METHOD FOR BASE EDITING IN PLANTS

Abstract

The present invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for base editing in plants. More particularly, the invention relates to a method for performing efficient base editing to a target sequence in the genome of a plant (such as a crop plant) by a Cas9-cytidine deaminase fusion protein, as well as plants produced through said method and progenies thereof.

Claims

1-23. (canceled)

24. A method for producing a genetically modified plant or plant cell, comprising introducing in the absence of any selective pressure into the plant or plant cell a composition for performing base editing to a target sequence in a plant genome, comprising at least one of the following (i) to (v): i) a base editing fusion protein, and a guide RNA; ii) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and a guide RNA; iii) a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA; iv) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA; v) an expression construct comprising a nucleotide sequence encoding base editing fusion protein and a nucleotide sequence encoding guide RNA; wherein said base editing fusion protein contains a nuclease-inactivated Cas9 domain and a deaminase domain, said guide RNA can target said base editing fusion protein to the target sequence in the plant genome, and thereby said guide RNA target said base editing fusion protein to a target sequence in the genome of said plant or plant cell, resulting in one or more C to T substitutions in said target sequence, wherein said deaminase is an APOBEC1 deaminase and said nuclease-inactivated Cas9 comprises amino acid substitutions of D10A and/or H840A relative to wild-type Cas9.

25. The method according to claim 24, wherein one or more C in the positions 3-9 of the protospacer sequence in said target sequence is substituted with T.

26. The method according to claim 24, further comprising screening for plants or plant cells with desired nucleotide substitution(s).

27. The method according to claim 24, wherein said plant or plant cell is selected from monocotyledon and dicotyledon.

28. The method according to claim 27, wherein said plant is a crop plant, such as wheat, rice, maize, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava, or potato.

29. The method according to claim 24, wherein said target sequence is associated with a plant trait such as an agronomy trait, and thereby said base editing results in a change of the trait in the plant relative to a wild-type plant.

30. The method according to claim 24, wherein said composition is introduced into said plant or plant cell through a method selected from particle bombardment, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, a pollen tube approach, and ovary injection approach.

31. The method according to claim 24, further comprising obtaining a progeny of the genetically modified plant.

32. A genetically modified plant or a progeny thereof or a part thereof, wherein said plant is obtained through the method according to claim 24.

33. A plant breeding method, comprising crossing a first plant genetically modified through the method according to claim 24 with a second plant that does not contain said genetic modification, and thereby introducing said genetic modification into said second plant.

34. The method according to claim 24, wherein said deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase having a deamination window of 7 nucleotides at positions 3-9 of the protospacer sequence

35. The method according to claim 34, wherein said APOBEC1 deaminase comprises an amino acid sequence set forth in SEQ ID NO: 11.

36. The method according to claim 24, wherein said nuclease-inactivated Cas9 comprises an amino acid sequence set forth in SEQ ID NO:13 or 14.

37. The method according to claim 24, wherein said deaminase domain is fused to the N-terminal of said nuclease-inactivated Cas9 domain, or wherein said deaminase domain is fused to the C-terminal of said nuclease-inactivated Cas9 domain.

38. The method according to claim 24, wherein said deaminase domain and said nuclease inactivated Cas9 domain is fused through a linker, for example said linker is XTEN linker set forth in SEQ ID NO: 12.

39. The method according to claim 24, wherein said base editing fusion protein further comprises an uracil DNA glycosylase inhibitor (UGI), for example said uracil DNA glycosylase inhibitor comprises an amino acid sequence set forth in SEQ ID NO: 15.

40. The method according to claim 24, wherein said base editing fusion protein further comprises a nuclear localization sequence (NLS), for example said NLS comprises an amino acid sequence set forth in SEQ ID NO: 30 or 31.

41. The method according to claim 24, wherein the base editing fusion protein has two NLSs, and the two NLSs are located at the N-terminus and C-terminus, respectively.

42. The method according to claim 24, wherein said base editing fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 22 or 23.

43. The method according to claim 24, wherein the nucleotide sequence encoding said base editing fusion protein is codon optimized for plants to be base edited, for example, said nucleotide sequence encoding said base editing fusion protein is set forth in SEQ ID NO: 19 or 20.

44. The method according to claim 24, wherein said guide RNA is a single guide RNA (sgRNA).

45. The method according to claim 24, wherein the nucleotide sequence encoding said base editing fusion protein and/or the nucleotide sequence encoding said guide RNA are operably linked to an expression regulatory element for the plant.

46. The method according to claim 45, wherein said expression regulatory element is a promoter, for example a 35S promoter, a maize Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, or a maize U3 promoter.

Description

DESCRIPTION OF DRAWINGS

[0015] FIG. 1A-1E. Establishment of a targeted-Plant Base Editing (PBE) mediated genome editing system in plant protoplasts by a BFP-to-GFP reporter system. FIG. 1A Schematic representation of n/dCas9-PBE expression vectors. APOBEC1, n/dCas9, XTEN, and UGI are all codon-optimized for wheat, and NLS is attached to both ends of n/dCas9-PBE. The fusion protein is driven by a maize Ubiquitin-1 promoter. FIG. 1B Diagram of the BFP-to-GFP reporter system for detecting Targeted-PBE mediated precision genome editing in plant protoplasts. FIG. 1C Measurement of Targeted-PBE mediated BFP to GFP mutation efficiency in wheat and maize protoplasts by flow cytometry. Four fields of protoplasts are shown. Protoplasts were transformed with the following DNA constructs (from left to right): (i) pnCas9-PBE, pUbi-BFPm, and pBFP-sgRNA; (ii) pdCas9-PBE, pUbi-BFPm and pBFP-sgRNA; (iii) only pUbi-BFPm and BFP-sgRNA; (iv) positive control pUbi-GFP for GFP expression, which is driven by the maize Ubiquitin-1 promoter. Scale bars, 800 μm. FIG. 1D The genotype and frequency of BFP target site induced by Targeted-PBE system. In the sequence, the target base is C located at the fourth position. The number underneath represents the location thereof in the target sequence. Following each sequence are the percentages of the corresponding base relative to total sequencing reads FIG. 1E. Three adjacent C (C3, C6, and C9) are also converted to T, but with no alteration to the protein sequence.

[0016] FIG. 2A-2D. The Targeted-PBE system mediates point mutation of endogenous genes in plant protoplasts. FIG. 2A Frequency of targeted single C to T substitution. The values and error bars reflect means and standard deviations (s.d.) of three biologically replicates on different days. FIG. 2B Frequency of multiple Cytidine substitution created by nCas9-PBE, based on the number of mutant reads relative to the total number of captured reads. “--” indicates not available. FIG. 2C Frequency of indels formation for seven target sites of endogenous genes in plant protoplasts. The percentage of total DNA sequencing reads with T or indels at the target positions indicated are shown for treatment with nCas9-PBE, dCas9-PBE, or wild type Cas9. The values and error bars reflect the mean and s.d. for three biological replicates performed on different days. FIG. 2D Spectrum of point mutations created by using nCas9-PBE in the CENP-A targeting domain (CATD) of ZmCENH3. The three consecutive residues, alanine-leucine-leucine (ALL), in wild type ZmCENH3 were mutated into AFL, VLL, ALF, AFF, VFL, VLF, or VFF. The CATD regions of Arabidopsis thaliana CENH3 (AtCENH3) and Hordeum vulgare L. CENH3 (HvβCENH3) were included in this figure to facilitate the comparison. The underlined leucine residue has previously been shown to be required for the proper function of AtCENH3 and HvCENH3. It has been found that the underlined leucine and alanine residues in AtCENH3 are substituted to result in haploid induction. The GenBank accession numbers for ZmCENH3, AtCENH3, and HvCENH3 are AF519807, AF465800 and JF419329, respectively.

[0017] FIG. 3A-3E. Genetically engineered wheat and rice plants obtained by application of the Targeted-PBE system. FIG. 3A Sequence of a sgRNA designed to target a site of exon 5 of TaLOX2. The targeted C in the deamination window is bold and italic. The PAM sequence is shown in italic, and the SalI restriction site is GTCGAC. FIG. 3B Results of T7E1 and PCR-RE assays analyzing two mutants with TaLOX2 point mutation. Lanes TO-1 to TO-12 show blots of PCR fragments amplified from independent wheat plants digested with T7E1 and SalI. Lanes labeled WT/D and WT/U are PCR fragments amplified from wild type plants digested with or without T7E1 and SalI, respectively. The bands marked by arrowheads are caused by Targeted-PBE-induced mutations. Genotypes of two mutants with point mutation were further identified by Sanger sequencing. FIG. 3C Schematic of the Agrobacterium expression vector pAG-n/dCas9-PBE, which is used for targeting the OsCDC48 gene. Hyg is driven by the 2×35S promoter. FIG. 3D Sequence of an sgRNA designed to target exon 9 of OsCDC48. The results of T7E1 assays analyzing twelve representative mutants with OsCDC48 point mutation are shown. FIG. 3E Genotype and frequency of rice mutants with OsCDC48 point mutation identified through Sanger sequencing. The frequency of point mutation for each genotype (each genotype mutant vs. rice mutants in total) is shown at right. Target bases are C.sub.3, C.sub.4, C.sub.7, and C.sub.8, the subscripted number represents its location in the target sequence. The sequences below are the sequencing results for rice mutants.

[0018] FIG. 4A-4B. Genotypes of 12 representative mutants with OsCDC48 point mutation identified through Sanger sequencing FIG. 4A. Italic and bold base C represents the Cs to be mutated at position 3, 4, 7, and 8 of the deamination window FIG. 4A. t represents successful C to T substitution in the target sites FIG. 4B.

[0019] FIG. 5A-5B. Base editing of ZmALS1/ZmALS2 through nCas9-PBE system. FIG. 5A Genome structure of ZmALS1/ZmALS2 and the common sgRNA target sequence; FIG. 5B detecting C to T substitution types of ZmALS1/ZmALS2 in different nCas9-PBE modified lines.

