Method for targeted modification of plant genome sequence

Abstract

Provided are a method for targeted modification of a specific sequence in a plant genome to a sequence of interest through a nuclease-reverse transcriptase fusion protein guided by a guide RNA, and genetically modified plants and progeny thereof produced by the method.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 25, 2020, application number 202080077133.2, and invention name “Method for targeted modification of plant genome sequences”. Technical Field

[0002] The present invention relates to the field of plant genetic engineering. Specifically, the present invention relates to a method for targeted modification of plant genome sequences. More specifically, the present invention relates to a method for targeted modification of a specific sequence in a plant genome into a target sequence of interest by a nuclease-reverse transcriptase fusion protein guided by a guide RNA, as well as a genetically modified plant produced by the method and its offspring. Background of the Invention

[0004] Many important agronomic traits depend on the sequence of the genome. By making directed changes to specific sequences of the genome, new heritable traits can be conferred to the organism, thereby providing possibilities for disease treatment and breeding improvement. At present, genome editing technology (such as CRISPR / Cas technology) can be used to cut specific sequences, thereby activating the repair pathway of the cell to repair the damaged part, thereby changing the sequence at the target site. Among them, based on the homologous recombination (HR) repair pathway, accurate changes to the genome sequence can be achieved. However, in most higher organisms, especially plants, the efficiency of homologous recombination is very low, which greatly limits the widespread application of this method. In addition, using a single base editing system, efficient conversion of cytosine to thymine (C→T) and conversion of adenine to guanine (A→G) at the target site can be achieved. However, the base conversion types of this method are limited, and it is also impossible to achieve accurate insertion or deletion of fragments. Therefore, there is still a need in the art for an efficient method for accurate directional modification of plant genome sequences.

[0005] Brief description of the invention

[0006] The present invention includes a novel plant DNA precision editing system, which is composed of a Cas nuclease (Cas9-H840A) fused with a reverse transcriptase with target chain nicking activity, and a pegRNA (prime editing gRNA, triggering editing gRNA) with a repair template (RT template) and a binding region (PBS) of a free single strand at the 3' end. The system combines the free single strand produced by the Cas nicking enzyme such as Cas9-H840A with PBS, and transcribes a single-stranded DNA sequence according to a given RT template. After cell repair, any change of the DNA sequence located downstream of the PAM sequence-3 position can be achieved in the genome. In addition, by introducing a new nicking sgRNA, it is nicked on the non-target chain of the pegRNA, which helps to promote the cell to repair according to the donor template. The experimental results show that the system effectively induces precise modification of the target site in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : Schematic diagram of the principle of the present invention

[0009] Figure 2 :Schematic diagram of the working of three different types of PPE (plant prime editor) systems. The system that does not provide additional nicking sgRNA is named (PPE2); the system that provides additional nicking sgRNA that helps cut the opposite strand of pegRNA is named (PPE3); when the PAM sequence of the nicking sgRNA that cuts the opposite strand is located within the spacer sequence of the pegRNA, the system is named (PPE3b).

[0010] Figure 3 : Schematic diagram of PPE construct and pegRNA construct.

[0011] Figure 4 : Working principle of the BFP-to-GFP reporter system for detecting precise editing in plant protoplasts.

[0012] Figure 5 : Flow cytometry results of the PPE system. "CK" is a protoplast control without plasmid transformation, "PBE" is the BE3 single-base editing reporter system, and "PPE3b (ΔM-MLV)" indicates a control group lacking M-MLV reverse transcriptase.

[0013] Figure 6:Flow cytometry was used to measure the efficiency of the PPE system. "CK" is a protoplast control without plasmid transformation, "PBE" is the BE3 single-base editing reporter system, and "PPE3b (ΔM-MLV)" indicates a control group lacking M-MLV reverse transcriptase.

[0014] Figure 7 : Editing of endogenous targets by the PPE system in rice.

[0015] Figure 8 : Editing of endogenous targets by the PPE system in wheat.

[0016] Fig. 9 : By-products produced by the PPE system and their proportions.

[0017] Fig.10 : Editing of endogenous targets in plants by the PPE-CaMV system.

[0018] Fig.11 : Schematic diagram of ribozyme-processed pegRNA driven by a type II promoter.

[0019] Fig.12 : Editing of endogenous genes by the PPE-R system in plants.

[0020] Fig.13 : Temperature processing improves PPE system editing efficiency.

[0021] Fig.14 : Effects of different PBS lengths on PPE systems.

[0022] Fig.15 : Effects of different RT template lengths on PPE systems.

[0023] Fig.16 : The influence of different RT template lengths on the precise editing ratio of the PPE system.

[0024] Fig.17 :The influence of different nicking sgRNA positions on the PPE system.

[0025] Fig.18 :The PPE system achieves different types of mutations in plant endogenous genes.

[0026] Fig.19 :The PPE system enables the insertion of fragments of different lengths into endogenous genes in plants.

[0027] Fig. 20 :The PPE system achieves deletion of fragments of different lengths in plant endogenous genes.

[0028] Fig.21 : Schematic diagram of PPE constructs used for Agrobacterium infection of rice.

[0029] Fig. 22 : Rice mutants obtained using the PPE system and their sequencing results. The arrows indicate the location of the target mutation.

[0030] Fig.23 : Single clone sequencing results of T0-9 mutant plants.

[0031] Fig.24 : The effects of different Tm-directed PBS lengths on editing efficiency were compared using published data for three target sites and newly obtained data for ten novel target sites in rice protoplasts.

[0032] Fig.25 : Normalization of initiated editing frequencies with different PBS melting temperatures. The highest editing frequency obtained at each target was normalized to 1, and the frequencies obtained at other PBS Tm were adjusted accordingly.

[0033] Fig.26 : Schematic diagram of priming editing using single-pegRNA and dual-pegRNA strategies. (a) Editing using only NGG-pegRNA (editing the forward DNA strand). (b) Editing using only CCN-pegRNA (editing the reverse DNA strand). (c) Editing using the dual-pegRNA strategy. The dual-pegRNA creates two edits simultaneously in both DNA strands.

[0034] Fig. 27 : Comparison of editing efficiencies induced by NGG-pegRNA, CCN-pegRNA, and dual-pegRNA strategies at 15 target sites.

[0035] Fig.28 : Product purity of NGG-pegRNA, CCN-pegRNA and dual-pegRNA when editing 15 endogenous sites in rice protoplasts.

[0036] Fig.29 : The percentage of rice genome bases that can theoretically be targeted for editing by single-pegRNA and dual-pegRNA priming. DETAILED DESCRIPTION

[0037] I. Definition

[0038] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0039] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0040] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0041] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0042] "Genetically modified plants" refers to plants that contain exogenous polynucleotides or modified genes or expression control sequences in their genome. For example, exogenous polynucleotides can be stably integrated into the genome of the plant and inherited for successive generations. Exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression control sequence is a gene or expression control sequence that contains one or more deoxynucleotide substitutions, deletions, and additions in the plant genome.

