Method for inserting exogenous sequence in genome at fixed point

Abstract

The invention belongs to the field of gene engineering. Specifically, the invention relates to a method for inserting an exogenous sequence in a genome at a fixed point. Specifically, on the basis of a guided editing system (PE), two adjacent pegRNAs with partially overlapped sequences on a reverse transcription template are used, and efficient and accurate exogenous sequence fixed-point insertion is realized in a genome, especially a plant genome. The system is further coupled with a recombinase system such as Cre / Lox or FLP / FRT and the like, and large-fragment exogenous sequence fixed-point insertion is realized in genomes, especially plant genomes.

Description

A method for inserting exogenous sequences into genomes

[0001] This application claims priority to Chinese patent application No. 202210580767.0 filed on May 25, 2022 and Chinese patent application No. 202310363943.X filed on April 6, 2023. Technical Field

[0002] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to a method for inserting an exogenous sequence at a specific site in a genome. Specifically, the present invention is based on a guide editing system (PE) and uses two adjacent pegRNAs with partially overlapping sequences on a reverse transcription template to achieve efficient and accurate site-specific insertion of exogenous sequences in genomes, particularly plant genomes. This system is further coupled with a recombinase system such as Cre / Lox or FLP / FRT to achieve site-specific insertion of large exogenous sequences in genomes, particularly plant genomes.

[0003] Background of the Invention

[0004] The rapid development of DNA sequencing technology has catapulted the life sciences into the genomic era. The emergence of technologies such as GWAS has significantly accelerated the development of genetics, particularly by unraveling the functions of numerous key genes in plants. This has created a significant opportunity for the development of molecular crop breeding. Traditional crop breeding methods, such as hybridization and backcrossing, are time-consuming and labor-intensive, making them inadequate for the rapidly growing demand for crop breeding. Therefore, the development of novel molecular breeding technologies has become increasingly important.

[0005] Transgenic technology, with its ability to rapidly and efficiently achieve desirable traits, has been rapidly adopted in plant molecular breeding. However, due to its inherent need to introduce foreign genes, it is subject to strict regulation. In contrast, genome editing technology allows for precise, targeted modification of functional genes without the introduction of foreign genes, resulting in more rapid and efficient acquisition of desirable traits. Currently, plant genome editing tools fall into three main categories: zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR / Cas). The CRISPR / Cas system is the most convenient and efficient, and has made significant contributions to genetic research and plant molecular breeding in recent years.

[0006] The widely used CRISPR / Cas system consists of a designed single-stranded guide RNA (sgRNA) and a site-specific nuclease, Cas9. The sgRNA targets a specific location in genomic DNA through complementary base pairing. Cas9 and the sgRNA form a ribonucleoprotein complex (RNP) within the cell. Simultaneously, the Cas9 conformation changes, and a domain within the complex (the PAM-interaction domain, or PI domain) continuously interacts with the NGG (PAM) motif at various locations on the genome until it finds a site that can base-pair with the sgRNA. At this point, the RNP complex interacts with the DNA to form a new complex. Cas9 unwinds the DNA double strand to form an R-loop, while undergoing another conformational change. The RuvC and HNH nuclease domains within the complex are activated, cleaving the non-target and target strands, respectively, creating a DNA double-strand break (DSB). This DSB triggers the cell's endogenous DNA repair mechanisms, typically through non-homologous end joining (NHEJ), which is the most frequent repair mechanism. NHEJ is an error-prone repair pathway, so during the repair process, some base insertions or deletions (Indels) may be randomly introduced near the DSB, resulting in the inability to express the gene normally. If a piece of exogenous DNA (donor) with homologous arms of genomic sequences on both sides of the DSB is provided during the process of DSB generation, the cell's endogenous repair mechanism may use this donor as a template for homologous recombination repair (HR). HR is a precise repair pathway that can introduce arbitrary point mutations, fragment insertions, and deletions in the genome. However, this repair pathway occurs very rarely in higher organisms, especially plant cells, and therefore has not been widely used. Subsequently, base editors (BEs) based on the CRISPR system were developed. The BE system utilizes a RuvC domain-inactivated Cas9 (nCas9-D10A) coupled to a deaminase (cytosine deaminase or adenine deaminase, corresponding to CBE and ABE, respectively). When the RNP complex binds to DNA to form an R-loop, the deaminase deaminates cytosine (C) or adenine (A) on the non-target strand to form uracil (U) or hypoxanthine (I). The intracellular repair machinery recognizes uracil as thymine (T) and hypoxanthine as guanine (G). At this point, nCas9 cleaves the target strand, promoting the base excision repair pathway (BER) in the cell to repair the cut or aig. The BE system can achieve efficient and precise point mutations without relying on the formation of DSBs or the HR pathway, and has therefore rapidly gained widespread application. The genome editing toolbox for coupling CRISPR systems with other effectors, represented by BE, has also developed rapidly, including coupling transcriptional activators, inhibitors or epigenetic modifiers for targeted activation, inhibition and epigenetic modification.

[0007] Despite the rapid development of the CRISPR molecular toolbox, from simple gene knockout to precise base editing, to transcriptional activation, repression, and epigenetic modification, the targeted and precise insertion of DNA fragments has been difficult to achieve in higher plant cells. Traditional strategies for achieving targeted insertion rely on the generation of DSBs. When an additional donor DNA segment without genomic homologous sequences is provided, this donor may be inserted near the DSB through the NHEJ repair pathway after the DSB is generated. However, this process is very imprecise and has low efficiency due to issues such as the donor provision method. When an additional donor DNA segment containing genomic homologous sequences is provided, the target fragment in this donor may be inserted into the target location through the HR repair pathway after the DSB is generated. However, this process is extremely inefficient and almost impossible to achieve in higher plant cells.

[0008] Due to the low efficiency of HR, site-specific large-fragment DNA integration can be achieved with the help of site-specific recombinases (SSRs). SSRs can specifically recognize and bind to a certain DNA sequence (recombination site, RS) and form a synaptonemal complex. A chain exchange process can occur between the two synaptonemal complexes and complete DNA recombination. This process is mediated by the tyrosine or serine residues in the SSR catalytic active center attacking the RS phosphate backbone to cause DNA cleavage. After cleavage, a covalent intermediate is formed and a chain exchange reaction occurs between the two RSs. This process does not require the participation of high-energy auxiliary factors and does not rely on the cell's endogenous DNA repair pathway because it is more efficient. Based on the differences in the residues in the SSR enzyme active center, it can be divided into the tyrosine recombinase family and the serine recombinase family. They are mainly derived from bacteriophages, bacteria and fungi, and play biological functions such as excision, inversion, integration, and transposition. Common tyrosine recombinases include Escherichia coli phage λ integrase, P1 phage Cre recombinase, and yeast FLP recombinase. They all utilize a conserved tyrosine residue to attack one strand of the RS backbone, exposing the 5' phosphate group and 3' hydroxyl group. The 5' phosphate and 3' hydroxyl groups of the two RSs then bind separately, achieving strand exchange. Simultaneously, the recombinase bound to the RS undergoes conformational changes, attacking the other strand and achieving strand exchange through the same pathway, thus completing the recombination process. Common serine recombinases include Tn3 transposase, Salmonella recombinase Hin, Streptomyces phage φC31 integrase, and Mycobacterium phage Bxb1 integrase. Their recombination processes are similar to those of tyrosine recombinases, but they utilize serine residues to simultaneously attack both strands of the RS backbone, achieving simultaneous exchange of the two RS strands and completing the recombination process. SSR has a wide range of applications: it is mainly used as a molecular cloning tool in vitro, and its high efficiency of recombination between DNA molecules makes the in vitro molecular cloning of large and multiple fragments very simple; in prokaryotic cells, it can be used as a gene or staining engineering tool to delete, invert, translocate or integrate large DNA fragments; in eukaryotic cells of higher organisms, it is mainly used as a tool for transgenic marker gene deletion, but since RS is difficult to knock in at a specific site, the integration of large DNA fragments at a specific site is very difficult.

[0009] Recently, a primed editing system (PE) capable of achieving arbitrary base mutations and short DNA insertions and deletions has been developed. Due to its robust and DSB-independent functionality, it has rapidly gained widespread application in genome editing for plants and animals. This system utilizes a Cas9 with an inactivated HNH domain (nCas9-H840A) coupled to a reverse transcriptase (MLV). Simultaneously, a reverse transcription template sequence (RT) and a reverse transcriptase primer binding site (PBS) are introduced at the 3' end of the sgRNA. The RT carries the target mutation sequence and flanked by genomic homology. This sgRNA is referred to as a pegRNA. After nCas9 cleaves the non-target strand, the PBS binds to its 5' end, acting as a starting primer for the reverse transcriptase. The reverse transcriptase then extends toward the 3' end of the RT and reverse transcribes the RT sequence into DNA, forming a 3' overhang containing the mutation sequence. After repair of the cell's endogenous DNA, this mutation sequence can be introduced into the genome, enabling genome editing of any length.

[0010] The efficiency of the guide editing system in higher plant cells is still too low to achieve efficient insertion, and the length of the inserted fragment is very limited. There are three main reasons for this. First, the repair pathway used by the guide editing system in higher plants occurs relatively infrequently, resulting in low final editing efficiency; second, RT and genomic homologous sequences competitively bind to genomic DNA, hindering the reverse transcription process; third, reverse transcriptase or pegRNA is easily degraded or has insufficient reverse transcription capacity. This field still needs systems and methods for achieving efficient insertion of exogenous nucleotide sequences, especially large exogenous nucleotide sequences, into plant genomes.

[0011] Summary of the Invention

[0012] In order to avoid the first two reasons for the low efficiency of PE in higher plants, the inventors first designed a dual-pegRNA strategy, in which the two pegRNAs target and bind to the two chains of genomic DNA respectively, and there is a certain distance between the PAMs (about 20bp-about 60bp). The RT of the two pegRNAs only contains the required insertion sequence and has a partially overlapping sequence at the 3' end. After the reverse transcription is completed, the two newly synthesized DNA chains bind to each other and anneal due to the overlapping sequences, and the insertion is completed through a DNA repair pathway different from the original PE system (according to some results of this application, this repair pathway may be SSA, a repair pathway that occurs more frequently and efficiently in plants).

[0013] Recently, an enhanced plant-based primed editing system (ePPE) has been established by fusing the retroviral nucleocapsid protein (NC) and deleting the RNaseH active domain of the MLV reverse transcriptase, which can enhance reverse transcription capacity or enhance reverse transcriptase stability, thereby greatly improving the efficiency of the plant-based primed editing system. In addition, by adding a secondary structure tevopre (epegRNA) to the 3' end of the pegRNA, it can also enhance reverse transcription capacity or enhance pegRNA stability and improve the efficiency of PE.

