNA molecule is phenotypically determined; or, (b) A method for determining the silencing specificity of the non-coding RNA molecule genotype-wise.

6. The method according to claim 5, wherein the phenotypic determination is brought about by the determination of at least one phenotype selected from the group consisting of cell size, growth rate / inhibition, cell shape, cell membrane integrity, tumor size, tumor shape, pigmentation of the organism, infection parameters and inflammation parameters.

7. The method according to claim 5, wherein the phenotype is determined before the genotype, or the genotype is determined before the phenotype.

8. The method according to any one of claims 1 to 7, wherein the non-coding RNA molecule is processed from a protein-coding RNA precursor.

9. The method according to any one of claims 1 to 8, wherein the non-coding RNA molecule is selected from the group consisting of nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), long non-coding RNA (lncRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), repeat-derived RNA, and transfer factor RNA.

10. The method according to any one of claims 1 to 9, wherein the modification of the gene is carried out by a modification selected from the group consisting of deletion, insertion, point mutation and combinations thereof.

11. The method according to claim 10, wherein the modification is in the stem region of the non-coding RNA molecule.

12. The method according to claim 10, wherein the modification is in the loop region of the non-coding RNA molecule.

13. The method according to claim 10, wherein the modification is located in the unstructured region of the non-coding RNA molecule.

14. The method according to claim 10, wherein the modification is in the stem region and loop region of the non-coding RNA molecule.

15. The method according to claim 10, wherein the modification is in the stem region, loop region, and unstructured region of the non-coding RNA molecule.

16. The method according to any one of claims 10 to 15, wherein the modification comprises a modification of up to 200 nucleotides.

17. The method according to any one of claims 1 to 16, further comprising introducing a donor oligonucleotide into the eukaryotic cell.

18. The method according to any one of claims 1 to 17, wherein the DNA editing agent comprises at least one gRNA operably linked to a promoter.

19. The method according to any one of claims 1 to 18, wherein the DNA editing agent comprises an endonuclease.

20. The method according to claim 19, wherein the endonuclease comprises Cas9.

21. The method according to any one of claims 1 to 20, wherein the DNA editing agent comprises a DNA editing system selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR.

22. A method according to any one of claims 1 to 21, wherein, (a) The DNA editing agent is applied to the cells as DNA, RNA, or RNP; and / or (b) A method comprising linking the DNA editing agent to a reporter for monitoring expression in eukaryotic cells.

23. The method according to claim 22, wherein the reporter is a fluorescent protein, in (b).

24. A method according to any one of claims 1 to 23, wherein, (a) The target RNA is endogenous to the eukaryotic cell; or, (b) A method wherein the target RNA is exogenous to the eukaryotic cell.

25. The method of claim 24, wherein the target RNA for the purpose is related to cancer, in (a).

26. The method of claim 24, wherein the target RNA for the purpose is related to an infectious disease, in (b).

27. ​​A method according to any one of claims 1 to 26, wherein, (a) The eukaryotic cells are obtained from eukaryotes selected from the group consisting of mammals, insects, nematodes, birds, reptiles, fish, crustaceans, fungi and algae; and / or (b) The eukaryotic cell is a mammalian cell; and / or, (c) A method wherein the eukaryotic cells are totipotent stem cells.

28. The method according to claim 27, wherein the mammalian cell is (i) a human cell; or (ii) a cell of a non-human organism.

29. A method for producing a eukaryotic non-human organism, wherein the organism is not a plant, and at least some of the cells of the organism contain a modified gene that encodes or processes a non-coding RNA molecule having silencing specificity for a target RNA of interest, and the method comprises the step of modifying a gene according to the method of any one of claims 1 to 28 to produce the eukaryotic non-human organism.

30. An isolated modified eukaryotic cell, wherein the eukaryotic cell is not a plant cell. Here, the eukaryotic cell is homozygous or heterozygous with respect to the DNA editing event, Here, the DNA editing event is a modification of a gene that encodes or processes non-coding RNA that does not have RNA silencing activity in a eukaryotic cell, using a method comprising the following steps: A step of introducing a DNA editing agent into the isolated eukaryotic cell that confers silencing specificity to the non-coding RNA to the target RNA of the desired purpose, thereby modifying the gene encoding the non-coding RNA.

