Isolated dsRNA molecules and methods of using same for silencing target molecules of interest

• US 10,041,068 B2
• Filed: 12/30/2013
• Issued: 08/07/2018
• Est. Priority Date: 01/01/2013
• Status: Expired due to Fees

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: A.B. Seed Ltd.
• Inventors: Avniel, Amir, Lidor-Nili, Efrat, Maor, Rudy, Meir, Ofir, Noivirt-Brik, Orly, Yanai-Azulay, Osnat
• Primary Examiner(s): Visone, Lee A
• Assistant Examiner(s): Fan, Weihua
• Application Number:
  US14/143,748
• Publication Number:
  US 20140296503A1
• Time in Patent Office: 1,681 Days
• CPC Class Codes: A01H 3/04 by treatment with chemicals
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8206 by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8279 for biotic stress resistance, pathogen resistance, disease resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

An isolated dsRNA molecule comprising an antisense RNA sequence for regulating a target gene of interest in a plant or a phytopathogen of the plant, wherein the dsRNA sequence is flanked by two complementary sites to an smRNA or smRNAs expressed in the plant and wherein the dsRNA molecule further comprises a helicase binding site positioned so as to allow unwinding of the strands of the isolated dsRNA molecule to single stranded RNA (ssRNA) and recruitment of an RNA-dependent RNA polymerase so as to amplify the dsRNA molecule in the plant cell and generate secondary siRNA products of the dsRNA sequence.

Claims

1. An isolated double-stranded RNA (dsRNA) molecule comprising

(a) a first RNA strand having

at least one antisense RNA sequence for suppressing expression of a target gene of interest in a corn, rice, tomato, or sorghum plant or an essential target gene of a phytopathogen of a corn, rice, tomato, or sorghum plant,

a first heterologous smRNA-binding sequence for binding to a first small RNA (smRNA) expressed in said corn, rice, tomato, sorghum plant, wherein said first smRNA comprises 15-30 nucleotides, and wherein said first smRNA comprises a nucleic acid sequence having at least 95% identity or complementarity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

41-43, 45, and 47-96,

a helicase-binding sequence comprising the helicase binding site of SEQ ID NO;

14; and

(b) a second RNA strand that is a reverse complement of said first RNA strand.

2. The isolated dsRNA molecule of claim 1, wherein said first heterologous smRNA-binding sequence is 5′

to said antisense RNA sequence.

3. The isolated dsRNA molecule of claim 1, wherein said first heterologous smRNA-binding sequence is 3′

to said antisense RNA sequence.

4. The isolated dsRNA molecule of claim 1, wherein said first heterologous smRNA-binding sequence is the complement of said first smRNA.

5. The isolated dsRNA molecule of claim 1, wherein said first RNA strand further comprises a second heterologous smRNA-binding sequence for binding a second smRNA comprising 15-30 nucleotides expressed in said corn, rice, tomato, or sorghum plant, wherein said second smRNA comprises a nucleic acid sequence having at least 95% identity or complementarity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

41-43, 45 and 47-96,and wherein said first heterologous smRNA-binding sequence and said second heterologous smRNA-binding sequence flank said at least one antisense RNA sequence.

6. The isolated dsRNA molecule of claim 5, wherein said first smRNA and said second smRNA comprise a nucleic acid sequence having 100% sequence identity or complementarity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

41-43, 45, and 47-96.

7. The isolated dsRNA molecule of claim 5, wherein said second heterologous smRNA-binding sequence is the complement of said second smRNA.

8. The isolated dsRNA molecule of claim 5, wherein said second smRNA is identical to said first smRNA.

9. The isolated dsRNA molecule of claim 5, wherein said second smRNA is non-identical to said first smRNA.

10. The isolated dsRNA molecule of claim 5, wherein said first heterologous smRNA-binding sequence and said second heterologous smRNA-binding sequence comprise a nucleotide sequence selected from the group consisting of:

a direct sequence of said first smRNA and a direct sequence of said second smRNA;

a reverse complement of said first smRNA and a direct sequence of said second smRNA;

a reverse complement of said first smRNA and a reverse complement of said second smRNA;

a direct sequence of said first smRNA and a reverse complement of said second smRNA;

a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA;

a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA;

a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA;

a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA;

a direct sequence of said first smRNA and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a reverse complement of said first smRNA and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a reverse complement of said first smRNA and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a direct sequence of said first smRNA and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;

a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage; and

a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage.

11. The isolated dsRNA molecule of claim 1, wherein said helicase binding sequence is positioned within said at least one antisense RNA sequence.

12. The isolated dsRNA molecule of claim 1, wherein said helicase binding sequence is positioned 5′

or 3′

of said at least one antisense RNA sequence.

13. The isolated dsRNA molecule of claim 12, wherein said first RNA strand further comprises a second heterologous smRNA-binding sequence for binding to a second smRNA expressed in said corn, rice, tomato, or sorghum plant, and wherein said first heterologous smRNA and said second heterologous smRNA-binding sequence flank said at least one antisense RNA sequence and said helicase binding sequence.

14. The isolated dsRNA molecule of claim 1, wherein said first smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a microRNA (miRNA) and an RNA sequence of an siRNA.

15. The isolated dsRNA molecule of claim 5, wherein said first smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA, and said second smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA.

16. The isolated dsRNA molecule of claim 13, wherein said first smRNA is selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA, and said second smRNA is selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA.

17. The isolated dsRNA molecule of claim 1, wherein said first smRNA is a miRNA.

18. The isolated dsRNA molecule of claim 5, wherein said first smRNA is a miRNA and said second smRNA is a miRNA.

19. The isolated dsRNA molecule of claim 13, wherein said first smRNA is a miRNA and said second smRNA is a miRNA.

20. The isolated dsRNA molecule of claim 1, wherein said first smRNA is miR390.

21. The isolated dsRNA molecule of claim 5, wherein said first smRNA is miR390.

22. The isolated dsRNA molecule of claim 13, wherein said first smRNA is miR390.

23. The isolated dsRNA molecule of claim 5, wherein said corn, rice, tomato, or sorghum plant or phytopathogen comprises a trans-acting-siRNA-producing (TAS) locus that comprises a binding site for binding said second smRNA.

24. The isolated dsRNA molecule of claim 5, wherein said corn, rice, tomato, or sorghum plant or phytopathogen comprises a TAS locus, wherein said TAS locus naturally comprises a binding site for binding said first smRNA and a binding site for binding said second smRNA, and wherein said binding sites flank said TAS locus in said corn, rice, tomato, or sorghum plant or phytopathogen.

25. The isolated dsRNA molecule of claim 5, wherein at least one of said first and second smRNA is an smRNA for which binding sites are naturally found flanking a TAS locus in a plant.

26. The isolated dsRNA molecule of claim 1, wherein said plant is a crop plant.

27. The isolated dsRNA molecule of claim 1, wherein said helicase binding site provides for the separation of the two strands of said dsRNA molecule to produce a region of single stranded RNA (ssRNA) when introduced into a plant cell or provided with a helicase and RNA Dependent RNA Polymerase (RDRP) in vitro.

28. The isolated dsRNA molecule of claim 1, wherein said helicase binding site provides for the recruitment of an RNA-dependent RNA Polymerase and transcription of a secondary small interfering RNA (siRNA) complementary to at least one strand of said dsRNA molecule when introduced into a plant cell or provided with a helicase in vitro.