Prime Editor for Modifying the EYS Gene and its Use

Abstract

One of the most important technical challenges to be addressed herein is to select a prime editor from among many theoretically designable prime editors that is effective in correcting the c.4957dupA mutation in the EYS gene in actual human retinal cells, to identify its composition, and thereby to provide a prime editor that can demonstrate actual therapeutic effects. To address the above technical challenge, this specification provides a prime editor and its components that are screened through a high-throughput screening method, validated in actual human retinal cells, and capable of actually correcting the c.4957dupA mutation in the EYS gene. The prime editor disclosed herein can act on human retinal cells containing the c.4957dupA mutation in the EYS gene, thereby correcting the EYS gene to a normal state. Therefore, the prime editor can be used as a therapeutic agent for retinitis pigmentosa caused by the c.4957dupA mutation in the EYS gene.

Description

Technical Field

[0001] The invention disclosed in the present specification relates to a technique for treating genetic diseases using a prime editor.

Background Art

[0002] On retinitis pigmentosa Retinitis pigmentosa Retinitis pigmentosa is a severe hereditary ocular disease that can cause night blindness and visual field constriction, and may even lead to bilateral blindness. Retinitis pigmentosa is a relatively common ocular disease with a high prevalence worldwide, affecting approximately 1 in 3,000 people. However, at present, there are few effective treatments for retinitis pigmentosa.

[0003] The c.4957dupA mutation in the EYS gene Recent advances in next-generation sequencing (NGS) technology have led to the discovery of more than 50 different genetic mutations that cause retinitis pigmentosa. Among these, mutations in the Eyes Shut Homolog (EYS) gene are known to be single-gene mutations that can cause retinitis pigmentosa. EYS gene mutations are particularly frequently found in Asian patients with retinitis pigmentosa.

[0004] Among the single-gene mutations that cause retinitis pigmentosa is the c.4957dupA mutation, which occurs when an adenine nucleotide is duplicated at position 4957 of the EYS gene. Studies have shown that a considerable number of Asian patients with retinitis pigmentosa, particularly those in Korea and Japan, develop the disease due to the c.4957dupA mutation in the EYS gene.

[0005] Therefore, it is expected that there are a large number of patients who can be treated for retinitis pigmentosa by correcting the c.4957dupA mutation in the EYS gene.

[0006] Limitations of existing treatment strategies Conventional therapeutic strategies for diseases caused by gene mutations primarily involve delivering wild-type genes of the mutated gene into cells. Methods for delivering these wild-type genes include 1) using adeno-associated viruses, 2) using lentiviruses, and 3) directly delivering mRNA into cells. However, these strategies have the following drawbacks: 1) Adeno-associated viruses can only deliver genes smaller than 4.8 kb, while the EYS gene exceeds 10 kb, requiring splitting into multiple viruses for delivery, thus significantly reducing delivery efficiency; 2) Lentiviruses offer flexibility in terms of the size of genes that can be delivered, but carry the risk of random integration into the cell genome; and 3) mRNA is unstable, thus limiting the duration of gene expression and requiring continuous administration of therapeutic agents.

[0007] Recently, CRISPR / Cas gene scissors have attracted attention as a therapeutic strategy that aims for a knockout or knockdown effect by 1) "damaging" the target gene by repeatedly cutting the double-stranded DNA of the target gene to generate indels in the target gene, and 2) using donor DNA to insert information from the donor DNA into the gene via homologous recombination (HDR) mechanism to show a corrective effect. However, 1) the gene knockout / knockdown strategy is not suitable for treating retinitis pigmentosa targeting mutations in the EYS gene, and 2) the method of modifying the gene using donor DNA is too inefficient to obtain a significant therapeutic effect, which are its limitations.

[0008] Prime Editor A prime editor refers to a protein-nucleic acid complex used in a gene editing technique called prime editing. A prime editor includes a prime editor protein containing a CRISPR / Cas protein with mutated double-strand break activity and reverse transcriptase, as well as prime editing guide RNA (pegRNA). Prime editing is a technique in which 1) the prime editor protein binds to the target gene after being induced to do so by pegRNA, 2) the prime editor protein reverse transcribes the modified sequence contained in the 3' end of the pegRNA into cDNA, and 3) the reverse-transcribed cDNA is inserted in place of the mutated sequence in the target gene, thereby modifying the gene as intended. [Overview of the initiative]

[0009] [Technical issues] This specification discloses a prime editor capable of correcting the c.4957dupA mutation in the EYS gene.

[0010] Applying this prime editor to correct only the c.4957dupA mutation in the EYS gene for retinitis pigmentosa caused by the c.4957dupA mutation is a highly effective therapeutic strategy that overcomes the limitations of existing treatment strategies. Theoretically, thousands or tens of thousands of prime editors, particularly pegRNAs, could be designed to correct the c.4957dupA mutation in the EYS gene. However, not all of these theoretically designed prime editors actually function to correct the c.4957dupA mutation in the EYS gene. To date, the specific configurations of prime editors that affect prime editing efficiency are largely unknown. Therefore, it is almost impossible to predict whether a theoretically designed prime editor will actually function based solely on its configuration.

[0011] Therefore, one of the most important technical challenges addressed herein is to identify a prime editor from among a large number of theoretically designable prime editors that demonstrates the effect of correcting the c.4957dupA mutation in the EYS gene in actual human retinal cells, and to provide a prime editor that can demonstrate actual therapeutic effects by identifying its composition.

[0012] [Technical solution] To address the above technical challenges, the present invention provides a prime editor and its components that can be screened using a high-throughput screening method, validated in actual human retinal cells, and capable of actually correcting the c.4957dupA mutation in the EYS gene.

[0013] In particular, the present invention provides a pegRNA capable of correcting the c.4957dupA mutation in the EYS gene by functioning in cooperation with a prime editing protein containing a nickas variant of the Cas9 protein derived from Streptococcus pyogenes.

[0014] [Advantageous effect] The prime editor disclosed herein may act on human retinal cells containing the c.4957dupA mutation in the EYS gene, thereby correcting the EYS gene to a normal state. Therefore, the prime editor may be used as a therapeutic agent for retinitis pigmentosa caused by the c.4957dupA mutation in the EYS gene. [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic diagram showing the structures of pegRNA and extended pegRNA.

[0016] [Figure 2]It is a schematic diagram showing the relationship between the guide domain, primer binding site (PBS) and reverse transcriptase template (RTT) of pegRNA, and a target nucleic acid. Specifically, FIG. 2 shows a state where the prime editor recognizes the protospacer adjacent motif (PAM) on the non-target strand of the target nucleic acid, and the guide domain complementarily binds to the target sequence on the target strand of the target nucleic acid.

[0017] [Figure 3] It is a schematic diagram showing the relationship between the guide domain, PBS and RTT of pegRNA, and a target nucleic acid. Specifically, FIG. 3 shows the positional relationship of the PBS and RTT sequences after a nick has been introduced into the target nucleic acid by the prime editor. MODES FOR CARRYING OUT THE INVENTION

[0018] Best Mode for Carrying Out the Invention Hereinafter, the best mode for carrying out the present invention is exemplarily disclosed. This includes several embodiments of the invention disclosed herein, but does not encompass all embodiments. The embodiments described in this section are merely examples, and should not be understood as the "best mode for carrying out the invention". Those skilled in the art can conceive of numerous variations and more preferred embodiments based on the embodiments described in this section, and such variations and embodiments should also be considered as part of the best mode for carrying out the present invention.

[0019] Disclosed herein is a pegRNA for a prime editor for correcting the c.4957dupA variant of the EYS gene, having the following structure: 5'-[guide domain]-[scaffold]-[prime editing domain]-3', wherein the scaffold is capable of interacting with the prime editor to form a complex, and the guide domain and the prime editing domain consist of a combination selected from the following: a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11534 to SEQ ID NO: 11552, and SEQ ID NO: 12104 to SEQ ID NO: 12122; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3343 to SEQ ID NO: 3482; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11553 to SEQ ID NO: 11571, and SEQ ID NO: 12123 to SEQ ID NO: 12141; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3483 to SEQ ID NO: 3573; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11572 to SEQ ID NO: 11590, and SEQ ID NO: 12142 and SEQ ID NO: 12160; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3574 to SEQ ID NO: 3710; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11591 to SEQ ID NO: 11609, and SEQ ID NO: 12161 to SEQ ID NO: 12179; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3711 to SEQ ID NO: 3829; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11610 to SEQ ID NO: 11628, and SEQ ID NO: 12180 to SEQ ID NO: 12198; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3830 to SEQ ID NO: 3888; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11629 to SEQ ID NO: 11647, and SEQ ID NO: 12199 to SEQ ID NO: 12217; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 3889 to SEQ ID NO: 4022; a guide domain that targets a target sequence of a nucleic acid sequence selected from SEQ ID NO: 11648 to SEQ ID NO: 11666, and SEQ ID NO: 12218 to SEQ ID NO: 12236; and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NO: 4023 to SEQ ID NO: 4169; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11667 to 11685 and SEQ ID NOs: 12237 to 12255, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4170 to 4309; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11686 to 11704 and sequence numbers 12256 to 12274, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 4310 to 4450; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11705 to 11723 and SEQ ID NOs: 12275 to 12293, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4451 to 4578; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11724 to 11742 and SEQ ID NOs: 12294 to 12312, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4579 to 4608; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11743 to 11761 and SEQ ID NOs: 12313 to 12331, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4609 to 4671; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11762 to 11780 and SEQ ID NOs: 12332 to 12350, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4672 to 4736; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11781 to 11799 and SEQ ID NOs: 12351 to 12369, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4737 to 4878; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11800 to 11818 and SEQ ID NOs: 12370 to 12388, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 4879 to 5024; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11819 to 11837 and SEQ ID NOs: 12389 to 12407, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5025 to 5101; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11838 to 11856 and SEQ ID NOs: 12408 to 12426, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5102 to 5214; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11857 ​​to 11875 and SEQ ID NOs: 12427 to 12445, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5215 to 5311; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11876 to 11894 and SEQ ID NOs: 12446 to 12464, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5312 to 5441; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11895 to 11913 and SEQ ID NOs: 12465 to 12483, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5442 to 5550; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11914 to 11932 and SEQ ID NOs: 12484 to 12502, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5551 to 5681; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11933 to 11951 and SEQ ID NOs: 12503 to 12521, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5682 to 5804; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11952 to 11970 and 12522 to 12540, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 5805 to 5831; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11971 to 11989 and 12541 to 12559, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 5832 to 5923; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11990 to 12008 and 12560 to 12578, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 5924 to 5966; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 12009 to 12027 and SEQ ID NOs: 12579 to 12597, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5967 to 5993; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 12028 to 12046 and SEQ ID NOs: 12598 to 12616, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 5994 to 6040; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 12047 to 12065 and SEQ ID NOs: 12617 to 12635, and a prime editing domain consisting of a nucleic acid sequence selected from SEQ ID NOs: 6041 to 6054; and A guide domain that targets a target nucleic acid sequence selected from sequence numbers 12066 to 12103, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 6055 to 6057.