[0043] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, a sequence that has been significantly altered in composition and / or locus from its native form through deliberate human intervention.

[0044] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally may contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "D" is A, T or G, "I" is inosine, and "N" is any nucleotide.

[0045] "Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0046] As used herein, "expression construct" refers to a vector such as a recombinant vector suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0047] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (such as mRNA), for example, RNA generated by in vitro transcription.

[0048] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0049] "Regulatory sequence" and "regulatory element" are used interchangeably and refer to nucleotide sequences located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation recognition sequences.

[0050] "Promoter" refers to a nucleic acid fragment that can control the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that can control the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0051] "Constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in one tissue or organ, and 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 an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

[0052] Examples of promoters include, but are not limited to, polymerase (pol) I, pol II or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoters. Examples of pol II promoters include, but are not limited to, cytomegalovirus immediate early (CMV) promoters, Rous sarcoma virus long terminal repeat (RSV-LTR) promoters, and simian virus 40 (SV40) immediate early promoters. Examples of pol III promoters include U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used. Other examples of promoters include T7 phage promoters, T3 phage promoters, β-galactosidase promoters, and Sp6 phage promoters. When used for plants, the promoter can be a cauliflower mosaic virus 35S promoter, a corn Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, a corn U3 promoter, a rice actin promoter.

[0053] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0054] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming an organism cell with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into a genome, resulting in stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell, performing a function without stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0055] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0056] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf green, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, plant total protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance and drought resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance and tillering number, etc.

[0057] 2. Plant Genome Editing System

[0058] In one aspect, the present invention relates to a genome editing system for targeted modification of a DNA sequence in the genome of an organism, comprising:

[0059] i) a fusion protein and / or an expression construct containing a nucleotide sequence encoding the fusion protein, wherein the fusion protein comprises a CRISPR nickase and a reverse transcriptase; and / or

[0060] ii) at least one pegRNA and / or an expression construct containing a nucleotide sequence encoding said at least one pegRNA,

[0061] The at least one pegRNA comprises a guide sequence, a scaffold sequence, a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence from 5' to 3' direction.

[0062] In some embodiments, the at least one pegRNA is capable of forming a complex with the fusion protein and targeting the fusion protein to a target sequence in the genome, resulting in a nick within the target sequence.

[0063] In some embodiments, the organism is a plant.

[0064] As used herein, "genome editing system" refers to a combination of components required for genome editing of a cell genome. The individual components of the system, such as fusion proteins, gRNA, etc., can exist independently of each other, or can exist in any combination as a composition.

[0065] As used herein, "target sequence" refers to a sequence of about 20 nucleotides in length characterized by a PAM (protospacer sequence adjacent motif) sequence on the 5' or 3' flank in the genome. Generally speaking, PAM is necessary for the complex formed by CRISPR nuclease or its variants and guide RNA to recognize the target sequence. For example, for Cas9 nuclease and its variants, its target sequence is adjacent to PAM at the 3' end, such as 5'-NGG-3'. Based on the presence of PAM, those skilled in the art can easily determine the target sequence that can be used for targeting in the genome. And depending on the position of PAM, the target sequence can be located on any chain of the genomic DNA molecule. For Cas9 or its derivatives such as Cas9 nickase, the target sequence is preferably 20 nucleotides.

[0066] In some embodiments, the CRISPR nickase in the fusion protein is capable of forming a nick in a target sequence in genomic DNA. In some embodiments, the CRISPR nickase is a Cas9 nickase.

[0067] In some embodiments, the Cas9 nickase is derived from SpCas9 of Streptococcus pyogenes (S. pyogenes) and comprises at least an amino acid substitution H840A relative to wild-type SpCas9. An exemplary wild-type SpCas9 comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the Cas9 nickase comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the Cas9 nickase in the fusion protein is capable of forming a nick between the -3 nucleotide (the first nucleotide at the 5' end of the PAM sequence is +1) and the -4 nucleotide of the PAM of the target sequence.

[0068] In some embodiments, the Cas9 nickase is a Cas9 nickase variant capable of recognizing an altered PAM sequence. In some preferred embodiments, the Cas9 nickase is a Cas9 variant that recognizes the PAM sequence 5'-NG-3'. In some embodiments, the Cas9 nickase variant that recognizes the PAM sequence 5'-NG-3' comprises the following amino acid substitutions H840A, R1335V, L1111R, D1135V, G1218R, E1219F, A1322R, T1337R relative to wild-type Cas9, wherein the amino acid numbering refers to SEQ ID NO: 1.

[0069] The nick formed by the Cas9 nickase of the present invention can cause the target sequence to form a free single strand with a 3' end (3' free single strand) and a free single strand with a 5' end (5' free single strand).

[0070] In some embodiments, the reverse transcriptase in the fusion protein of the present invention can be derived from different sources. In some embodiments, the reverse transcriptase is a reverse transcriptase derived from a virus. For example, in some embodiments, the reverse transcriptase is an M-MLV reverse transcriptase or a functional variant thereof. An exemplary wild-type M-MLV reverse transcriptase sequence is shown in SEQ ID NO:3. In some embodiments, the reverse transcriptase is an enhanced M-MLV reverse transcriptase, for example, the amino acid sequence of the enhanced M-MLV reverse transcriptase is shown in SEQ ID NO:4. In some embodiments, the reverse transcriptase is CaMV-RT from cauliflower mosaic virus (CaMV, Cauliflower mosaic virus), and its amino acid sequence is shown in SEQ ID NO:5. In some embodiments, the reverse transcriptase is a reverse transcriptase derived from bacteria, for example, retron-RT from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO:6.

[0071] In some embodiments, the CRISPR nickase and the reverse transcriptase in the fusion protein are connected by a joint. As used herein, a "joint" can be a non-functional amino acid sequence of 1-50 (e.g., 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, no secondary or higher structure. For example, the joint can be a flexible joint, such as GGGGS, GS, GAP, (GGGGS) x 3, GGS and (GGS) x7, etc. For example, it can be a joint shown in SEQ ID NO: 7.

[0072] In some embodiments, the CRISPR nickase in the fusion protein is fused directly or via a linker to the N-terminus of the reverse transcriptase. In some embodiments, the CRISPR nickase in the fusion protein is fused directly or via a linker to the C-terminus of the reverse transcriptase.

[0073] In some embodiments of the present invention, the fusion protein of the present invention may further include a nuclear localization sequence (NLS). In general, one or more NLS in the fusion protein should have sufficient strength to drive the fusion protein in the nucleus of the cell to accumulate in an amount that can realize its base editing function. In general, the intensity of nuclear localization activity is determined by the number, position, one or more specific NLS used in the fusion protein, or a combination of these factors.

