Methods and compositions for multiplexed editing of plant cell genomes
AssigneePioneer Hi-Bred International, Inc.
InventorsNicholas Doane Chilcoat, Joseph Evans, Yi Jia, Eli Rodgers-Melnick, Joshua K Young
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Abstract
Methods and compositions are provided for massively parallel base editing in the genome of an organism, such as a plant. Multiple loci are selected based on one or more specific criteria, such as evolutionary conservation, recombination frequency, nonsense mutations, and/or missense mutations. Predictive models for deleterious alleles and biologically-informed phenotype prediction may be developed and refined based on the editing results, and improved plant lines for commercial or breeding purposes may be obtained.
Claims44
A method of base editing in a plant cell, the method comprising:
- selecting a plurality of polymorphic sites in the genome of the plant, wherein selecting the plurality of polymorphic sites comprises:
- (a) calculating a selection score for each polymorphic site of the plurality of polymorphic sites, wherein the selection score is based on evolutionary conservation of each polymorphic site;
- and
- (b) selecting polymorphic sites with selection scores that meet a user-defined criterion and comprise a nucleotide sequence capable of being modified by base editing;
- and
- introducing into the plant cell a base editing agent, wherein the base editing agent alters a nucleobase in one or more selected polymorphic sites.
A method of altering a characteristic of a plant, the method comprising:
- (a) selecting a plurality of target sites in a plant cell, wherein the plurality of target sites each comprise a single nucleotide polymorphism, wherein selecting the plurality of target sites comprises:
- calculating a selection score for a single nucleotide polymorphism within each of the plurality of target sites, wherein the selection score is based on evolutionary conservation of the single nucleotide polymorphism;
- and
- selecting the plurality of target sites, wherein each selected target site comprises a single nucleotide polymorphism with a selection score that meets a user-defined criterion, wherein the single nucleotide polymorphism is capable of being modified by base editing;
- (b) introducing into the plant cell a composition comprising:
- (i) a plurality of different guide RNAs that each share sufficient homology with a different selected target site;
- (ii) an RNA-guided protein that forms a complex with a guide RNA of the plurality of different guide RNAs, and
- (iii) a base editing agent;
- wherein the composition alters the single nucleotide polymorphism within one or more selected target sites;
- (c) regenerating a plant from the plant cell;
- and
- (d) assessing the plant for the presence of an altered characteristic, as compared to an isoline plant that did not have the composition introduced.
Dependencies are inferred from the claim text.