[0020] In one embodiment, the scaffold may be a pegRNA composed of nucleic acid sequences selected from SEQ ID NOs: 11531 to 11533.

[0021] Disclosed herein is a prime editor composition for modifying the c.4957dupA variant of the EYS gene, comprising: a prime editor protein, or a nucleic acid encoding the prime editor protein; and pegRNA, or a nucleic acid encoding pegRNA, wherein the prime editor protein comprises a Cas9 protein or a variant thereof derived from Streptococcus pyogenes, and a reverse transcriptase, and the pegRNA scaffold interacts with the prime editor protein to form a complex.

[0022] In one embodiment, the prime editor composition may include a prime editor protein and pegRNA, where the prime editor protein and pegRNA may be combined to form a complex.

[0023] In one embodiment, the prime editor composition may include a nucleic acid encoding the prime editor and a nucleic acid encoding pegRNA.

[0024] In one embodiment, the prime editor protein may be selected from: NRCH-PE2max; NRCH-hyPE2max; NRCH-PE2; and PE2max. [Mode of the invention]

[0025] The present invention will be described in more detail below through specific embodiments and examples with reference to the accompanying drawings. It should be noted that the accompanying drawings include some, but not all, embodiments of the present invention. The invention disclosed herein can be carried out in a variety of ways and is not limited to the specific embodiments described herein. These embodiments should be considered as being provided to satisfy the legal requirements applicable herein. Those skilled in the art can conceive of many variations and other embodiments of the invention disclosed herein. Therefore, it should be understood that the invention disclosed herein is not limited to the specific embodiments described herein, and that variations and other embodiments thereof are also included within the scope of the claims.

[0026] Definition of Terms about As used herein, the term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies within the range of 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% based on a baseline quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0027] Nucleic acid sequence notation The symbols A, T, C, G, and U used herein are to be interpreted as having meanings understandable to those skilled in the art. Depending on the context and the art, these symbols may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA. For example, when referring to bases, the symbols may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), respectively. When referring to nucleosides, the symbols may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively. When referring to nucleotides in a sequence, the symbols should be interpreted as referring to nucleotides containing the respective nucleosides.

[0028] Target gene or target nucleic acid As used herein, the terms “target gene” and “target nucleic acid” primarily refer to the cellular gene or nucleic acid that is the target of gene editing. The terms “target gene” and “target nucleic acid” may be used interchangeably and may refer to the same entity. Unless otherwise specified, a target gene or target nucleic acid may refer to either an endogenous gene or nucleic acid of a target cell, or an exogenous gene or nucleic acid, and is not particularly limited as long as it can be a target of gene editing. A target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and / or RNA. Furthermore, the terms encompass all meanings that can be recognized by those skilled in the art and may be appropriately interpreted in context.

[0029] Target chain and non-target chain In this specification, when the terms “target strand” and “non-target strand” are used to refer to the target nucleic acid, the terms should be understood in relation to the Cas9 protein (and the prime editor protein containing it) and guide RNA (and pegRNA). When the target nucleic acid is a double-stranded nucleic acid targeted by the prime editor, the target strand and non-target strand are distinguished based on the following criteria: The non-target strand is the strand that contains a protospacer adjacent motif (PAM) sequence that can be recognized by the Cas9 protein and to which the guide RNA (and pegRNA) does not complementarily bind. The target strand is the strand to which the guide domain of the guide RNA (and pegRNA) complementarily binds after the Cas9 protein recognizes the PAM sequence on the non-target strand. In addition, the terms encompass all meanings that can be recognized by those skilled in the art and can be appropriately interpreted in context.

[0030] EYS Gene Modification Prime Editor Overview of EYS Gene Modification Prime Editor Disclosed herein are a prime editor capable of correcting the c.4957dupA mutation in the EYS gene and its components. The prime editor comprises a prime editor protein and pegRNA, where the prime editor protein comprises a Cas9 nickerse variant and reverse transcriptase derived from Streptococcus pyogenes. The pegRNA comprises a guide domain, a scaffold, and a prime editing domain, which are sequentially ligated from the 5' end to the 3' end, where the prime editing domain comprises a reverse transcriptase template (RTT) and a primer binding site (PBS). The pegRNA forms a complex with the prime editor protein via the scaffold. The pegRNA 1) guides the prime editor protein to the target nucleic acid surrounding the c.4957dupA mutation in the EYS gene, and 2) provides a normal gene sequence template so that the c.4957dupA mutation in the EYS gene can be corrected via the reverse transcriptase contained in the prime editor protein. Prime editor proteins 1) generate nicks in the target nucleic acid so that PBS of pegRNA can bind to the target nucleic acid and position it appropriately, and 2) reverse transcribe the DNA using RTT as a template so that gene modification can be induced.

[0031] When the sequences of the mutated gene to be modified and the normal gene are clearly identified, pegRNAs that enable gene modification using prime editors can be theoretically designed. When designing pegRNAs, the main design variables are: 1) a guide domain capable of targeting the target nucleic acid surrounding the modification target; 2) an RTT sequence and its length depending on the sequence and nick position of the target nucleic acid; and 3) a PBS sequence length depending on the sequence and nick position of the target nucleic acid. Theoretically, thousands or even tens of thousands of pegRNAs can be designed by changing these variables. However, since little is known about which pegRNA configurations exhibit prime editing effects, it is virtually impossible to select pegRNA sequences that are actually applicable to gene modification based solely on theoretically designed sequence information.

[0032] Therefore, the inventors chose a method of selecting pegRNAs by confirming through actual experiments whether each pegRNA truly exhibits a prime editing effect. Specifically, in order to select pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene, the inventors synthesized almost all pegRNAs that could be theoretically designed and screened for gene-editing pegRNAs using a high-throughput screening method. Furthermore, the inventors verified that the pegRNAs that showed editing efficiency as a result of the screening could correct the c.4957dupA mutation in the EYS gene in actual human retinal cells, thereby completing the present invention by identifying the sequences of pegRNAs that can be used to correct the c.4957dupA mutation in the EYS gene.

[0033] Prime Editor Protein The prime editor disclosed herein, capable of correcting the c.4957dupA mutation in the EYS gene, comprises a prime editor protein. The prime editor protein is an artificial protein comprising a Cas protein with nickase activity and a reverse transcriptase. In particular, the Cas protein with nickase activity is a variant of the Cas9 protein derived from Streptococcus pyogenes. The prime editor protein can cleave only one of the two strands of a target double-stranded nucleic acid (hereinafter referred to as "nick"). Specifically, the prime editor protein introduces a nick into the non-target strand of the target double-stranded nucleic acid. Here, when the location of the target double-stranded nucleic acid is identified, the location where the prime editor protein creates the nick can also be known, and thus each component of the pegRNA for prime editing modification (e.g., RTT and PBS sequences) can be designed based on it.

[0034] The composition of the prime editor protein is specifically illustrated in the "Prime Editor Protein" section of the "Possible Embodiments of the Invention" below. pegRNA for EYS gene modification The prime editor disclosed herein, capable of correcting the c.4957dupA mutation in the EYS gene, comprises a pegRNA. The pegRNA comprises a guide domain, a scaffold, an RTT, and a PBS, where the RTT and PBS are collectively referred to as the prime editing domain. Optionally, the pegRNA may further comprise an RNA linker and a tmpknot at its 3' end.

[0035] The structures of pegRNA and extended pegRNA are schematically shown in Figure 1.

[0036] PegRNA can be designed in countless forms by altering the guide domain sequence, RTT sequence, and PBS sequence.

[0037] The composition of pegRNA is specifically illustrated in the "pegRNA" section of the "Possible Embodiments of the Invention" below.

[0038] Method for designing pegRNA for gene modification pegRNAs for gene modification can be designed based on 1) the target gene region (the c.4957dupA mutation site in the EYS gene) and 2) the prime editor protein used, as well as the nick location in the target nucleic acid.

[0039] Specifically, pegRNAs for gene modification can be designed using the following methods. The following description serves as an example to illustrate the design method. Although numbered sequentially, the steps do not need to be performed in the specified order, and alternative methods may be used as long as the intended purpose is achieved. 1) The sequences of the target gene before and after modification are identified. 2) Depending on the prime editor protein used, the guide domain of the pegRNA is designed so that the prime editor can be guided to a predetermined target nucleic acid near the target gene region. 3) Depending on the sequence of the target nucleic acid, the location of the nick formed by the prime editor protein is identified. 4) Based on the nick position, the PBS of the pegRNA is designed to bind complementaryally to the 5' terminal nucleotide of the non-target strand. 5) The RTT of pegRNA is designed so that the modified sequence can be reverse transcribed by reverse transcriptase toward the 3' end of the non-target strand to which the primer binding site binds. The relationship between the pegRNA guide domain, PBS, RTT, and target nucleic acid is schematically shown in Figures 2 and 3.

