US 2014/0373196 A1Published application

Method to trigger rna interference

AssigneeOregon State University

InventorsJames C. Carrington, Edwards Allen

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Abstract

A method to generate siRNAs in vivo is described, as are constructs and compositions useful in the method. The method does not depend on the use of DNA or synthetic constructs that contain inverted duplications or dual promoters so as to form perfect or largely double-stranded RNA. Rather, the method depends on constructs that yield single-stranded RNA transcripts, and exploits endogenous or in vivo-produced miRNAs or siRNAs to initiate production of siRNAs. The miRNAs or siRNAs guide cleavage of the transcript and set the register for production of siRNAs (usually 21 nucleotides in length) encoded adjacent to the initiation cleavage site within the construct. The method results in specific formation of siRNAs of predictable size and register (phase) relative to the initiation cleavage site. The method can be used to produce specific siRNAs in vivo for inactivation or suppression of one or more target genes or other entities, such as pathogens.

Claims18

Dependent claims

A method of inhibiting activity of at least one target gene in a plant cell, the method comprising providing in the plant cell:

an engineered single-stranded RNA transcript comprising:
an engineered initiator sequence of about 20 to 25 nucleotides with an initiation cleavage site between the tenth and eleventh or eleventh and twelfth nucleotides counted from the 3′ end of the initiator sequence, wherein the initiator sequence is recognized by a RNA-induced Silencing Complex guided by an siRNA or a miRNA that has sufficient complementarity to the initiator sequence to initiate cleavage in 21-nucleotide register from the initiation cleavage site;
and
at least one gene suppressing segment engineered to be in about 21-nucleotide register counted either upstream or downstream from the initiation cleavage site, wherein the gene suppressing segment or its complement is complementary to RNA transcribed from the at least one target gene selected for siRNA inhibition, wherein at least one gene suppressing segment of transcript is specific for the target gene;
and
an miRNA or siRNA that anneals to said initiator sequence at said initiation cleavage site.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/468,159Filed 25 Aug 2014
Priority: 7 Jan 2005
Published: 18 Dec 2014
Term ends: Not estimatedThe source records don’t support an estimate.

Classification19 codes

C12N 15/8279
For stress resistance, e.g. heavy metal resistance: for biotic stress resistance, pathogen resistance, disease resistance
C12N 15/111
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: general methods applicable to biologically…
C12N 15/113
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: non-coding nucleic acids modulating the…
C12N 15/8218
Methods for controlling, regulating or enhancing expression of transgenes in plant cells: antisense, co-suppression, viral induced gene silencing…

Family

Application60/642,126Us provisional application · filed 7 Jan 2005
Application11/334,776Continuation · filed 6 Jan 2006
Application13/216,942Continuation · filed 24 Aug 2011
Application13/827,176Continuation · filed 14 Mar 2013
Application14/468,159Continuation
US 2006/0174380 A1Published application
US 2011/0314571 A1Published application
US 2013/0185818 A1Published application
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From the US filing history. Foreign family members aren’t listed.