[0014] To further improve insertion efficiency, the inventors simultaneously employed the aforementioned ePPE system and epegRNA, achieving highly efficient, site-specific insertion of short fragments into plant somatic cells. They also evaluated the DNA integration abilities of the Cre / Lox and FLP / FRT systems from the tyrosine recombinase family, along with the φC31 and Bxb1 recombinase systems from the serine family, in rice somatic cells. They found that the Cre / Lox and FLP / FRT systems were more effective, and thus combined them with the aforementioned efficient insertion systems. By providing an additional donor carrying the desired insertion gene, RS, they achieved site-specific insertion of large exogenous nucleotide sequences in a single step.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Five constructs were tested for insertion efficiency into Lox66 or FRT1 using dual pegRNA in rice protoplasts.

[0017] Figure 2. Testing the efficiency of RS insertion using PPE+pegRNA, ePPE+pegRNA, PPE+epegRNA, and ePPE+epegRNA.

[0018] Figure 3. The relationship between the insertion length (30bp-100bp) and the distance between the two pegRNAs (PAM distance 20bp-80bp) and the effect of the overlap length between the two RTs (10bp-50bp) on the insertion efficiency were evaluated using the ePPE+epegRNA combination.

[0019] Figure 4. Efficiency of site-directed insertion at NG PAM when Cas9 is replaced by SpG-Cas9 or SpRY-Cas9.

[0020] Figure 5. Effect of different promoters on pegRNA insertion efficiency.

[0021] Figure 6. Effects of 37°C temperature treatment (6B) and the MS2-MCP MLV recruitment system (6C) on long fragment insertion efficiency.

[0022] Figure 7. A) Schematic diagram of the GFP reporter system; B) Evaluation of the editing effects of eight recombinases and the corresponding recombinase site sequences. Microscope images of rice protoplasts transformed with or without the corresponding recombinases; C) Verification of recombinase editing effects using a fluorescent reporter system; D) Schematic diagram of constructs used to evaluate the DNA integration ability of recombinases using a fluorescent reporter system; F) Schematic diagram of constructs for one-step large-fragment insertion using a combination of recombinases and ePPE.

[0023] Figure 8. Insertion efficiency of the PrimeROOT.v1 system detected by ddPCR.

[0024] Figure 9. A) Flow cytometry analysis of the percentage of GFP-positive plant protoplast cells reflects the efficiency of "one-step" large-fragment insertion using different recombinase combinations; B) ddPCR determination of the GFP insertion efficiency of OsALS in rice protoplasts.

[0025] Figure 10. Fluorescence microscopy and flow cytometry were used to verify the editing efficiency of different base editing systems.

[0026] Figure 11. ddPCR detection of the insertion percentage of different donors into four endogenous sites.

[0027] Figure 12. Percentage of insertions at six endogenous loci in maize using the PrimeROOT.v2C-Cre system as detected by ddPCR.

[0028] Figure 13. ddPCR detection of the insertion percentage of large fragments by different gene editing systems.

[0029] Figure 14. Comparison of the precise editing efficiency of PrimeROOT.v2C-Cre and NHEJ using base sequencing results.

[0030] Figure 15. A) Schematic diagram of inserting the Act1 promoter into the OsHPPD locus using PrimeROOT.v2C-Cre; B) Screening of pegRNA pairs; C) Insertion efficiency.

[0031] Figure 16. GSH sites obtained by high-throughput sequencing and the insertion efficiency of the recombination site in GSH1 detected by high-throughput sequencing.

[0032] Figure 17. Schematic diagram of PrimeROOT.v3 and the efficiency of precise insertion through PrimeROOT.v3.

[0033] Figure 18. Efficiency and sequencing results of precise insertions in human HEK293 cells using the PrimeROOT system.

[0034] Detailed Description of the Invention

[0035] 1. Definition

[0036] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology 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 literature: 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, definitions and explanations of relevant terms are provided below.

[0037] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed 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."

[0038] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed 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, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0039] As used herein, a "genome editing system" refers to a combination of components required for genome editing in a cell. The individual components of the system, such as the guide editing fusion protein or its expression construct, the pegRNA or its expression construct, and the donor construct, can exist independently or in any combination as a composite.

[0040] "Genome" as used herein encompasses not only the chromosomal DNA present in the nucleus of a cell, but also the organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0041] As used herein, "genetically modified plants" refer to plants that contain an inserted exogenous polynucleotide in their genome. For example, the exogenous polynucleotide can be stably integrated into the genome of the plant and inherited for successive generations.

[0042] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, has been significantly altered from its native form in composition and / or locus through deliberate human intervention.

[0043] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter designations as follows: "A" for adenosine or deoxyadenosine (RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "D" for A, T, or G, "I" for inosine, and "N" for any nucleotide. Although nucleotide sequences herein may be presented as DNA sequences (including T), when reference is made to RNA, one skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0044] "Polypeptide," "peptide," 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 residues is an artificial chemical analog 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" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0045] As used herein, an "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, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0046] 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 RNA (such as mRNA) that can be translated, for example, RNA generated by in vitro transcription.

[0047] 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.

[0048] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0049] Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. When used in plants, the promoter can be cauliflower mosaic virus 35S promoter, maize Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, maize U3 promoter, rice actin promoter.

[0050] "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 the cells of the organism 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 the genome, resulting in the 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 to perform a function without the stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0051] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0052] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf greenness, 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 tiller number, etc.

[0053] 2. Genome editing systems for site-specific modification in the genome of an organism, such as site-specific insertion of exogenous nucleotide sequences

[0054] In one aspect, the present invention relates to a genome editing system for site-specific modification, such as site-specific insertion of an exogenous nucleotide sequence, in the genome of an organism, comprising:

[0055] i) a) a CRISPR nuclease and / or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and / or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase, or

[0056] b) a guide editing fusion protein and / or an expression construct containing a nucleotide sequence encoding the guide editing fusion protein, wherein the guide editing fusion protein comprises a CRISPR nuclease and a reverse transcriptase;

[0057] ii) a first pegRNA and / or an expression construct containing a nucleotide sequence encoding said first pegRNA, and

[0058] iii) a second pegRNA and / or an expression construct containing a nucleotide sequence encoding said second pegRNA,

[0059] The first pegRNA comprises a first guide sequence, a first scaffold sequence, a first reverse transcription template (RT) sequence and a first primer binding site (PBS) sequence from 5' to 3' direction,

[0060] wherein the second pegRNA comprises a second guide sequence, a first scaffold sequence, a second reverse transcription template (RT) sequence and a second primer binding site (PBS) sequence from 5' to 3' direction,

[0061] Wherein the first pegRNA targets a first target sequence on the sense strand of the organism's genomic DNA, and the second pegRNA targets a second target sequence on the antisense strand of the organism's genomic DNA. In some embodiments, the organism is a plant.

[0062] As used herein, "target sequence" refers to a sequence of approximately 20 nucleotides in length characterized by a PAM (protospacer adjacent motif) sequence flanking the 5' or 3' ends of the genome. Generally speaking, a PAM is necessary for the complex formed by the CRISPR nuclease or its variants and the guide RNA to recognize the target sequence. For example, for the Cas9 nuclease and its variants, the target sequence is adjacent to the PAM at the 3' end, such as 5'-NGG-3'. Based on the presence of the 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 the PAM, the target sequence can be located on either strand of the genomic DNA molecule, and the strand where the target sequence is located is called the target strand. For Cas9 or its derivatives, such as Cas9 nickase, the target sequence is preferably 20 nucleotides. Depending on the different CRISPR nucleases or their different variants, the PAM sequence will be different.

[0063] In some embodiments, the 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 on the target strand (eg, within the target sequence).

[0064] In some embodiments, the PAMs of the first target sequence and the second target sequence are spaced apart by about 1 to about 300 bp, such as 10 bp to about 100 bp, such as about 20 bp to about 60 bp. In some embodiments, the PAMs of the first target sequence and the second target sequence can be spaced apart by about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 100 bp, about 150 bp, or about 300 bp.

[0065] In some embodiments, the CRISPR nuclease is a Cas9 nuclease, such as SpCas9 derived from Streptococcus pyogenes (S. pyogenes). An exemplary wild-type SpCas9 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0066] In some embodiments, the CRISPR nuclease is a CRISPR nickase. The CRISPR nickase in the fusion protein is capable of forming a nick within the target sequence on the target strand of the genomic DNA (the strand where the target sequence is located). 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 the amino acid substitution H840A relative to wild-type SpCas9. 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 the +1 position) and the -4 nucleotide of the PAM of the target sequence.

[0068] In some embodiments, the Cas9 nuclease, such as a nickase, is a Cas9 nuclease or nickase variant that can recognize an altered PAM sequence. Many Cas9 nickase variants that can recognize an altered PAM sequence are known in the art. In some embodiments, the Cas9 nuclease, such as a 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, D1135L, S1136W, G1218K, E1219Q, R1335Q, T1337R relative to wild-type Cas9, wherein the amino acid numbering is with reference to SEQ ID NO: 1. In some embodiments, the Cas9 nickase variant (SpG-Cas9 nickase) comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the Cas9 nickase variant that recognizes the PAM sequence 5'-NG-3' comprises the following amino acid substitutions relative to wild-type Cas9: H840A, A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R, wherein the amino acid numbering is with reference to SEQ ID NO: 1. In some embodiments, the Cas9 nickase variant (SpRY-Cas9 nickase) comprises the amino acid sequence shown in SEQ ID NO: 43.

[0069] The nick formed by the Cas9 nuclease of the present invention, such as a nickase, can cause the target strand 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 CRISPR nuclease, such as Cas9 nickase, and the reverse transcriptase in the guide editing fusion protein are connected by a linker.

[0071] In some embodiments, the reverse transcriptase of the present invention can be derived from various sources. In some embodiments, the reverse transcriptase is a reverse transcriptase derived from a virus. For example, in some embodiments, the reverse transcriptase is 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.

[0072] In some embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof,

[0073] (a) comprising a mutation at position 155, 156, 200 and / or 524, for example comprising a mutation selected from any one of F155Y, F155V, F156Y, D524N, N200C, or a combination thereof, wherein the amino acid positions are referenced to SEQ ID NO: 3;

[0074] (b) the connection sequence is missing; and / or

[0075] (c) The RNase H domain is mutated or deleted.

[0076] In some preferred embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase, or a functional variant thereof comprises a mutation selected from D524N, wherein the amino acid position is referenced to SEQ ID NO: 3.

[0077] In some preferred embodiments, the RNase H domain of the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is deleted.

[0078] In some embodiments, the connection sequence comprises the amino acid sequence shown in SEQ ID NO:4.

[0079] In some embodiments, the RNase H domain comprises the amino acid sequence shown in SEQ ID NO:5.

[0080] In some embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase, or a functional variant thereof comprises the sequence of any one of SEQ ID NOs: 9-15, preferably comprises the amino acid sequence shown in SEQ ID NO: 14.

[0081] In some embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is fused directly or via a linker to a nucleocapsid protein (NC), a hydrolase (PR) or an integrase (IN) at the N-terminus or C-terminus. The nucleocapsid protein (NC), a hydrolase (PR) or an integrase (IN) is, for example, from M-MLV.

[0082] In some embodiments, the nucleocapsid protein (NC) comprises the amino acid sequence shown in SEQ ID NO:6.

[0083] In some embodiments, the hydrolase (PR) comprises the amino acid sequence shown in SEQ ID NO:7.