[0074] The guide sequence (also called seed sequence or spacer sequence) in at least one pegRNA of the present invention is set to have sufficient sequence identity (preferably 100% identity) with the target sequence, so that it can bind to the complementary chain of the target sequence through base pairing to achieve sequence-specific targeting.

[0075] A variety of scaffold sequences suitable for gRNAs for genome editing based on CRISPR nucleases (e.g., Cas9) are known in the art, which can be used in the pegRNA of the present invention. In some specific embodiments, the scaffold sequence of the gRNA is shown in SEQ ID NO:8.

[0076] In some embodiments, the primer binding sequence is set to be complementary to at least a portion of the target sequence (preferably completely matched with at least a portion of the target sequence), preferably, the primer binding sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA chain where the target sequence is located (preferably completely matched with at least a portion of the 3' free single strand), in particular, complementary to the nucleotide sequence at the 3' end of the 3' free single strand (preferably completely matched). When the 3' free single strand of the chain binds to the primer binding sequence through base pairing, the 3' free single strand can serve as a primer, and reverse transcription is performed under the action of the reverse transcriptase in the fusion protein using the reverse transcription (RT) template sequence adjacent to the primer binding sequence as a template, thereby extending a DNA sequence corresponding to the reverse transcription (RT) template sequence.

[0077] The primer binding sequence depends on the length of the free single strand formed by the CRISPR nickase used in the target sequence, however, it should have a minimum length to ensure specific binding. In some embodiments, the primer binding sequence length can be 4-20 nucleotides, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length.

[0078] In some embodiments, the primer binding sequence is configured to have a Tm (melting temperature) of no more than about 52° C. In some embodiments, the Tm (melting temperature) of the primer binding sequence is about 18° C.-52° C., preferably about 24° C.-36° C., more preferably about 28° C.-32° C., and more preferably about 30° C.

[0079] Methods for calculating the Tm of a nucleic acid sequence are well known in the art, and can be calculated using, for example, the Oligo Analysis Tool online analysis tool. An exemplary calculation formula is Tm = N G:C *4+N A:T *2, where N G:C is the number of G and C bases in the sequence, N A:T is the number of A and T bases in the sequence. A suitable Tm can be obtained by selecting a suitable length of the PBS. Alternatively, a PBS sequence with a suitable Tm can be obtained by selecting a suitable target sequence.

[0080] In some embodiments, the RT template sequence can be any sequence. Through the above-mentioned reverse transcription, its sequence information can be integrated into the DNA chain where the target sequence is located (i.e., the chain containing the target sequence PAM), and then through the DNA repair of the cell, a DNA double strand containing the RT template sequence information is formed. In some embodiments, the RT template sequence contains a desired modification. For example, the desired modification includes substitution, deletion and / or addition of one or more nucleotides. For example, the modification comprises one or more substitutions selected from the group consisting of C to T substitutions, C to G substitutions, C to A substitutions, G to T substitutions, G to C substitutions, G to A substitutions, A to T substitutions, A to G substitutions, A to C substitutions, T to C substitutions, T to G substitutions, T to A substitutions; and / or comprises a deletion of one or more nucleotides, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions; and / or comprises an insertion of one or more nucleotides, such as 1 to about 100 or more, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide insertions.

[0081] In some embodiments, the RT template sequence is set to correspond to the sequence downstream of the target sequence nick (e.g., complementary to at least a portion of the sequence downstream of the target sequence nick), and comprises a desired modification. The desired modification includes substitution, deletion and / or addition of one or more nucleotides. For example, the modification comprises one or more substitutions selected from the group consisting of C to T substitutions, C to G substitutions, C to A substitutions, G to T substitutions, G to C substitutions, G to A substitutions, A to T substitutions, A to G substitutions, A to C substitutions, T to C substitutions, T to G substitutions, T to A substitutions; and / or comprises a deletion of one or more nucleotides, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions; and / or comprises an insertion of one or more nucleotides, such as 1 to about 100 or more, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide insertions.

[0082] In some embodiments, the RT template sequence length can be about 1-300 or more nucleotides, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300 nucleotides or more polynucleotides in length. Preferably, the RT template sequence length is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 nucleotides.

[0083] In some embodiments, the plant genome editing system further comprises a nicking gRNA (nicking gRNA, for producing additional nicks) and / or an expression construct containing a nucleotide sequence encoding the nicking gRNA, wherein the nicking gRNA comprises a guide sequence and a scaffold sequence. In some preferred embodiments, the nicking gRNA does not comprise a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence.

[0084] The guide sequence (also called seed sequence or spacer sequence) in the nicking gRNA of the present invention is set to have sufficient sequence identity (preferably 100% identity) with the nicking target sequence in the genome, so that the fusion protein of the present invention can be targeted to the nicking target sequence and cause a nick in the nicking target sequence, and the nicking target sequence and the target sequence targeted by the pegRNA (pegRNA target sequence) are located on the opposite strand of the genomic DNA. In some embodiments, the nick formed by the nicking RNA and the nick formed by the pegRNA are about 1-about 300 or more nucleotides apart, for example, 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300 nucleotides or more nucleotides apart. In some embodiments, the nick formed by the nicking RNA is located upstream or downstream of the pegRNA (the upstream or downstream refers to the DNA chain where the pegRNA target sequence is located). In some embodiments, the guide sequence in the nicking gRNA has sufficient sequence identity (preferably 100% identity) with the relative chain (modified) of the pegRNA target sequence after the editing event occurs, so that the nicking gRNA only targets the nicking target sequence generated after the pegRNA-induced target sequence targeting and modification are completed. In some embodiments, the PAM of the nicking target sequence is located within the complementary sequence of the pegRNA target sequence.

[0085] In some embodiments, the pegRNA and / or nicked gRNA can be precisely processed using a self-processing system. In some specific embodiments, the 5' end of the pegRNA and / or nicked gRNA is connected to the 3' end of the first ribozyme, and the first ribozyme is designed to cut the fusion at the 5' end of the pegRNA and / or nicked gRNA; and / or the 3' end of the pegRNA and / or nicked gRNA is connected to the 5' end of the second ribozyme, and the second ribozyme is designed to cut the fusion at the 3' end of the pegRNA and / or nicked gRNA. The design of the first or second ribozyme is within the capabilities of those skilled in the art. For example, see Gao et al., JIPB, Apr, 2014; Vol 56, Issue 4, 343-349. Methods for precisely processing gRNA can be found, for example, in WO 2018 / 149418.