DETAILED DESCRIPTION OF THE INVENTION

1. Definition

[0020] In the present invention, unless indicated otherwise, the scientific and technological terminologies used herein refer to meanings commonly understood by a person skilled in the art. Also, the terminologies and experimental procedures used herein relating to protein and nucleotide chemistry, molecular biology, cell and tissue cultivation, microbiology, immunology, all belong to terminologies and conventional methods generally used in the art. For example, the standard DNA recombination and molecular cloning technology used herein are well known to a person skilled in the art, and are described in details in the following references: Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter refers to as “Sambrook et al”). In the meantime, in order to better understand the present invention, definitions and explanations for the relevant terminologies are provided below.

[0021] “Cas9 nuclease” and “Cas9” can be used interchangeably herein, which refer to a RNA directed nuclease, including the Cas9 protein or fragments thereof (such as a protein comprising an active DNA cleavage domain of Cas9 and/or a gRNA binding domain of Cas9). Cas9 is a component of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats and its associated system) genome editing system, which targets and cleaves a DNA target sequence to form a DNA double strand breaks (DSB) under the guidance of a guide RNA.

[0022] “guide RNA” and “gRNA” can be used interchangeably herein, which typically are composed of crRNA and tracrRNA molecules forming complexes through partial complement, wherein crRNA comprises a sequence that is sufficiently complementary to a target sequence for hybridization and directs the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single guide RNA (sgRNA) can be designed, which comprises the characteristics of both crRNA and tracrRNA.

[0023] “Deaminase” refers to an enzyme that catalyzes the deamination reaction. In some embodiments of the present invention, the deaminase refers to a cytidine deaminase, which catalyzes the deamination of a cytidine or a deoxycytidine to a uracil or a deoxyuridine, respectively.

[0024] “Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.

[0025] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.

[0026] Plant parts include harvestable parts and parts useful for propagation of progeny plants. Plant parts useful for propagation include, for example and without limitation: seed; fruit; a cutting; a seedling; a tuber; and a rootstock. A harvestable part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

[0027] A plant cell is the structural and physiological unit of the plant, and includes protoplast cells without a cell wall and plant cells with a cell wall. A plant cell may be in the form of an isolated single cell, or an aggregate of cells (e.g., a friable callus and a cultured cell), and may be part of a higher organized unit (e.g., a plant tissue, plant organ, and plant). Thus, a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a “plant cell” in embodiments herein.

[0028] The term “protoplast”, as used herein, refers to a plant cell that had its cell wall completely or partially removed, with the lipid bilayer membrane thereof naked, and thus includes protoplasts, which have their cell wall entirely removed, and spheroplasts, which have their cell wall only partially removed, but is not limited thereto. Typically, a protoplast is an isolated plant cell without cell walls which has the potency for regeneration into cell culture or a whole plant.

[0029] “Progeny” of a plant comprises any subsequent generation of the plant.

[0030] A “genetically modified plant” includes a plant which comprises within its genome an exogenous polynucleotide. For example, the exogenous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression regulatory sequence means that, in the plant genome, said sequence comprises one or more nucleotide substitution, deletion, or addition. For example, a genetically modified plant obtained by the present invention may contain one or more C to T substitutions relative to the wild type plant (corresponding plant that is not genetically modified).

[0031] The term “exogenous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

[0032] “Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid fragment” are used interchangeably to refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

[0033] “Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0034] As used herein, an “expression construct” refers to a vector suitable for expression of a nucleotide sequence of interest in a plant, such as a recombinant vector. “Expression” refers to the production of a functional product. For example, the expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (such as transcribe to produce an mRNA or a functional RNA) and/or translation of RNA into a protein precursor or a mature protein.

[0035] “Expression construct” of the invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, an RNA that can be translated (such as an mRNA).

[0036] “Expression construct” of the invention may comprise regulatory sequences and nucleotide sequences of interest that are derived from different sources, or regulatory sequences and nucleotide sequences of interest derived from the same source, but arranged in a manner different than that normally found in nature.

[0037] “Regulatory sequence” or “regulatory element” are used interchangeably and refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. A plant expression regulatory element refers to a nucleotide sequence capable of controlling the transcription, RNA processing or stability or translation of a nucleotide sequence of interest in a plant.

[0038] Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0039] “Promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment. In some embodiments of the invention, the promoter is a promoter capable of controlling gene transcription in a plant cell whether or not its origin is from a plant cell. The promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0040] “Constitutive promoter” refers to a promoter that generally causes gene expression in most cell types in most circumstances. “Tissue-specific promoter” and “tissue-preferred promoter” are used interchangeably, and refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell or cell type. “Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events. “Inducible promoter” selectively expresses a DNA sequence operably linked to it in response to an endogenous or exogenous stimulus (environment, hormones, or chemical signals, and so on).

[0041] As used herein, the term “operably linked” means that a regulatory element (for example but not limited to, a promoter sequence, a transcription termination sequence, and so on) is associated to a nucleic acid sequence (such as a coding sequence or an open reading frame), such that the transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0042] “Introduction” of a nucleic acid molecule (such as a plasmid, a linear nucleic acid fragment, RNA, and so on) or protein into a plant means transforming the plant cell with the nucleic acid or protein so that the nucleic acid or protein can function in the plant cell. “Transformation” as used herein includes stable transformation and transient transformation.

[0043] “Stable transformation” refers to introducing an exogenous nucleotide sequence into a plant genome, resulting in genetically stable inheritance. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the plant and any successive generations thereof.

[0044] “Transient transformation” refers to introducing a nucleic acid molecule or protein into a plant cell, performing its function without stable inheritance. In transient transformation, the exogenous nucleic acid sequence is not integrated into the plant genome.

[0045] “Trait” refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell. In some embodiments, the characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield. In some embodiments, trait also includes ploidy of a plant, such as haploidy which is important for plant breeding. In some embodiments, trait also includes resistance of a plant to herbicides.

[0046] “Agronomic trait” is a measurable parameter including but not limited to, leaf greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt tolerance, and tiller number and so on.

2. Base Editing System for Plants

[0047] The present invention provides a system for performing base editing to a target sequence in the genome of a plant, comprising at least one of the following (i) to (v):

[0048] i) a base editing fusion protein, and a guide RNA;

[0049] ii) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and a guide RNA;

[0050] iii) a base editing fusion protein, and an expression construction comprising a nucleotide sequence encoding a guide RNA;

[0051] iv) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

[0052] v) an expression construct comprising a nucleotide sequence encoding base editing fusion protein and a nucleotide sequence encoding guide RNA;

[0053] wherein said base editing fusion protein contains a nuclease-inactivated Cas9 domain and a deaminase domain, said guide RNA can target said base editing fusion protein to the target sequence in the plant genome.

[0054] The DNA cleavage domain of Cas9 nuclease is known to contain two subdomains: the HNH nuclease subdomain and the RuvC subdomain. HNH subdomains cleave the chain that is complementary to gRNA, whereas the RuvC subdomain cleaves the non-complementary chain. Mutations in these subdomains can inactivate Cas9 nuclease to form “nuclease-inactivated Cas9”. The nuclease-inactivated Cas9 retains DNA binding capacity directed by gRNA. Thus, in principle, when fused with an additional protein, the nuclease-inactivated Cas9 can simply target said additional protein to almost any DNA sequence through co-expression with appropriate guide RNA.

[0055] Cytidine deaminase can catalyze the deamination of cytidine (C) in DNA to form uracil (U). If nuclease-inactivated Cas9 is fused with Cytidine deaminase, the fusion protein can target a target sequence in the genome of a plant through the direction of a guide RNA. The DNA double strand is not cleaved due to the loss of Cas9 nuclease activity, whereas the deaminase domain in the fusion protein is capable of converting the cytidine of the single-strand DNA produced during the formation of the Cas9-guide RNA-DNA complex into a U, and then C to T substitution may be achieved by base mismatch repair.

[0056] Therefore, in some embodiments of the invention, the deaminase is a cytidine deaminase, such as an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. Particularly, the deaminase described herein is a deaminase that can accept single-strand DNA as the substrate.

[0057] Examples of cytidine deaminase can be used in the present invention include but are not limited to APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, or CDA 1.

[0058] In some embodiments of the present invention, the cytidine deaminase comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0059] The nuclease-inactivated Cas9 of the present invention can be derived from Cas9 of different species, for example, derived from S. pyogenes Cas9 (SpCas9, the nucleotide sequence of which is shown in SEQ ID NO: 18; the amino acid sequence is shown in SEQ ID NO: 21). Mutations in both the HNH nuclease subdomain and the RuvC subdomain of the SpCas9 (includes, for example, D10A and H840A mutations) inactivate S. pyogenes Cas9 nuclease, resulting in a nuclease dead Cas9 (dCas9). Inactivation of one of the subdomains by mutation allows Cas9 to gain nickase activity, i.e., resulting in a Cas9 nickase (nCas9), for example, nCas9 with a D10A mutation only.

[0060] Therefore, in some embodiments of the invention, the nuclease-inactivated Cas9 of the invention comprises amino acid substitutions D10A and/or H840A relative to wild-type Cas9.

[0061] In some preferred embodiments of the invention, the nuclease-inactivated Cas9 of the invention has nickase activity. Without being bound by any theory, it is believed that Eukaryotic mismatch repair uses nicks on a DNA strand for the removal and repair of the mismatched base in the DNA strand. The U: G mismatch formed by cytidine deaminase may be repaired into C: G. Through the introduction of a nick on the chain containing unedited G, it will be possible to preferentially repair the U: G mismatch to the desired U:A or T:A. Therefore, preferably, the nuclease-inactivated Cas9 is a Cas9 nickase that retains the cleavage activity of the HNH subdomain of Cas9, whereas the cleavage activity of the RuvC subdomain is inactivated. For example, the nuclease-inactivated Cas9 contains an amino acid substitution D10A relative to wild-type Cas9.

[0062] In some embodiments of the present invention, the nuclease-inactivated Cas9 comprises the amino acid sequence of SEQ ID NO:14. In some preferred embodiments, the nuclease-inactivated Cas9 comprises the amino acid sequence of SEQ ID NO: 13.

