Compositions and methods for the production and delivery of double stranded RNA
Compositions and methods for efficiently producing and delivering double stranded RNA (dsRNA) are provided. Vector constructs useful for in vitro and in vivo expression of dsRNA are described. Also described are cell expression systems for efficient and cost-effective production of dsRNA in living cells and methods and compositions for providing the expressed dsRNA to target organisms. The described compositions and methods can be used to produce RNA molecules for screening or other uses, and to amplify RNA sequences for analysis.
1. An engineered dsRNA expression construct comprising:
a. a promoter;
b. a dsRNA encoding region positioned transcriptionally downstream of the promoter, wherein the dsRNA encoding region comprises a first, sense-oriented, nucleotide sequence, which substantially corresponds to a target sequence, a second, anti-sense-oriented nucleotide sequence, which is substantially complementary to the target sequence, and a third nucleotide sequence, which is flanked by the first and second nucleotide sequences and which encodes one or more nucleotides of a loop-region of an RNA transcript;
c. a transcription terminator sequence, positioned 3′ to the dsRNA encoding region, wherein said transcription terminator sequence comprises a PTH terminator sequence, a first pET-T7 terminator sequence, and a second pET-T7 terminator sequence, wherein at least one pET-T7 terminator sequence is positioned 3′ to the PTH-terminator, wherein the transcription terminator sequence comprises nucleotides 659-764 of SEQ ID NO: 4; and
wherein the dsRNA encoding region and the transcription terminator are operably linked to the promoter.
2. The engineered dsRNA expression construct of claim 1 , wherein the engineered dsRNA expression construct further comprises one or more Zinc finger nuclease (ZFN), TAL-effector nuclease (TALEN) or meganuclease restriction sites positioned 3′ to the second transcription terminator sequence.
3. The engineered dsRNA expression construct of claim 1 , wherein the engineered dsRNA expression construct further comprises 1, 2, 3 or more additional transcription terminator sequence(s) positioned 3′ to the dsRNA encoding region.
4. The engineered dsRNA expression construct of claim 3 , wherein the additional transcription terminator sequences are each, independently, selected from a group consisting of PTH-terminator, pET-T7 terminator, T3-Tφ terminator, pBR322-P4 terminator, vesicular stomatitus virus terminator, rrnB-T1 terminator, rrnC terminator, and TTadc transcriptional terminator, such that the promoter and transcription terminator sequences form a functional combination.
5. The engineered dsRNA expression construct of claim 1 , wherein the promoter is a bacteriophage promoter.
6. The engineered dsRNA expression construct of claim 1 , wherein the dsRNA encoding region comprises SEQ ID NO 2.
7. A vector comprising the engineered dsRNA expression construct of claim 1 , wherein the vector is a plasmid vector.
8. A bacterial host cell comprising the vector of claim 7 .
9. The bacterial host cell of claim 8 , wherein the bacterial host cell does not express RNAse A.
10. A cell culture system for in vivo synthesis of dsRNA comprising the bacterial host cell of claim 8 and a growth media.
11. The cell culture system of claim 10 , wherein the growth media comprises 3.2% Tryptone, 2% Yeast Extract, 0.5% NaCl, 1% glycerol, 0.1% glucose, 0.4% alpha-lactose, 50 mM (NH4)2SO4, 10 mM KH2PO4, 40 mM Na2HPO4, 2 mM MgSO4.
12. A composition for controlling an invertebrate pest infestation comprising the bacterial host cell of claim 8 , wherein the bacterial host cell is dead and un-lysed.
13. A composition for controlling an invertebrate pest infestation comprising a lysate of the bacterial host cell of claim 8 .
14. A method for controlling an invertebrate pest infestation comprising applying the composition of claim 12 to a plant.
15. A composition for inhibiting the spread of a viral disease in a population of plants comprising a lysate of the bacterial host cell of claim 8 .
16. A method for inhibiting the spread of a viral disease in a population of plants comprising applying the composition of claim 15 to a plant, wherein the plant is a food source for an insect or nematode vector of the virus.