[0086] In some embodiments, the genome editing system comprises at least one pair of pegRNA and / or an expression construct containing a nucleotide sequence encoding the at least one pair of pegRNA. In some embodiments, the two pegRNAs in the pegRNA pair are set to target different target sequences on the same strand of the genomic DNA. In some embodiments, the two pegRNAs in the pegRNA pair are set to target sequences on different strands of the genomic DNA. In some embodiments, the PAM of the target sequence of one pegRNA in the pegRNA pair is located in the sense strand, while the PAM of the other pegRNA is located in the antisense strand. In some embodiments, the induced cuts of the two pegRNAs are located on both sides of the site to be modified. In some embodiments, the cut induced by the pegRNA for the sense strand is located upstream (5' direction) of the site to be modified, and the cut induced by the pegRNA for the antisense strand is located downstream (3' direction) of the site to be modified. The upstream or downstream is relative to the sense strand. In some embodiments, the induced cuts of the two pegRNAs are about 1-about 300 or more nucleotides apart, for example, 1-15 nucleotides apart.

[0087] In some embodiments, the two pegRNAs in the pegRNA pair are configured to introduce the same desired modification. For example, one pegRNA is configured to introduce an A to G substitution in the sense strand, while the other pegRNA is configured to introduce a T to C substitution in the corresponding position of the antisense strand. For another example, one pegRNA is configured to introduce a deletion of two nucleotides in the sense strand, while the other pegRNA is configured to also introduce a deletion of two nucleotides in the corresponding position of the antisense strand. Other types of modifications can be deduced by analogy. The same desired modification can be achieved by designing a suitable RT template sequence to enable pegRNAs that target two different chains, respectively.

[0088] In order to achieve efficient expression in plants, in some embodiments of the present invention, the nucleotide sequence encoding the fusion protein is codon-optimized for the plant species whose genome is to be modified.

[0089] Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted for use in different ways. See, Nakamura et al., 2001. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0090] In some embodiments, the fusion protein of the present invention is encoded by a nucleotide sequence shown in any one of SEQ ID NOs: 9-11 or comprises an amino acid sequence shown in any one of SEQ ID NOs: 12-14.

[0091] Plants that can be genome-modified by the genome editing system of the present invention include monocotyledons and dicotyledons, for example, the plants are crop plants, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato.

[0092] In one aspect, the present invention provides a method for determining a PBS sequence in a pegRNA of a genome editing system of the present invention, the method comprising:

[0093] a) determining at least one candidate target sequence based on the PAM recognized by the CRISPR nickase used and the site to be modified,

[0094] b) obtaining a series of PBS sequences according to the nick positions produced by the CRISPR nickase at the at least one candidate target sequence;

[0095] c) calculating the Tm of the PBS sequence;

[0096] d) selecting a PBS sequence with a Tm not exceeding 52°C, for example a Tm of about 18°C-52°C, preferably about 24°C-36°C, more preferably about 28°C-32°C, more preferably about 30°C.

[0097] III. Methods for producing genetically modified plants

[0098] In another aspect, the present invention provides a method for producing a genetically modified plant, comprising introducing the genome editing system of the present invention into at least one of the plants, thereby causing a modification in the genome of the at least one plant. The modification comprises substitution, deletion and / or addition of one or more nucleotides. For example, the modification comprises one or more substitutions selected from the group consisting of C to T substitutions, C to G substitutions, C to A substitutions, G to T substitutions, G to C substitutions, G to A substitutions, A to T substitutions, A to G substitutions, A to C substitutions, T to C substitutions, T to G substitutions, T to A substitutions; and / or comprises a deletion of one or more nucleotides, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions; and / or comprises an insertion of one or more nucleotides, such as 1 to about 100 or more, such as 1 to about 100 or more, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide insertions.

[0099] In some embodiments, the method further comprises screening the at least one plant for plants having the desired modification.

[0100] In the method of the present invention, the genome editing system can be introduced into plants by various methods well known to those skilled in the art. Methods that can be used to introduce the genome editing system of the present invention into plants include, but are not limited to, gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and ovary injection method. Preferably, the genome editing system is introduced into plants by transient transformation.

[0101] In the method of the present invention, the genome modification can be achieved by simply introducing or producing the fusion protein and gRNA in plant cells, and the modification can be stably inherited without the need to stably transform the exogenous polynucleotide encoding the components of the genome editing system into the plant. This avoids the potential off-target effects of the stably existing (continuously produced) genome editing system and also avoids the integration of exogenous nucleotide sequences into the plant genome, thereby having higher biosafety.

[0102] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0103] In some embodiments, the introduction includes transforming the genome editing system of the present invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into complete plants. Preferably, the regeneration is performed in the absence of selection pressure, that is, no selection agent for the selection gene carried on the expression vector is used during tissue culture. Not using a selection agent can improve the regeneration efficiency of the plant and obtain a modified plant without exogenous nucleotide sequences.

[0104] In other embodiments, the genome editing system of the present invention can be transformed into specific parts of the whole plant, such as leaves, stem tips, pollen tubes, panicles or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0105] In some embodiments of the invention, the in vitro expressed protein and / or in vitro transcribed RNA molecule (e.g., the expression construct is an in vitro transcribed RNA molecule) is directly transformed into the plant. The protein and / or RNA molecule can achieve genome editing in plant cells and is subsequently degraded by the cells, thereby avoiding the integration of exogenous nucleotide sequences in the plant genome.

[0106] Therefore, in some embodiments, genetic modification and breeding of plants using the methods of the present invention can obtain plants whose genomes have no exogenous polynucleotides integrated, ie, non-transgenic (transgene-free) modified plants.

[0107] In some embodiments, the method further comprises culturing the plant cell, tissue or whole plant into which the genome editing system has been introduced at an elevated temperature, such as 37°C.

[0108] In some embodiments of the present invention, the modified genomic region is associated with a plant trait such as an agronomic trait, whereby the modification substitution results in the plant having an altered (preferably improved) trait, such as an agronomic trait, relative to a wild-type plant.

[0109] In some embodiments, the method further comprises the step of screening plants for desired modifications and / or desired traits, such as agronomic traits.

[0110] In some embodiments of the present invention, the method further comprises obtaining offspring of the genetically modified plant. Preferably, the genetically modified plant or its offspring has a desired modification and / or a desired trait such as an agronomic trait.

[0111] On the other hand, the present invention also provides a genetically modified plant or its offspring or its part, wherein the plant is obtained by the above-mentioned method of the present invention. In some embodiments, the genetically modified plant or its offspring or its part is non-transgenic. Preferably, the genetically modified plant or its offspring has a desired genetic modification and / or a desired trait such as an agronomic trait.

[0112] In another aspect, the present invention also provides a plant breeding method, comprising hybridizing a first genetically modified plant obtained by the above method of the present invention with a second plant not containing the modification, thereby introducing the modification into the second plant. Preferably, the first genetically modified plant has a desired trait such as an agronomic trait.