[0063] In some embodiments of the invention, the deaminase domain is fused to the N-terminus of the nuclease-inactivated Cas9 domain. In some embodiments, the deaminase domain is fused to the C-terminus of the nuclease-inactivated Cas9 domain.

[0064] In some embodiments of the invention, the deaminase domain and the nuclease inactivated Cas9 domain are fused through a linker. The linker can be a non-functional amino acid sequence having no secondary or higher structure, which is 1 to 50 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-25, 25-50) or more amino acids in length. For example, the linker may be a flexible linker, such as GGGGS (SEQ ID NO: 67), GS, GAP, (GGGGS)×3 (SEQ ID NO: 68), GGS, (GGS)×7 (SEQ ID NO: 69), and the like. In some preferred embodiments, the linker is an XTEN linker as shown in SEQ ID NO: 12.

[0065] In cells, uracil DNA glycosylase catalyzes the removal of U from DNA and initiates base excision repair (BER), which results in the repair of U: G to C: G. Therefore, without any theoretical limitation, including uracil DNA glycosylase inhibitor in the base editing fusion protein of the invention or the system of the present invention will be able to increase the efficiency of base editing.

[0066] Accordingly, in some embodiments of the invention, the base editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the uracil DNA glycosylase inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0067] In some embodiments of the invention, the base editing fusion protein of the invention further comprises a nuclear localization sequence (NLS). In general, one or more NLSs in the base editing fusion protein should have sufficient strength to drive the accumulation of the base editing fusion protein in the nucleus of a plant cell in an amount sufficient for the base editing function. In general, the strength of the nuclear localization activity is determined by the number and position of NLSs, and one or more specific NLSs used in the base editing fusion protein, or a combination thereof.

[0068] In some embodiments of the present invention, the NLSs of the base editing fusion protein of the invention may be located at the N-terminus and/or the C-terminus. In some embodiments, the base editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the base editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the N-terminus. In some embodiments, the base-editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the C-terminus. In some embodiments, the base editing fusion protein comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. Where there are more than one NLS, each NLS may be selected as independent from other NLSs. In some preferred embodiments of the invention, the base-editing fusion protein comprises two NLSs, for example, the two NLSs are located at the N-terminus and the C-terminus, respectively.

[0069] In general, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of a protein, but other types of NLS are also known in the art. Non-limiting examples of NLSs include KKRKV (SEQ ID NO: 70) (nucleotide sequence 5′-AAGAAGAGAAAGGTC-3′) (SEQ ID NO: 71), PKKKRKV (SEQ ID NO: 30) (nucleotide sequence 5′-CCCAAGAAGAAGAGGAAGGTG-3′ (SEQ ID NO: 72) or CCAAAGAAGAAGAGGAAGGTT) (SEQ ID NO: 73), or SGGSPKKKRKV (SEQ ID NO: 31) (nucleotide sequence 5′-TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG-3′) (SEQ ID NO: 74).

[0070] In some embodiments of the invention, the N-terminus of the base editing fusion protein comprises an NLS with an amino acid sequence shown by PKKKRKV (SEQ ID NO: 30). In some embodiments of the invention, the C-terminus of the base-editing fusion protein comprises an NLS with an amino acid sequence shown by SGGSPKKKRKV (SEQ ID NO: 31).

[0071] In addition, the base editing fusion protein of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, and the like, depending on the location of the DNA to be edited.

[0072] In some embodiments of the present invention, the base editing fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or 23.

[0073] In order to obtain efficient expression in plants, in some embodiments of the invention, the nucleotide sequence encoding the base editing fusion protein is codon optimized for the plant to be base edited.

[0074] Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the“Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y, et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).

[0075] In some embodiments of the invention, the codon-optimized nucleotide sequence encoding the base editing fusion protein is set forth in SEQ ID NO: 19 or 20.

[0076] In some embodiments of the invention, the guide RNA is a single guide RNA (sgRNA). Methods of constructing suitable sgRNAs according to a given target sequence are known in the art. See e.g., Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947-951 (2014); Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013); Liang, Z. et al. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics. 41, 63-68 (2014).

[0077] In some embodiments of the invention, the nucleotide sequence encoding the base-edited fusion protein and/or the nucleotide sequence encoding the guide RNA is operably linked to a plant expression regulatory element, such as a promoter.

[0078] Examples of promoters that can be used in the present invention include but not limited to the cauliflower mosaic virus 35S promoter (Odell et al. (1985) Nature 313: 810-812), a maize Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, a maize U3 promoter, a rice actin promoter, a TrpPro5 promoter (U.S. patent application Ser. No. 10/377,318; filed on Mar. 16, 2005), a pEMU promoter (Last et al. Theor. Appl. Genet. 81: 581-588), a MAS promoter (Velten et al. (1984) EMBO J. 3: 2723-2730), a maize H3 histone promoter (Lepetit et al. Mol. Gen. Genet. 231: 276-285 and Atanassova et al. (1992) Plant J. 2 (3): 291-300), and a Brassica napus ALS3 (PCT Application WO 97/41228) promoters. Promoters that can be used in the present invention also include the commonly used tissue specific promoters as reviewed in Moore et al. (2006) Plant J. 45 (4):

3. The Method of Producing a Genetically Modified Plant

[0079] In another aspect, the present invention provides a method of producing a genetically modified plant, comprising introducing a system for performing base editing to a target sequence in a plant genome of the invention into a plant, and thereby said base editing fusion protein is targeted to the target sequence in said plant genome by the guide RNA, and results in one or more C to T substitutions in said target sequence.

[0080] The design of the target sequence that can be recognized and targeted by a Cas9 and guide RNA complex is within the technical skills of one of ordinary skill in the art. In general, the target sequence is a sequence that is complementary to a leader sequence of about 20 nucleotides comprised in guide RNA, and the 3′-end of which is immediately adjacent to the protospacer adjacent motif (PAM) NGG.

[0081] For example, in some embodiments of the invention, the target sequence has the structure: 5′-N.sub.X-NGG-3′, wherein N is selected independently from A, G, C, and T; X is an integer of 14≤X≤30; N.sub.X represents X contiguous nucleotides, and NGG is a PAM sequence. In some specific embodiments of the invention, X is 20.

[0082] The base editing system of the present invention has a broad deamination window in plants, for example, a deamination window with a length of 7 nucleotides. In some embodiments of the methods of the invention, one or more C bases within positions 3 to 9 of the target sequence are substituted with Ts. For example, if present, any one, two, three, four, five, six, or seven Cs within positions 3 to 9 in the target sequence can be replaced with Ts. For example, if there are four Cs within positions 3 to 9 of the target sequence, any one, two, three, four Cs can be replaced by Ts. The C bases may be contiguous or separated by other nucleotides. Therefore, if there are multiple Cs in the target sequence, a variety of mutation combinations can be obtained by the method of the present invention. In some embodiments of the methods of the invention, further comprises screening plants having the desired nucleotide substitutions. Nucleotide substitutions in plants can be detected by T7EI, PCR/RE or sequencing methods, see e.g., Shan, Q., Wang, Y, Li, J. & Gao, C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat. Protoc. 9, 2395-2410 (2014).

[0083] In the methods of the invention, the base editing system can be introduced into plants by various methods well known to people skilled in the art. Methods that can be used to introduce the base editing system of the present invention into plants include but not limited to particle bombardment, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube approach, and ovary injection approach.

[0084] In the methods of the present invention, modification of the target sequence can be accomplished simply by introducing or producing the base editing fusion protein and guide RNA in plant cells, and the modification can be stably inherited without the need of stably transformation of plants with the base editing system. This avoids potential off-target effects of a stable base editing system, and also avoids the integration of exogenous nucleotide sequences into the plant genome, and thereby resulting in higher biosafety.

[0085] In some preferred embodiments, the introduction is performed in the absence of a selective pressure, thereby avoiding the integration of exogenous nucleotide sequences in the plant genome.

[0086] In some embodiments, the introduction comprises transforming the base editing system of the invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into an intact plant. Preferably, the regeneration is performed in the absence of a selective pressure, i.e., no selective agent against the selective gene carried on the expression vector is used during the tissue culture. Without the use of a selective agent, the regeneration efficiency of the plant can be increased to obtain a modified plant that does not contain exogenous nucleotide sequences.

[0087] In other embodiments, the base editing system of the present invention can be transformed to a particular site on an intact plant, such as leaf, shoot tip, pollen tube, young ear, or hypocotyl. This is particularly suitable for the transformation of plants that are difficult to regenerate by tissue culture.

[0088] In some embodiments of the invention, proteins expressed in vitro and/or RNA molecules transcribed in vitro are directly transformed into the plant. The proteins and/or RNA molecules are capable of achieving base-editing in plant cells, and are subsequently degraded by the cells to avoid the integration of exogenous nucleotide sequences into the plant genome.

[0089] Plant that can be base-edited by the methods of the invention includes monocotyledon and dicotyledon. For example, the plant may be a crop plant such as wheat, rice, maize, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava, or potato.

[0090] In some embodiments of the invention, the target sequence is associated with plant traits such as agronomic traits, and thereby the base editing results in the plant having altered traits relative to a wild type plant.

[0091] In the present invention, the target sequence to be modified may be located anywhere in the genome, for example, within a functional gene such as a protein-coding gene or, for example, may be located in a gene expression regulatory region such as a promoter region or an enhancer region, and thereby accomplish the functional modification of said gene or accomplish the modification of a gene expression. Accordingly, in some embodiments of the invention, C to T substitution(s) results in amino acid substitutions in a target protein or the truncation of a target protein (resulting in a stop codon). In other embodiments of the invention, C to T substitution(s) results in a change in the expression of a target gene.

[0092] In some embodiments, the gene modified by the methods of the invention can be wheat LOX2, rice CDC48, NRT1.1B and SPL14, maize CENH3, and ALS gene.

[0093] In some embodiments of the invention, the method further comprises obtaining progeny of the genetically modified plant.

[0094] In a further aspect, the invention also provides a genetically modified plant or progeny thereof or parts thereof, wherein the plant is obtained by the method of the invention described above.