[0084] In some embodiments, the integrase (IN) comprises the amino acid sequence shown in SEQ ID NO:8.

[0085] In some preferred embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is fused to the nucleocapsid protein (NC) at the N-terminus directly or through a linker.

[0086] In some preferred embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is fused to the nucleocapsid protein (NC) at the C-terminus directly or through a linker.

[0087] In some embodiments, the reverse transcriptase can also be fused to an RNA aptamer binding protein sequence (e.g., an MCP protein sequence) via a linker or directly. Thus, the reverse transcriptase can be recruited to the CRISPR nuclease by the interaction of an RNA aptamer binding protein sequence (e.g., an MCP protein sequence) and one or more RNA aptamer sequences (e.g., an MS2 sequence) present on the pegRNA. In this case, there is no need to fuse the CRISPR nuclease to the reverse transcriptase. An exemplary MCP protein comprises the amino acid sequence of SEQ ID NO:44.

[0088] As used herein, a "linker" 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 in length, without secondary or higher structure. For example, the linker can be a flexible linker, such as GGGGS, GS, GAP, (GGGGS) x 3, GGS, and (GGS) x 7. For example, it can be the linker shown in SEQ ID NO: 16.

[0089] In some embodiments, the CRISPR nuclease, such as a CRISPR nickase, in the fusion protein is located at the N-terminus of the reverse transcriptase. In some embodiments, the CRISPR nuclease, such as a CRISPR nickase, in the fusion protein is located at the C-terminus of the reverse transcriptase.

[0090] In some embodiments of the present invention, the CRISPR nuclease, reverse transcriptase, recombinase or fusion protein of the present invention may further comprise one or more nuclear localization sequences (NLS). In general, the one or more NLSs in the CRISPR nuclease, reverse transcriptase or fusion protein should have sufficient strength to drive the CRISPR nuclease, reverse transcriptase or fusion protein to accumulate in the nucleus of the cell in an amount that can achieve its base editing function. In general, The intensity of nuclear localization activity is determined by the number, position, one or more specific NLSs used, or a combination of these factors in the CRISPR nuclease, reverse transcriptase or fusion protein.

[0091] In some preferred embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, the CRISPR nuclease, such as a nickase, the nucleocapsid protein (NC), and the reverse transcriptase, connected by or without a linker. In some preferred embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a nuclear localization sequence-the CRISPR nuclease, such as a nickase-linker-the nucleocapsid protein (NC)-nuclear localization sequence-linker-the reverse transcriptase-nuclear localization sequence.

[0092] In some preferred embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 19 (ePPE).

[0093] In some embodiments, the fusion protein comprises a nuclease portion and a reverse transcriptase portion, wherein the nuclease portion comprises the CRISPR nuclease such as CRISPR nickase and one or more NLS, and the reverse transcriptase portion comprises an RNA aptamer binding protein sequence (e.g., MCP protein sequence), the reverse transcriptase, one or more NLS and optionally the nucleocapsid protein (NC), wherein the nuclease portion and the reverse transcriptase portion are connected by a self-cleaving peptide. When the fusion protein is translated in vivo, a separate nuclease portion polypeptide and a nuclease portion polypeptide are formed, and the reverse transcriptase portion is recruited to the nuclease portion by the interaction of an RNA aptamer binding protein sequence (e.g., MCP protein sequence) and one or more RNA aptamer sequences (e.g., MS2 sequences) present on the pegRNA. Exemplary MCP protein comprises the amino acid sequence of SEQ ID NO:44.

[0094] In some embodiments, the pegRNA of the present invention further comprises one or more RNA aptamer sequences (e.g., MS2 sequences). An exemplary one or more MS2 sequences are shown in SEQ ID NO: 45. In some embodiments, the one or more RNA aptamer sequences (e.g., MS2 sequences) are located at the 3' end of the pegRNA. In some embodiments, the one or more RNA aptamer sequences (e.g., MS2 sequences) are located in the middle of the pegRNA, for example, between the scaffold sequence and the RT sequence. The one or more RNA aptamer sequences (e.g., MS2 sequences) can be used to recruit a reverse transcriptase comprising an RNA aptamer binding protein sequence (e.g., MCP protein sequence) to the CRISPR nuclease-pegRNA complex.

[0095] The guide sequence (also called seed sequence or spacer sequence) in the 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.

[0096] For example, the guide sequence in the first pegRNA can have sufficient sequence identity (preferably 100% identity) to the first target sequence such that its complex with a CRISPR nuclease, such as a nickase, results in a nick in the first target sequence; the guide sequence in the second pegRNA can have sufficient sequence identity (preferably 100% identity) to the second target sequence on the opposite strand such that its complex with a CRISPR nuclease, such as a nickase, results in a nick in the second target sequence, whereby the two pegRNAs result in nicks on different strands of the genomic DNA.

[0097] A variety of scaffold sequences suitable for gRNAs for genome editing based on CRISPR nucleases (e.g., Cas9) are known in the art and can be used in the pegRNA of the present invention. In some embodiments, the scaffold sequence of the gRNA is shown in SEQ ID NO: 17.

[0098] In some embodiments, the primer binding sequence is configured to be complementary to at least a portion of the target sequence (preferably completely paired 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 resulting from the nick in the DNA chain containing the target sequence (preferably completely paired 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 paired with). 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, using the reverse transcription template (RT) sequence immediately adjacent to the primer binding sequence as a template, and reverse transcription is performed under the action of the reverse transcriptase in the fusion protein, thereby extending a DNA sequence corresponding to the reverse transcription template (RT) sequence.

[0099] 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.

[0100] 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.

[0101] Methods for calculating the Tm of nucleic acid sequences are well known in the art, for example, the Oligo Analysis Tool online analysis tool can be used for calculation. 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 an appropriate length of the PBS. Alternatively, a PBS sequence with a suitable Tm can be obtained by selecting an appropriate target sequence.

[0102] 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 (that is, the chain containing the target sequence PAM), and then through the DNA repair effect of the cell, a DNA double-stranded chain containing the RT template sequence information is formed. In some embodiments, the RT template sequence contains the desired modification. For example, the desired modification includes the substitution, deletion and / or addition of one or more nucleotides. In some embodiments, the RT template sequence is set to a sequence corresponding to the downstream of the target sequence nick (for example, complementary to at least a portion of the sequence downstream of the target sequence nick), but contains the desired modification. The desired modification includes the substitution, deletion and / or addition of one or more nucleotides.

[0103] In some embodiments, the two pegRNAs 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 the pegRNAs targeting two different chains to achieve the same desired modification.

[0104] In some embodiments, the RT sequence is configured to generate the exogenous nucleotide sequence to be inserted into the genome or a portion thereof, or to generate a complementary sequence of the exogenous nucleotide sequence to be inserted into the genome of an organism, such as a plant, or a portion thereof, after reverse transcription using the RT sequence as a template. In some embodiments, the RT sequence does not contain a genomic sequence near the target sequence or a complementary sequence of a genomic sequence near the target sequence. In some embodiments, the RT sequence does not contain sequence information other than the exogenous nucleotide sequence to be inserted.

[0105] In some embodiments, the first RT sequence and the second RT sequence are used to insert a first exogenous nucleotide sequence, for example, between the first target sequence and the second target sequence (such as between the nick of the first target sequence and the nick of the second target sequence).

[0106] In some embodiments, the first RT sequence of the first pegRNA is set to generate a first fragment of the first exogenous nucleotide sequence to be inserted into the genome after reverse transcription using it as a template; the second RT sequence of the second pegRNA is set to generate a complementary sequence of the second fragment of the first exogenous nucleotide sequence to be inserted into the genome after reverse transcription using it as a template.

[0107] In some embodiments, the first segment and the second segment of the first exogenous nucleotide sequence to be inserted at least partially overlap. In some embodiments, the first segment and the second segment overlap by at least about 10 bp to about 50 bp, such as at least about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, or about 50 bp. In some embodiments, the first segment and the second segment of the first exogenous nucleotide sequence to be inserted completely overlap.

[0108] In some embodiments, the length of the first exogenous nucleotide sequence to be inserted is about 1 bp to about 700 bp, such as about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 600 bp, about 700 bp, or any value therebetween.

[0109] In some embodiments, the pegRNA further comprises a tevopre sequence at the 3' end of the PBS. The design of the tevopre sequence can be found in James W. Nelson et al., Engineered pegRNAs improve prime editing efficiency. 2022, Nature Biotech. Volume 40, Pages 402–410. An exemplary tevopre sequence is shown in SEQ ID NO: 20.

[0110] In some embodiments, the pegRNA further comprises a polyA sequence at the 3' end. The polyA sequence, for example, comprises approximately 10-30 consecutive adenosine nucleotides (A).

[0111] In some embodiments, the pegRNA comprises, from 5' to 3' direction, a guide sequence, a scaffold sequence, a reverse transcription template (RT) sequence, a primer binding site (PBS) sequence, a tevopre sequence, and a polyA sequence.

[0112] In some embodiments, the pegRNA can be precisely processed using a self-processing system. In some specific embodiments, the 5' end of the pegRNA is connected to a first ribozyme or tRNA, which is designed to cut the fusion at the 5' end of the pegRNA; and / or the 3' end of the pegRNA is connected to a second ribozyme or tRNA, which is designed to cut the fusion at the 3' end of the pegRNA. The design of the first or second ribozyme or tRNA 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.

[0113] In some embodiments, the first pegRNA and the second pegRNA are transcribed by different promoters. For example, the first pegRNA is expressed by the OsU3 promoter, and the second pegRNA is expressed by the TaU3 promoter.

[0114] In some embodiments, the pegRNA is transcribed by a type II promoter, i.e., in an expression construct comprising a nucleotide sequence encoding the pegRNA, the pegRNA encoding nucleotide sequence is operably linked to a type II promoter. In some specific embodiments, the type II promoter is a GS promoter. The sequence of an exemplary GS promoter is shown in SEQ ID NO: 21.

[0115] In some embodiments, the first target sequence, the second target sequence and / or the desired modification, such as the first exogenous nucleotide sequence, is associated with an organism, such as a plant trait, such as an agronomic trait, whereby insertion of the desired modification, such as the first exogenous nucleotide sequence, results in the organism, such as a plant, having an altered (preferably improved) trait, such as an agronomic trait, relative to a wild-type organism, such as a plant.

[0116] In some embodiments, the first exogenous nucleotide sequence comprises one or more recombinase recognition sites (RS).

[0117] In some embodiments, the recombinase is a recombinase of the tyrosine recombinase family or a recombinase of the serine recombinase family, preferably a recombinase of the tyrosine recombinase family. Exemplary tyrosine recombinases include but are not limited to Escherichia coli phage lambda integrase, P1 phage Cre recombinase (cyclization recombinase), yeast FLP recombinase (flippase recombinase). Exemplary serine recombinases include but are not limited to Tn3 transposase, Salmonella recombinase Hin, Streptomyces phage ΦC31 integrase and mycobacteriophage Bxb1 integrase. Different recombinases and their corresponding recombinase recognition sites (RS) are known in the art, and those skilled in the art can select as needed.