[0040] Figures 2 and 3 show only a portion of the target nucleic acid and pegRNA, with the portions labeled 3' and 5' being shown for orientation purposes only and not the actual nucleic acid ends. Figure 2 illustrates the state in which the prime editor recognizes the PAM on the non-target strand of the target nucleic acid and the guide domain binds complementaryly to the target sequence on the target strand of the target nucleic acid. In this case, a single-strand break (nick) occurs at a specific location on the non-target strand. Figure 3 shows the positional relationship of the PBS and RTT sequences after the nick has been generated on the target nucleic acid by the prime editor. The PBS of the pegRNA binds complementaryly to the 5' end of the non-target strand of the cleaved target nucleic acid, thereby positioning the PBS and enabling prime editing to occur using the RTT as a template.

[0041] As described above, guide domains, PBS, and RTT of pegRNA can be designed.

[0042] Furthermore, 1) various guide domains can be designed by changing the position and length of the target nucleic acid; 2) PBS can be designed in various ways by changing the length of the complementary binding to the 5' terminal nucleotide of the non-target strand; and 4) RTT can be designed in various ways by changing the length of the modified sequence reverse-transcribed using reverse transcriptase. Therefore, theoretically, thousands or even tens of thousands of pegRNAs can be designed by changing each of the guide domain, PBS, and RTT.

[0043] The difficulty of selecting pegRNAs that are actually functional. As described above, once 1) the target gene site for modification (the c.4957dupA mutation site in the EYS gene) is identified, and 2) the prime editor protein to be used and the nick location in the target nucleic acid are identified, a pegRNA for modifying the target gene site can be designed. However, while thousands or tens of thousands of pegRNAs can be theoretically designed, the pegRNA patterns that have high gene editing efficiency and the factors that determine this efficiency are still not fully understood. Therefore, it is extremely difficult to select a pegRNA that can be used for actual gene editing based solely on the sequence information of the designed pegRNA.

[0044] Our approach - Selection and validation by high-throughput screening The inventors used a method to select pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene through actual experiments. First, the inventors designed almost all pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene using prime editor proteins that can recognize various PAMs. Subsequently, the inventors experimentally confirmed whether each pegRNA exhibited a prime editing effect and selected pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene. To efficiently carry out this work, a high-throughput screening method was introduced. This high-throughput screening method was based on the method disclosed by Kim et al. (Predicting the efficiency of prime editing guide RNAs in human cells, Nature Biotechnology, Vol. 39, Feb. 2021). Furthermore, by verifying whether the pegRNAs showing a corrective effect in the high-throughput screening method can correct the endogenous EYS gene in actual human retinal cells (ARPE19), it was shown that the pegRNAs selected by the high-throughput screening method can correct the c.4957dupA mutation in the EYS gene in actual retinal cells.

[0045] The following examples and experimental illustrations illustrate in detail a prime editor capable of correcting the c.4957dupA mutation in the EYS gene and its components.

[0046] [Possible embodiments of the invention] Possible embodiments of the invention are disclosed below. The following description illustrates embodiments that may be derived from the technical concepts described herein. It should be understood that the invention disclosed herein is not limited to the specific embodiments described below, and that variations thereof and other embodiments are also included within the scope of the invention disclosed herein.

[0047] Prime Editor Protein Example 1: SpCas9-based prime editor protein Prime Editor proteins including the following: Cas9 protein derived from Streptococcus pyogenes, variants of Cas9 protein derived from Streptococcus pyogenes, or Cas9 proteins exhibiting nickase activity among variants of Cas9 protein derived from Streptococcus pyogenes; and Reverse transcriptase.

[0048] Example 2, Identification of Nick Location In Example 1, the Cas9 protein exhibiting nickase activity can recognize the protospacer adjacent motif (PAM) sequence on the non-target strand of the target double-stranded nucleic acid and cleave the PAM sequence between the third and fourth nucleotides in the 5' direction from the 5' terminal nucleotide.

[0049] Example 3, SpCas9-derived nickase In any one of Examples 1-2, the Cas9 protein exhibiting nickase activity contains one or more mutations selected from the following in the amino acid sequence of the Streptococcus pyogenes-derived Cas9 protein (SEQ ID NO: 1): H840A; R222K; and N394K.

[0050] Example 4, PAM variant In any one of Examples 1 to 3, the Cas9 protein exhibiting nickase activity can recognize a PAM sequence different from 5'-NGG-3'.

[0051] Example 5, Identification of PAM In Example 4, the Cas9 protein exhibiting nickase activity is capable of recognizing one or more PAM sequences selected from the following group: 5'-NGG-3';5'-NG-3';5'-TAG-3';5'-AAT-3';5'-TTG-3';5'-TGA-3';5'-GTA-3';5'-TAC-3';5'-GAT-3';5'-GAA-3';5'-AGA-3';5'-GGA-3';5'-AGT- 3';5'-AAA-3';5'-CAT-3';5'-AAG-3';5'-CAA-3';5'-CCC-3';5'-TAT-3';5'-TAA-3';5'-GTC-3';5'-AAC-3';5'-CTA-3';5'-ATA-3'; and 5'-NGAG-3'.

[0052] Example 6, Identification of PAM mutations In any of Examples 4-5, the Cas9 protein, the Cas9 protein variant, or the Cas9 protein exhibiting nickase activity of the Cas9 protein variant contains one or more mutations selected from the following in the amino acid sequence of the Streptococcus pyogenes-derived Cas9 protein (SEQ ID NO: 1): I322V, S409I, E427G, R654L, R753G, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, and H1349R; L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, and T1337R; and D1135V, G1218R, R1335Q, and T1337R.

[0053] Example 7, Limitation of reverse transcriptase In any one of Examples 1 to 6, the reverse transcriptase is selected from the following: Sequence numbers: 13187 to 13188.

[0054] Example 8, Other Domains In any one of Examples 1 to 7, the prime editor protein further comprises one or more domains selected from the following: One or more nuclear localization signals (NLSs); Rad51; and linker.

[0055] Example 9, Limitation of NLS sequence In Example 8, one or more NLSs each independently include an array selected from the following: Sequence IDs: 13189 to 13190.

[0056] Example 10, Limitation of Rad51 sequence In any one of Examples 8 to 9, Rad51 contains the amino acid sequence AMQMQLEANADTSVEEESFGPQPISRLEQCGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAKADKILAEAAKLVPMGFTTATEFHQRRSEIIQITTGSKELDKLLQ (Sequence ID: 13200).

[0057] Example 11, Limitation of the linker In any one of Examples 8 to 9, the linker includes an array selected from Sequence ID: 13193.

[0058] Example 12, Limitation of Prime Editor Protein In any one of Examples 1 to 11, the prime editor protein is selected from the following group: PE2; PE2max; NRCH-PE2; NRCH-PE2max; NRCH-hyPE2max; VRQR-PE2; and VRQR-PE7max.

[0059] Example 13, Limitation of Prime Editor Protein Sequence In Example 12, the prime editor protein contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, SEQ ID NOs: 13195 to 13196, and SEQ ID NO: 13203.

[0060] Example 14, Prime Editor, Structural Limitation Prime Editor proteins including the following: [NLS1]-[Cas9 protein]-[Linker 1]-[Rad51]-[Linker 2]-[Reverse transcriptase]-[NLS2] Here, NLS1 is either the first nucleus localization signal (NLS) or it is not present. NLS2 is either the second NLS or does not exist. The Cas9 protein contains sequences selected from SEQ ID NO: 13204 and SEQ ID NO: 13207 to SEQ ID NO: 13209. Linker 1 is either an amino acid linker or absent. Rad51 is either a Rad51 domain or does not exist. Linker 2 is either an amino acid linker or absent. The reverse transcriptase contains sequences selected from sequence numbers 13187 and 13188.

[0061] Example 15, Limitation of NLS and Linker Sequence In Example 14, NLS1 and NLS2 each independently contain an amino acid sequence selected from SEQ ID NOs: 13189 to 13188, and the amino acid linker contains an amino acid sequence selected from SEQ ID NOs: 13193 and 13201.

[0062] Example 16, Limitation of Prime Editor Full-Length Array In Example 14, the prime editor protein contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, SEQ ID NOs: 13195 to 13196, and SEQ ID NO: 13203.

[0063] pegRNA Example 17, pegRNA pegRNA for prime editor to correct the c.4957dupA variant of the EYS gene.

[0064] Example 18, Limitation of Prime Editor Protein In Example 17, the prime editor comprises a prime editor protein selected from Examples 1 to 16.

[0065] Example 19, pegRNA structure In any one of Examples 17-18, the pegRNA has the following structure: 5'-[Guide Domain]-[Scaffold]-[Prime Editing Domain]-3', Here, the guide domain is capable of targeting target nucleic acids contained in the EYS gene. The scaffold can interact with the prime editor protein of the prime editor to form a complex. The prime editing domain includes a reverse transcriptase template (RTT) and a primer binding site (PBS).

[0066] Example 20, pegRNA structure #1 In any one of Examples 17-19, the pegRNA comprises a guide domain, a scaffold, and a prime editing domain, and the prime editing domain comprises a reverse transcriptase template (RTT) and a primer binding site (PBS). pegRNA contains a guide domain, scaffold, and prime editing domain that are sequentially linked from the 5' end to the 3' end. The guide domain is capable of targeting target nucleic acids contained in the EYS gene. The scaffold can interact with the prime editor protein of the prime editor to form a complex. The prime editing domain includes RTT and PBS sequentially concatenated from the 5' end to the 3' end.

[0067] Example 21, Meaning of Targeted Nucleic Acid Targets In any one of Examples 17 to 20, The target nucleic acid refers to a portion of the double-stranded DNA in the EYS gene. The target nucleic acid includes the target strand and the non-target strand. Since the target strand sequence and the non-target strand sequence are complementary sequences, the target nucleic acid sequence can be identified solely by the target strand sequence or the non-target strand sequence. The statement that the guide domain targets target sequences contained in the EYS gene has one of the following meanings: The guide domain may bind complementaryally to and / or hybridize with the target strand of the target nucleic acid; The guide domain sequence contains a sequence that is complementary to all or part of the sequence of the target strand of the target nucleic acid; The sequence of the guide domain includes a sequence that is identical, coincident, homologous, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid; and A suitable combination of the above-mentioned items, within the scope that can be recognized by a person skilled in the art.