[0095] In another aspect, the present invention also provides a plant breeding method comprising crossing a first genetically modified plant obtained by the above-mentioned method of the present invention with a second plant not containing said genetic modification, thereby introducing said genetic modification into said second plant.

4. Production Method of Maize Haploid Plants and the Use Thereof

[0096] CENH3 encodes centromeric histones, which are essential for the normal functioning of centromeres in animals and plants. TILLING studies have shown that amino acid substitutions of several residues in the C-terminal region of Arabidopsis CENH3 (AtCENH3) can result in haploid induction, which is advantageous for accelerating crop breeding. Substitution of a highly conserved leucine residue in the CENP-A targeting domain (CATD, FIG. 2d) of the plant CENH3 protein with a phenylalanine (F) results in a haploid induction in Arabidopsis, whereas in barley, although the loading of CENH3 into centromere is impaired, no haploid induction is occurred. The present inventors found that the mutation of the CATD domain in ZmCENH3 by the base editing method of the present invention, particularly the mutation of the conserved leucine residue, can be used for the induction of maize haploid, and can be widely used in the improvement of maize varieties.

[0097] The present invention provides a method of producing a maize haploid inducer line comprising modifying the ZmCENH3 gene in a maize plant by the base editing method of the invention, resulting in one or more amino acid substitutions in the CATD domain in ZmCENH3, the one or more amino acid substitutions confer a haploid inducer activity to the maize plant.

[0098] In a specific embodiment, the modification results in one or more amino acid substitutions in the conserved motif alanine-leucine-leucine (ALL) at positions 109-111 in ZmCENH3 of SEQ ID NO: 25. For example, the ALL motif is modified as single substitution: AFL, VLL, or ALF; double substitution: AFF, VFL, or VLF; or triple substitution: VFF.

[0099] In some embodiments, the target sequence for modification of ZmCENH3 by the base editing method of the invention is AGCCCTCCTTGCGCTGCAAGAGG (SEQ ID NO: 75), wherein the underlined sequence is a PAM sequence.

[0100] In some embodiments, the maize plant is a Zong31 variety. In some embodiments, the maize plant is a HiII variety.

[0101] The present invention provides a method of producing maize haploid comprising crossing a maize haploid inducer line obtained by the method of the present invention with a wild-type maize plant, and harvesting the hybrid progeny to obtain a maize haploid plant. In some embodiments, the maize haploid inducer line is used as a male parent and the wild-type maize plant is used as a maternal parent for cross.

[0102] The present invention also encompasses the maize haploid inducer line and the maize haploid plant obtained by the methods of the invention and their use in maize breeding.

5. Methods of Producing Herbicide-Resistant Maize Plants

[0103] The present invention provides a method of producing a herbicide-resistant maize plant comprising modifying the ZmALS gene (encoding acetolactate synthase) in a maize plant by the base editing method of the invention, resulting in one or more amino acid substitutions in ZmALS, said one or more amino acid substitutions confer herbicide resistance to the maize plant.

[0104] In a specific embodiment, the modification simultaneously results in one or more amino acid substitutions of both ZmALS1 of SEQ ID NO: 27 and ZmALS2 of SEQ ID NO: 29. For example, the 165th residues of ZmALS1 and ZmALS2 are substituted.

[0105] In some embodiments, the target sequence for the modification of ZmALS1 and ZmALS2 by the base editing methods of the present invention is CAGGTGCCGCGACGCATGATTGG (SEQ ID NO: 56), wherein the underlined sequence is a PAM sequence.

[0106] In some embodiments, the maize plant is a Zong31 variety. In some embodiments, the maize plant is a HiII variety.

[0107] The present invention also provides a method of breeding a herbicide-resistant maize plant, comprising crossing a first herbicide-resistant maize plant obtained by the above-described method of the present invention with a second plant so as to introduce the herbicide resistance into the second plant.

[0108] The present invention also encompasses herbicide-resistant maize plants or progeny thereof obtained by the methods of the invention.

[0109] A method of controlling undesired plants in a maize plant growing area, comprising applying an ALS inhibitor herbicide to the plants in the area, wherein the maize plant is a herbicide-resistant maize plant obtained by the method of the invention.

Examples

Materials and Methods

[0110] Construction of pn/dCas9-PBE Expression Vector

[0111] The APOBEC1, XTEN, nCas9(D10A), dCas9, and UGI sequences were codon-optimized for wheat (SEQ ID NO:1-5) and ordered from GenScript (Nanjing). The full length n/dCas9 fragment was amplified using the primer set AflII-F (with AflII restriction site) and MluI-R (with MluI restriction site). The PCR products were digested with AflII and MluI, and then inserted into the both enzymes-digested pUC57-APOBEC1-XTEN-UGI vector (the sequence of the vector is set forth in SEQ ID NO: 10) to generate the fusion cloning vector pUC57-APOBECI-XTEN-n/dCas9-UGI. Then the primer set BamHI-F and Bsp1047I-R was used to amplify the fragment of APOBECI-XTEN-n/dCas9-UGI. Products were digested with BamHI and Bsp1047I, and further inserted into BamHI and Bsp1047I-digested pUbi-GFP (the sequence of the vector is set forth in SEQ ID NO: 8) to generate the fusion expression vector pn/dCas9-PBE.

Construction of sgRNA Expression Vectors

[0112] sgRNA target sequences in the experiment are shown in the Table 1 below:

TABLE-US-00001 TABLE 1 Target genes and gRNA target sequences SEQ SEQ SEQ ID ID ID sgRNA NO: target sequence NO: Oligo-F NO: Oligo-R sgRNA- 77 ACCCACGGCGTG  84 GGCAACCC  94 AAACAA OsBFPm CAGTGCTTCGG ACGG GCACTG CGTGCAGTG CACGCC CTT GTGGGT sgRNA- 78 ACCCACGGCGTG  85 CTTGACCCA  94 AAACAA TaBFPm CAGTGCTTCGG CGG GCACTG CGTGCAGTG CACGCC CTT GTGGGT sgRNA- 79 ACCCACGGCGTG  86 AGCAACCC  94 AAACAA ZmBFPm CAGTGCTTCGG ACGG GCACTG CGTGCAGTG CACGCC CTT GTGGGT sgRNA- 61 GACCAGCCAGCG  87 GGCAGACC  95 AAACGC OsCDC48 TCTGGCGCCGG AGCC GCCAGA AGCGTCTGG CGCTGG CGC CTGGTC sgRNA- 80 CGGCGACGGCGA  88 GGCACGGC  96 AAACCC OsNRT1.1B GCAAGTGGAGG GACG ACTTGC GCGAGCAA TCGCCGT GTGG CGCCG sgRNA- 81 CTCTTCTGTCAAC  89 GGCACTCTT  97 AAACTG OsSPL14 CCAGCCATGG CTG GCTGGG TCAACCCAG TTGACA CCA GAAGAG sgRNA- 65 GTCGACATCAAC  90 CTTGGTCGA  98 AAACTC TaLOX2-S1 AACCTCGACGG CAT GAGGTT CAACAACCT GTTGAT CGA GTCGAC sgRNA- 82 CTTCCTGGGCTAC  91 CTTGCTTCC  99 AAACTG TaLOX2-S2 ACGCTCAAGG TGG AGCGTG GCTACACGC TAGCCC TCA AGGAAG sgRNA- 83 AAGGACCTCATC  92 CTTGAAGGA 100 AAACCC TaLOX2-S3 CCCATGGGCGG CCT CATGGG CATCCCCAT GATGAG GGG GTCCTT sgRNA- 75 AGCCCTCCTTGCG 93 AGCAAGCC 101 AAACCT ZmCENH3 CTGCAAGAGG CTCC TGCAGC TTGCGCTGC GCAAGG AAG AGGGCT The italic Cs represent Cs to be mutated in the deamination window from positions 3-9; bold letters represents PAM sequence.

[0113] According to the previous description (Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947-951,2014; Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688, 2013; and Liang, Z. et al. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics. 41, 63-68, 2014), sgRNA expression vectors were constructed based on pTaU6-sgRNA (Addgene ID53062), pOsU3-sgRNA (Addgene ID53063), or pZmU3-sgRNA (Addgene ID53061):

[0114] pTaU6-BFP-sgRNA, pOsU3-BFP-sgRNA, pZmU3-BFP-sgRNA, pTaU6-LOX2-S1-sgRNA, pTaU6-LOX2-S2-sgRNA, pTaU6-LOX2-S3-sgRNA, pOsU3-CDC48-sgRNA, pOsU3-NRT1.1-sgRNA, pOsU3-SPL14-sgRNA, and pZmU3-CENH3-sgRNA.

BFP and GFP Expression Vectors

[0115] The sequence of pUbi-BFPm is set forth in SEQ ID NO: 9. The amino acid sequence of BFP is set forth in SEQ ID NO: 17.

[0116] The sequence of pUbi-GFP is set forth in SEQ ID NO: 8. The amino acid sequence of GFP is set forth in SEQ ID NO: 16.

Construction of pAG-n/dCas9-PBE-CDC48-sgRNA Expression Vector

[0117] The APOBECI-XTEN-d/nCas9-UGI fragment was fused to the StuI and SacI-digested pHUE411 (Addgen #62203) with primer set Gibson-F and Gibson-R through Gibson cloning method, generating the vector pHUE411-APOBECI-XTEN-d/nCas9-UGI without sgRNA target sites. Paired oligonucleotides (oligos) comprising OsCDC48 targeting sequences were synthesized, annealed and cloned into BsaI-digested pHUN411 vector, obtaining the pHUE411-sgRNA-CDC48. Then the fragment resulted from the digestion of pHUE411-sgRNA-CDC48 with PmeI and AvrII was inserted into pHUE411-APOBECI-XTEN-d/nCas9-UGI which was also digested with PmeI and AvrII, to finally obtain the Agrobacterium-mediated transformation vector pAG-n/dCas9-PBE-CDC48-sgRNA.

Protoplast Assays

[0118] Wheat Bobwhite variety, rice Nipponbare, and maize inbred line variety Zong31 were used in this study. Protoplasts transformation is performed as described below. The average transformation efficiency is 55-70%. Transformation is carried out with 10 μg of each plasmid by PEG-mediated transfection. Protoplasts were collected after 48 h and DNA was extracted for T7E1 and PCR-RE assay.