[0118] In some embodiments, the recombinase is Dre recombinase. An exemplary Dre recombinase comprises the amino acid sequence of SEQ ID NO: 56. Accordingly, the one or more recombinase recognition sites (RS) include but are not limited to rox (SEQ ID NO: 57, 58).

[0119] In some embodiments, the recombinase is ΦC31 integrase. An exemplary ΦC31 integrase comprises the amino acid sequence of SEQ ID NO: 22. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, aTTP (SEQ ID NO: 38) and / or aTTB (SEQ ID NO: 39).

[0120] In some preferred embodiments, the recombinase is Bxb1 integrase. An exemplary Bxb1 integrase comprises the amino acid sequence of SEQ ID NO: 23. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, aGTP (SEQ ID NO: 40) and / or aGTB (SEQ ID NO: 41).

[0121] In some preferred embodiments, the recombinase is a Cre recombinase. An exemplary Cre recombinase comprises the amino acid sequence of SEQ ID NO: 24. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, loxP (SEQ ID NO: 26), Lox2272 (SEQ ID NO: 29), Lox71 (SEQ ID NO: 27), Lox66 (SEQ ID NO: 28), or variants thereof, and any combination thereof.

[0122] In some preferred embodiments, the recombinase is FLP recombinase. An exemplary FLP recombinase comprises the amino acid sequence of SEQ ID NO: 25. Accordingly, the one or more recombinase recognition sites (RS) include but are not limited to FRT1 (SEQ ID NO: 30), FRT6 (SEQ ID NO: 31), or variants thereof, and any combination thereof. In some embodiments, the one or more recombinase recognition sites (RS) are variants of FRT1, for example, comprising a sequence described in one of SEQ ID NOs: 32-37.

[0123] In some embodiments, the recombinase is a B2 recombinase. An exemplary B2 recombinase comprises the amino acid sequence of SEQ ID NO: 50. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, the nucleotide sequence of SEQ ID NO: 53.

[0124] In some embodiments, the recombinase is a KD recombinase. An exemplary KD recombinase comprises the amino acid sequence of SEQ ID NO: 51. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, the nucleotide sequence shown in SEQ ID NO: 54.

[0125] In some embodiments, the recombinase is pSR1 recombinase. An exemplary pSR1 recombinase comprises the amino acid sequence of SEQ ID NO: 52. Accordingly, the one or more recombinase recognition sites (RS) include, but are not limited to, the nucleotide sequence of SEQ ID NO: 55.

[0126] Based on one or more recombinase recognition sites (RS) in the first exogenous nucleotide sequence inserted into the genome, a donor comprising RS and a second exogenous nucleotide sequence is provided, and the second exogenous nucleotide sequence can be inserted into an organism such as a plant genome by recombination using a corresponding recombinase. The recombinase can be a recombinase expressed alone or included in the guide editing fusion protein. Those skilled in the art can select a suitable combination of RS located in the first exogenous polynucleotide inserted into the genome and RS located in the donor to insert the second exogenous nucleotide sequence into the genome by recombination.

[0127] Therefore, in some embodiments, the genome editing system further comprises:

[0128] iv) a recombinase and / or an expression construct containing a nucleotide sequence encoding said recombinase, and

[0129] v) a donor construct comprising one or more recombinase recognition sites (RS) and a second exogenous polynucleotide sequence to be inserted into the plant genome.

[0130] In some preferred embodiments, the recombinase is contained within the guide editing fusion protein. In some embodiments, the recombinase is located at the N-terminus of the guide editing fusion protein relative to the CRISPR nuclease and reverse transcriptase. In some embodiments, the recombinase is located at the C-terminus of the guide editing fusion protein relative to the CRISPR nuclease and reverse transcriptase.

[0131] The second exogenous polynucleotide sequence can be of any length. The second exogenous polynucleotide sequence can be from 1 bp to about 10 kb or longer. Preferably, the second exogenous polynucleotide is a long fragment, for example, at least 300 bp, at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb or longer. In some embodiments, the second exogenous polynucleotide can be a full-length gene.

[0132] In some embodiments, the second exogenous nucleotide sequence is associated with a trait, such as an agronomic trait, of an organism, such as a plant, whereby insertion of the second exogenous nucleotide sequence results in the organism, such as a plant, having an altered (preferably improved) trait, such as an agronomic trait, relative to a wild-type organism, such as a plant.

[0133] Different components of the genome editing system of the present invention, such as the coding sequences of CRISPR nuclease, reverse transcriptase, guide editing fusion protein, pegRNA and / or recombinase and the second exogenous polynucleotide sequence, can be located in the same construct in different combinations, or respectively in different constructs.

[0134] The organisms that can be subjected to site-directed modification, such as site-directed insertion of exogenous nucleotide sequences, by the genome editing system of the present invention can be non-human animals, humans, or plants, preferably plants. Suitable plants include monocots and dicots, for example, crop plants, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, and potato.

[0135] In order to achieve efficient expression in organisms such as plants, in some embodiments of the present invention, the nucleotide sequence encoding the fusion protein is codon-optimized for the organism, such as the plant species, whose genome is to be modified.

[0136] Codon optimization refers to a process 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 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. Thus, 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 in various ways. See, Nakamura et al., 2002, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0137] 3. Methods for site-directed modification in plant genomes, such as site-directed insertion of exogenous nucleotide sequences

[0138] In another aspect, the present invention provides a method for site-directed modification of a plant genome, comprising introducing the genome editing system of the present invention into at least one plant. The site-directed modification comprises substitution, deletion, and / or addition of one or more nucleotides. For example, the site-directed modification comprises site-directed insertion of an exogenous nucleotide sequence.

[0139] In another aspect, the present invention provides a method for producing a genetically modified plant comprising a site-directed modification, the method comprising introducing the genome editing system of the present invention into at least one of the plants. The site-directed modification comprises substitution, deletion, and / or addition of one or more nucleotides. For example, the site-directed modification comprises site-directed insertion of an exogenous nucleotide sequence.

[0140] In some embodiments, the method further comprises screening the at least one plant for plants having a desired site-directed modification, such as a site-directed insertion of an exogenous nucleotide sequence.

[0141] In the methods of the present invention, the genome editing system can be introduced into plants using various methods well known to those skilled in the art. Methods for introducing the genome editing system of the present invention into plants include, but are not limited to, biolistic methods, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection. Preferably, the genome editing system is introduced into plants via transient transformation.

[0142] In some embodiments, the components of the sgRNA editing system are introduced into the plant simultaneously. In some embodiments, the components of the sgRNA editing system are introduced into the plant separately or sequentially.

[0143] In some embodiments, the method comprises the following steps:

[0144] 1) transforming components i)-iv) of the genome editing system into isolated plant cells or tissues to obtain plant cells or tissues into which a first exogenous nucleotide sequence comprising one or more recombinase recognition sites (RS) is inserted;

[0145] 2) transforming component v) of the genome editing system into the plant cell or tissue obtained in step 1), thereby obtaining a plant cell or tissue comprising the inserted second exogenous polynucleotide sequence; and

[0146] 3) Regenerating a whole plant from the plant cells or tissues obtained in step 2).

[0147] In some embodiments, wherein the exogenous nucleotide sequence is inserted into a safe harbor site in the plant genome, the safe harbor site in the plant genome

[0148] 1) At least 5 kb away from the protein coding region;

[0149] 2) at least 30 kb away from the miRNA coding region;

[0150] 3) at least 20 kb away from the lncRNA coding region;

[0151] 4) at least 20 kb away from the tRNA coding region;

[0152] 5) at least 5 kb from the promoter and / or enhancer;

[0153] 6) at least 20 kb away from the LTR repeat;

[0154] 7) at least 200 bp away from non-LTR repeats; and

[0155] 8) At least 10 kb away from the centromere.

[0156] In some embodiments, the plant is rice, and the safe harbor site is selected from the sites shown in Tables 1 and 2.

[0157] In some embodiments, the introduction comprises 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 whole plants. Preferably, no selection agent for the selection gene carried on the expression vector is used during tissue culture.

[0158] In other embodiments, the genome editing system of the present invention can be transformed into specific parts of intact plants, such as leaves, stem tips, pollen tubes, young ears, or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0159] In some embodiments of the invention, an in vitro expressed protein and / or in vitro transcribed RNA molecule (eg, the expression construct is an in vitro transcribed RNA molecule) and / or a donor DNA molecule is directly transformed into the plant.

[0160] In some embodiments, the method further comprises treating (e.g., culturing) the plant cells, tissues, or whole plants into which the genome editing system has been introduced at an elevated temperature (relative to conventional culture temperatures, such as room temperature), for example, 37°C.

[0161] In some embodiments of the present invention, the site-directed modification, e.g., the site-directed inserted exogenous nucleotide sequence and / or the target sequence is associated with a plant trait, such as an agronomic trait, thereby causing the site-directed modification, e.g., the site-directed insertion, to result in the plant having an altered (preferably improved) trait, e.g., an agronomic trait, relative to a wild-type plant.

[0162] In some embodiments, the method further comprises the step of screening plants for a desired site-directed modification, such as a site-directed insertion, and / or a desired trait, such as an agronomic trait.

[0163] 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 (such as site-directed exogenous polynucleotide insertion) and / or a desired trait such as an agronomic trait.

[0164] In another aspect, the present invention also provides a genetically modified plant or its offspring or part thereof, wherein the plant is obtained by the above-mentioned method of the present invention. Preferably, the genetically modified plant or its offspring has a desired genetic modification (such as site-directed exogenous polynucleotide insertion) and / or a desired trait such as an agronomic trait.

[0165] In another aspect, the present invention also provides a plant breeding method, comprising crossing a first genetically modified plant obtained by the above-described method of the present invention with a second plant that does not contain the modification, thereby introducing the modification (e.g., site-directed exogenous polynucleotide insertion) into the second plant. Preferably, the genetically modified first plant and the second plant have desired traits, such as agronomic traits.

[0166] Plants that can be subjected to site-directed modification, such as site-directed insertion of exogenous nucleotide sequences, 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.

[0167] In another aspect, the present invention provides a method for producing a genetically modified plant comprising a site-specifically inserted exogenous nucleotide sequence, the method comprising inserting the exogenous nucleotide sequence into a safe harbor site in the plant genome, the safe harbor site in the plant genome

[0168] 1) At least 5 kb away from the protein coding region;

[0169] 2) at least 30 kb away from the miRNA coding region;

[0170] 3) at least 20 kb away from the lncRNA coding region;

[0171] 4) at least 20 kb away from the tRNA coding region;

[0172] 5) at least 5 kb from the promoter and / or enhancer;

[0173] 6) at least 20 kb away from the LTR repeat;

[0174] 7) at least 200 bp away from non-LTR repeats; and

[0175] 8) At least 10 kb away from the centromere.

[0176] In some embodiments, the plant is rice, and the safe harbor site is selected from the sites shown in Tables 1 and 2.