[0068] Example 22, limitation of non-target chain sequence In Example 21, the sequence of the non-target strand of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 11534 to 12084 and SEQ ID NOs: 12085 to 12635. The sequence of the guide domain of pegRNA contains sequences that are identical, coincident, homologous, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid.

[0069] Example 23, Limitation of sequences similar to the non-target chain In Example 21, the sequence of the non-target strand of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 11534 to 12084 and SEQ ID NOs: 12085 to 12635. The sequence of the guide domain of the pegRNA is identical, coincident, homologous, and / or equivalent to, in whole or in part, the sequence of the non-target strand of the target nucleic acid by approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0070] Example 24: Identical to the non-target chain but containing a mismatch. In Example 21, the sequence of the non-target strand of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 11534 to 12084 and SEQ ID NOs: 12085 to 12635. The sequence of the guide domain of pegRNA includes a sequence that is identical, coincident, homologous, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid, except for nucleotide bases 1, 2, 3, 4, or 5.

[0071] Example 25, Restriction of target chain sequence and restriction of complementary sequence In Example 21, the target strand sequence of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 12636 to 13186, and the guide domain sequence of the pegRNA includes a sequence complementary to all or part of the target strand sequence.

[0072] Example 26, Limitation of sequences similar to the target chain sequence In Example 21, the target strand sequence of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 12636 to 13186, and the guide domain sequence of the pegRNA includes a sequence that is approximately 80% or more, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more complementary to all or part of the target strand sequence.

[0073] Example 27: Complementary to the target chain but containing a mismatch. In Example 21, the target strand sequence of the target nucleic acid includes a sequence selected from the group consisting of SEQ ID NOs: 12636 to 13186, and The guide domain sequence of pegRNA contains a sequence that is complementary to all or part of the target chain sequence, with the exception of nucleotide bases 1, 2, 3, 4, or 5.

[0074] Example 28, Limitation of mismatch patterns In Example 27, the mismatch between the sequence of the guide domain of pegRNA and the sequence of the target strand is a base mismatch, resulting in a bulge forming in either the pegRNA or the target strand.

[0075] Example 29, limitation of target chain sequence and limitation of binding relationship In any one of Examples 17 to 28, the guide domain of the pegRNA may bind and / or hybridize complementarily with the target strand.

[0076] Example 30, Limitation of Guide Domain Length In any one of Examples 17 to 29, the guide domain has a nucleotide length of 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt, or a length within a range composed of the above numerical ranges (for example, a length of 12nt to 30nt).

[0077] Example 31, Limitation of the guide domain In any one of Examples 17-30, the guide domain sequence includes a nucleic acid sequence selected from the following group: Nucleic acid sequences selected from SEQ ID NO: 48 to SEQ ID NO: 627; and A sequence that is approximately 80% or more identical, coincident, homologous, and / or equivalent to a nucleic acid sequence selected from SEQ ID NO: 48 to SEQ ID NO: 627, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more.

[0078] Example 32, Inclusion of G or GG at the 5' end In Example 31, the sequence of the guide domain further includes G or GG at its 5' end.

[0079] Example 33, Limitation of scaffold sequence In any one of Examples 17 to 32, the scaffold sequence includes a nucleic acid sequence selected from the group consisting of: Nucleic acid sequences selected from SEQ ID NO: 11531 to SEQ ID NO: 11533; and A sequence that is approximately 80% or more identical, coincident, homologous, and / or equivalent to, a nucleic acid sequence selected from SEQ ID NO: 11531 to SEQ ID NO: 11533, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more, and is capable of interacting with a prime editor protein to form a complex.

[0080] Example 34, limitations of RTT and PBS In any one of Examples 17 to 33, The prime editing domain sequence contains the following sequence, sequentially concatenated from the 5' end to the 3' end: RTT sequences selected from sequence numbers 8816 to 11530; and A PBS sequence selected from sequence numbers 628 to 3342.

[0081] Example 35: Matching of guide domain, RTT, and PBS In any one of Examples 17-34, the sequences of the guide domain and prime editing domain of the pegRNA are represented by a combination selected from the following group: A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 48 to 67, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 8816 to 8955 and a primer binding site sequence selected from SEQ ID NOs: 628 to 767, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68 to 87, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 8956 to 9046 and a primer binding site sequence selected from SEQ ID NOs: 768 to 858, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 88 to 107, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9047 to 9183 and a primer binding site sequence selected from SEQ ID NOs: 859 to 995, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 108 to 127, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9184 to 9302 and a primer binding site sequence selected from SEQ ID NOs: 996 to 1114, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 128 to 147, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9303 to 9361 and a primer binding site sequence selected from SEQ ID NOs: 1115 to 1173, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 148 to 167, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9362 to 9495 and a primer binding site sequence selected from SEQ ID NOs: 1174 to 1307, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 168 to 187, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9496 to 9642 and a primer binding site sequence selected from SEQ ID NOs: 1308 to 1454, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 188 to 207, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9643 to 9782 and a primer binding site sequence selected from SEQ ID NOs: 1455 to 1594, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 208 to 227, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9783 to 9923 and a primer binding site sequence selected from SEQ ID NOs: 1595 to 1735, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 228 to 247, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 9924 to 10051 and a primer binding site sequence selected from SEQ ID NOs: 1736 to 1863, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 248 to 267, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10052 to 10081 and a primer binding site sequence selected from SEQ ID NOs: 1864 to 1893, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 268 to 287, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10082 to 10144 and a primer binding site sequence selected from SEQ ID NOs: 1894 to 1956, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 288 to 307, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10145 to 10209 and a primer binding site sequence selected from SEQ ID NOs: 1957 to 2021, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 308 to 327, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10210 to 10351 and a primer binding site sequence selected from SEQ ID NOs: 2022 to 2163, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 328 to 347, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10352 to 10497 and a primer binding site sequence selected from SEQ ID NOs: 2164 to 2309, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 348 to 367, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10498 to 10574 and a primer binding site sequence selected from SEQ ID NOs: 2310 to 2386, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 368 to 387, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10575 to 10687 and a primer binding site sequence selected from SEQ ID NOs: 2387 to 2499, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 388 to 407, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10688 to 10784 and a primer binding site sequence selected from SEQ ID NOs: 2500 to 2596, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 408 to 427, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10785 to 10914 and a primer binding site sequence selected from SEQ ID NOs: 2597 to 2726, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 428 to 447, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 10915 to 11023 and a primer binding site sequence selected from SEQ ID NOs: 2727 to 2835, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 448 to 467, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11024 to 11154 and a primer binding site sequence selected from SEQ ID NOs: 2836 to 2966, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 468 to 487, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11155 to 11277 and a primer binding site sequence selected from SEQ ID NOs: 2967 to 3089, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 488 to 507, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11278 to 11304 and a primer binding site sequence selected from SEQ ID NOs: 3090 to 3116, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 508 to 527, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11305 to 11396 and a primer binding site sequence selected from SEQ ID NOs: 3117 to 3208, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 528 to 547, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11397 to 11439 and a primer binding site sequence selected from SEQ ID NOs: 3209 to 3251, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 548 to 567, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11440 to 11466 and a primer binding site sequence selected from SEQ ID NOs: 3252 to 3278, sequentially ligated from the 5' end to the 3' end; A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 568 to 587, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11467 to 11513 and a primer binding site sequence selected from SEQ ID NOs: 3279 to 3325, sequentially ligated from the 5' end to the 3' end; and A sequence of a guide domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 588 to 607, and a sequence of a prime editing domain containing a reverse transcriptase template sequence selected from SEQ ID NOs: 11514 to 11527 and a primer binding site sequence selected from SEQ ID NOs: 3326 to 3339, sequentially ligated from the 5' end to the 3' end.

[0082] Example 36, Restriction of Prime Editing Domain In any one of Examples 17 to 35, the prime editing domain sequence includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3343 to 6057.

[0083] Example 37: Matching of Guide Domain and Prime Editing Domain In any one of Examples 17 to 36, the sequence of the guide domain and the sequence of the prime editing domain of the pegRNA are represented by a combination selected from the following group: A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 48 to 67, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3343 to 3482; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68 to 87, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3483 to 3573; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 88 to 107, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3574 to 3710; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 108 to 127, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3711 to 3829; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 128 to 147, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3830 to 3888; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 148 to 167, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3889 to 4022; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 168 to 187, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4023 to 4169; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 188 to 207, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4170 to 4309; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 208 to 227, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4310 to 4450; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 228 to 247, and a prime editing domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4451 to 4578; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 248 to 267, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4579 to 4608; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 268 to 287, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4609 to 4671; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 288 to 307, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4672 to 4736; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 308 to 327, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4737 to 4878; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 328 to 347, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4879 to 5024; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 348 to 367, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5025 to 5101; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 368 to 387, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5102 to 5214; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 388 to 407, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5215 to 5311; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 408 to 427, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5312 to 5441; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 428 to 447, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5442 to 5550; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 448 to 467, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5551 to 5681; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 468 to 487, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5682 to 5804; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 488 to 507, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5805 to 5831; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 508 to 527, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5832 to 5923; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 528 to 547, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5924 to 5966; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 548 to 567, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5967 to 5993; A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 568 to 587, and a prime editing domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5994 to 6040; and A guide domain sequence containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 588 to 607, and a prime-edit domain containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6041 to 6054.

[0084] Example 38, Restriction of the full-length pegRNA sequence In any one of Examples 17 to 37, the pegRNA sequence includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6058 to 8815.

[0085] Example 39, Addition of tmpknot In any one of Examples 17-38, the pegRNA further contains a tmpknot at its 3' end.