Preparation and Transformation of Wheat (Maize) Protoplasts

[0119] 1) The middle parts of wheat (maize) tender leaves were cut into strips of 0.5-1 mm in width. The strips were placed into 0.6M Mannitol solution for 10 minutes, filtered, and then placed in 50 ml enzyme solution 20-25° C. in darkness, with gently shaking (10 rmp) for 5 hours.

[0120] 2) 10 ml W5 was added to dilute the enzymolysis products and the products were filtered with a 75 μm nylon filter in a round bottom centrifuge tube (50 ml).

[0121] 3) 23° C. 100 g centrifugation for 3 min, and the supernatant was discarded.

[0122] 4) The products were gently suspended with 10 ml W5, placed on the ice for 30 min to allow the protoplasts gradually settling, and the supernatant was discarded.

[0123] 5) Protoplasts were suspended by adding an appropriate amount of MMG, placed on ice until transformation.

[0124] 6) 10-20 μg plasmid, 200 μl protoplasts (about 4×10.sup.5 cells), 220 μl fresh PEG solution were added into a 2 ml centrifuge tube, mixed up, and placed under room temperature in darkness for 10-20 minutes to induce transformation.

[0125] 7) After the induction of transformation, 880 μl W5 solution was slowly added, and the tubes were gently turned upside down for mixing, then 100 g horizontal centrifuged for 3 min, and the supernatant was discarded.

[0126] 8) The products were resuspended in 2 ml W5 solution, transfered to a six-well plate, cultivated under room temperature (or 25° C.) in darkness. For protoplast genomic DNA extraction, the products need to be cultivated for 48 h.

Preparation and Transformation of Rice Protoplast

[0127] 1) Leaf sheath of the seedlings were used for protoplasts isolation, and cut into about 0.5 mm wide with a sharp blade.

[0128] 2) Immediately after incision, transfered into 0.6M Mannitol solution, and placed in the dark for 10 min.

[0129] 3) Mannitol solution was removed by filtration, and the products were transfered into enzymolysis solution, and evacuated for 30 min.

[0130] 4) Enzymolysis was performed for 5-6 h in darkness with gently shaking (decolorization shaker, speed 10).

[0131] 5) After enzymolysis completion, an equal volume of W5 was added, horizontal shake for 10 s to release protoplasts.

[0132] 6) Protoplasts were filtered into a 50 ml round bottom centrifuge tube with a 40 μm nylon membrane and washed with W5 solution.

[0133] 7) 250 g horizontal centrifugation for 3 min to precipitate the protoplasts, the supernatant was discarded.

[0134] 8) Protoplasts were resuspended by adding 10 ml W5, and then centrifuged at 250 g for 3 min, and the supernatant was discarded.

[0135] 9) An appropriate amount of MMG solution was added to resuspend the protoplasts to a concentration of 2×10.sup.6/ml.

[0136] Note: All the above steps were carried out at room temperature.

[0137] 10) 10-20 μg plasmid, 200 μl protoplasts (about 4×10.sup.5 cells), and 220 μl fresh PEG solution were added into a 2 ml centrifugal tube, mixed, and placed at room temperature in darkness for 10-20 minutes to induce transformation.

[0138] 11) After the completion of the transformation, 880 μl W5 solution was slowly added, and the tubes were gently turned upside down for mixing, 250 g horizontal centrifuged for 3 min, and the supernatant was discarded.

[0139] 12) The products were resuspended in 2 ml WI solution, transfered to a six-well plate, cultivated in room temperature (or 25° C.) in darkness. For protoplast genomic DNA extraction, the products need to be cultivated for 48 h.

Transformation of DNA Constructs into Wheat Calli by Particle Bombardment

[0140] Plasmid DNA (pnCas9-PBE and pTaU6-LOX2-S1-sgRNA) was used to bombard Bobwhite immature embryos. Particle bombardment transformation was performed as described previously (Zhang, K., Liu, J., Zhang, Y, Yang, Z. & Gao, C. Biolistic genetic transformation of a wide range of Chinese elite wheat (Triticum aestivum L.) varieties. J. Genet Genomics 42, 39-42 (2015)). After bombardment, the embryos are treated according to the literature, but no selection agents were used during tissue culture.

Transformation of pAG-n/dCas9-PBE-CDC48-sgRNA into Rice Calli by Agrobacterium

[0141] pAG-n/dCas9-PBE-CDC48-sgRNA binary vector was transformed into Agrobacterium AGL1 strain by electroporation. Agrobacterium-mediated transformation, tissue culture, and regeneration of rice Nipponbare were performed according to Shan et al. (Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013)). Hygromycin (50 μg/ml) was used in all the subsequent tissue culture process for screen.

Identification of Mutations by T7EI and PCR/RE assay

[0142] DNA from individual rice plant was extracted to detect the mutations by T7EI assay, and then mutations were confirmed by Sanger sequencing. In wheat, in order to save costs and labor, 3-4 plants were randomly selected as a group to detect mutations by T7EI and PCR/RE. Once a group showed a positive result, all the plants in the group were further tested by T7EI and PCR/RE, and then the results were confirmed by Sanger sequencing.

[0143] T7EI detect method:

[0144] 1) Genomic DNA was extracted from a plant, amplified by PCR and detected by electrophoresis.

[0145] 2) PCR products were added into T7EI buffer as follows:

TABLE-US-00002 10 × T7EI buffer 1.1 μl PCR product 5 μl ddH.sub.2O 4.4 μl

[0146] 3) The mixture was heated in the PCR device to 95° C. for 5 min, and then cooled to room temperature such that the PCR products re-anneal to form heteroduplex DNA.

[0147] 4) T7EI endonuclease was added as follows:

TABLE-US-00003 Anneal product in 3) 10.5 μl T7EI, 5 units/μl 0.5 μl Total volume 11 μl

[0148] 5) 37° C. for 1 h, all 11 μl products was subjected to gel electrophoresis. Cleavage of the PCR products indicates that the products contain indel mutations.

[0149] PCR/RE:

[0150] 1) Plant genomic DNA was extracted.

[0151] 2) Fragments containing the target sites, the length of which is between 350-1000 bp, were amplified with synthetic gene-specific primers:

TABLE-US-00004 10 × EasyTaq Buffer 5 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 2 μl Forward primer (10 μM) 2 μl Easy Taq 0.5 μl DNA 2 μl ddH.sub.2O To 50 μl

[0152] 3) The general reaction conditions are: denaturation at 94° C. for 5 min; denaturation at 94° C. for 30 s; anneal at 58° C. for 30 s, extension at 72° C. for 30 s, amplification for 30 to 35 cycles; incubation at 72° C. for 5 min; incubation at 12° C. 5 μl PCR products were subjected to electrophoresis.

[0153] 4) PCR products were digested with restriction endonuclease as follows:

TABLE-US-00005 10 × Fastdigest Buffer 2 μl Restriction enzymes 1 μl PCR product 3-5 μl ddH.sub.2O To 20 μl

[0154] 5) Digestion at 37° C. for 2-3 h. Products were analyzed by 1.2% agarose gel electrophoresis.

[0155] 6) The uncut mutant bands in the PCR products were recovered and purified, and subjected to TA cloning as follows:

TABLE-US-00006 pEasy-T Vector 1 μl Recovered uncleaved PCR product 4 μl

[0156] 7) The ligation was performed at 22° C. for 12 min. And the products were transformed into E. coli competent cells, which were then plated on LB plates (Amp100, IPTG, and X-gal), incubated at 22° C. for 12-16 h. White colonies were picked for identifying positive clones and sequencing.

In-depth Sequencing

[0157] Different sgRNA expression vectors in combination with pnCas9-PBE, pdCas9-PBE, and pwCas9 respectively were transformed into wheat, rice, or maize protoplasts for 48 hours. After that, protoplasts were collected and DNA was extracted for in-depth sequencing. In the first round of PCRs, the target regions were amplified with site-specific primers (Table 5). In the second round of PCRs, forward and reverse tags were added to the end of the PCR products for library construction (Table 5). Equal amount of different PCR products were pooled. The samples were then sequenced with Illumina High-Seq 4000 at Beijing Genomics Institute.

Example 1. Base Editing of BFP in Plant Protoplasts by nCas9-PBE and dCas9-PBE

[0158] In the nCas9-PBE fusion protein, from N-terminal to C-terminal are NLD (SEQ ID NO: 30), APOBEC1 (SEQ ID NO: 11), XTEN linker (SEQ ID NO: 12), Cas9 nickase (nCas9, SEQ ID NO: 13), uracil DNA glycosylase inhibitors (UGI, SEQ ID NO: 15) and NLS (SEQ ID NO: 31) respectively; whereas in the dCas9-PBE fusion protein, from N-terminal to C-terminal are NLS, APOBEC1, XTEN linkers, catalytically deactivated Cas9 (dCas9, SEQ ID NO: 14), UGI, and NLS, respectively. Codon-optimized fusion protein coding sequences for efficient expression in cereal crops were placed downstream of the Ubiquitin-1 gene promoter Ubi-1 in the plasmid constructs pnCas9-PBE and pdCas9-PBE (FIG. 1a).

[0159] The ability to convert blue fluorescent protein (BFP) into green fluorescent protein (GFP) of the two constructs in wheat and rice protoplasts was compared. This conversion involves mutating the first nucleotide of the 66th codon of the BFP encoding gene from C to T, resulting in alteration of CAC (histidine) to TAC (tyrosine).

[0160] The target region of BFP-specific sgRNA is designed to cover codons ranging from codon 65 to 71 and the first two nucleotides of the codon 72, and the last three bases (CAG) constitute the protospacer adjacent motif (PAM) (FIG. 1b). The C base to be mutated is located at position 4 of the target sequence. The sgRNAs are transcribed using the promoters TaU6 and OsU3, respectively (FIG. 1b). The construction of the sgRNA expression vectors pTaU6-BFP-sgRNA and pOsU3-BFP-sgRNA is described above.