[0177] IV. Methods for site-directed modification, such as site-directed insertion of exogenous nucleotide sequences, in human or non-human animal genomes

[0178] In another aspect, the present invention provides a method for site-directed modification of a human or non-human animal genome, comprising introducing the genome editing system of the present invention into at least one of the human or non-human animal cells. The site-directed modification comprises substitution, deletion, and / or addition of one or more nucleotides. For example, the site-directed modification comprises site-directed insertion of an exogenous nucleotide sequence.

[0179] On the other hand, the present invention provides a kind of site-directed modification of human or non-human animal genome in vivo, exogenous gene therapy purposes, can realize the deletion, addition, up-regulation, down-regulation, inactivation, activation or mutation correction of disease-related genes, so as to realize the prevention and / or treatment of disease.For example, the target nucleic acid region described in the present invention can be located in the protein coding region of disease-related genes, or for example can be located in gene expression regulatory region such as promoter region or enhancer region, so as to realize the modification of disease-related gene function or the modification of disease-related gene expression.Therefore, the modification disease-related genes described herein include modification of disease-related genes themselves (such as protein coding regions), and also include modification of their expression regulatory regions (such as promoters, enhancers, introns, etc.).

[0180] On the other hand, the present invention provides a method for producing a genetically modified human or non-human animal somatic cell, wherein the genetically modified somatic cell comprises a site-directed modification, and the method comprises introducing the genome editing system of the present invention into at least one of the human or animal somatic cells. The site-directed modification comprises substitution, deletion and / or addition of one or more nucleotides. For example, the site-directed modification comprises site-directed insertion of an exogenous nucleotide sequence.

[0181] Therefore, the present invention also provides a method for treating a disease in a subject in need thereof, comprising delivering an effective amount of the genome editing system of the present invention to the subject to modify a gene associated with the disease. The present invention also provides the use of a genome editing system in the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the genome editing system is used to modify a gene associated with the disease. The present invention also provides a pharmaceutical composition for treating a disease in a subject in need thereof, comprising the genome editing system of the present invention, and an optional pharmaceutically acceptable carrier, wherein the genome editing system is used to modify a gene associated with the disease. In some embodiments, the subject is a human.

[0182] 5. Test Kit

[0183] The present invention also includes a kit for the method of the present invention, which kit includes at least components of the genome editing system of the present invention. The kit may also include reagents for introducing the genome editing system into an organism or an organism cell. The kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit. Example

[0184] Example 1. Design of a novel genome editing system

[0185] 1.1. Boot Editor (PE) System Screening

[0186] Primer editing (PE) is a precise genome editing technology that can generate base changes and short DNA insertions and deletions without forming DSBs. It has been widely used across species such as humans, mice, rice, wheat, and maize. To develop a novel genome editing system, this example first screened the efficiency of reported PE systems for editing endogenous targets using a dual-pegRNA strategy. Five PE system constructs were compared: PPE, Art-PPE (Cas9 fused to the 5' end of the mouse exonuclease Artemis), PPE-NCV1, ePPE (Zong, Y., Liu, Y., Xue, C. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat Biotechnol 40, 1394–1402 (2022).), and ePPE-wtCas9 (H840A-Cas9 in ePPE replaced with wtCas9). The efficiency of inserting two recombinase recognition sites (RS) of Lox66 (34 bp in length) and / or FRT1 (48 bp in length) into the endogenous target site using a dual pegRNA strategy was shown in Figure 1A.

[0187] Rice protoplasts were selected as model cells. The constructs described above were transformed into rice protoplasts via PEG transformation. Five guide editing system constructs were tested for their efficiency in inserting Lox66 or FRT1 into endogenous loci in rice protoplasts using five pairs of pegRNAs. The next-generation sequencing results are shown in Figure 1B.

[0188] The results showed that when using the dual pegRNA strategy for site-directed insertion, ePPE was the most efficient, and could increase the precise insertion efficiency by 10-50 times compared to PPE.

[0189] 1.2. Guide RNA Screening

[0190] We further compared the editing efficiency of conventional pegRNA with that of tevoPre-containing epegRNA, which has been reported to improve prime editing efficiency (Nelson, JW, Randolph, PB, Shen, SP et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol 40, 402–410 (2022)). Four combinations were tested: PPE+pegRNA, ePPE+pegRNA, PPE+epegRNA, and ePPE+epegRNA. Vector construction is shown in Figure 2A.

[0191] Rice protoplasts were also used to test the efficiency of the above four combinations in site-directed insertion of RS using eight pairs of pegRNA / epegRNA. The results of next-generation sequencing are shown in Figure 2B.

[0192] The results show that the site-directed insertion efficiency mediated by the dual-pegRNA strategy using a combination of ePPE+epegRNA (hereinafter referred to as "dual-ePPE") is the highest, and the maximum efficiency of some sites can reach more than 50%, which is more than 100 times higher than that of ordinary PPE+pegRNA combinations, and has a higher efficiency in most inefficient target sites. It is worth noting that while the editing efficiency of the editing tool at the target site is improved, it is also accompanied by an increase in the probability of non-precise editing or insertion or deletion of other sites. While dual-ePPE has a significant improvement in the precise editing efficiency of the target site compared to other combinations, there is no significant change in the efficiency of insertion or deletion of other sites.

[0193] 1.3.dual-ePPE insertion

[0194] We further used dual-ePPE to evaluate the relationship between insert length (30-100 bp) and the distance between the two pegRNAs (PAM distance 20-80 bp), as well as the effect of the overlap length between the two RTs (10-50 bp) on insertion efficiency. The results of next-generation sequencing using rice protoplasts are shown in Figure 3.

[0195] The results showed that there was a non-significant linear relationship between the insertion length and the distance between the pegRNAs. The efficiency was higher when the insertion length was greater than the distance between the pegRNAs, and the overlap length between the two RTs was between 10bp and 50bp, which showed a high site-specific insertion efficiency. These results show that the ePPE+epegRNA system of the present invention can meet the requirements of efficient and site-specific insertion of tag sequences such as Flag and Tag.

[0196] Example 2. Optimization of the dual-ePPE system

[0197] In order to further verify and optimize the effects of the dual-ePPE system of the present invention in different usage environments and thus obtain an optimal technical solution, this embodiment verifies and analyzes possible improvements to the various components of the dual-ePPE system.

[0198] 2.1. CRISPR system effector proteins

[0199] In this example, SpG-Cas9 with an NGN PAM recognition sequence and a SpRY-Cas9 variant that is almost unrestricted by the PAM sequence (Christie KA, Guo JA, Silverstein RA, Doll RM, Mabuchi M, Stutzman HE, Lin J, Ma L, Walton RT, Pinello L, Robb GB, Kleinstiver BP. Precise DNA cleavage using CRISPR-SpRYgests. Nat Biotechnol. 2023 Mar; 41(3): 409-416.) were designed into dual-ePPE and their insertion efficiency at NGN-containing PAMs was evaluated in order to expand the targeting range of dual-ePPE. The vector construction is shown in Figure 4A.

[0200] The efficiency of site-directed insertion using three PAM combinations (NGA, NGC, and NGT) was tested using rice protoplasts. The next-generation sequencing results are shown in Figure 4B.

[0201] The results showed that SpG-ePPE and SpRY-ePPE also had high efficiency in site-directed insertion at PAMs NGA, NGC, and NGT, further verifying that the dual-ePPE system can be applied to a variety of CRISPR system effector proteins and effectively exert their functions. This result demonstrates that the dual-ePPE system of the present invention can effectively insert RS sequences in plants.

[0202] 2.2. Synonymous mutations in RT sequences

[0203] Reports have shown that introducing synonymous mutations (SM) into the RT sequence can improve recombineering editing efficiency (Xu, W., Yang, Y., Yang, B. et al. A design optimized prime editor with expanded scope and capability in plants. Nat. Plants 8, 45–52 (2022).). This example tests the editing efficiency of this system using SM on the RT. The vector construction is shown in Figure 4A.

[0204] The efficiency of point mutagenesis using two RTs at four target sites was tested using rice protoplasts. The next-generation sequencing results are shown in Figure 4C. The results demonstrate that uniform mismatches between the RT sequence and the genomic sequence significantly improve point mutagenesis efficiency (4-20-fold).

[0205] 2.3. Promoter driving epegRNA expression

[0206] We further investigated the efficiency of inserting longer fragments (150 bp-300 bp) using the above system and tested the editing efficiency when the guide RNA was expressed from the U3 promoter and the composite type II promoter (pGS promoter). The construction of the pGS-epegRNA vector is shown in Figure 5A.

[0207] The efficiency of site-directed insertion of fragments of different lengths using the U3 promoter and the pGS promoter was compared in rice protoplasts. The ddPCR test results are shown in Figures 5B and C. The results showed that there was no significant difference between the U3 promoter and the pGS promoter when inserting small fragments (Figure 5B). When the insertion length reached more than 150bp, the efficiency of the pGS promoter driving epegRNA was higher than that of the U3 promoter. Using the pGS promoter to express epegRNA can increase the efficiency of site-directed insertion of large fragments by 2-5 times, and accurate insertion can still be achieved when the insertion fragment length reaches 700bp.

[0208] Effects of MS2-MCP and Temperature Shift on Editing Efficiency

[0209] The MS2-MCP system for recruiting MLVs and the 37°C temperature treatment were used to further improve the efficiency of long fragment insertion. The vector construction is shown in FIG6A .

[0210] The efficiency of the above two recruitment methods for site-directed insertion of large fragments was tested using rice protoplasts. The efficiency of 37°C treatment (TT, normal culture for 12 h → 37°C culture for 12 h → normal culture for 24 h) was also tested. The ddPCR results are shown in Figure 6B.

[0211] The results showed that treatment with 37°C temperature could increase the efficiency of large fragment insertion by approximately 1.2-5 times (Figure 6B), and recruiting MLVs using the MS2-MCP system could increase the efficiency of large fragment insertion by approximately 2-4 times (Figure 6C).

[0212] Example 3. Using the PrimeROOT system to achieve large DNA insertion without double-strand breaks in plants

[0213] In this example, dual-ePPE is combined with recombinase as a prime editing-mediated precise targeted DNA recombination system (Prime editing-mediated Recombination of Opportune Targets, hereinafter referred to as PrimeROOT), and its ability to insert DNA fragments in plants is verified.

[0214] 3.1. Construction of fluorescence reporter system

[0215] To verify the DNA recombination ability of various recombinases in plant base editing. First, the inventors constructed a fluorescent reporter system to characterize the DNA recombination efficiency of commonly used site-specific recombinases in rice protoplasts. The reporter system divides GFP into two domains, the N-terminus (GFP-N) and the C-terminus (GFP-C), which are encoded on two separate plasmids (Figure 7A). Each of the two plasmids carries a recombinase site. See Figure 7A for a schematic diagram of the plasmid construction. After the recombinase is expressed and recombined, GFP-N and GFP-C are linked through an intron linker, thereby expressing GFP in the protoplasts. GFP fluorescence can then be observed by fluorescence microscopy and detected by flow cytometry to characterize the recombinase activity in the protoplasts.

[0216] 3.2. PrimeROOT build for detection

[0217] The inventors constructed independent fluorescent reporter systems for six different tyrosine recombinases and two serine recombinases (all codon-optimized for expression in rice). GFP fluorescence microscopy (Figure 7B) and flow cytometry (Figure 7C) revealed that the Cre and FLP recombinase systems produced the strongest fluorescence, making them the optimal recombinase systems for validating and optimizing the effectiveness of the present invention.