[0086] Example 40, limitation of the tmpknot sequence In Example 39, the tmpknot sequence includes the nucleic acid sequence of sequence number 6.

[0087] Example 41, tmpknot connected linker In any one of Examples 39 to 40, the pegRNA further comprises an RNA linker, and the tmpknot is attached to the 3' end of the pegRNA via the RNA linker.

[0088] Example 42, Limitation of Linker Arrangement In Example 41, the RNA linker sequence includes the nucleic acid sequences from SEQ ID NO: 7 to SEQ ID NO: 47.

[0089] Example 43, containing poly-U In any one of Examples 17 to 42, the pegRNA further includes a poly-U sequence at its 3' end.

[0090] Example 44, limitation of poly-U sequence In Example 43, the poly-U sequence is 5'-UUUUUU-3'.

[0091] Example 45, Marcush type claim The following pegRNA for prime editor to modify the c.4957dupA variant of the EYS gene is represented by the following structure: 5'-[5'-terminus addition]-[guide domain]-[scaffold]-[reverse transcriptase template (RTT)]-[primer binding site (PBS)]-[linker]-[tmpknot]-3' Here, the sequence of the 5' appendix is ​​either 5'-G-3' or 5'-GG-3', or the 5' appendix does not exist. The sequence of the guide domain includes a sequence selected from the group consisting of SEQ ID NOs: 48 to 627. The sequence of the scaffold includes a sequence selected from the group consisting of SEQ ID NOs: 11531 to 11533. The sequence of the RTT includes a sequence selected from the group consisting of SEQ ID NOs: 8816 to 11530. The sequence of the PBS includes a sequence selected from the group consisting of SEQ ID NOs: 628 to 3342. The linker sequence includes a sequence selected from the group consisting of SEQ ID NOs: 7 to 47, or the linker does not exist. The sequence of the tmpknot is SEQ ID NO: 6, or the tmpknot does not exist.

[0092] Example 46, DNA encoding pegRNA DNA encoding one of the pegRNAs from Examples 17 to 45.

[0093] Prime Editor Complex Example 47, Prime Editor Complex The Prime Editor complex includes the following: Any one of the prime editor proteins from Examples 1 to 16; and One pegRNA from any of Examples 17 to 46, Here, the pegRNA scaffold interacts with the prime editor protein to form a complex.

[0094] Example 48, Verification, PE2max In Example 47, the prime editor protein is PE2max, and the pegRNA has the following structure: 5'-[Guide Domain]-[Scaffold]-[Prime Editing Domain]-3', Here, the ]-[ part means that the 3' and 5' ends of each domain are connected directly or via an appropriate linker. Here, the guide domain and the prime editing domain are combinations selected from the following group: The target nucleic acid sequences targeted by the guide domain containing the sequence ACAATTTTATCCTCATCCT (Sequence ID: 407); and TCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATA (Sequence ID: 5302), AATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAAT (Sequence ID: 5303), CAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTG (Sequence ID: 5304), AATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGTT (Sequence ID: 5305), AAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGTT (Sequence ID: 5306), A prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAA (SEQ ID NO: 5307), TCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGT (SEQ ID NO: 5308), ATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGT (SEQ ID NO: 5309), ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTG (SEQ ID NO: 5310), and ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGT (SEQ ID NO: 5311); and A sequence of a target nucleic acid targeted by the guide domain, including the nucleic acid sequence CATCCTTGGAAGAATCCAT (SEQ ID NO: 107), and a sequence of a prime editing domain, including a nucleic acid sequence selected from the group consisting of CCAAATTACTTGATAGGGTAATGGATTCTTCCA (SEQ ID NO: 3707), CATCCAAATTACTTGATAGGGTAATGGATTCTTCCA (SEQ ID NO: 3708), TTAACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGA (SEQ ID NO: 3709), and TAACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGA (SEQ ID NO: 3710); The scaffold can interact with PE2max to form a complex. The prime editing domain contains sequences selected from the group consisting of sequence numbers 3707 to 3710 and sequence numbers 5302 to 5311.

[0095] Example 49, Verification, NRCH-PE2 In Example 47, the prime editor protein is NRCH-PE2 or NRCH-PE2max, and the pegRNA has the following structure: 5'-[Guide Domain]-[Scaffold]-[Prime Editing Domain]-3', Here, the ]-[ part means that the 3' and 5' ends of each domain are connected directly or via an appropriate linker. Here, the guide domain and the prime editing domain are combinations selected from the following group: A target nucleic acid sequence targeted by the guide domain, including the sequence AACAATTTTATCCTCATCC (SEQ ID NO: 267), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of ATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGAT (SEQ ID NO: 4605), CAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATT (SEQ ID NO: 4606), CATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAAATTGTTC (SEQ ID NO: 4607), and TCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATA (SEQ ID NO: 4608); A target nucleic acid sequence targeted by a guide domain containing the sequence ATTTTATCCTCATCCTTGG (SEQ ID NO: 367), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of AATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 5098), TAACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGA (SEQ ID NO: 5099), ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 5100), and AACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 5101); A target nucleic acid sequence targeted by a guide domain containing the sequence CATCCTTGGAAGAATCCAT (SEQ ID NO: 107), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of AATTACTTGATAGGGTAATGGATTCTTCCAAGGA (SEQ ID NO: 3703), AATTACTTGATAGGGTAATGGATTCTTCCAAGGAT (SEQ ID NO: 3704), AACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGA (SEQ ID NO: 3705), and AAATTACTTGATAGGGTAATGGATTCTTCCAAGGA (SEQ ID NO: 3706); A target nucleic acid sequence targeted by a guide domain containing the sequence CCTCATCCTTGGAAGAATC (Sequence ID: 127), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of AATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAG (Sequence ID: 3826), AAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAG (Sequence ID: 3827), CATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGG (Sequence ID: 3828), and TAACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAG (Sequence ID: 3829); A target nucleic acid sequence targeted by a guide domain containing the sequence TCCATTACCCTATCAAAGT (SEQ ID NO: 67), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of AAATTACTTGATAGGGT (SEQ ID NO: 3479), AAATTACTTGATAGGGTAAT (SEQ ID NO: 3480), AACATCCAAATTACTTGATAGGGTA (SEQ ID NO: 3481), and AAATTACTTGATAGGGTA (SEQ ID NO: 3482); and A target nucleic acid sequence targeted by a guide domain containing the sequence TTTTATCCTCATCCTTGGA (SEQ ID NO: 87), and a prime editing domain sequence containing a nucleic acid sequence selected from the group consisting of CCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 3568), TCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 3569), ATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAA (SEQ ID NO: 3570), TCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAAA (SEQ ID NO: 3571), ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGATAA (SEQ ID NO: 3572), and ACATCCAAATTACTTGATAGGGTAATGGATTCTTCCAAGGATGAGGA (SEQ ID NO: 3573); A target nucleic acid sequence targeted by the guide domain, including the sequence AGAATCCATTACCCTATCA (SEQ ID NO: 626), and a prime editing domain sequence including a nucleic acid sequence selected from the group consisting of CAAATTACTTGATAGGGTAATGGAT (SEQ ID NO: 6055), CAAATTACTTGATAGGGTAATGGATT (SEQ ID NO: 6056), and AAATTACTTGATAGGGTAATGGAT (SEQ ID NO: 6057); The scaffold can interact with PE2max to form a complex. The prime editing domain contains sequences selected from the group consisting of SEQ ID NOs: 3479-3482, 3568-3573, 3703-3706, 3826-3829, 4605-4608, 5098-5101, and 6055-6057.

[0096] Prime Editor Expression Vector Example 50, Prime Editor Expression Vector Prime editor expression vectors including the following: A nucleic acid encoding any one of the prime editor proteins from Examples 1 to 16; and A nucleic acid encoding any one of the pegRNAs from Examples 17 to 46, Here, the pegRNA scaffold can interact with the prime editor protein to form a complex. The Prime Editor expression vector allows for the expression of both the Prime Editor protein and pegRNA within cells.

[0097] Example 51, Verification In Example 50, the prime editor expression vector can express the prime editor protein and pegRNA contained in the prime editor complex of Example 48 or Example 49, respectively.

[0098] Example 52, Vectors of two or more molecules In Example 50, the prime editor expression vector comprises a vector of two or more molecules, each containing sequence information for a nucleic acid encoding the prime editor protein and sequence information for a nucleic acid encoding pegRNA.

[0099] Example 53, Limitation of Prime Editor Codon In any one of Examples 50 to 52, the nucleic acid encoding the prime editor protein comprises a nucleic acid sequence selected from SEQ ID NOs: 13210 to 13213.

[0100] Example 54, Limitation of nucleic acids In any one of Examples 50 to 53, nucleic acid refers to either DNA or RNA. Prime Editor Composition Example 55, Prime Editor Composition Prime Editor composition including the following: One of the prime editor proteins from Examples 1 to 16, or a nucleic acid encoding a prime editor protein; and One of the pegRNAs or nucleic acids encoding pegRNAs from Examples 17-46, Here, the pegRNA scaffold can interact with the prime editor protein to form a complex.

[0101] Example 56, Composition containing the complex In Example 55, the prime editor composition comprises a prime editor protein and pegRNA in the form of one of the complexes from Examples 47 to 49.

[0102] Example 57, Composition containing a vector In any one of Examples 55 to 56, the prime editor composition comprises a nucleic acid encoding a prime editor protein and a nucleic acid encoding pegRNA in the form of a vector in any one of Examples 50 to 54.

[0103] Example 58, Composition containing mRNA In any one of Examples 55 to 57, the prime editor composition comprises mRNA encoding the prime editor protein and pegRNA or nucleic acid encoding pegRNA.

[0104] Pharmaceutical composition containing Prime Editor Example 59, Pharmaceutical Composition Pharmaceutical composition containing the following: A therapeutically effective amount of a prime editor complex selected from any one of Examples 47-49, a prime editor expression vector selected from any one of Examples 50-54, or a prime editor composition selected from any one of Examples 55-58; and A pharmaceutically acceptable carrier.

