US 11,718,845 B2
Methods for increasing gene-editing frequencies in maize cells
Erik William Clement, Somerville, MA (US); Yajie Niu, Lexington (CN); and Frank Anthony Skraly, Watertown, MA (US)
Assigned to INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
Filed by INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
Filed on Sep. 27, 2021, as Appl. No. 17/449,011.
Application 17/449,011 is a continuation of application No. 15/908,191, filed on Feb. 28, 2018, granted, now 11,168,319.
Claims priority of provisional application 62/516,600, filed on Jun. 7, 2017.
Claims priority of provisional application 62/464,991, filed on Feb. 28, 2017.
Prior Publication US 2022/0002706 A1, Jan. 6, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12N 5/00 (2006.01); C12N 15/11 (2006.01); C12N 9/22 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 5/0025 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2500/02 (2013.01); C12N 2500/14 (2013.01); C12N 2500/16 (2013.01); C12N 2800/80 (2013.01)] 4 Claims
 
1. A method for making a maize plant protoplast having a genomic modification comprising:
(a) providing genome editing molecules to a maize plant protoplast lacking a cell wall wherein the genome editing molecules are designed for homology-directed repair of a target gene in the plant cell's genome and comprise: (i) an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof,
(b) exposing the maize plant protoplast from step (a) to plant cell culture medium comprising a reactive oxygen species (ROS) concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar and Ca2+ and/or Mg2+ at a concentration of about 40 mM to 150 mM for at least one hour to effect homology-directed repair of the target gene in the plant cell's genome at a frequency that is increased by at least 2-fold in comparison to a control method wherein a control plant cell is provided with the genome editing molecules but is not subjected to a ROS concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar, wherein the ROS concentration lowering agent is a ROS scavenging agent or an antioxidant; and,
(c) isolating, selecting, identifying, and/or propagating a maize plant protoplast comprising the genome modification, thereby making the maize plant protoplast having a genomic modification.