[0218] In another set of parallel experiments, the inventors constructed fluorescent reporter systems for the Cre / Lox system and FLP / FRT system of the tyrosine recombinase family, and the ФC31 and Bxb1 recombinases of the serine family. The vector construction is shown in FIG7D .

[0219] The above reporter system was transformed into rice protoplasts, and fluorescence microscopy and flow cytometry were performed. The results are shown in Figure 7E.

[0220] The results showed that the Cre / Lox system and the FLP / FRT system had stronger DNA integration capabilities. Therefore, they were combined with the above-mentioned site-directed insertion system, and all components (ePPE, two epegRNAs, recombinase, and the gene to be inserted with a recombination site) were transferred into rice cells through a "one-step method" to achieve site-directed insertion of large fragments at the gene level, as shown in Figure 7F. The above-mentioned "one-step method" composition containing dual-ePPE, recombinase, and the gene to be inserted with a recombination site was named PrimeROOT.v1, and was named PrimeROOT.v1-Cre and PrimeROOT.v1-FLP according to whether the recombinase was the Cre / Lox system or the FLP / FRT system, respectively.

[0221] 3.3. PrimeROOT.v1 Large Fragment Insertion Capability Verification

[0222] To verify PrimeROOT.v1's ability to insert large DNA fragments, the inventors tested the integration efficiency of PrimeROOT.v1-Cre and PrimeROOT.v1-FLP for GFP (720 kp) at four endogenous sites in rice protoplasts using ddPCR. The experimental results are shown in Figure 8. The results showed that both PrimeROOTs achieved precise, targeted insertion of large fragments at all four sites.

[0223] 3.4. Reorganization system optimization

[0224] Due to the presence of short repeat sequences in FRT1, some FRT1 mutants have been reported to promote the efficiency of FLP recombinase (Bruckner, RC & Cox, MM Specific Contacts between the Flp Protein of the Yeast 2-Micron Plasmid and Its Recombination Site. Journal of Biological Chemistry 261, 1798-1807 (1986).; Senecoff, JF, Rossmeissl, PJ & Cox, MM DNA recognition by the FLP recombinase of the yeast 2mu plasmid. A mutational analysis of the FLP binding site. J Mol Biol 201, 405-421 (1988).). In order to further optimize the editing system and obtain a more preferred technical solution. The inventors artificially designed multiple FRT1 mutants (F1m1, F1m2 and F1m3) and two truncated FRT1 (tFRT1) sequence mutants (tF1m2 and tF1m3). When PrimeROOT was used for integration, ddPCR was used to evaluate the efficiency of the one-step large-fragment insertion of the above recombinases, as well as constructs such as FRT variants, at the endogenous target site. Furthermore, a one-step method was used to insert GFP into an endogenous rice gene, resulting in luminescence in protoplast cells. The ddPCR results, shown in Figure 9, show that the combination of FRT1 mutants exhibited a higher mutation efficiency than the wild type.

[0225] 3.5. PrimeROOT system optimization

[0226] Based on PrimeROOT.v1, the inventors further optimized it to obtain a more optimal technical solution. In this technical solution, the inventors fused the ePPE of the PrimeROOT combination species with the recombinase and created two structural schemes based on different fusion sites. The example sequences are shown in Figure 10A:

[0227] In protocol 1, the recombinase was linked to the N-terminus of the ePPE system via the SV40 NLS and a 32-amino acid flexible linker, designated PrimeROOT.v2N. Protocol 2, the recombinase was linked to the C-terminus of the ePPE system via the same pathway, designated PrimeROOT.v2C. Fluorescence microscopy and flow cytometry revealed that the PrimeROOT.v2N and PrimeROOT.v2C systems achieved higher GFP insertion efficiency at the four endogenous sites compared to PrimeROOT.v1 (Figure 10).

[0228] 3.6. PrimeROOT.v2 Large Fragment Insertion Capability Verification

[0229] To validate PrimeROOT.v2's ability to insert large DNA fragments, the inventors constructed vector constructs containing any one or a combination of three genes (pigmR, OsMYB30, and OsHPPD), with donor lengths of 1.4 kb, 4.9 kb, 7.7 kb, and 11.1 kb, respectively. The vector construction is shown in Figure 11A. The inventors tested the insertion efficiency of the four donors at four endogenous loci using ddPCR and found that as the donor length gradually increased, precise and targeted large fragment insertion was achieved, without a significant decrease in editing efficiency (Figure 11B).

[0230] Example 4. Using the PrimeROOT system to achieve large DNA insertion without double-strand breaks in maize

[0231] In addition to rice protoplasts, the inventors also evaluated the editing efficiency of dual-ePPE and its PrimeROOT in maize protoplasts.

[0232] The inventors first tested the precise RS insertion editing efficiency of dual-ePPE at six endogenous gene loci in maize protoplasts. The experimental results showed that it can achieve an editing efficiency of up to 40% ( FIG12A ).

[0233] The inventors then tested the editing efficiency of PrimeROOT.v2C-Cre on large GFP DNA fragments. The experimental results showed that it achieved a GFP sequence editing efficiency of up to 4% at the endogenous site ( Figure 12B ).

[0234] The experimental results are similar to the editing efficiency in rice, which shows that the dual-ePPE of the present invention and the PrimeROOT system composed of it have broad and universal application prospects in plant synthetic biology and gene editing engineering, and can accurately insert the required DNA sequence without introducing the donor backbone sequence.

[0235] Example 5. Editing Capabilities of PrimeROOT and CRISPR-Mediated NHEJ Systems

[0236] The CRISPR-mediated NHEJ system is a currently reported system that can perform targeted large fragment insertion in plants (Li, J. et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nature Plants 2 (2016).; Dong, OXO et al. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. Nature Communications 11 (2020).). In this example, PrimeROOT.v2C-Cre was used as an example to compare the targeted insertion capabilities of PrimeROOT and CRISPR-mediated NHEJ systems for GFP (720 bp), Act1 promoter (Act1P, 1.4 kb), Act1P-pigmR gene cassette (4.9 kb), and Act1P-pigmR-Act1P-OsMYB30 gene cassette (7.7 kb). The results showed that for the insertion of GFP and Act1P, the two had similar insertion efficiencies. However, for longer donor insertions, the average efficiency of the PrimeROOT.v2C-Cre system is 2-4 times that of the NHEJ system (see Figure 13A for a schematic diagram of the construct and Figure 13B for the editing efficiency graph).

[0237] In terms of editing precision, the inventors observed that Act1P events inserted by the PrimeROOT.v2C-Cre system showed clear Sanger sequencing results, but mixed peaks appeared in the results of insertion using NHEJ (Figure 14A, underlined to indicate imprecise insertion). This indicates that the PrimeROOT system has superior editing precision compared to the traditional CRISPR-mediated NHEJ system.

[0238] The inventors then cloned the edited insertion events from protoplasts into bacteria and sequenced the junctions between the endogenous genome and the individual cloned inserts. When the inventors randomly selected 20 clones from the PrimeROOT and NHEJ-treated Act1P insertion samples, the inventors found that all 20 PrimeROOT-generated insertions contained the precise inserted sequence as expected, while all 20 NHEJ-generated insertions contained random DNA base insertions and deletions / deletions at their junctions (Figure 14A, B).

[0239] Next, the inventors used PrimeROOT and CRISPR-mediated NHEJ to insert Act1P and Act1P-pigmR sequences into the genomic site of rice callus (Figure 14C). After transfer and induction of callus, the inventors analyzed 95 callus clones from each treatment to compare editing efficiency and precision. PrimeROOT generated 2 precise Act1P insertions and 2 precise Act1P-pigmR insertions, while NHEJ generated 3 imprecise Act1P insertions and 1 imprecise Act1P-pigmR insertion (Figure 14C underlined imprecise insertions, Figure 14D). These results show that compared to the NHEJ system that relies heavily on double-strand DNA breaks as intermediate products, PrimeROOT is an effective editing tool that can be used to create large, targeted, precise DNA insertions.

[0240] Example 6: Using PrimeROOT tool to achieve precise and targeted insertion of the actin promoter

[0241] Many desirable agronomic traits are quantitative traits that depend on the upregulation or downregulation of specific genes or on tissue-specific expression. This example utilizes the PrimeROOT system to precisely insert a favorable promoter upstream of a target gene, thereby enabling the application of the PrimeROOT tool in plant trait improvement.

[0242] Specifically, the inventors used PrimeROOT.v2C-Cre to knock a strong promoter into the 5'UTR region of OsHPPD (Figure 15A). The inventors first designed 16 pairs of pegRNAs in the 5'UTR and compared their RS insertion editing efficiency in rice protoplasts, determining that the RS insertion frequency of the optimal pegRNA pair was 30% (Figure 15B). Next, the inventors used PrimeROOT.v2C-Cre and this pegRNA pair to bombard the rice Actin1 promoter (Act1P) into rice callus tissue. The inventors identified the edited plants by amplifying the connection between the genome and the inserted donor sequence, and assessed the insertion accuracy by Sanger sequencing. A total of 12 accurate Act1P insertion events (2.4%) were detected in 507 regenerated rice plants (Figure 15C). These results show that PrimeROOT can be used as an effective genome insertion tool to introduce new genetic regulatory elements into the plant genome for breeding.

[0243] Example 7: Precise gene insertion in the GSH region

[0244] In order to ensure that transgenic can be safely inserted into the plant genome, the inventors predicted the genomic safe harbor (GSH) region of the entire Kitaake rice genome. Based on the previous research method for GSH (Aznauryan, E. et al. Discovery and validation of human genomic safe harbor sites for gene and cell therapies. Cell Rep Methods 2, 100154 (2022).;Sadelain, M., Papapetrou, EP & Bushman, FD Safe harbors for the integration of new DNA in the human genome. Nat Rev Cancer 12, 51-58 (2011).), the inventors used a variety of algorithms to identify regions with a certain distance from some elements (such as gene coding regions, small RNA, miRNA, lncRNA, tRNA, promoters, enhancers, LTR, etc.). In this way, the inventors generated a set of new GSH regions, consisting of 30 regions, totaling 40kb (Figure 16A). All GSH regions of Kitaake are shown in Table 1. In addition, the inventors also determined the GSH of 33 rice genomes, and their mutually mapped GSH regions are shown in Table 2.

[0245] The inventors selected GSH1 (kitaake, Chr1:7660637-7661671) as a proof-of-concept region and designed 4 pairs of pegRNA for inserting RS in this region (Table 3). When comparing the RS insertion efficiency using dual-ePPE in GSH1, the highest RS insertion efficiency was >40% (Figure 16B). The inventors then detected the insertion of the 4.9kb ActP1P-pigmRdonor box in the GSH1 region. Gel electrophoresis and Sanger sequencing results showed that 19 Act1-pigmR insertion events (2.6%) were identified in 744 regenerated plants. Importantly, all 19 connections produced amplification products of the same size and were shown by sequencing to be the result of precise insertion events, with the ends of the donor box completely consistent with predictions.

