Generation of heritable chimeric plant traits
View Patent ↗The present invention provides methods and compositions for targeting enzymes involved in lignin or xylan biosynthesis using genome editing nucleases to specifically reduce content in a desired plant cell type(s).
1. A method of engineering a plant having reduced lignin content, the method comprising:
introducing a nucleic acid construct into a plant, wherein the construct encodes a gene editing nuclease and comprises a polynucleotide encoding (i) a nuclear-targeted Cas9 domain operably linked to a fiber-specific NST3 promoter, and (ii) a sequence encoding at least a first chimeric RNA comprising a targeting region that selectively hybridizes to a target site in a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) gene linked to a Cas9 handle;
culturing the plant under conditions in which the nucleic acid construct is expressed and the Cas9 domain cleaves the gene at the target site and reduces or inactivates expression of the HCT gene; and
evaluating lignin content in plant fibers and selecting a plant that has reduced lignin content and does not exhibit reduction in biomass yield compared to wild-type plants.
2. The method of claim 1 , wherein the fiber-specific NST3 promoter comprises SEQ ID NO:24.
3. The method of claim 1 , wherein the nucleic acid construct further comprises a sequence encoding a second chimeric RNA that comprises a targeting region that selectively hybridizes to a second site in the HCT gene different from the site targeted by the first chimeric RNA.
4. The method of claim 1 , wherein the nucleic acid construct further comprises a sequence encoding a second chimeric RNA that comprises a targeting region that selectively hybridizes to a site in a cinnamate 4-hydroxylase (C4H) gene, a coumaryol shikimate 3-hydroxylase (C3H) gene, a cinnamoyl-CoA reductase 1 (CCR1) gene, an irregular xylem 8 (IRX8) gene, an IRX14 gene, an IRX9 gene, an IRX gene7, an IRX10 gene, an IRX15 gene, an F8H gene, a PARVUS gene, a Myb gene, an RWA gene or a TBL gene.
5. The method of claim 4 , wherein the nucleic acid construct further comprises a sequence encoding a second chimeric RNA that comprises a targeting region that selectively hybridizes to a site in a C4H gene, a C3H gene, a CCR gene, a Myb63 gene, an IRX7 gene or an IRX8 gene.
6. A plant generated by the method of claim 1 .
7. A method of obtaining an increased amount of soluble sugars from a plant in a saccharification reaction, the method comprising:
subjecting the plant of claim 6 to a saccharification reaction, thereby increasing the amount of soluble sugars that can be obtained from the plant as compared to a wild-type plant.
8. Seedlings obtained from a plant of claim 6 , wherein the seedlings have fiber-specific reduction in lignin content.