[0161] Because CAG is not the optimal PAM for CRISPR/Cas9, CAG was artificially mutated into CGG, and the resulting BFP sequence (BFPm) was cloned downstream of the Ubi-1 promoter to form the expression construct pUbi-BFPm (FIG. 1b) as the target to be edited in protoplasts.

[0162] The combination of pnCas9-PBE, pUbi-BFPm, and pOsU3-BFP-sgRNA resulted in the expression of GFP in 5.8% of cells when introduced into rice protoplasts, whereas the replacement of pnCas9-PBE with pdCas9-PBE resulted in only 0.5% GFP-expressing cells; no GFP-expressing cell was detected when pnCas9-PBE or pdCas9-PBE were not included. 58.4% of cells expressed GFP (FIG. 1c) in parallel positive controls (cells were transformed with GFP-expressing construct pUbi-GFP).

[0163] In wheat protoplasts, more GFP-expressing cells were produced by using pnCas9-PBE (6.8%) than pdCas9-PBE (0.3%).

[0164] In-depth sequencing of rice (wheat or maize) protoplasts transformed with pnCas9-PBE, pUbi-BFPm, and pOsU3-BFP-sgRNA (pTaU6-BFP-sgRNA or pZmU3-BFP-sgRNA) showed that about 4.00% of the total DNA reads carrying C to T mutation (FIG. 1d). Mutation only occurred to C bases at position 3, 4, 6, and 9 in the protospace sequence (target sequence), and the mutation frequencies are 2.48-3.92%, 3.06-3.79%, 5.86-8.75%, and 6.47-7.86% respectively (FIG. 1d). The desired C (position 4) was also mutated, although its mutation frequency is not the highest. When pdCas9-PBE is replaced with pnCas9-PBE, the C bases at the above positions were also mutated, but the mutation frequency (0.06-0.22%) is significantly lower than pnCas9-PBE (2.48-8.75%) (FIG. 1d).

[0165] Therefore, the results of the fluorescent protein reporter assay show that both nCas9-PBE and dCas9-PBE are able to convert C to T in the target region in wheat, rice, and maize protoplasts, and the deamination window encompasses position 3-9 of the protospace sequence (target sequence). And the activity of nCas9-PBE is stronger than that of dCas9-PBE.

Example 2. Base Editing of Endogenous Genes in Plant Protoplasts by nCas9-PBE and dCas9-PBE

[0166] In this example, the activity of pnCas9-PBE or pdCas9-PBE on wheat, rice, and maize endogenous genes was further studied. As a control for the induction of indel, a construct pwCas9 (Addgene ID53064) expressing wild-type Cas9 was also used in this experiment.

[0167] Three different sgRNA target sites (S1, S2, and S3) were designed in the TaLOX2 gene of wheat. For each of the three rice genes OsCDC48, OsNRT1.1B, and OsSPL14, one sgRNA target site was designed. One sgRNA target site was designed in maize ZmCENH3 gene (see Table 1).

[0168] Each sgRNA expression construct was combined with pnCas9-PBE, pdCas9-PBE, and pwCas9, respectively, and co-expressed in wheat, rice, and maize protoplasts, respectively. Protoplast DNA was extracted. PCR amplicons for seven different targets were prepared and sequenced. 100000 to 270000 sequencing reads were used for detailed analysis of the mutagenicity.

[0169] For pnCas9-PBE, C to T transitions were observed in all 7 targets, and the deamination window encompasses positions 3-6 of the protospace sequence (target sequence) (FIG. 2a). The frequency of a single substitution of C to T is 0.57% to 7.07%, the C base at position 7 or near position 7 has the highest substitution frequency, and the editing event is independent of the sequence structure (FIG. 2a). The frequency of multiple C editing (including 2 to 5 Cs) is between 0.31% and 12.48%, while the frequency of editing two or three Cs is higher (FIG. 2b). The pnCas9-PBE-induced indel frequency is very low (0.01%-0.34%) at 7 target sites compared to pwCas9-induced indel frequency (6.27%-11.68%) (FIG. 2c and Table 3).

[0170] On the other hand, the expression of pdCas9-PBE produced only a low frequency of single C-editing (<0.96%) or multiple Cs-editing (<1.29%) at 7 target sites (FIG. 2a and Table 2), whereas the frequency of the appearance of indel (<0.06%) is comparable to pnCas9-PBE (FIG. 2c and Table 3). It is clear that the results of this experiment verify the previous result in reporter gene assays that nCas9-PBE is superior over dCas9-PBE, and verify the size of the deamination window in cereal plant cells (a 7 nucleotides window at positions 3-9). It also verifies that nCas9-PBE does not rely on target site context sequences to edit a single C and multiple Cs in high efficiency.

[0171] Using ZmCENH3 as an example, the amino acid mutation caused by nCas9-PBE in the target genome region was analyzed. As shown in FIG. 2d, in the wild-type ZmCENH3, the conserved residue is leucine residue located in the middle of an alanine-leucine-leucine (ALL, residues 109-111) segment. In the amplicons of ZmCENH3 target site obtained from the protoplasts treated with nCas-PBE, it is easy to identify A109V, L110V, and/or L111F substitutions (FIG. 2d). The ability of nCas9-PBE to edit a single C and multiple Cs allows a total of 7 different types of substitution to ALL, including a single substitution for each residue (AFL, VLL, and ALF), double substitutions of two residues (AFF, VFL, and VLF) and triple substitutions of all three residues (VFF) (FIG. 2d). Substitution event ZmCENH3-AFL has been studied in Arabidopsis and barley, but the remaining single, double, and triple substitutions events have not been reported in any research previously.

TABLE-US-00007 TABLE 2 Efficiency of dCas9-PBE treatment induced multiple C substitution 2 Cs 3 Cs 4 Cs 5 Cs Total Mutant Mutagenesis Mutant Mutagenesis Mutant Mutagenesis Mutant Mutagenesis Gene Name reads reads frequency reads frequency reads frequency reads frequency SgRNA-TaLOX2-s1 197064  72 0.04% 4 Mutant — — — — SgRNA-TaLOX2-s2 149200  420 0.28% — reads — — — — SgRNA-TaLOX2-s3  96396 1244 1.29% — — — — — — SgRNA-OsCDC48 154760  40 0.03% 0 0.00% 0 0.00% — — SgRNA-OsNRT1.1B 142130   0 0.00% — — — — — — SgRNA-OsSPL14  96659  36 0.04% — — — — — — SgRNA-ZmCENH3 275466  36 0.01% 0 0.00% 0 0.00% 0 0.00% “—” represents not applicable; .sup.a The number of mutant reads relative to the number of total reads.

TABLE-US-00008 TABLE 3 Efficiency of Indel Induction nCas9-PBE dCas9-PBE wCas9 Untreated TaLOX2-S1 0.01 0.01 11.68 0.01 TaLOX2-S2 0.03 0.02 6.92 0.02 TaLOX2-S3 0.04 0.03 7.10 0.06 OsCDC48 0.22 0.13 9.04 0.02 OsNRT1.1B 0.06 0.05 7.12 0.03 OsSPL14 0.08 0.14 7.34 0.04 ZmCENH3 0.02 0.01 6.27 0.01

Example 3. Analysis for Mutant Plants

[0172] This example further investigated the functional differences between nCas9-PBE and dCas9-PBE and tested the functional properties of nCas9-PBE by analyzing mutant plants.

[0173] For wheat transformation, pnCas9-PBE and pTaU6-sgRNA-LOX2-s1 (FIG. 3a) were co-transformed into a bread wheat variety, Bobwhite by particle bombardment, and plants were regenerated without herbicide selection. The target site of sgRNA-LOX2-s1 was amplified from the genomic DNA of the regenerated plants using specific primers (Table 4). Two lines carrying nucleotide substitutions were identified from 160 immature embryos by T7EI and restriction enzyme digestion (PCR-RE). The mutation efficiency is 1.25% (2/160) (FIG. 3b). Sanger sequencing confirmed that the mutant TO-3 contains all three C to T substitutions at positions 3, 6, and 9 downstream of the PAM, whereas the mutant TO-7 carries a C to T mutation at position 3 (FIG. 3b). We failed to detect indels in these two mutant plants.

[0174] Rice variety Nipponbare (Japonica) was used for rice transformation, because this variety is highly responsive to genetic manipulation and its whole genome sequence is available. The OsCDC48 gene, which has been found to regulate rice senescence and cell death, is adopted as a target (FIG. 3c and Table 1). The pAG-n/dCas9-PBE-CDC48-sgRNA binary vector was transformed into rice by Agrobacterium-mediated gene transfer method.

[0175] 92 and 87 independent transgenic TO plants were obtained for nCas9-PBE and dCas9-PBE, respectively. After T7EI analysis and Sanger sequencing, it is found that among the 92 nCas9-PBE plants, 40 of them carry at least one C to T substitution in the OsCDC48 target region, and the mutant production efficiency is 43.48% (40/92) (FIG. 3d). A representative sequencing result is shown in FIG. 4. Among the 40 mutants, mutations are detected only for C bases at positions 3, 4, 7, and 8 of the target region, and a total of seven different types of point mutations are identified (FIG. 3e). Specifically, there are four single nucleotide mutations (C3T, C4T, C7T, and C8T), two double nucleotide mutations (C3C4 to T3T4 and C7C8 to T7T8), and one triple nucleotide mutation (C3C4C7 to T3T4T7) (FIG. 3e). Therefore, nCas9-PBE encompasses 7 nucleotides (positions 3-9) in the deamination window in these rice mutants. The mutation frequency for each C ranges between 5.00% (C3, 2/40) to 32.50% (C7, 13/40) with the highest editing frequency for C at position 7. No indel is observed in the target region of the 40 mutants. Surprisingly, when using T7EI assay and Sanger sequencing analysis, no point mutation or indel was detected in the target region of OsCDC48 for the 87 dCas9-PBE plants.

[0176] The potential off-target effect of the base editing method of the present invention was investigated based on the obtained 40 strains of nCas-PBE mutants. Five possible off-target sites are found in the Nipponbare genomic sequence, each of them has three nucleotide mismatches with the sgRNA target region of OsCDC48 (Table 4). Amplicons of these 5 sites are carefully analyzed using T7EI assay and Sanger sequencing. No point mutation or indel was detected, indicating that base editing by nCas9-PBE is highly specific.