[0246] Example 8: PrimeROOT and donor transfer method

[0247] In order to detect the insertion efficiency of the transformation method of PrimeROOT and donor components in the plant editing process. The inventors used Lox66 and FRT mutant F1m2 as landing sites to test the recovery efficiency of PrimeROOT and donor components in rice callus tissue (the system of sequential transformation is called PrimeROOT.v3) on the overall edited plant. The inventors first evaluated the dual-ePPE-mediated RS insertion in rice callus and achieved an editing efficiency of up to 84.7% (Figure 17A). In the first round of transformation, the inventors transformed PrimeROOT reagent (without donor) into callus tissue by Agrobacterium. After 1 month of hygromycin selection, the inventors enriched callus tissue containing the desired RS insertion. These calli were then used as substrates for a second round of transformation, which contained donor vectors delivered by particle bombardment or Agrobacterium. After G418 selection and regeneration, the inventors examined the regenerated plants and measured the editing frequency of the desired insertion events (Figure 17B). The inventors found that the Cre-Lox66 and FLP-F1m2 sites achieved editing efficiencies of 7.1% and 8.3% for precise insertion of OsHPPD 5'UTR into Act1P, respectively, representing 3-fold and 3.5-fold improvements over the efficiency achieved using the one-step transformation method. When evaluating the editing efficiency of Act1P-pigmR for precise insertion into GSH1, the inventors achieved efficiencies of 4.2% at the Cre-Lox66 site and 6.3% at the FLP-F1m2 site, respectively, 1.6-fold and 2.4-fold higher than those achieved using the integrated plant transformation method. When the inventors delivered the donor via Agrobacterium transformation, they achieved a 3.9% efficiency for precise insertion of Act1P-pigmR into the GSH1 locus. These results demonstrate that PrimeROOT.v3 can be used with different delivery methods, further improving the efficiency of precisely targeted gene insertion in plants.

[0248] Example 9: Testing of PrimeROOT for large insertions in human cells

[0249] In order to test whether PrimeROOT works in human cells, the inventors first replaced the promoters of PrimeROOT.V2N-Cre and PrimeROOT.V2C-Cre with the CMV promoter, a commonly used expression promoter in human cells. The inventors designed pegRNA in four regions, hAAVS1, hACTB, hCCR5, and hLMNB1, respectively, and constructed the pegRNA on the hU6 expression vector. The above plasmids and the donor plasmid containing GFP were then transferred into the HEK293 cell line by the method of plastid transformation. Cell DNA was extracted after 72 hours, and the efficiency was subsequently detected by ddPCR (Figure 18A). Junction PCR was also performed for first-generation sequencing detection, and it was found that the site-specific integration of GFP in the genome was completely accurate and predictable (Figure 18B). This example shows that the PrimeROOT system has the effect of precise targeted gene insertion in human cells.

[0250] Table 1: Summary of GSH regions

[0251] Table 2: Mutually mapped GSH regions in 33 rice genomes

[0252] Table 3 Information of pegRNA designed for GSH1 verification

[0253] Sequence information

[0254] >Wild-type SpCas9 amino acid sequence (SEQ ID NO: 1)

[0255] >nCas9(H840A) amino acid sequence (SEQ ID NO:2)

[0256] >Wild-type M-MLV-RT amino acid sequence (SEQ ID NO.3)

[0257] >M-MLV-RT-connection(SEQ ID NO.4)

[0258] >RT-RNase H (SEQ ID NO.5)

[0259] >NC (SEQ ID NO.6)

[0260] >PR (SEQ ID NO.7)

[0261] >IN (SEQ ID NO.8)

[0262] >M-MLV-RT-F155Y (SEQ ID NO.9)

[0263] >M-MLV-RT-F155V (SEQ ID NO. 10)

[0264] >M-MLV-RT-F156Y (SEQ ID NO.11)

[0265] >M-MLV-RT-D524N (SEQ ID NO.12)

[0266] >M-MLV-RT-N200C (SEQ ID NO.13)

[0267] >M-MLV-RT-ΔRNase H (SEQ ID NO.14)

[0268] >M-MLV-RT-ΔRNase H-ΔConnection (SEQ ID NO.15)

[0269] >Adapter sequence (SEQ ID NO:16)

[0270] >gRNA scaffold (SEQ ID NO:17)

[0271] >PPE (SEQ ID NO:18)

[0272] >ePPE (SEQ ID NO:19)

[0273] LAEAHGTRPDLTDQPLSGGSPKKKRKV

[0274] >tevopre (SEQ ID NO:20)

[0275] >pGS(SEQ ID NO:21)

[0276] >ФC31(SEQ ID NO:22)

[0277] >Bxb1(SEQ ID NO:23)

[0278] >Cre(SEQ ID NO:24)

[0279] >FLP(SEQ ID NO:25)

[0280] >LoxP(SEQ ID NO:26)

[0281] Lox66(SEQ ID NO:27)

[0282] Lox71(SEQ ID NO:28)

[0283] Lox2272(SEQ ID NO:29)

[0284] FRT1(SEQ ID NO:30)

[0285] FRT6(SEQ ID NO:31)

[0286] FRT1m1(SEQ ID NO:32)

[0287] FRT1m2(SEQ ID NO:33)

[0288] FRT1m3(SEQ ID NO:34)

[0289] mFRT1(SEQ ID NO:35)

[0290] mFRT1m1(SEQ ID NO:36)

[0291] mFRT1m2(SEQ ID NO:37)

[0292] aTTP (SEQ ID NO: 38)

[0293] aTTB (SEQ ID NO: 39)

[0294] aGTP (SEQ ID NO: 40)

[0295] aGTB (SEQ ID NO:41)

[0296] SpG-nCas9 (SEQ ID NO: 42)

[0297] SpRY-nCas9: (SEQ ID NO: 43)

[0298] MCP (SEQ ID NO: 44)

[0299] 2×MS2 (SEQ ID NO: 45)

[0300] PrimeROOT.v2N-Cre(SEQ ID NO:46)

[0301] PrimeROOT.v2N-FLP(SEQ ID NO:47)

[0302] PrimeROOT.v2C-Cre(SEQ ID NO:48)

[0303] PrimeROOT.v2C-FLP(SEQ ID NO:49)

[0304] B2 recombinase amino acid sequence (SEQ ID NO: 50)

[0305] KD recombinase amino acid sequence (SEQ ID NO: 51)

[0306] pSR1 recombinase amino acid sequence (SEQ ID NO: 52)

[0307] B2 recombinase recognition site (SEQ ID NO: 53)

[0308] KD recombinase recognition site (SEQ ID NO: 54)

[0309] pSR1 recombinase recognition site (SEQ ID NO: 55)

[0310] Dre recombinase amino acid sequence (SEQ ID NO: 56)

[0311] Dre recombinase recognition site rox (SEQ ID NO: 57)

[0312] Dre recombinase recognition site rox (SEQ ID NO: 58)

[0313] Figure 14A PrimeROOT.v2-Cre inserted into S20-T2 (SEQ ID NO: 59)

[0314] Figure 14A PrimeROOT.v2-Cre inserted into S20-T4 (SEQ ID NO: 60)

[0315] FIG14A NHEJ insertion of S20-T2 (SEQ ID NO: 61)

[0316] FIG14A NHEJ insertion of S20-T4 (SEQ ID NO: 62)

[0317] FIG14A NHEJ insertion S20-T2-80 bp (SEQ ID NO: 63)

[0318] CGTGGAACTGATGTTT / / GA… / / … / / AAGGTGGTATA

[0319] FIG14A NHEJ insertion S20-T2+35bp (SEQ ID NO: 64)

[0320] FIG14A NHEJ insertion S20-T2+34 / -1 bp (SEQ ID NO: 65)

[0321] FIG14A NHEJ insertion S20-T2+44 / -79 bp (SEQ ID NO: 66)

[0322] FIG14A NHEJ insertion S20-T2+62 / -4 bp (SEQ ID NO: 67)

[0323] FIG14A NHEJ insertion S20-T2+289 / -1 bp (SEQ ID NO: 68)

[0324] FIG14A NHEJ insertion S20-T2+271 / -7 bp (SEQ ID NO: 69)

[0325] FIG14A NHEJ insertion S20-T2+151 bp (SEQ ID NO: 70)

[0326] Figure 14A NHEJ insertion S20-T2+72bp (SEQ ID NO: 71)

[0327] FIG14A NHEJ insertion S20-T2+183 / -92 bp (SEQ ID NO: 72)

[0328] Figure 14A NHEJ insertion S20-T4+15bp (1) (SEQ ID NO: 73)

[0329] FIG14A NHEJ insertion S20-T4+12 bp (SEQ ID NO: 74)

[0330] FIG14A NHEJ insertion S20-T4+109 / -2bp (SEQ ID NO: 75)

[0331] Figure 14A NHEJ insertion S20-T4+15bp (2) (SEQ ID NO: 76)

[0332] FIG14A NHEJ insertion S20-T4+172 / -5bp (SEQ ID NO: 77)

[0333] Figure 14A NHEJ insertion S20-T4+15bp (3) (SEQ ID NO: 78)

[0334] FIG14A NHEJ insertion S20-T4+47 / -2 bp (SEQ ID NO: 79)

[0335] FIG14A NHEJ insertion S20-T4+143 / -2 bp (SEQ ID NO: 80)

[0336] Figure 14A NHEJ insertion S20-T4+15bp (4) (SEQ ID NO: 81)

[0337] Figure 14A NHEJ insertion S20-T4+15bp (5) (SEQ ID NO: 82)

[0338] Figure 14C PrimeROOT.v2C-Cre-Act1P or PrimeROOT.v2C-Act1P-pigmT insertion (SEQ ID NO: 83)

[0339] Figure 14C NHEJ strategy-ActP insertion ins 36bp (SEQ ID NO: 84)

[0340] Figure 14C NHEJ strategy-ActP insertion ins 12 / del 1 bp (SEQ ID NO: 85)

[0341] Figure 14C NHEJ strategy-ActP insertion ins 131bp (SEQ ID NO: 86)

[0342] Figure 14C NHEJ strategy-ActP-pigmR insertion ins 38 / del 11 bp (SEQ ID NO: 87)

[0343] Figure 18B GFP-N-terminal-hLMNB1-F (SEQ ID NO: 88)

[0344] Figure 18B GFP-C-terminal-hLMNB1-R (SEQ ID NO: 89)

Claims

1. A genome editing system for inserting an exogenous nucleotide sequence into a plant genome, comprising: i) a) a CRISPR nuclease and / or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and / or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase, or b) a guide editing fusion protein and / or an expression construct containing a nucleotide sequence encoding the guide editing fusion protein, wherein the guide editing fusion protein comprises a CRISPR nuclease and a reverse transcriptase; ii) a first pegRNA and / or an expression construct containing a nucleotide sequence encoding said first pegRNA, and iii) a second pegRNA and / or an expression construct containing a nucleotide sequence encoding said second pegRNA, The first pegRNA comprises a first guide sequence, a first scaffold sequence, a first reverse transcription template (RT) sequence and a first primer binding site (PBS) sequence from 5' to 3' direction, wherein the second pegRNA comprises a second guide sequence, a first scaffold sequence, a second reverse transcription template (RT) sequence and a second primer binding site (PBS) sequence from 5' to 3' direction, wherein the first pegRNA targets a first target sequence on the sense strand of the plant genomic DNA, and the second pegRNA targets a second target sequence on the antisense strand of the plant genomic DNA, The first RT sequence and the second RT sequence are used to insert a first exogenous nucleotide sequence.

