US 12,305,176 B2
Polynucleotides for expression of RNA-guided nucleases and DNA binding proteins in soybean
Scott A. Bevan, Cambridge, MA (US); and Adam Patrick Joyce, Stow, MA (US)
Assigned to INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
Appl. No. 17/906,947
Filed by INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
PCT Filed Mar. 29, 2021, PCT No. PCT/US2021/024681
§ 371(c)(1), (2) Date Sep. 21, 2022,
PCT Pub. No. WO2021/202397, PCT Pub. Date Oct. 7, 2021.
Claims priority of provisional application 63/075,395, filed on Sep. 8, 2020.
Claims priority of provisional application 63/072,585, filed on Aug. 31, 2020.
Claims priority of provisional application 63/001,806, filed on Mar. 30, 2020.
Prior Publication US 2023/0175001 A1, Jun. 8, 2023
Int. Cl. C12N 15/82 (2006.01)
CPC C12N 15/8213 (2013.01) 10 Claims
 
1. A method of modifying an endogenous soybean gene in a soybean genome comprising:
(a) introducing a guide RNA or a polynucleotide encoding a guide RNA directed to a target editing site in the endogenous soybean gene and optionally a donor template DNA molecule having homology to the target editing site into a soybean plant cell comprising a synthetic polynucleotide encoding an Cas9 RNA-guided endonuclease (RGE) or Cas9 RNA guided nickase (RGN) having at least 99% sequence identity to SEQ ID NO: 1, wherein said synthetic polynucleotide:
(i) has more than 80% identity across the entire length of SEQ ID NO: 3 and a GC (guanine and cytosine) content greater than 48% and a soybean codon adaptation index (sCAI) which is decreased by a value of 0.02 to 0.03 in comparison to the sCAI of the soybean codon-optimized reference polynucleotide of SEQ ID NO: 2; or
(ii) has more than 80% sequence identity across the entire length of SEQ ID NO: 3 and a soybean codon adaptation index (sCAI) which is decreased by a value of 0.02 to 0.03 in comparison to the sCAI of a soybean codon-optimized reference polynucleotide of SEQ ID NO: 2; and
(b) selecting a modified soybean plant cell, soybean plant, soybean plant part, soybean tissue, or soybean callus comprising a modification of the endogenous soybean gene.