[0105] Therapeutic uses of Prime Editor and methods for treating retinitis pigmentosa Example 60, Use for the treatment of retinitis pigmentosa

[0106] Use of a prime editor complex selected from any one of Examples 47-49, a prime editor expression vector selected from any one of Examples 50-54, a prime editor composition selected from any one of Examples 55-58, or a pharmaceutical composition selected from any one of Examples 59 for the treatment of retinitis pigmentosa.

[0107] Example 61: Use in the manufacture of a drug for treating retinitis pigmentosa. Use of a prime editor complex selected from any one of Examples 47-49, a prime editor expression vector selected from any one of Examples 50-54, a prime editor composition selected from any one of Examples 55-58, or a pharmaceutical composition selected from any one of Examples 59 in the manufacture of a drug for the treatment of retinitis pigmentosa.

[0108] Example 62: Method for treating retinitis pigmentosa A method for treating retinitis pigmentosa, comprising administering, introducing, injecting, and / or delivering to a patient a prime editor complex selected from any one of Examples 47-49, a prime editor expression vector selected from any one of Examples 50-54, a prime editor composition selected from any one of Examples 55-58, or a pharmaceutical composition selected from any one of Examples 59.

[0109] Example 63, Limitation of Retinitis Pigmentosa In any one of Examples 60-62, retinitis pigmentosa is caused by the c.4957dupA mutation in the EYS gene. [Example of experiment]

[0110] The inventions provided herein will be described in more detail below through experimental examples and embodiments. These embodiments are intended solely to illustrate the subject matter disclosed herein, and it will be apparent to those skilled in the art that the scope of the subject matter disclosed herein is not limited by these embodiments.

[0111] Experimental Example 1. Experimental methods and materials Experimental Example 1.1. Method for preparing a plasmid library Plasmid libraries containing pegRNA coding sequences and their corresponding target sequences were prepared using the following two-step cloning process: (Step I) Gibson assembly process; and (Step II) Restriction enzyme-induced cleavage and ligation process.

[0112] The two-step cloning process described above effectively prevents the separation of pegRNA and their corresponding target sequences during PCR amplification. This two-step process was carried out using a previously reported method with appropriate modifications.

[0113] Step I: Construction of an initial plasmid library containing pegRNA coding and target sequence pairs. Oligonucleotide pools containing each pegRNA coding sequence and its corresponding target sequence were amplified by PCR for 20 cycles using Phusion polymerase (New England Biolabs Inc.), and the amplicons were then gel-purified. The Lenti_gRNA-Puro vector (Addgene #84752) was digested with BsmBI enzyme (New England Biolabs, Inc.) at 55°C for more than 6 hours. After digestion, the linearized vector was gel-purified. The linearized Lenti_gRNA-Puro vector and the amplified oligonucleotide pool were assembled by Gibson assembly. After column purification, the assembled product was transformed into electrocompetent cells (Lucigen) using MicroPulser (Bio-Rad). Luria-Bertani (LB) medium (1 ml) was added to the transformation mixture and incubated at 37°C for 1 hour. Subsequently, the cells were seeded on LB agar plates containing 100 μg / ml carbenicillin and cultured. To determine library coverage, aliquots (10, 1, and 0.1 μl) of each culture were seeded separately onto plates. Plasmids were extracted from the recovered colonies. The initial plasmid library coverage was calculated to be 25X.

[0114] Step II: Insertion of sgRNA scaffold The initial plasmid library generated in Step I was digested in BsmBI for 9 hours. The digested products were size-selected and gel-purified on a 1% agarose gel. The sgRNA-optimized scaffold sequence (Dang et al., 2015) was chemically synthesized as oligonucleotide (Integrated DNA Technologies, Inc.) and PCR-amplified for 20 cycles using pairs of Pfu polymerase and primers containing a BsmBI restriction site in each pair. The PCR amplification product was inserted into a PCR-Blunt II-TOPO vector (Solgent CO., LTD.), digested with BsmBI for 4 hours, and gel-purified on a 4% agarose gel. The purified insert was ligated overnight at room temperature (25 °C) with T4 ligase (Enzynomics CO., LTD.) in a 20:1 molar ratio with the digested initial plasmid library vector. The ligation product was purified by isopropanol precipitation and electroporated into Endura electrocompetent cells (Lucigen). Colonies were collected, and the final plasmid library was extracted using the QIAGEN Plasmid Maxi kit. The coverage of the final plasmid library was calculated to be 112X.

[0115] Experimental Example 1.2. Construction of Prime Editor protein expression vectors Blastocydin S deaminase (BSD) Selection Vector The pLenti-PE2max-BSD plasmid (Addgene #191102) was used for the PE2max prime editor protein, the pLenti-NRCH-hyPE2max-BSD plasmid was used for the NRCH-hyPE2max prime editor protein, and the pLenti-NRCH-PE2-BSD plasmid (Addgene #191101) was used for the NRCH-PE2 prime editor protein.

[0116] The vector construction method was based on the method described in Yu et al., "Prediction of eficiencies for diverse prime editing systems in multiple cell types," 2023, Cell 186, 2256-2272. The NRCH-hyPE2max plasmid was constructed based on research showing that adding a rad51 sequence between NRCH nickase and MMLV RT increases prime editing efficiency (Song et al., "Generation of a more efficient prime editor 2 by addition of the RAD51 DNA-binding domain," 2021, Nat Commun 12, 5617).

[0117] Puromycin Selection Vector The pLenti-PE2max-puro plasmid was used for the PE2max prime editor protein, the pLenti-NRCH-PE2max-puro plasmid was used for the NRCH-PE2max prime editor protein, and the pLenti-NRCH-PE2-puro plasmid was used for the NRCH-PE2 prime editor protein.

[0118] The construction methods for each plasmid are described below.

[0119] Using the pLenti-PE2max-BSD plasmid (Addgene #191102), the pLenti-NRCH-PE2max-BSD plasmid for the NRCH-PE2max prime editor protein (Addgene #191103), and the pLenti-NRCH-PE2-BSD plasmid for the NRCH-PE2 prime editor (Addgene #191101), the BSD antibiotic resistance gene regions in these plasmids were converted to puromycin (puro) antibiotic resistance gene sequences. The puromycin antibiotic resistance gene was amplified by PCR for 20 cycles using lentiGuide-puro (Addgene #52963) and inserted into the plasmids from which the BSD antibiotic resistance gene region had been truncated using the Gibson assembly method.

[0120] Experimental Example 1.3. Lentivirus Preparation HEK293T cells (1.0 × 10 7The cells were seeded in 150 mm cell culture dishes containing Dulbecco's Modified Eagle Medium (DMEM). After 21 hours, the library plasmid and psPAX2 (Addgene #12260) were mixed with pMD2.G (Addgene #12259) in a mass ratio of 4:3:1 and co-transfected HEK293T cells using polyethyleneimine. 20 hours after transfection, the medium was replaced with maintenance medium. 48 hours after transfection, the supernatant containing the lentivirus was collected and treated with benzonase (50 U / ml) and 10X benzonase buffer (500 mM Tris-HCl, 10 mM MgCl2, 1 mg / ml recombinant albumin, and ultrapure water). The benzonase-treated supernatant was filtered through a Millex-HV 0.45 μm low protein-binding membrane (Millipore), aliquoted, and stored at -80°C. To determine viral titer, serial dilutions of aliquot virus were introduced into HEK293T cells in the presence of polyblen (10 μg / ml). Both unintroduced cells and cells treated with serial dilutions were cultured in the presence of 2 μg / ml puromycin (Invitrogen). When the majority of unintroduced cells had died, the number of viable cells in the virus-treated population was counted to estimate viral titer. Lentivirus production containing prime editor expression plasmids (PE2max, NRCH PE2max) was carried out according to the same protocol as described above.

[0121] Experimental Example 1.4. Creation of Prime Editor protein-expressing cell lines HEK293T cells were placed in 150 mm cell culture dishes, with 1.0 × 10⁶ cells per dish. 7 Cells were seeded at a specified density and cultured overnight. Lentiviruses expressing the prime editor (PE2max, NRCH-PE2max) prepared in Experimental Example 1.5 were introduced into the cells at a multiple of infection (MOI) of 0.1–0.3. After introduction, the cells were cultured for 19 hours and maintained in 20 μg / ml BSD for the next 7 days to remove non-introduced cells. After BSD selection, each cell line was continuously maintained in 10 μg / ml BSD.

[0122] Experimental Example 1.5. Library Introduction Lentiviruses containing libraries prepared according to Experimental Example 1.5 were introduced into prime editor protein-expressing cell lines prepared according to Experimental Example 1.6. The specific process is described below. 1) The cell line prepared according to Experimental Example 1.6 was placed in a dish of 2.0 × 10⁶ units. 7 Based on cell density, each cell line was seeded in two 150mm dishes and cultured for 21 hours. 2) The lentiviral library prepared according to Experimental Example 1.5 was introduced into cells prepared according to Experimental Example 1.5 at an MOI of 0.5, while ensuring library coverage > 500x in the presence of 10 μg / ml polyblen. 3) Cells prepared according to Experimental Example 2) were cultured overnight, and then cultured for 6 days in the presence of 2 μg / ml puromycin to remove non-transduced cells. 4) The cells were harvested and used 8 days after introduction.

[0123] Experimental Example 1.6. Deep Sequencing For Experiment Example 2, genomic DNA was extracted from the recovered cells using the Wizard Genomic DNA Purification Kit (Promega). For the high-throughput experiment, the incorporated barcode and target sequence were PCR amplified using 2X Taq PCR Smart Mix (SolGent CO., LTD.). For each cell library, the first PCR product contained a total of 240 μg of genomic DNA per cell line screening. This is 10 6 Assuming 10 μg of genomic DNA per cell, this represents at least a 1,000-fold increase in library size. Forty-eight independent 50 μl PCR reactions were performed at an initial genomic DNA concentration of 5 μg per reaction. The products were pooled and gel-purified using the MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology, Inc.).