2. The genome editing system of claim 1, wherein the pegRNA is capable of forming a complex with the CRISPR nuclease or fusion protein and targeting the CRISPR nuclease or fusion protein to a target sequence in the genome, resulting in a nick on the target strand within the target sequence.

3. The genome editing system of claim 1 or 2, wherein the PAMs of the first target sequence and the second target sequence are spaced apart by about 20 bp to about 80 bp, such as about 20 bp to about 60 bp.

4. The genome editing system of any one of claims 1-3, wherein the CRISPR nuclease is a Cas9 nuclease or a variant thereof.

5. The genome editing system of any one of claims 1 to 4, wherein the CRISPR nuclease is a CRISPR nickase, such as a Cas9 nickase or a variant thereof, such as a Cas9 nickase or a variant thereof comprising an amino acid sequence selected from SEQ ID NOs: 2 and 42-43.

6. The genome editing system of any one of claims 1-5, wherein the CRISPR nuclease, such as Cas9 nickase, and the reverse transcriptase are connected via a linker.

7. The genome editing system of any one of claims 1-6, wherein the reverse transcriptase is M-MLV reverse transcriptase or a functional variant thereof.

8. The genome editing system of any one of claims 1 to 7, wherein the RNase H domain of the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is deleted.

9. The genome editing system of any one of claims 1 to 8, wherein the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is fused to the nucleocapsid protein (NC) at the N-terminus or C-terminus directly or through a linker.

10. The genome editing system of claim 9, wherein the nucleocapsid protein (NC) comprises the amino acid sequence shown in SEQ ID NO:

6.

11. The genome editing system of any one of claims 1 to 10, wherein the reverse transcriptase is fused to an RNA aptamer binding protein sequence, such as an MCP protein sequence, via a linker or directly, and the pegRNA comprises one or more RNA aptamer sequences, such as an MS2 sequence.

12. The genome editing system of any one of claims 1-11, wherein the CRISPR nuclease, such as a CRISPR nickase, in i)-b) is fused to the reverse transcriptase via a self-cleaving peptide.

13. The genome editing system of any one of claims 1-12, wherein the CRISPR nuclease, such as a CRISPR nickase, in i)-b) is fused to the N-terminus of the reverse transcriptase.

14. The genome editing system of any one of claims 1 to 13, wherein the fusion protein in i)-b) comprises the amino acid sequence shown in SEQ ID NO:

19.

15. The genome editing system of any one of claims 1 to 14, wherein the guide sequence in the first pegRNA has sufficient sequence identity (preferably 100% identity) to the first target sequence on the sense strand, and its complex with a CRISPR nuclease such as a nickase results in a nick in the first target sequence; the guide sequence in the second pegRNA has sufficient sequence identity (preferably 100% identity) to the second target sequence on the antisense strand, and its complex with a CRISPR nuclease such as a nickase results in a nick in the second target sequence.

16. The genome editing system of any one of claims 1-15, wherein the scaffold sequence of the gRNA is shown in SEQ ID NO:

17.

17. The genome editing system of any one of claims 1 to 16, wherein the primer binding sequence is configured to be complementary to 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.

18. The genome editing system of any one of claims 1 to 17, wherein the RT sequence is configured to generate a first exogenous nucleotide sequence or a portion thereof to be inserted into the genome after reverse transcription using it as a template, or to generate a complementary sequence of the first exogenous nucleotide sequence or a portion thereof to be inserted into the plant genome.

19. The genome editing system of any one of claims 1 to 18, wherein the first RT sequence of the first pegRNA is configured to generate a first fragment of the first exogenous nucleotide sequence to be inserted into the genome after reverse transcription using it as a template; the second RT sequence of the second pegRNA is configured to generate a complementary sequence of the second fragment of the first exogenous nucleotide sequence to be inserted into the genome after reverse transcription using it as a template.

20. The genome editing system of claim 19, wherein the first fragment and the second fragment of the first exogenous nucleotide sequence to be inserted at least partially overlap.

21. The genome editing system of claim 20, wherein the first fragment and the second fragment overlap by at least about 10 bp to about 50 bp.

22. The genome editing system of any one of claims 1-21, wherein the pegRNA further comprises a tevopre sequence at the 3' end of the PBS.

23. The genome editing system of any one of claims 1-22, wherein the pegRNA further comprises a polyA sequence at the 3' end.

24. The genome editing system of any one of claims 1 to 23, wherein the length of the first exogenous nucleotide sequence to be inserted is about 1 bp to about 700 bp.

25. The genome editing system of any one of claims 1-24, wherein the 5' end of the pegRNA is linked to a first ribozyme or tRNA, which is designed to cut the fusion at the 5' end of the pegRNA; and / or the 3' end of the pegRNA is linked to a second ribozyme or tRNA, which is designed to cut the fusion at the 3' end of the pegRNA.

26. The genome editing system of any one of claims 1-25, wherein the pegRNA is transcribed by a type II promoter, for example, the type II promoter is a GS promoter.

27. The genome editing system of any one of claims 1-26, wherein the first exogenous nucleotide sequence comprises one or more recognition sites (RS) for a recombinase.

28. The genome editing system of claim 27, further comprising: iv) said recombinase and / or an expression construct containing a nucleotide sequence encoding said recombinase, and v) a donor construct comprising one or more recognition sites (RS) for the recombinase and a second exogenous polynucleotide sequence to be inserted into the plant genome.

29. The genome editing system of claim 27 or 28, wherein the recombinase is a recombinase of the tyrosine recombinase family or a recombinase of the serine recombinase family, preferably a recombinase of the tyrosine recombinase family, For example, the recombinase of the tyrosine recombinase family is selected from B2, KD, pSR1, Dre, Cre, and FLP; or the recombinase of the serine recombinase family is selected from phiC31 and Bxb1.

30. The genome editing system of any one of claims 27-29, wherein the recombinase is Cre recombinase.

31. The genome editing system of claim 30, wherein the one or more recombinase recognition sites (RS) are selected from loxP, Lox2272, Lox71, Lox66 or variants thereof, and any combination thereof.

32. The genome editing system of any one of claims 27-29, wherein the recombinase is FLP recombinase.

33. The genome editing system of claim 32, wherein the one or more recombinase recognition sites (RS) are selected from FRT1, FRT3, FRT5, FRT6 or variants thereof such as a FRT1 variant comprising a sequence described in one of SEQ ID NOs: 32-37, and any combination thereof.

34. The genome editing system of any one of claims 28 to 33, wherein the recombinase is contained in the guide editing fusion protein of i)-b), For example, the recombinase is located at the N-terminus or C-terminus of the fusion protein, and is connected to the other parts of the fusion protein directly or through a linker. Preferably, the guide editing fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 46-49 or an amino acid sequence having 85%, 90%, or 95% identity thereto.

35. The genome editing system of any one of claims 28-34, wherein the second exogenous polynucleotide sequence can be 1 bp to about 10 kb or longer.

36. The genome editing system of any one of claims 1 to 35, wherein the first target sequence, the second target sequence, the first exogenous nucleotide sequence and / or the second exogenous nucleotide sequence are associated with plant traits such as agronomic traits, whereby insertion of the first and / or second exogenous nucleotide sequence results in the plant having altered (preferably improved) traits, such as agronomic traits, relative to wild-type plants.

37. The genome editing system of any one of claims 1-36, wherein the plant includes monocotyledonous plants and dicotyledonous plants, for example, the plant is a crop plant, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato.

38. A method for producing a genetically modified plant comprising a site-specifically inserted exogenous nucleotide sequence, the method comprising introducing the genome editing system of any one of claims 1-37 into at least one of the plants.

39. The method of claim 38, further comprising screening the at least one plant for plants having the desired exogenous nucleotide sequence inserted therein.

40. The method of claim 38 or 39, wherein the genome editing system is introduced into the plant by a method selected from the group consisting of: biolistic method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and ovary injection method.

41. The method of any one of claims 38-40, wherein introducing comprises transforming the genome editing system into an isolated plant cell or tissue, and then regenerating the transformed plant cell or tissue into a whole plant.

42. The method of any one of claims 38-40, wherein the introducing comprises transforming the genome editing system into a specific part of the intact plant, such as a leaf, a stem tip, a pollen tube, a young ear, or a hypocotyl.

43. The method of any one of claims 38-42, further comprising treating (e.g., culturing) the plant cells, tissues, or whole plants into which the genome editing system has been introduced at an elevated temperature, e.g., 37°C.

44. The method of any one of claims 38-43, wherein the components of the genome editing system are introduced into the plant simultaneously.

45. The method of any one of claims 38-43, comprising introducing the genome editing system of any one of claims 28-37 into at least one of the plants, and comprising the steps of: 1) transforming components i)-iv) of the genome editing system into isolated plant cells or tissues to obtain plant cells or tissues into which the first exogenous nucleotide sequence comprising one or more recombinase recognition sites (RS) is inserted; 2) transforming component v) of the genome editing system into the plant cell or tissue obtained in step 1), thereby obtaining a plant cell or tissue comprising the inserted second exogenous polynucleotide sequence; and 3) Regenerating a whole plant from the plant cells or tissues obtained in step 2).

46. ​​The method of any one of claims 38-45, wherein the exogenous nucleotide sequence is inserted into a safe harbor site in the plant genome, wherein the safe harbor site is located in the plant genome. 1) At least 5 kb away from the protein coding region; 2) at least 30 kb away from the miRNA coding region; 3) at least 20 kb away from the lncRNA coding region; 4) at least 20 kb away from the tRNA coding region; 5) at least 5 kb from the promoter and / or enhancer; 6) at least 20 kb away from the LTR repeat; 7) at least 200 bp away from non-LTR repeats; and 8) At least 10 kb away from the centromere.

47. The method of claim 46, wherein the plant is rice and the safe harbor site is selected from the group consisting of: sites shown in Table 1 or Table 2.

48. A method for producing a genetically modified plant comprising a site-specifically inserted exogenous nucleotide sequence, the method comprising inserting the exogenous nucleotide sequence into a safe harbor site in the plant genome, the safe harbor site in the plant genome 1) At least 5 kb away from the protein coding region; 2) at least 30 kb away from the miRNA coding region; 3) at least 20 kb away from the lncRNA coding region; 4) at least 20 kb away from the tRNA coding region; 5) at least 5 kb from the promoter and / or enhancer; 6) at least 20 kb away from the LTR repeat; 7) at least 200 bp away from non-LTR repeats; and 8) At least 10 kb away from the centromere.

49. The method of claim 48, wherein the plant is rice and the safe harbor site is selected from the group consisting of: sites shown in Table 1 or Table 2.

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