[0113] Example

[0114] Materials and Methods

[0115] 1. Vector construction

[0116] The nCas9(H840A)-M-MLV construct, nCas9(H840A)-CaMV construct, and nCas9(H840A)-retron construct were constructed by Suzhou Genewise Co., Ltd. The M-MLV used in this example has 5 amino acid mutations compared to the wild-type M-MLV reverse transcriptase. M-MLV, RT-CaMV, and RT-retron have all been codon-optimized for monocots.

[0117] The pegRNA fragment (including RT and PBS sequences) was constructed into a vector driven by the OsU3 promoter using the Gibson method to obtain an OsU3-pegRNA construct suitable for rice. The pegRNA fragment (including RT and PBS sequences) was constructed into a vector driven by the TaU6 promoter using the Gibson method to obtain a TaU6-pegRNA construct suitable for wheat. The pegRNA fragment (including RT and PBS sequences) with ribozymes at both the 5' and 3' ends was constructed into a vector driven by the maize Ubiquitin-1 (Ubi-1) promoter using the Gibson method to obtain an Ubi-pegRNA-R construct.

[0118] The nicking gRNA was constructed into the vector driven by the TaU3 promoter using T4 ligase to obtain the TaU3-nick vector.

[0119] Table 1. List of pegRNA targeting sites and RT and PBS sequences

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] The PAM sequence is shown in bold.

[0127] 2. Protoplast isolation and transformation

[0128] The protoplasts used in the present invention are from rice variety Zhonghua 11 and wheat variety Kenong 199.

[0129] 2.1 Rice seedling cultivation

[0130] The seeds were first rinsed with 75% ethanol for 1 minute, then treated with 4% sodium hypochlorite for 30 minutes, washed with sterile water for more than 5 times, and cultured on M6 medium for 3-4 weeks at 26°C in the dark.

[0131] 2.2 Rice protoplast isolation

[0132] (1) Cut off the rice stalks, cut the middle part into 0.5-1 mm silk with a blade, put them into 0.6 M mannitol solution in the dark for 10 min, filter them with a filter, put them into 50 mL of enzymatic solution (filtered with a 0.45 μm filter membrane), evacuate (pressure about 15 KPa) for 30 min, take them out and place them on a shaker (10 rpm) for enzymatic hydrolysis at room temperature for 5 h;

[0133] (2) Add 30-50 mL of W5 to dilute the enzymatic hydrolysate, and filter the enzymatic hydrolysate through a 75 μm nylon filter into a round-bottom centrifuge tube (50 mL);

[0134] (3) 23°C, 250 g (rcf), centrifuge for 3 min, up 3 and down 3, and discard the supernatant;

[0135] (4) Gently suspend the cells with 20 mL of W5 and repeat step (3).

[0136] (5) Add appropriate amount of MMG to suspend and wait for transformation.

[0137] 2.3 Rice protoplast transformation

[0138] (1) Add 10 μg of each desired transformation vector to a 2 mL centrifuge tube, mix well, then use a sharpened pipette to draw up 200 μL of protoplasts, flick to mix, add 220 μL of PEG4000 solution, flick to mix, and induce transformation at room temperature in the dark for 20-30 min;

[0139] (2) Add 880 μL of W5 and gently invert to mix, centrifuge at 250 g (rcf), up 3 times and down 3 times, and discard the supernatant;

[0140] (3) Add 1 mL of WI solution, gently invert to mix, gently transfer to a flow tube, and culture in the dark at 26°C for 48 hours. If protoplasts need to be treated at 37°C, transfer to the flow tube and culture in the dark at 26°C for 12 hours, then transfer to the dark at 37°C for 8 hours, and finally transfer to 26°C and continue to culture for a total of 48 hours.

[0141] 2.4 Wheat seedling culture

[0142] The wheat seeds were potted and planted in a culture room, and cultured for about 1-2 weeks (about 10 days) at a temperature of 25±2°C, a light intensity of 1000Lx, and a light intensity of 14-16h / d.

[0143] 2.5 Wheat protoplast isolation

[0144] (1) Take young wheat leaves, cut the middle part into 0.5-1 mm strips with a blade, put them into 0.6 M mannitol solution in the dark for 10 min, filter them with a filter, put them into 50 mL of enzymatic solution (filtered with a 0.45 μm filter membrane), evacuate (pressure about 15 KPa) for 30 min, take them out and place them on a shaker (10 rpm) for enzymatic hydrolysis at room temperature for 5 h;

[0145] (2) Add 30-50 mL of W5 to dilute the enzymatic hydrolysate, and filter the enzymatic hydrolysate through a 75 μm nylon filter into a round-bottom centrifuge tube (50 mL);

[0146] (3) Centrifuge for 3 min at 23°C and 100 g (rcf), up 3 times and down 3 times, and discard the supernatant; (4) Gently resuspend in 10 mL of W5 and place on ice for 30 min; the protoplasts gradually settle and the supernatant is discarded; (5) Add an appropriate amount of MMG to resuspend and place on ice for transformation.

[0147] 2.6 Wheat protoplast transformation

[0148] (1) Add 10 μg of each plasmid to be transformed into a 2 mL centrifuge tube and mix well.

[0149] (2) Use a sharpened pipette tip to draw 200 μL of protoplasts, flick gently to mix, immediately add 250 μL of PEG4000 solution, flick gently to mix, and induce transformation at room temperature in the dark for 20-30 min;

[0150] (3) Add 800 μL of W5 (room temperature) and gently invert to mix. Centrifuge at 100 g (rcf) for 3 min, up 3 times and down 3 times, and discard the supernatant.

[0151] (4) Add 1 mL of W5, gently invert to mix, and gently transfer to a 6-well plate that has been pre-added with 1 mL of W5. Wrap the 6-well plate with tin foil and incubate in the dark at 26°C for 48 h.

[0152] 3. Observe cell fluorescence by flow cytometry

[0153] The flow cytometry analysis of protoplasts was performed using a FACSAria III (BD Biosciences) instrument. The specific steps are as follows:

[0154] (1) After turning on the instrument, open the BD FACSDiva Software and perform instrument calibration and other operations.

[0155] (2) Click “New Protocol” to create a suitable experimental protocol.

[0156] (3) Select “density plot” and draw a FSC / SSC scatter plot, and then draw a GFP / PE-Texas Red scatter plot.

[0157] (4) Adjust the FSC / SSC voltage so that the cell population appears in the center of the scatter plot, adjust the FL1 voltage so that the wild-type control protoplast population appears in the center of the scatter plot, and the GFP-positive protoplast population will appear at a location with a stronger GFP fluorescence channel signal (this population is not present in the wild-type control sample). If necessary, adjust the compensation to make the GFP-negative and -positive protoplast populations more distinct.