[0124] The first PCR was performed under the following conditions: 1 minute at 95°C; followed by 22 cycles of 1 minute at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C; followed by a final extension of 2 minutes at 72°C.

[0125] Subsequently, 50 ng of purified DNA was amplified by PCR using primers containing both the Illumina adapter sequence and the barcode sequence.

[0126] The second PCR was performed under the following conditions: 1 minute at 95°C; followed by 10 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C; followed by a final extension of 30 seconds at 72°C.

[0127] The product of the second PCR was gel-purified and subjected to deep sequencing.

[0128] For Experimental Example 3, to measure the prime editing efficiency at endogenous sites in ARPE19 cells, an independent first PCR was performed in a 50 μL reaction volume containing 50 ng of initial genomic DNA template per sample. A second PCR was performed for 20 cycles in a 30 μL reaction volume containing 1 μL of template from the first PCR product to add the Illumina adapter and barcode sequence. After gel purification, the obtained amplicons were analyzed using HiSeq or iSeq (Illumina, Inc., San Diego, CA).

[0129] Experimental Example 1.7. Prime editing efficiency analysis Each pair of pegRNA and target sequence was validated through a 21nt sequence (a 17nt barcode and a 4nt sequence located upstream of the barcode). Reads containing specified edits without unintended mutations within a broad range of target sequences were considered to represent prime editor-induced mutations. To exclude background prime edit frequencies resulting from array synthesis and PCR amplification procedures, the background prime edit frequency derived in the absence of the prime editor was subtracted from the observed prime edit frequency, as shown below.

number

[0130] Experimental Example 1.8. Customize Prime Editing Efficiency Prediction For prediction using DeepPrime, we used the web tool (http: / / deepcrispr.info / DeepPrime / ) (Kim et al., 2023). We performed experiments by predicting the efficiency of prime editing guide RNAs that can restore the EYS c.4957dupA gene mutation to its normal form and selecting the pegRNA sequence that was expected to be the most efficient.

[0131] Experimental Example 2. Screening for pegRNAs capable of correcting the EYS c.4957dupA mutation Experimental Example 2.1. Preparation of a pegRNA screening library To identify the most efficient pegRNAs and target sequences for modifying the EYS c.4957dupA target, pegRNA screening was performed by designing all possible pegRNAs for a total of 28 guide sequences. Specifically, for each target sequence (guide sequence), pegRNAs were designed for all possible combinations of RTT sequence length (minimum editable length ~40 nt) and PBS sequence length (6~17 nt) and included in the library.

[0132] Following the design described above, an oligonucleotide pool containing 4,116 pairs of pegRNAs and target sequences prepared in Experimental Example 1.1 was synthesized by Twist Bioscience (San Francisco, CA). Specifically, each oligonucleotide contained a 19nt guide sequence, BsmBI restriction site #1, a 15nt barcode stuffer sequence, BsmBI restriction site #2, an RTT sequence, a PBS sequence, a poly-T sequence, a 17nt barcode sequence (identifier barcode), and a corresponding 64nt target sequence containing PAM and RTT binding regions. Here, the barcode stuffer sequence was later removed by cleavage via BsmBI, and the identifier barcode sequence was located upstream of the target sequence, enabling identification of individual pegRNA and target sequence pairs after deep sequencing. Oligonucleotides containing unintended BsmBI restriction sites in their sequences were excluded from screening. To find the most efficient pegRNA for targeting, pegRNAs were prepared for a total of 61 pairs of guide sequences, using all possible combinations of RTT sequence lengths (minimum editable length ~40 nt) and PBS sequence lengths (6 nt ~ 17 nucleotides).

[0133] Experimental Example 2.2. High-throughput evaluation of cell libraries Lentiviruses containing the library from Experimental Example 2.1 were prepared according to Experimental Example 1.5, and then introduced into the prime editor protein-expressing cell line from Experimental Example 1.6 according to Experimental Example 1.7. The resulting products were deep-sequenced according to Experimental Example 1.7, and prime editing efficiency was analyzed according to Experimental Example 1.8. Prime editing efficiency was measured twice, and the average value was used.

[0134] The pegRNA screening results are summarized in the following section, "Experimental Example 4.1. pegRNA and screening result data used in Experimental Example 2".

[0135] Based on the results of the experiment in Experiment Example 2, if the prime editing efficiency (value excluding background prime editing frequency) is greater than 0, it can be concluded that pegRNA is capable of prime editing the c.4957dupA mutation in the EYS gene.

[0136] Experimental Example 3. Measurement of prime editing efficiency at endogenous sites in the EYS c.4957dupA ARPE19 cell line Experimental Example 3.1. Prime editor and pegRNA information used in Experimental Example 3 Selected experimental results from Experimental Example 2 were used to test whether the c.4957dupA mutation in the endogenous EYS gene of ARPE19 could be corrected by prime editing.

[0137] The prime editors and pegRNAs used in the experimental examples are: 1) sequences that demonstrate prime editing efficiency as a result of the screening experiment in Experiment Example 2, and 2) pegRNAs that exhibit high prime editing efficiency, selected based on the Deeprime efficiency prediction according to Experiment Example 1.9.

[0138] The prime editor and pegRNA information used in the experiment are summarized in the following section, "Experimental Example 4.2. PegRNA and Result Data Used in Experimental Example 3".

[0139] Experimental Example 3.2. Measurement of prime editing efficiency in the ARPE19 cell line To measure the prime editing efficiency for the endogenous EYS gene c.4957dupA mutation in the ARPE19 cell line, the following experiment was performed: 1) ARPE19 cells carrying the c.4957dupA mutation in the endogenous EYS gene were prepared and cultured in DMEM / F12 (1:1) medium containing 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin). 2) To transfect the ARPE19 cells from 1) with pegRNA and prime editor according to Experimental Example 3.1, divide the cells into 1.0 x 10⁶ wells. 5 The seeds were sown in 12-well plates at this density. 3) 24 hours after seeding, cells were transfected with a prime editor protein expression vector (pLenti-PE2max-puro, pLenti-NRCH PE2max-puro, or pLenti-NRCH PE2 puro) according to Experimental Example 1.4 and with a pegRNA coding plasmid according to Experimental Example 3.1. Here, the prime editor protein expression vector was transfected at 1.0 x 10⁻⁶ 5 Add 600 ng per cell and 1.0 x 10⁶ pegRNA coding plasmid. 5 200 ng was added per cell, and these were treated with 1.6 μL of Lipofectamine 2000 per 800 ng of DNA, according to the manufacturer's instructions. 4) After culturing the mixture from 3) overnight, the culture medium was replaced with DMEM / F12 medium containing puromycin (4 μg / mL). 5) Three days after transfection, cells were collected from 4). 6) The prime editing efficiency in the recovered cells was measured according to Experimental Examples 1.7 and 1.8.

[0140] The experimental results are summarized in the following section, "Experimental Example 4.2. pegRNA and result data used in Experimental Example 3".

[0141] The results of Experimental Example 3 showed that the pegRNA determined to be suitable for prime editing based on the screening results from Experimental Example 2 could correct the c.4957dupA mutation in the endogenous EYS gene in human retinal epithelial ARPE19 cells.

[0142] Based on the results of this experiment, the pegRNA (Experimental Example 2.3) that showed an editing efficiency of more than 0% in the screening experiment of Experimental Example 2 is expected to exhibit a prime editing effect in human retinal cells as well.

[0143] Experimental Example 4. Summary of results from Experimental Examples 2 and 3 Experimental Example 4.1. pegRNA and screening result data used in Experimental Example 2 The pegRNA and screening results data used in Experimental Example 2 are summarized in the table below: [Table 1] [Table 1] TIFF2026530498000004.tif214152TIFF2026530498000005.tif214152TIFF2026530498000006.tif214152TIFF2026530498000007.tif214152TIFF2026530498000008.tif214152TIFF2026530498000009.tif214152TIFF2026530498000010.tif214152TIFF2026530498000011.tif214152TIFF2026530498000012.tif214152TIFF2026530498000013.tif214152TIFF2026530498000014.tif214152TIFF2026530498000015.tif214152TIFF2026530498000016.tif214152TIFF2026530498000017.tif214152TIFF2026530498000018.tif214152TIFF2026530498000019.tif214152TIFF2026530498000020.tif214152TIFF2026530498000021.tif214152TIFF2026530498000022.tif214152TIFF2026530498000023.tif214152TIFF2026530498000024.tif214152TIFF2026530498000025.tif214152TIFF2026530498000026.tif214152TIFF2026530498000027.tif214152TIFF2026530498000028.tif214152TIFF2026530498000029.tif214152TIFF2026530498000030.tif214152TIFF2026530498000031.tif214152TIFF2026530498000032.tif214152TIFF2026530498000033.tif214152TIFF2026530498000034.tif214152TIFF2026530498000035.tif214152TIFF2026530498000036.tif214152TIFF2026530498000037.tif214152TIFF2026530498000038.tif214152TIFF2026530498000039.tif214152TIFF2026530498000040.tif214152TIFF2026530498000041.tif214152TIFF2026530498000042.tif214152TIFF2026530498000043.tif214152TIFF2026530498000044.tif214152TIFF2026530498000045.tif214152TIFF2026530498000046.tif214152TIFF2026530498000047.tif214152TIFF2026530498000048.tif214152TIFF2026530498000049.tif214152TIFF2026530498000050.tif214152TIFF2026530498000051.tif214152TIFF2026530498000052.tif214152TIFF2026530498000053.tif214152TIFF2026530498000054.tif214152TIFF2026530498000055.tif214152TIFF2026530498000056.tif214152[Table 2]. Table 2 TIFF2026530498000058.tif214152TIFF2026530498000059.tif214152TIFF2026530498000060.tif214152TIFF2026530498000061.tif214152TIFF2026530498000062.tif214152TIFF2026530498000063.tif214152TIFF2026530498000064.tif214152TIFF2026530498000065.tif214152TIFF2026530498000066.tif214152TIFF2026530498000067.tif214152TIFF2026530498000068.tif214152TIFF2026530498000069.tif214152TIFF2026530498000070.tif214152TIFF2026530498000071.tif214152TIFF2026530498000072.tif214152TIFF2026530498000073.tif214152TIFF2026530498000074.tif214152TIFF2026530498000075.tif214152TIFF2026530498000076.tif214152TIFF2026530498000077.tif214152TIFF2026530498000078.tif214152TIFF2026530498000079.tif214152TIFF2026530498000080.tif214152TIFF2026530498000081.tif214152TIFF2026530498000082.tif214152TIFF2026530498000083.tif214152TIFF2026530498000084.tif214152TIFF2026530498000085.tif214152TIFF2026530498000086.tif214152TIFF2026530498000087.tif214152TIFF2026530498000088.tif214152TIFF2026530498000089.tif214152TIFF2026530498000090.tif214152TIFF2026530498000091.tif214152TIFF2026530498000092.tif214152TIFF2026530498000093.t if214152TIFF2026530498000094.tif214152TIFF2026530498000095.tif214152TIFF2026530498000096.ti f214152TIFF2026530498000097.tif214152TIFF2026530498000098.tif214152TIFF2026530498000099.tif 214152TIFF2026530498000100.tif214152TIFF2026530498000101.tif214152TIFF2026530498000102.tif21 4152TIFF2026530498000103.tif214152TIFF2026530498000104.tif214152TIFF2026530498000105.tif214 152TIFF2026530498000106.tif214152TIFF2026530498000107.tif214152TIFF2026530498000108.tif2141 52TIFF2026530498000109.tif214152TIFF2026530498000110.tif214152TIFF2026530498000111.tif21415 2TIFF2026530498000112.tif214152TIFF2026530498000113.tif214152TIFF2026530498000114.tif214152.