31. The modified eukaryotic cell according to claim 30, wherein the method comprises the step of introducing a donor oligonucleotide, which includes a modification for use as a repair template to generate the modification in the gene, into the isolated eukaryotic cell.

32. The modified eukaryotic cell according to claim 30 or 31, wherein the DNA editing agent comprises an endonuclease and at least one gRNA.

33. The modified eukaryotic cell according to claim 32, wherein the endonuclease is Cas9.

34. The modified eukaryotic cell according to any one of claims 30 to 33, wherein the DNA editing event is an insertion, deletion, insertion-deletion, inversion, substitution, or a combination thereof.

35. The modified eukaryotic cell according to any one of claims 30 to 34, wherein the cell does not contain a DNA sequence encoding the DNA editing agent.

36. The modified eukaryotic cell according to claim 35, wherein the absence of the DNA sequence encoding the DNA editing agent is detected by loss of expression of the DNA editing agent.

37. The modified eukaryotic cell according to claim 36, wherein the loss of expression of the DNA editing agent is determined by fluorescence detection of green fluorescent protein (GFP), quantitative polymerase chain reaction (q-PCR), or high-performance liquid chromatography (HPLC).

38. The modified eukaryotic cell according to any one of claims 30 to 37, wherein the DNA editing event is detected by: DNA and RNA sequencing, electrophoresis, enzyme-based mismatch detection assay, hybridization assay such as PCR, RT-PCR, RNase protection, In Situ hybridization, primer extension, Southern blotting, Northern blotting, or dot blotting analysis, or a mismatch cleavage assay utilizing a structure-selective enzyme that recognizes and cleaves mismatch DNA.

39. A modified eukaryotic cell according to any one of claims 30 to 38, wherein, (a) The cells are obtained from eukaryotes selected from the group consisting of mammals, insects, nematodes, birds, reptiles, fish, crustaceans, fungi and algae; or, (b) The cells are human cells; or, (c) The cells are totipotent stem cells; or, (d) Modified eukaryotic cells in which the cells are immune cells.

40. The modified eukaryotic cell according to claim 39, wherein the immune cell in (d) is a T cell, a B cell, a macrophage, or a natural killer cell.

41. A pharmaceutical composition for treating an infectious disease in a target subject, comprising a DNA editing agent, wherein the DNA editing agent modifies a gene that encodes or processes a non-coding RNA molecule that does not have RNA silencing activity in eukaryotic cells, thereby conferring silencing specificity to the non-coding RNA molecule for a target RNA of interest, wherein the target RNA of interest is associated with the onset or progression of the infectious disease.

42. A pharmaceutical composition comprising a DNA editing agent for treating a monogene recessive genetic disorder in a target subject of interest, wherein the DNA editing agent modifies a gene that encodes or processes a non-coding RNA molecule that does not have RNA silencing activity in eukaryotic cells, thereby conferring silencing specificity to the non-coding RNA molecule for a target RNA of interest, wherein the target RNA of interest is associated with the onset or progression of the monogene recessive genetic disorder.

43. A pharmaceutical composition for treating an autoimmune disease in a target area of ​​interest, comprising a DNA editing agent, wherein the DNA editing agent modifies a gene that encodes or processes a non-coding RNA molecule that does not have RNA silencing activity in eukaryotic cells, thereby conferring silencing specificity to the non-coding RNA molecule for a target RNA of interest, wherein the target RNA of interest is associated with the onset or progression of the autoimmune disease.

44. A pharmaceutical composition for treating a cancerous disease in a target subject, comprising a DNA editing agent, wherein the DNA editing agent modifies a gene that encodes or processes a non-coding RNA molecule that does not have RNA silencing activity in eukaryotic cells, thereby conferring silencing specificity to the non-coding RNA molecule for a target RNA of interest, wherein the target RNA of interest is associated with the onset or progression of the cancerous disease.

45. The pharmaceutical composition according to any one of claims 41 to 44, wherein the DNA editing agent comprises an endonuclease and at least one gRNA.

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