[0158] (5) Set a gate to enclose the GFP-positive population. A protoplast negative control should be prepared to determine the boundaries of the gate.

[0159] (6) Right-click the cell population to be sorted and select "left sort". Set the analysis conditions and analysis mode according to the experimental needs and the percentage of target cells.

[0160] (7) Load the prepared protoplast samples cultured in the flow tubes one by one, record the relevant data, and perform analysis.

[0161] (8) Close the software and shut down the instrument.

[0162] 4. Protoplast DNA extraction and amplicon sequencing analysis

[0163] 4.1 Protoplast DNA extraction

[0164] The protoplasts were collected in 2 mL centrifuge tubes, and the protoplast DNA (~30 μL) was extracted using the CTAB method. The concentration (30-60 ng / μL) was determined using a NanoDrop ultra-micro spectrophotometer and stored at -20°C.

[0165] 4.2 Amplicon sequencing analysis

[0166] (1) Use genomic primers to perform PCR amplification on the protoplast DNA template. The 20μL amplification system contains 4μL 5×Fastpfu buffer, 1.6μL dNTPs (2.5mM), 0.4μL Forward primer (10μM), 0.4μL Reverseprimer (10μM), 0.4μL FastPfu polymerase (2.5U / μL), and 2μL DNA template (~60ng). Amplification conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 50-64℃ annealing for 30s, 72℃ extension for 30s, 35 cycles; 72℃ full extension for 5min, 12℃ storage;

[0167] (2) The above amplification product was diluted 10 times, and 1 μL was taken as the template for the second round of PCR amplification. The amplification primer was a sequencing primer containing a barcode. The 50 μL amplification system contained 10 μL 5×Fastpfu buffer, 4 μL dNTPs (2.5 mM), 1 μL Forward primer (10 μM), 1 μL Reverse primer (10 μM), 1 μL FastPfu polymerase (2.5 U / μL), and 1 μL DNA template. The amplification conditions were the same as above, and the number of amplification cycles was 35 cycles.

[0168] (3) The PCR products were separated by 2% agarose gel electrophoresis, and the target fragments were recovered by gel extraction using AxyPrep DNA Gel Extraction kit. The recovered products were quantitatively analyzed using NanoDrop ultra-micro spectrophotometer; 100 ng of the recovered products were mixed and sent to Sangon Biotech Co., Ltd. for amplicon sequencing library construction and amplicon sequencing analysis.

[0169] (4) After sequencing is completed, the raw data are split according to the sequencing primers, and the WT is used as a control. The editing type and editing efficiency of the products at different gene targeting sites in three repeated experiments are compared and analyzed.

[0170] Example 1: Cas9 (H840A) nickase-reverse transcriptase fusion system for precise modification of BFP-to-GFP reporter system in rice protoplasts

[0171] To test whether Cas9(H840A) nickase-reverse transcriptase fusions (PPEs, plant prime editors) can be used to precisely modify target sequences ( Figure 1-2 ), nCas9(H840A)-M-MLV construct (PPE-M-MLV), nCas9(H840A)-CaMV construct (PPE-CaMV), nCas9(H840A)-retron (PPE-retron), OsU3 / TaU6 promoter-driven pegRNA construct with target guide RNA and RT and PBS sequences, and TaU3 promoter-driven nicking gRNA construct that can generate nicks on the non-target strand ( Figure 3 ). By combining the BFP-to-GFP reporter system ( Figure 4 ) to observe the working of PPE in protoplasts. When the "CC" in the BFP gene sequence is changed to "GT", the 66th amino acid can be changed from histidine (H, Histidine) to tyrosine (Y, Tyrosine), so that the gene encodes GFP fluorescent protein, thereby causing the cells to emit green fluorescence. The working efficiency of PPE can then be analyzed using a flow cytometer. The results showed that PPE lacking reverse transcriptase (i.e., PPE3b (ΔM-MLV)) could not make cells glow, but after PPE-M-MLV, pegRNA, nicking gRNA and BFP-to-GFP reporter system were co-transformed into protoplasts (i.e., PPE3b), it was clearly observed that the cells emitted green fluorescence, with an average efficiency of 4.4% ( Figure 5-6 Replacing the M-MLV reverse transcriptase in this system with a reverse transcriptase derived from the CaMV virus (i.e., PPE3b-CaMV) or a reverse transcriptase derived from the bacterial retron system (i.e., PPE3b-retron) can also cause cells to emit green fluorescence, with efficiencies of 3.7% and 2.4%, respectively ( Figure 5-6 ). Therefore, the above results show that PPE has the ability to modify the target sequence of the reporter system as desired in plants, which preliminarily proves that PPE can work in plants. Moreover, PPE can be a reverse transcriptase of other forms and sources.

[0172] Example 2: Cas9 (H840A) nickase-reverse transcriptase fusion system can accurately modify genome sequences in rice and wheat protoplasts

[0173] To test whether the PPE system can work at endogenous loci in rice, 21 pegRNAs were designed and constructed targeting 10 endogenous loci in rice (OsCDC48-T1, OsCDC48-T2, OsCDC48-T3, OsALS-T1, OsALS-T2, OsDEP1, OsEPSPS-T1, OsEPSPS-T2, OsLDAMR and OsGAPDH) and 7 endogenous loci in wheat (TaUbi10-T1, TaUbi10-T2, TaGW2, TaGASR7, TaLOX2, TaMLO and TaDME), and whether PPE2 and PPE3 (or PPE3b) could work at these endogenous loci were tested. The results showed that both PPE2 and PPE3 (or PPE3b) systems can achieve specific single base changes including C to T, G to T, A to G, G to A, T to A and C to A at endogenous sites in rice, as well as adding or deleting specific bases. The highest efficiency of this system in rice is 8.2% ( Figure 7 The PPE2 and PPE3 (or PPE3b) systems can also achieve single base changes in endogenous loci in wheat, including A to T, C to G, G to C, T to G, and C to A, with a maximum efficiency of 1.4% ( Figure 8 ). The overall efficiency of PPE2 and PPE3 (or PPE3b) in the tested sites is not much different. In addition, a certain proportion of byproducts of the PPE system was observed, mainly pegRNA backbone insertion or replacement ( Fig. 9 ). The above results indicate that the PPE system, including PPE2, PPE3 and PPE3b, can achieve targeted modifications including precise single-base mutation, precise insertion and precise deletion at endogenous sites in plants.

