COMPOSITIONS FOR REDUCED LIGNIN CONTENT IN SORGHUM AND IMPROVING CELL WALL DIGESTIBILITY, AND METHODS OF MAKING THE SAME

• US 20150291969A1
• Filed: 01/30/2015
• Published: 10/15/2015
• Est. Priority Date: 01/30/2014
• Status: Abandoned Application

• Current Assignee: Chromatin, Inc. (S&W Seed Company)
• Original Assignee: Chromatin, Inc. (S&W Seed Company)
• Inventors: Nair, Ramesh B., Lee, Hyeran
• Application Number:
  US14/611,168
• Publication Number:
  US 20150291969A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12Y 304/22062 Caspase-9 (3.4.22.62)

Abstract

RNAi vectors comprising a fragment of the SbCSE polynucleotide sequence and transgenic plants, e.g. transgenic sorghum plants, comprising said RNAi vectors are described. Aspects of the technology are further directed to methods of using the RNAi vectors of the present technology to silence SbCSE gene expression or activity in a transgenic plant, such as a transgenic sorghum plant. Silencing the SbCSE gene leads to reduced lignin content in a transgenic plant.

Claims

1. -30. (canceled)

31. A method for converting a recessive trait to a dominant trait in a eukaryotic organism, the method comprising:

introducing into a cell of the eukaryotic organism a CRISPR-Cas vector system, wherein the CRISPR-Cas vector system is configured to generate a first guide sequence, a second guide sequence and a Cas endonuclease; and

introducing into the cell a donor arm comprising an antisense sequence of a first portion of a targeted sequence in a genomic locus of a DNA molecule encoding a targeted gene product having the recessive trait,

wherein the first guide sequence, the second guide sequence and the Cas endonuclease facilitate homologous recombination of the donor arm within the DNA molecule and at a location spaced apart from the first portion in a manner that modifies the DNA molecule, and

wherein expression of the modified DNA molecule is modified, thereby converting the recessive trait to the dominant trait.

32. A method for modifying expression of a targeted gene product in an eukaryotic cell, comprising:

introducing into the eukaryotic cell a vector system comprising one or more vectors comprising—

(a) a first regulatory element operably linked to a first guide sequence, wherein the first guide sequence hybridizes with a first target sequence in a genomic locus of a DNA molecule encoding the targeted gene product,

(b) a second regulatory element operably linked to a second guide sequence, wherein the second guide sequence hybridizes with a second target sequence in the genomic locus of the DNA molecule encoding the targeted gene product, and wherein the first target sequence is non-overlapping with the second target sequence, and

(c) a third regulatory element operably linked to a DNA sequence encoding a Cas endonuclease, wherein the Cas endonuclease induces double strand breaks at or near the first and second target sequences; and

introducing into the eukaryotic cell a donor arm comprising—

an antisense sequence of at least a portion of a targeted sequence in the genomic locus, wherein the portion of the targeted sequence is spaced apart from the first and second target sequences, and

a first homologous region and a second homologous region, wherein the first and second homologous regions flank the antisense sequence, and wherein the first homologous region hybridizes at or near the first target sequence and the second homologous region hybridizes at or near the second target sequence,

whereby, introduction of the vector system and the donor arm causes gene modification of the DNA molecule in a manner that modifies expression of the targeted gene product.

33. The method of claim 32 wherein gene modification includes homologous recombination of the donor arm and the DNA molecule at or near the first and second target sequences.

34. The method of claim 32 wherein gene modification of the DNA molecule generates a transcript having a hairpin structure.

35. The method of claim 32 wherein gene modification of the DNA molecule silences expression of a targeted gene product.

36. The method of claim 32 wherein the cell is a plant cell.

37. The method of claim 32 wherein the cell is a sorghum plant cell.

38. The method of claim 32 wherein the DNA molecule is at least one of sorghum sbCAD2 and sbCSE.