Methods of eliminating or reducing expression of genes in filamentous fungal strains by transitive RNA interference

Patent metadata

Field	Value
Publication number	US-9499820-B2
Application number	US-52006707-A
Country	US
Kind code	B2
Filing date	Dec 20, 2007
Priority date	Dec 21, 2006
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

The present invention relates to methods of reducing or eliminating expression of a target gene in a filamentous fungal strain by transitive RNA interference.

First claim

What is claimed is:

1. A method for reducing or eliminating expression of a target gene encoding a biological substance in an Aspergillus niger strain, an Aspergillus oryzae strain, or a Trichoderma reesei strain, comprising:
(a) inserting into the genome of the Aspergillus niger strain, the Aspergillus oryzae strain, or the Trichoderma reesei strain a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene or a homologue thereof encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene or the homologue thereof, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation relative to each other and the first and second transcribable regions are transcribed as a single mRNA molecule; and
(b) inducing production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, by cultivating the Aspergillus niger strain, the Aspergillus oryzae strain, or the Trichoderma reesei strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene or the homologue thereof to reduce or eliminate expression of the target gene or the homologue thereof encoding the biological substance.

2. The method of claim 1 , wherein the first transcribable region with homology to the target gene comprises at least 19 nucleotides of the target gene.

3. The method of claim 1 , wherein the second transcribable region with no effective homology to the target gene comprises at least 19 nucleotides.

4. The method of claim 1 , wherein the first and second polynucleotides are separated by an intervening sequence.

5. The method of claim 1 , wherein the two segments complementary to each other in reverse orientation are separated by a linking sequence.

6. The method of claim 1 , wherein expression of the target gene is reduced by at least 20%.

7. The method of claim 1 , wherein the short interfering RNAs interact with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene.

8. The method of claim 1 , wherein expression of the one or more homologues of the target gene is reduced by at least 20%.

Assignees

• Brody Howard
• Moyer Donna
• Fisher Amanda
• Novozymes Inc

Inventors

• Brody Howard
• Moyer Donna
• Fisher Amanda

Classifications

• C12P1/02

  by using fungi · CPC title

• C12Y302/01037

  Xylan 1,4-beta-xylosidase (3.2.1.37) · CPC title

• C12Y604/00

  Ligases forming carbon-carbon bonds (6.4) · CPC title

• C12N2310/111

  spanning the whole gene, or a large part of it · CPC title

• C12N15/1137 Primary

  against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138) · CPC title