US 2016/0257969 A1 · METHOD TO TRIGGER RNA INTERFERENCE

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.

Claims23

Dependent claims

An expression vector comprising a recombinant siRNA-triggering or RNAi-triggering nucleic acid cassette operably linked to a promoter, the nucleic acid cassette comprising:

an initiator sequence that consists of 20 to 25 nucleotides and has an initiation cleavage site between the tenth and eleventh nucleotides counted from the 3′ end of the initiator sequence, wherein the initiator sequence is recognized by an RNA-Induced Silencing Complex (RISC) guided by an siRNA or miRNA which binds to the initiation sequence by complementarity;
and wherein said initiator sequence is linked to or overlaps with
at least one 21-nucleotide gene suppressing segment in 21-nucleotide register counted either upstream or downstream from the initiation cleavage site, wherein when the cassette is transcribed in a plant, RNA transcribed from the gene suppressing segment or its complement is complementary to RNA transcribed from a target gene selected for siRNA inhibition.

A transgenic plant cell that expresses RNA for siRNA-based suppression of a target gene, wherein said plant cell comprises an expression vector comprising a recombinant siRNA-triggering or RNAi-triggering nucleic acid cassette operably linked to a promoter, the nucleic acid cassette comprising recombinant DNA from which there is transcribed a first RNA comprising:

an initiator sequence that consists of 20-25 nucleotides and has an initiation cleavage site located between the tenth and eleventh nucleotides counted from the 3′ end of the initiator sequence, wherein the initiator sequence is recognized by an RNA-Induced Silencing Complex (RISC) guided by an siRNA or miRNA which binds to the initiation sequence by complementarity;
and wherein said initiator sequence is linked to or overlaps with
at least one gene suppressing segment of 21-nucleotides in 21-nucleotide register counted either upstream or downstream from the initiation cleavage site, wherein said gene suppressing segment or its complement is complementary to RNA transcribed from said target gene;
wherein said recombinant DNA comprises a promoter functional in said plant operably linked to DNA coding for said first RNA.

1 more element

A seed for a transgenic plant that expresses RNA for siRNA-based suppression of a target gene, wherein said seed and plant comprise an expression vector comprising a recombinant siRNA-triggering or RNAi-triggering nucleic acid cassette operably linked to a promoter, the nucleic acid cassette comprising recombinant DNA from which there is transcribed a first RNA comprising:

an initiator sequence that consists of 20-25 nucleotides and has an initiation cleavage site located between the tenth and eleventh nucleotides counted from the 3′ end of the initiator segment sequence, wherein the initiator sequence is recognized by an RNA-Induced Silencing Complex (RISC) guided by an siRNA or miRNA which binds to the initiation sequence by complementarity;
and wherein said initiator sequence is linked to or overlaps with
at least one gene suppressing segment of 21-nucleotides in 21-nucleotide register counted either upstream or downstream from the initiation cleavage site, wherein said gene suppressing segment or its complement thereof is complementary to RNA transcribed from said target gene;
wherein said recombinant DNA comprises a promoter functional in said plant operably linked to DNA coding for said first RNA.

1 more element

Dependencies are inferred from the claim text.

Facts

Application

15/163,573Filed 24 May 2016

Priority

7 Jan 2005

Published

8 Sep 2016

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 · filed 25 Aug 2014

Application15/163,573Continuation

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