[0174] Example 3: Improvement of Cas9 (H840A) nickase-reverse transcriptase fusion system

[0175] To further improve the PPE editing system, the PPE-CaMV system was tested at endogenous sites in rice. The results showed that PPE-CaMV can also achieve precise targeted modification of endogenous sequences in rice, with an efficiency of up to 5.8% ( Fig.10 ), which indicates that PPE systems derived from other species or other forms of reverse transcriptase can also modify endogenous plant genes in a targeted manner.

[0176] In addition, the work of ribozyme-processed pegRNA for PPE system was also tested. A Ubi-1-driven ribozyme-processed pegRNA with target guide RNA and RT and PBS sequences was constructed to replace the OsU3-driven pegRNA construct in the original system, and the system was named (PPE-R, R represents ribozyme Ribozyme) ( Fig.11 The results of endogenous targets showed that the strategy of ribozyme processing can also achieve precise changes in endogenous sequences. PPE-R is better than PPE at some sites, with the highest efficiency reaching 9.7% ( Fig.12 ). This result indicates that ribozyme-processed pegRNA or pegRNA initiated by type II promoter are both suitable for the PPE system.

[0177] In order to improve the editing efficiency of the PPE system, the protoplasts were cultured at 37°C to test whether it could improve the editing efficiency. Two endogenous rice sites (OsCDC48-T2 and OsALS-T2) were selected for testing. After the transformed protoplasts were cultured at 26°C overnight, they were incubated at 37°C for 8 hours, and then returned to 26°C for continued culture, and the efficiency was compared with that of the treatment group. The results showed that 37°C treatment can significantly improve the editing efficiency of the PPE system (including PPE2, PPE3 and PPE3b forms), with an average increase of 1.6 times (from 3.9% to 6.3%), and a maximum increase of 2.9 times ( Fig.13 ).

[0178] Example 4: Testing the effects of different PBS, RT template lengths and nicking gRNA positions on the PPE system

[0179] The effects of different PBS, RT template lengths and nicking gRNA positions on the PPE system were tested. The results using OsCDC48-T1 as the test site showed that the different PBS lengths (6-16nt) and RT template lengths (7-23nt) tested could produce targeted sequence modifications at specific sites ( Figure 14-15 ), the efficiency at OsCDC48-T1 site was 3.4% to 15.3%, the efficiency at OsCDC48-T2 site was 0.9% to 8.1%, and the efficiency at OsALS-T2 site was 1.1% to 10.5%. The results showed that different lengths of RT templates and PBS had a significant effect on the editing efficiency, and it was found that different RT template lengths had a significant effect on the proportion and type of byproducts produced by the PPE system ( Fig.16 In addition, different nicking gRNAs also affect the efficiency of the PPE system, ranging from 3.2% to 19.2% at the OsCDC48-T1 site and from 2.9% to 8.6% at the OsCDC48-T2 site ( Fig.17 ).

[0180] Example 5: PPE system achieves precise modification of multiple types of endogenous sites

[0181] In order to test whether the PPE system can achieve multiple types of precise modifications, 12 types of single-base conversions (N to N, where N represents four base types, A, T, C or G), multi-base conversions, and base insertions and deletions of different lengths were designed using four rice sites (OsCDC48-T1, OsCDC48-T2, OsALS-T2, and OsGAPDH) as examples. The results showed that the PPE system can achieve all types of single-base conversions with a maximum efficiency of 8.0%. The system can also achieve simultaneous conversion of multiple bases with a maximum efficiency of 1.5% ( Fig.18 The efficiency of base insertion and deletion decreases as the required modification length increases. The maximum base-directed insertion efficiency can reach 3.0%, and the maximum insertion length can reach 15nt ( Fig.19 ); the base-directed deletion efficiency can reach up to 19.2%, and the longest deletion length can reach 40nt ( Fig. 20 ). Therefore, the system can efficiently achieve the addition and deletion of small fragments. Therefore, the PPE system can achieve various types of targeted modifications of endogenous sites.

[0182] Example 6: PPE system to obtain targeted editing plants

[0183] In order to obtain targeted editing plants, the OsCDC48-T1 and OsALS-T2 loci were selected for testing, and a binary vector in the form of PPE3 was constructed ( Fig.21 ) and infected rice by Agrobacterium transformation. Finally, 12 rice seedlings with the expected precise deletion of OsCDC48-T1 locus were detected, with an editing efficiency of 21.8% (12 / 55); 2 rice seedlings with the expected single base mutation of G to T at OsALS-T2 locus, with an editing efficiency of 14.3% (2 / 14); and 1 rice seedling with the expected precise simultaneous mutation of 3 base pairs at OsCDC48-T1 locus, with an editing efficiency of 2.6% (1 / 38) ( Fig. 22 )(Table 2). It was also found that a small amount of unexpected by-products were produced in plants ( Fig.23 ). The above results indicate that the PPE system can be used to obtain targeted modified mutant plants in plants.

[0184] Table 2. PPE system to obtain targeted editing of rice plants

[0185]

[0186] Example 7: Tm of PBS affects the efficiency of the PPE system

[0187] We first evaluated the effect of PBS length controlling Tm on plant-primed editing based on publicly available data ( Fig.24 The results showed that the editing efficiency was highest when the PBS Tm was close to 30°C (30°C for OsCDC48-T1, 28°C for OsCDC48-T2, and 30°C for OsALS-T1).

[0188] The editing efficiency of PPE using PBS with Tm ranging from 18°C ​​to 52°C (corresponding to PBS lengths of 6nt to 17nt) at four target sites (OsACC-T1, OsCDC48-T3, OsEPSPS-T1, and OsPDS-T1) in rice protoplasts was then evaluated. Fig.24 . When the PBS Tm was close to 30°C (24-30°C in OsACC-T1, 26-34°C in OsEPSPS-T1, 28-36°C in OsCDC48-T3, and 30°C in OsPDS-T1), the pegRNA had higher activity. At these PBS Tms, the editing efficiency was 1.5 to 4.3 times higher than that of other PBS Tms. Six additional target sites were also tested (see Fig.24 ), including OsALS-T2, OsDEP1-T1, OsEPSPS-T2, OsAAT-T1, OsGAPDH-T1, and OsLDMAR-T1, and five of the six target sites (OsEPSPS-T2 was the exception) were found to have similar performance.

[0189] Then, the overall editing efficiency of PPE at different PBS Tm for all 13 target sites was normalized and compared. The results showed that the editing efficiency followed a normal distribution (P>0.1)( Fig.25 ), usually reaching a maximum at PBS Tm 30°C, followed by PBS Tm 32°C and 28°C, while the efficiency decreased with increasing or decreasing PBS Tm.

[0190] It can be concluded that the melting temperature of the PBS sequence is closely related to the PPE editing efficiency ( Fig.25 ), and is likely to be the main factor affecting plant pegRNA design. It is recommended to use the PBS Tm of 30°C to guide the design of pegRNA in PPE.

