Methods and compositions for targeting RNA polymerases and non-coding RNA biogenesis to specific loci
View Patent ↗The present disclosure relates to recombinant proteins that induce epigenetic gene silencing and to methods of using such proteins for reducing the expression of genes in plants.
1. A method for producing a plant with reduced expression of one or more target nucleic acids, comprising:
(a) providing a plant comprising a recombinant nucleic acid, wherein the recombinant nucleic acid encodes a recombinant polypeptide comprising a first amino acid sequence comprising a DNA-binding domain and a second amino acid sequence comprising a plant DMS3 polypeptide, wherein the second amino acid sequence comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 41; and
(b) growing the plant under conditions whereby the recombinant polypeptide encoded by the recombinant nucleic acid is expressed and binds to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids to produce the plant with reduced expression of the one or more target nucleic acids.
2. The method of claim 1 , wherein the DNA-binding domain comprises a zinc finger domain.
3. The method of claim 1 , wherein the DNA-binding domain is selected from the group consisting of a TAL effector targeting domain, a helix-turn-helix family DNA-binding domain, a basic domain, a ribbon-helix-helix domain, a TBP domain, a barrel dimer domain, a real homology domain, a BAH domain, a SANT domain, a Chromodomain, a Tudor domain, a Bromodomain, a PHD domain, a WD40 domain, and a MBD domain.
4. The method of claim 1 , wherein the DNA-binding domain comprises a TAL effector targeting domain.
5. The method of claim 1 , wherein the second amino acid sequence comprises an amino acid sequence that is 100% identical to SEQ ID NO: 41.
6. The method of claim 1 , wherein the one or more target nucleic acids are endogenous nucleic acids.
7. The method of claim 1 , wherein the one or more target nucleic acids are heterologous nucleic acids.
8. The method of claim 1 , wherein expression of the one or more target nucleic acids is silenced.
9. A plant cell comprising a recombinant nucleic acid encoding a recombinant polypeptide comprising a first amino acid sequence comprising a DNA-binding domain and a second amino acid sequence comprising a plant DMS3 polypeptide, wherein the second amino acid sequence comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 41.
10. The plant cell of claim 9 , wherein the second amino acid sequence comprises an amino acid sequence that is 100% identical to SEQ ID NO: 41.
11. The plant cell of claim 9 , wherein the DNA-binding domain comprises a zinc finger domain.
12. The plant cell of claim 9 , wherein the DNA-binding domain is selected from the group consisting of a TAL effector targeting domain, a helix-turn-helix family DNA-binding domain, a basic domain, a ribbon-helix-helix domain, a TBP domain, a barrel dimer domain, a real homology domain, a BAH domain, a SANT domain, a Chromodomain, a Tudor domain, a Bromodomain, a PHD domain, a WD40 domain, and a MBD domain.
13. The plant cell of claim 9 , wherein the DNA-binding domain comprises a TAL effector targeting domain.
14. The method of claim 1 , further comprising:
(c) crossing the plant with reduced expression of the one or more target nucleic acids to a second plant to produce one or more F1 plants with reduced expression of the one or more target nucleic acids.
15. The method of claim 14 , further comprising:
(d) selecting from the one or more F1 plants with reduced expression of the one or more target nucleic acids an F1 plant that (i) lacks the recombinant nucleic acid, and (ii) has reduced expression of the one or more target nucleic acids.