Methods and compositions for multiplexed editing of plant cell genomes
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.
1 . 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.
2 . 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.
3 . The method of claim 2 , further comprising crossing the plant with a second plant to obtain a third plant.
4 . The method of claim 2 , further comprising self-crossing the plant to obtain progeny.
5 . The method of claim 3 , wherein the second plant is a wild type plant.
6 . The method of claim 3 , wherein the second plant is a sibling plant.
7 . The method of claim 3 , wherein the second plant comprises a plurality of edited nucleobases.
8 . The method of claim 3 , wherein the third plant is crossed with a fourth plant.
9 . The method of claim 8 , wherein the fourth plant is a plant obtained from step (c).
10 . The method of claim 8 , wherein the fourth plant is a wild type plant.
11 . The method of claim 8 , wherein the fourth plant is a sibling plant.
12 . The method of claim 8 , wherein the fourth plant comprises a plurality of edited nucleobases.
13 . The method of claim 8 , wherein the fourth plant is a parental plant.
14 . A method of creating a population of plants with an altered characteristic, the method comprising: crossing a plant obtained from step (c) of claim 2 with a second plant to obtain a population of plants.
15 . The method of claim 1 , wherein the one or more selected polymorphic sites is at least 5.
16 . The method of claim 1 , wherein the nucleobase in the one or more selected polymorphic sites is cytosine.
17 . The method of claim 1 , wherein the nucleobase in the one or more selected polymorphic sites is adenine.
18 . The method of claim 1 , wherein the base editing agent is a composition comprising:
(i) an RNA-guided protein;
(ii) a plurality of different guide RNAs that each share sufficient homology with a different selected polymorphic site; and
(iii) a deaminase.
19 . The method of claim 18 , wherein the base editing agent further comprises:
(iv) a uracil glycosylase inhibitor.
20 . The method of claim 18 , wherein the deaminase is a cytosine deaminase.
21 . The method of claim 18 , wherein the deaminase is an adenine deaminase.
22 . The method of claim 18 , wherein the RNA-guided protein is a nickase.
23 . The method of claim 22 , wherein the nickase is a Cas nickase (nCas).
24 . The method of claim 18 , wherein the RNA-guided protein is a catalytically inactive endonuclease.
25 . The method of claim 24 , wherein the catalytically inactive endonuclease is a deactivated Cas endonuclease (dCas).
26 . The method of claim 2 , wherein the composition further comprises:
(iv) a uracil glycosylase inhibitor.
27 . The method of claim 2 , wherein the base editing agent is a deaminase.
28 . The method of claim 27 , wherein the deaminase is a cytosine deaminase.
29 . The method of claim 27 , wherein the deaminase is an adenine deaminase.
30 . The method of claim 2 , wherein the RNA-guided protein is a nickase.
31 . The method of claim 30 , wherein the nickase is a Cas nickase (nCas).
32 . The method of claim 2 , wherein the RNA-guided protein is a catalytically inactive endonuclease.
33 . The method of claim 32 , wherein the catalytically inactive endonuclease is a deactivated Cas endonuclease (dCas).
34 . The method of claim 2 , wherein the one or more selected target sites is at least 5.
35 . The method of claim 2 , wherein the single nucleotide polymorphism is cytosine.
36 . The method of claim 1 , wherein the single nucleotide polymorphism is adenine.
37 . The method of claim 1 , wherein the one or more selected polymorphic sites constitute a rare allele.
38 . The method of claim 2 , wherein the one or more selected target sites constitute a rare allele.
39 . The method of claim 1 , wherein the one or more selected polymorphic sites comprises a deleterious missense mutation.
40 . The method of claim 2 , wherein the one or more selected target sites comprises a deleterious missense mutation.
41 . The method of claim 1 , wherein the one or more selected polymorphic sites are in a coding region of the plant cell genome.
42 . The method of claim 2 , wherein the one or more selected target sites are in a coding region of the plant cell genome.
43 . The method of claim 1 , wherein the one or more selected polymorphic sites are in a noncoding region of the plant cell genome.
44 . The method of claim 2 , wherein the one or more selected target sites are in a noncoding region of the plant cell genome.