[0191] Example 8: Using a dual pegRNA strategy to significantly improve the efficiency of the PPE system

[0192] To optimize for optimal editing, the inventors developed a dual-pegRNA strategy that uses different pegRNAs targeting the forward and reverse DNA strands (referred to as NGG-pegRNA and CCN-pegRNA, respectively) that simultaneously encode the same editing ( Fig.26 ). Fifteen target sites were selected from ten rice genes, and a pair of pegRNAs was designed for each target (Table 3). Then, the editing activities of NGG-pegRNA only, CCN-pegRNA only, and dual-pegRNA were compared at the same position.

[0193] Table 3. Target sequences, RT templates and PBS sequences of dual-pegRNA

[0194]

[0195]

[0196] The PAM for each target site is shown in bold and the PBS is underlined.

[0197] As expected, the dual-pegRNA strategy had the highest activity in most target sites (13 out of 15). They generated point mutations of C-to-A, G-to-A, G-to-T, A-to-G, T-to-A, C-to-G, and CT-AG, 1 bp (T) or 2 bp (AT) deletions, and 1 bp (A) insertions, with a maximum editing efficiency of 24.5% ( Fig. 27 ).

[0198] The editing efficiency of dual-pegRNA at all tested sites was about 4.2 times higher than that of single NGG-pegRNA (up to 27.9 times for OsNRT1.1B (insertion of A)) and 1.8 times higher than that of single CCN-pegRNA on average (up to 7.2 times for OsALS (A to G)). In addition, the proportion of byproducts using dual-pegRNA was no higher than that of single-pegRNA ( Fig.28 ).

[0199] Computational analysis based on the rice reference genome (Os-Nipponbare reference IRGSP-1.0) showed that dual-pegRNA could theoretically target 20.0% of genomic bases when the priming editing window was from +1 to +15. However, when combined with the SpCas9-NG variant with NG PAM, the dual-pegRNA strategy was able to target 87.9% of rice bases ( Fig.29 ).

[0200] Sequence Description

[0201] >SEQ ID NO:1 Wild-type SpCas9 amino acid sequence

[0202] >SEQ ID NO:2nCas9(H840A) amino acid sequence

[0203] >SEQ ID NO:3 Wild-type M-MLV amino acid sequence

[0204] >SEQ ID NO:4 amino acid sequence of M-MLV of the present invention

[0205] >SEQ ID NO:5CaMV reverse transcriptase amino acid sequence

[0206] >SEQ ID NO:6retron reverse transcriptase amino acid sequence

[0207] >SEQ ID NO:7 32aa linker amino acid sequence

[0208] >SEQ ID NO:8 gRNA scaffold sequence

[0209] >SEQ ID NO:9 Gene sequence of the fusion protein nCas9(H840A)-M-MLV of the present invention

[0210] >SEQ ID NO: 10 Gene sequence of the fusion protein nCas9 (H840A)-CaMV of the present invention

[0211] >SEQ ID NO: 11 Gene sequence of the fusion protein nCas9(H840A)-retron of the present invention

[0212] >SEQ ID NO:12 Amino acid sequence of the fusion protein nCas9(H840A)-M-MLV of the present invention

[0213] >SEQ ID NO:13 Amino acid sequence of the fusion protein nCas9(H840A)-CaMV of the present invention

[0214] >SEQ ID NO: 14 Amino acid sequence of the fusion protein nCas9(H840A)-retron of the present invention.

Claims

1. A genome editing system for targeted modification of plant genomes, comprising: i) a fusion protein and / or an expression construct containing a nucleotide sequence encoding the fusion protein, wherein the fusion protein comprises a CRISPR nickase and a reverse transcriptase; and ii) at least one pair of pegRNAs or an expression construct containing a nucleotide sequence encoding said at least one pair of pegRNAs, The two pegRNAs in the pegRNA pair are configured to target target sequences on different strands of genomic DNA, wherein the at least one pegRNA comprises a guide sequence, a scaffold sequence, a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence from 5' to 3' direction, The guide sequence in the pegRNA is set to have sufficient sequence identity with the target sequence so that it can bind to the complementary strand of the target sequence through base pairing to achieve sequence-specific targeting; wherein the at least one pegRNA is capable of forming a complex with the fusion protein and targeting the fusion protein to a target sequence in the genome, resulting in a nick within the target sequence; wherein the RT template sequence is configured to contain a desired modification, which comprises substitution, deletion and / or addition of one or more nucleotides, The two pegRNAs in the pegRNA pair are configured to introduce the same desired modification.

2. The system of claim 1, wherein the CRISPR nickase is a Cas9 nickase comprising the amino acid sequence shown in SEQ ID NO:

2.

3. The system of claim 1, wherein the reverse transcriptase is an enhanced M-MLV reverse transcriptase having an amino acid sequence as shown in SEQ ID NO:4, or the reverse transcriptase is a CaMV reverse transcriptase as shown in SEQ ID NO:5 or a retron reverse transcriptase as shown in SEQ ID NO:

6.

4. The system of claim 1, wherein the scaffold sequence of the pegRNA comprises the sequence described in SEQ ID NO:

8.

5. The system of any one of claims 1 to 4, wherein the primer binding sequence is complementary to the nucleotide sequence of the 3' free single strand resulting from the nick.

6. The system of any one of claims 1 to 4, wherein the Tm (melting temperature) of the primer binding sequence is 24°C to 36°C.

7. The system of claim 6, wherein the PAM of the target sequence of one pegRNA in the pegRNA pair is located on the sense strand, and the PAM of the other pegRNA is located on the antisense strand.

8. The system of claim 7, wherein the induced cuts of the two pegRNAs are located on both sides of the site to be modified.

9. The system of claim 8, wherein the nick induced by the pegRNA for the sense strand is located upstream (5' direction) of the site to be modified, and the nick induced by the pegRNA for the antisense strand is located downstream (3' direction) of the site to be modified.

10. The system of claim 9, wherein the nicks induced by the two pegRNAs are 1-300 nucleotides apart.

11. A method for producing a genetically modified plant, comprising introducing a genome editing system as defined in any one of claims 1 to 10 into at least one of the plants, thereby causing modifications in the genome of the at least one plant, the modifications comprising substitution, deletion and / or addition of one or more nucleotides.

12. The method of claim 11, wherein the introducing comprises transforming the genome editing system defined in any one of claims 1 to 10 into an isolated plant cell or tissue, and then regenerating the transformed plant cell or tissue into a whole plant; or The introduction comprises transforming the genome editing system defined in any one of claims 1 to 10 into a specific part of the whole plant, such as leaves, stem tips, pollen tubes, young ears or hypocotyls.

13. The method of claim 12, further comprising culturing the plant cell, tissue or whole plant into which the genome editing system has been introduced at an elevated temperature.

Images