TABLE-US-00009 TABLE 4 Potential off-target effect analysis for OsCDC48 SEQ Number Site ID of Mutagenesis Sequencing name NO: Sequence mismatch Gene frequency method On-target  61 GACCAGCCAGCGT LOC_ CTGGCGCCGG Os03g05730 OT-1 102 GACCAGCCgGCGTg 3 LOC_ 0 T7EI/Sanger TGGtGCAGG Os12g09720 Sequencing OT-2 102 GACCAGCCgGCGTg 3 LOC_ 0 T7EI/Sanger TGGtGCAGG Os12g09700 Sequencing OT-3 102 GACCAGCCgGCGTg 3 LOC_ 0 T7EI/Sanger TGGtGCAGG Os12g14440 Sequencing OT-4 103 GACCAGCCAGCGT 3 LOC_ 0 T7EI/Sanger CTGaaGgCGG Os03g19390 Sequencing OT-5 104 GACCAagCAGCGgC 3 LOC_ 0 T7EI/Sanger TGGCGCCGG Os04g34030 Sequencing Lowercase bases are bases mismatched with OsCDC48. The bold letters show the PAM sequence.

Example 4. Base Editing of Maize ALS Gene

[0177] Using AtALS (AT3G48560.1) as a seed sequence, two ZmALS homologous genes are obtained by BLASTN alignment analysis in a maize database (https://phytozomejgi.doe.gov), wherein the two ZmALS homologous genes are named as ZmALS1 (Locus Name: GRMZM2G143357) and ZmALS2 (Locus Name: GRMZM2G143008). ZmALS1 and ZmALS2 have a sequence identify of 93.84%. A common sgRNA target sequence was designed in the conserved region of ZmALS1 (SEQ ID NO: 26) and ZmALS2 (SEQ ID NO: 28) (Svitashev et al., Plant Physiol., 2015), which is CAGGTGCCGCGACGCATGATTGG (SEQ ID NO: 56). A base editing system was constructed as described above, and transformed into maize variety Zong31 by particle bombardment method. After PCR/RE detection, plants with C7 to T7 substitution on ZmALS1 and ZmALS2 were obtained (FIG. 5).

[0178] Based on the data shown above, the modified and optimized nCas9-PBE induces highly efficient and specific C to T base editing in wheat, rice, and maize cells, and the point mutation produced by nCas9-PBE is unique comparing to the ones produced by TILLING. nCas9-PBE in combination of a properly engineered sgRNA can make it possible to perform a point mutation to a desired residue and adjacent residues, and thus facilitates the analysis for the effect of a single or combined mutations of amino acids located in a key domain of a protein. On the other hand, point mutations identified by TILLING usually only appear to a single amino acid residue, and accordingly it is difficult to simultaneously obtain mutations of the target residue and its adjacent residues by TILLING. Therefore, nCas9-PBE is clearly advantageous for the rapid generation of multiple mutations, and can be used for the detail analysis of the function of one or more amino acids in a key protein domain. Important functional properties of nCas9-PBE include a relatively large deamination window (covering 7 bases of the protospace sequence/target sequence), its base editing is independent of the sequence context structure of the target region, and there are few indel mutations. nCas9-PBE has a larger deamination window in cereal plants, which is advantageous for generating more diverse mutations.

[0179] The present invention demonstrates that C to T base-editing, which is mediated by the Cas9 variant-cytidine deaminase fusion protein, is a highly efficient tool for producing site-directed point mutations in the plant genome, and thereby increases the efficiency of improving crops through genomic engineering.

TABLE-US-00010 TABLE 5 All of the Primers Used in the Examples SEQ ID Primer Sequence Primer Name NO: (5′-3′) Application BFPm-F 105 ACGGCGTGCAGTGCTTCGGCCGCT Construct BFPm-R 106 ACCCCGACCA pJIT163-Ubi-BFPm TGGTCGGGGTAGCGGCCGAAGCAC TGCACGCCGT AflII-n/dCas9-F 107 GGCTTAAGGACAAGAAGTACTCGA Amplify n/dCas9 MluI-n/dCas9-R 108 TCGGCCT segment GCGACGCGTCTTCTTCTTCTTTGCT TGCCCTGC BamHI-n/dCas9-F 109 CGGGATCCATGCCAAAGAAGAAGA Amplify Bsp1047I-n/dCas9- 110 GGAAGGTTTCATC APOBEC1-XTEN- R CCGTGTACACTACACCTTCCGCTTC n/dCas9 segment TTCTTTGGGCTC Gibson-F 111 AATACTTGTATGGCCGCGGCCATG Amplify Gibson-R 112 CCAAAGAAGAAGAGG APOBEC1-XTEN- ACTTGTATGGAGGCCTGAGCTCTA n/dCas9 segment CACCTTCCGCTTCTT BFP-F 113 ATGGTGAGCAAGGGCGAGGAG Amplify BFP gene BFP-R 114 CCTCGATGTTGTGGCGGATCT target site and for first-round PCR for in-depth sequencing OsBFP-nCas9-F 115 CGATGTCGAGGGCGATGCCACCTA Second-round PCR OsBFP-nCas9-R 116 C for BFP in-depth TGGTCAAAGTCGTGCTGCTTCATGT sequencing in GG nCas9-PBE treated rice protoplasts OsBFP-dCas9-F 117 ATCACGCGAGGGCGATGCCACCTA Second-round PCR OsBFP-dCas9-R 118 C for BFP in-depth GCCTAAAAGTCGTGCTGCTTCATGT sequencing in GG dCas9-PBE treated rice protoplasts OsBFP-Cas9-F 119 AGTTCCCGAGGGCGATGCCACCTA Second-round PCR OsBFP-Cas9-R 120 C for BFP in-depth CTCTACAAGTCGTGCTGCTTCATGT sequencing in GG wild-type Cas9 treated rice protoplasts OsBFP-CK-F 121 CACTCACGAGGGCGATGCCACCTA Second-round PCR OsBFP-CK-R 122 C for BFP in-depth TGTTGGAAGTCGTGCTGCTTCATGT sequencing in GG control rice protoplasts TaBFP-nCas9-F 123 GTGGCCCGAGGGCGATGCCACCTA Second-round PCR TaBFP-nCas9-R 124 C for BFP in-depth CGAAACAAGTCGTGCTGCTTCATG sequencing in TGG nCas9-PBE treated wheat protoplasts TaBFP-dCas9-F 125 CGTACGCGAGGGCGATGCCACCTA Second-round PCR TaBFP-dCas9-R 126 C for BFP in-depth CCACTCAAGTCGTGCTGCTTCATGT sequencing in GG dCas9-PBE treated wheat protoplasts TaBFP-Cas9-F 127 GGTAGCCCGAGGGCGATGCCACCT Second-round PCR TaBFP-Cas9-R 128 AC for BFP in-depth ATCAGTAAGTCGTGCTGCTTCATGT sequencing in GG wild-type Cas9 treated wheat protoplasts TaBFP-CK-F 129 CACCGGCCGAGGGCGATGCCACCT Second-round PCR TaBFP-CK-R 130 AC for BFP in-depth ATCGTGAAGTCGTGCTGCTTCATGT sequencing in GG control wheat protoplasts ZmBFP-nCas9-F 131 ATGAGCCGAGGGCGATGCCACCTA Second-round PCR ZmBFP-nCas9-R 132 C for BFP in-depth AGGAATAAGTCGTGCTGCTTCATG sequencing in TGG nCas9-PBE treated maize protoplasts ZmBFP-dCas9-F 133 CAAAAGGCGAGGGCGATGCCACCT Second-round PCR ZmBFP-dCas9-R 134 AC for BFP in-depth TAGTTGAAGTCGTGCTGCTTCATGT sequencingin GG dCas9-PBE treated maize protoplasts ZmBFP-Cas9-F 135 TCGGCACGAGGGCGATGCCACCTA Second-round PCR ZmBFP-Cas9-R 136 C for BFP in-depth GAATGAAAGTCGTGCTGCTTCATG sequencing in TGG wild-type Cas9 treated maize protoplasts ZmBFP-CK-F 137 TCCCGACGAGGGCGATGCCACCTA Second-round PCR ZmBFP-CK-R 138 C for BFP in-depth CTTCGAAAGTCGTGCTGCTTCATGT sequencing in GG control maize protoplasts OsCDC48-F 139 TTCAGGACATCGAGATGGAGAAG Amplify OsCDC48 OsCDC48-R 140 ACAACGCAAATCTATCCATGCTC target site and for first-round PCR for in-depth sequencing OsCDC48-nCas9-F 141 CGATGTGCCGACATCCGCAAGTAC Second-round PCR OsCDC48-nCas9-R 142 CAG for OsCDC48 TGGTCATCATCATCGTCAGCTGCGG in-depth sequencing C in nCas9-PBE treated rice protoplasts OsCDC48-dCas9-F 143 ATCACGGCCGACATCCGCAAGTAC Second-round PCR OsCDC48-dCas9-R 144 CAG for OsCDC48 GCCTAATCATCATCGTCAGCTGCG in-depth sequencing GC in dCas9-PBE treated rice protoplasts OsCDC48-Cas9-F 145 AGTTCCGCCGACATCCGCAAGTAC Second-round of OsCDC48-Cas9-R 146 CAG PCR for OsCDC48 CTCTACTCATCATCGTCAGCTGCGG in-depth sequencing C in wild-type Cas9 treated rice protoplasts OsCDC48-CK-F 147 CACTCAGCCGACATCCGCAAGTAC Second-round PCR OsCDC48-CK-R 148 CAG for OsCDC48 deep TGTTGGTCATCATCGTCAGCTGCGG sequencing in C control rice protoplasts OsNRT1.1B-F 149 GATGTCACCTGATGATCTGAAGTA Amplify OsNRT1.1B-R 150 GC OsNRT1.1B target ATGATGGTGGTCGCCCAGAT site and for first-round PCR for in-depth sequencing OsNRT1.1B-nCas9- 151 CGATGTGGTGCAGGTTCCTGGACC Second-round PCR F 152 AT for OsNRT1.1B OsNRT1.1B-nCas9- TGGTCAATGATGGTGGTCGCCCAG in-depth sequencing R AT in nCas9-PBE treated rice protoplasts OsNRT1.1B-dCas9- 153 ATCACGGGTGCAGGTTCCTGGACC Second-round PCR F 154 AT for OsNRT1.1B OsNRT1.1B-dCas9 GCCTAAATGATGGTGGTCGCCCAG in-depth sequencing R- AT in dCas9-PBE treated rice protoplasts OsNRT1.1B-Cas9- 155 AGTTCCGGTGCAGGTTCCTGGACC Second-round PCR F 156 AT for OsNRT1.1B OsNRT1.1B-Cas9- CTCTACATGATGGTGGTCGCCCAG in-depth sequencing R AT in wild-type Cas9 treated rice protoplasts OsNRT1.1B-CK-F 157 CACTCAGGTGCAGGTTCCTGGACC Second-round PCR OsNRT1.1B-CK-R 158 AT for OsNRT1.1B TGTTGGATGATGGTGGTCGCCCAG in-depth sequencing AT in control rice protoplasts OsSPL14-F 159 CGCTGATGTGTTGTTTGTTGCGA Amplify OsSPL14 OsSPL14-R 160 CCTGCAGAGCAAGCTCAAGCTCA target site and for first-round PCR for in-depth sequencing OsSPL14-nCas9-F 161 CGATGTTCGCTGGCCCAAATCTCCC Second-round PCR OsSPL14-nCas9-R 162 T for OsSPL14 TGGTCAGACATGGCTGCAGCCTGG in-depth sequencing TT in nCas9-PBE treated rice protoplasts OsSPL14-dCas9-F 163 ATCACGTCGCTGGCCCAAATCTCCC Second-round PCR OsSPL14-dCas9-R 164 T for OsSPL14 GCCTAAGACATGGCTGCAGCCTGG in-depth sequencing TT in dCas9-PBE treated rice protoplasts OsSPL14-Cas9-F 165 AGTTCCTCGCTGGCCCAAATCTCCC Second-round PCR OsSPL14-Cas9-R 166 T for OsSPL14 CTCTACGACATGGCTGCAGCCTGG in-depth sequencing TT in wild-type Cas9 treated rice protoplasts OsSPL14-CK-F 167 CACTCATCGCTGGCCCAAATCTCCC First-round PCR for OsSPL14-CK-R 168 T OsSPL14 in-depth TGTTGGGACATGGCTGCAGCCTGG sequencing in TT control rice protoplasts TaLOX2-S1-F 169 ACTCCGTCTACCGACCATTGAG Amplify TaLOX2 TaLOX2-S1-R 170 TAGACCATGGAGGACATGGGCAT S1 target site and for first-round PCR for in-depth sequencing TaLOX2-S1-nCas9- 171 GTGGCCAGGGCCTCACCGTGGAGC Second-round PCR F 172 AGA for TaLOX2-S1 TaLOX2-S1-nCas9- CGAAACTCCCCTCGCAGGAAGAGC in-depth sequencing R AG in nCas9-PBE treated wheat protoplasts TaLOX2-S1-dCas9- 173 CGTACGAGGGCCTCACCGTGGAGC Second-round PCR F 174 AGA for TaLOX2-S1 TaLOX2-S1-dCas9- CCACTCTCCCCTCGCAGGAAGAGC in-depth sequencing R AG in dCas9-PBE treated wheat protoplasts TaLOX2-S1-Cas9- 175 GGTAGCAGGGCCTCACCGTGGAGC Second-round PCR F 176 AGA for TaLOX2-S1 TaLOX2-S1-Cas9- ATCAGTTCCCCTCGCAGGAAGAGC in-depth sequencing R AG in Cas9 treated wild-type wheat protoplasts TaLOX2-S1-CK-F 177 CGGAATAGGGCCTCACCGTGGAGC Second-round PCR TaLOX2-S1-CK-R 178 AGA for TaLOX2-S1 TCTGAGTCCCCTCGCAGGAAGAGC in-depth sequencing AG in control wheat protoplasts TaLOX2-S2-F 179 CAATCATCGATGTACTAGTGTGGTC Amplify TaLOX2 TaLOX2-S2-R 180 CAG S2 target site and GGATGTCGGCGAAGGAGTCGAACT for first-round PCR for in-depth sequencing TaLOX2-S2-nCas9- 181 ATGAGCTATGTATGGCTGGCGCAG Second-round PCR F 182 AGC for TaLOX2-S2 TaLOX2-S2-nCas9- AGGAATGTATGATCCCGTCCACCA in-depth sequencing R GC in nCas9-PBE treated wheat protoplasts TaLOX2-S2-dCas9- 183 CAAAAGTATGTATGGCTGGCGCAG Second-round PCR F 184 AGC for TaLOX2-S2 TaLOX2-S2-dCas9- TAGTTGGTATGATCCCGTCCACCAG in-depth sequencing R C in dCas9-PBE treated wheat protoplasts TaLOX2-S2-Cas9- 185 CACCGGTATGTATGGCTGGCGCAG Second-round PCR F 186 AGC for TaLOX2-S2 TaLOX2-S2-Cas9- ATCGTGGTATGATCCCGTCCACCA in-depth sequencing R GC in Cas9 treated wild-type wheat protoplasts TaLOX2-S2-CK-F 187 CTAGCTTATGTATGGCTGGCGCAG Second-round PCR TaLOX2-S2-CK-R 188 AGC for TaLOX2-S2 TCTGAGGTATGATCCCGTCCACCAG in-depth sequencing C in control wheat protoplasts TaLOX2-S3-F 189 GTCCCCTTCCTTCCGATCTAATCTC Amplify TaLOX2 TaLOX2-S3-R 190 TGCACGCAGTCAAATAATGGTACG S3 target site and A for first-round PCR for in-depth sequencing TaLOX2-S3-nCas9- 191 CGATGTCATCAAGCTGCCCAACAT Second-round PCR F 192 CCC for TaLOX2-S3 TaLOX2-S3-nCas9- TGGTCATCGGTCATCCATGCCTTCT in-depth sequencing R CGT in nCas9-PBE treated wheat protoplasts TaLOX2-S3-dCas9- 193 ATCACGCATCAAGCTGCCCAACAT Second-round PCR F 194 CCC for TaLOX2-S3 TaLOX2-S3-dCas9- GCCTAATCGGTCATCCATGCCTTCT in-depth sequencing R CGT in dCas9-PBE treated wheat protoplasts TaLOX2-S3-Cas9- 195 AGTTCCCATCAAGCTGCCCAACAT Second-round PCR F 196 CCC for TaLOX2-S3 TaLOX2-S3-Cas9- CTCTACTCGGTCATCCATGCCTTCT in-depth sequencing R CGT in Cas9 treated wild-type wheat protoplasts TaLOX2-S3-CK-F 197 CTATACCATCAAGCTGCCCAACAT Second-round PCR TaLOX2-S3-CK-R 198 CCC for TaLOX2-S3 TCTGAGTCGGTCATCCATGCCTTCT in-depth sequencing CGT in control wheat protoplasts ZmCENH3-F 199 AATGTGCCAGTTCCATGTGGGTGT Amplify ZmCENH3 ZmCENH3-R 200 GCAGGCCATAATGCTGTCGGGTAT target site and for first-round PCR for in-depth sequencing ZmCENH3-nCas9- 201 ATGAGCATGGAAAGTTATTCTTCTG Second-round PCR F 202 AGAA for ZmCENH3 ZmCENH3-nCas9- AGGAATTATGAAGAGGATCTTAAC in-depth sequencing R AGAGAG in nCas9-PBE treated maize protoplasts ZmCENH3-dCas9- 203 CAAAAGATGGAAAGTTATTCTTCT Second-round PCR F 204 GAGAA for ZmCENH3 ZmCENH3-dCas9- TAGTTGTATGAAGAGGATCTTAAC in-depth sequencing R AGAGAG in dCas9-PBE treated maize protoplasts ZmCENH3-Cas9-F 205 TCGGCAATGGAAAGTTATTCTTCTG Second-round PCR ZmCENH3-Cas9-R 206 AGAA for ZmCENH3 GAATGATATGAAGAGGATCTTAAC in-depth sequencing AGAGAG in Cas9 treated wild-type maize protoplasts ZmCENH3-CK-F 207 CTATACATGGAAAGTTATTCTTCTG Second-round PCR ZmCENH3-CK-R 208 AGAA for ZmCENH3 TCTGAGTATGAAGAGGATCTTAAC in-depth sequencing AGAGAG in control maize protoplasts Off-target-1F 209 ATGTCGGCCAGCAACAACAA Potential off-target Off-target-1R 210 AGTAGTGTATCCATCCTCGTGCAT effect for detection site 1 Off-target-2F 211 AATAGCCCATTCACCTTGTTCAACA Potential off-target Off-target-2R 212 CAGCCATAGACCATAGTACTACAC effect for detection CAC site 2 Off-target-3F 213 TCATCCTCGAACACTAGGCTGAAG Potential off-target Off-target-3R 214 TACTACTCGCAGCGCATCACTCA effect for detection site 3 Off-target-4F 215 ATTGAACGGTGTCACTTCAGACCA Potential off-target Off-target-4R 216 TATTGAGCTGATCAGCTGAACAGA effect for detection AC site 4 Off-target-5F 217 ACTCGCTGGAACTATCCATCTTGGC Potential off-target Off-target-5R 218 AAGCGCTCGACGGCGTGGA effect for detection site 5 ZmALS1-F 219 CTCCGACATCCTCGTCGAGGCT For maize ZmALS1 ZmALS1-R 220 GATTCACCAACAAGACGCAGCA PCR/RE test ZmALS2-F 221 AACCACCTCTTCCGCCACGAG For maize ZmALS2 ZmALS2-R 222 ACGCAGCACCTGCTCAAGCAAC PCR/RE test