[0144] Experimental Example 4.2. pegRNA and result data used in Experimental Example 3 The pegRNAs and result data used in Experimental Example 2 are summarized in the table below: [Table 3] [Table 3] TIFF2026530498000116.tif214152TIFF2026530498000117.tif214152

[0145] [Industrial applicability] The Prime Editor disclosed herein acts on human retinal cells containing the c.4957dupA mutation in the EYS gene, thereby correcting the EYS gene to a normal state. Therefore, the Prime Editor can be used as a therapeutic agent for retinitis pigmentosa caused by the c.4957dupA mutation in the EYS gene.

[0146] [Sequence List] The following is a list of sequences containing fewer than 10 nucleotides or fewer than 4 amino acids. The sequence listing files attached to this specification provide lists of sequences containing 10 or more nucleotides or 4 or more amino acids. In accordance with the ST26 sequence listing standard, both DNA and RNA are denoted using the symbols A, T, C, and G. In the following tables, if the Type column is RNA, the base corresponding to T in the sequence should be understood to represent uracil (U). [Table 4] [Table 4] TIFF2026530498000119.tif214152TIFF2026530498000120.tif214152TIFF2026530498000121.tif214152TIFF2026530498000122.tif214152TIFF2026530498000123.tif214152TIFF2026530498000124.tif214152TIFF2026530498000125.tif214152TIFF2026530498000126.tif214152TIFF2026530498000127.tif214152TIFF2026530498000128.tif214152TIFF2026530498000129.tif214152TIFF2026530498000130.tif214152TIFF2026530498000131.tif214152TIFF2026530498000132.tif214152TIFF2026530498000133.tif214152TIFF2026530498000134.tif214152TIFF2026530498000135.tif214152TIFF2026530498000136.tif214152TIFF2026530498000137.tif214152TIFF2026530498000138.tif214152TIFF2026530498000139.tif214152TIFF2026530498000140.tif214152TIFF2026530498000141.tif214152TIFF2026530498000142.tif214152TIFF2026530498000143.tif214152TIFF2026530498000144.tif214152TIFF2026530498000145.tif214152TIFF2026530498000146.tif214152TIFF2026530498000147.tif214152TIFF2026530498000148.tif214152TIFF2026530498000149.tif214152TIFF2026530498000150.tif214152TIFF2026530498000151.tif214152TIFF2026530498000152.tif214152TIFF2026530498000153.tif214152TIFF2026530498000154.tif214152TIFF2026530498000155.tif214152TIFF2026530498000156.tif214152TIFF2026530498000157.tif214152TIFF2026530498000158.tif214152TIFF2026530498000159.tif214152TIFF2026530498000160.tif214152TIFF2026530498000161.tif214152TIFF2026530498000162.tif214152TIFF2026530498000163.tif214152TIFF2026530498000164.tif214152TIFF2026530498000165.tif214152TIFF2026530498000166.tif214152TIFF2026530498000167.tif214152TIFF2026530498000168.tif214152TIFF2026530498000169.tif214152TIFF2026530498000170.tif214152TIFF2026530498000171.tif214152TIFF2026530498000172.tif214152TIFF2026530498000173.tif214152TIFF2026530498000174.tif214152TIFF2026530498000175.tif214152TIFF2026530498000176.tif214152TIFF2026530498000177.tif214152TIFF2026530498000178.tif214152TIFF2026530498000179.tif214152TIFF2026530498000180.tif214152TIFF2026530498000181.tif214152TIFF2026530498000182.tif214152TIFF2026530498000183.tif214152TIFF2026530498000184.tif214152TIFF2026530498000185.tif214152TIFF2026530498000186.tif214152TIFF2026530498000187.tif214152TIFF2026530498000188.tif214152TIFF2026530498000189.tif214152TIFF2026530498000190.tif214152.

Claims

1. A pegRNA for a prime editor to modify the c.4957dupA variant of the EYS gene, It has the structure 5'-[guide domain]-[scaffold]-[prime editing domain]-3', Here, the scaffold can interact with the prime editor to form a complex, The aforementioned guide domain and the aforementioned prime editing domain are as follows: A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11534 to 11552 and sequence numbers 12104 to 12122, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 3343 to 3482; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11553 to 11571 and sequence numbers 12123 to 12141, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 3483 to 3573; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11572 to 11590, and sequence numbers 12142 and 12160, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 3574 to 3710; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11591 to 11609 and SEQ ID NOs: 12161 to 12179, and a prime editing domain composed of nucleic acid sequences selected from SEQ ID NOs: 3711 to 3829; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11610 to 11628 and sequence numbers 12180 to 12198, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 3830 to 3888; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 11629 to 11647 and SEQ ID NOs: 12199 to 12217, and a prime editing domain composed of nucleic acid sequences selected from SEQ ID NOs: 3889 to 4022; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11648 to 11666 and sequence numbers 12218 to 12236, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4023 to 4169; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11667 to 11685 and sequence numbers 12237 to 12255, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4170 to 4309; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11686 to 11704 and sequence numbers 12256 to 12274, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4310 to 4450; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11705 to 11723 and sequence numbers 12275 to 12293, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4451 to 4578; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11724 to 11742 and 12294 to 12312, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4579 to 4608; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11743 to 11761 and sequence numbers 12313 to 12331, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4609 to 4671; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11762 to 11780 and sequence numbers 12332 to 12350, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 4672 to 4736; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11781 to 11799 and sequence numbers 12351 to 12369, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 4737 to 4878; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11800 to 11818 and sequence numbers 12370 to 12388, and a prime editing domain consisting of nucleic acid sequences selected from sequence numbers 4879 to 5024; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11819 to 11837 and sequence numbers 12389 to 12407, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5025 to 5101; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11838 to 11856 and sequence numbers 12408 to 12426, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5102 to 5214; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11857 ​​to 11875 and sequence numbers 12427 to 12445, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5215 to 5311; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11876 to 11894 and sequence numbers 12446 to 12464, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5312 to 5441; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11895 to 11913 and sequence numbers 12465 to 12483, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5442 to 5550; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11914 to 11932 and sequence numbers 12484 to 12502, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5551 to 5681; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11933 to 11951 and sequence numbers 12503 to 12521, and a prime editing domain consisting of nucleic acid sequences selected from sequence numbers 5682 to 5804; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11952 to 11970 and sequence numbers 12522 to 12540, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5805 to 5831; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11971 to 11989 and sequence numbers 12541 to 12559, and a prime editing domain consisting of nucleic acid sequences selected from sequence numbers 5832 to 5923; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 11990 to 12008 and 12560 to 12578, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5924 to 5966; A guide domain that targets a target sequence of nucleic acid sequences selected from sequence numbers 12009 to 12027 and 12579 to 12597, and a prime editing domain composed of nucleic acid sequences selected from sequence numbers 5967 to 5993; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 12028 to 12046 and SEQ ID NOs: 12598 to 12616, and a prime editing domain consisting of nucleic acid sequences selected from SEQ ID NOs: 5994 to 6040; A guide domain that targets a target sequence of nucleic acid sequences selected from SEQ ID NOs: 12047 to 12065 and SEQ ID NOs: 12617 to 12635, and a prime editing domain composed of nucleic acid sequences selected from SEQ ID NOs: 6041 to 6054; and A guide domain that targets a target nucleic acid sequence selected from sequence numbers 12066 to 12103, and a prime editing domain consisting of a nucleic acid sequence selected from sequence numbers 6055 to 6057. pegRNA consisting of combinations selected from the above.

2. The pegRNA according to claim 1, wherein the scaffold is composed of nucleic acid sequences selected from sequence numbers 11531 to 11533.

3. A prime editor composition for correcting the c.4957dupA variant of the EYS gene: Prime editor protein, or nucleic acid encoding the prime editor protein; and pegRNA according to claim 1, or nucleic acid encoding the pegRNA Includes, Here, the prime editor protein comprises a Cas9 protein or a variant thereof derived from Streptococcus pyogenes, and a reverse transcriptase. The scaffold of the pegRNA interacts with the prime editor protein to form a complex. Prime Editor composition.

4. The prime editor composition according to claim 3, comprising the prime editor protein and the pegRNA, wherein the prime editor protein and the pegRNA are combined to form a complex.

5. The prime editor composition according to claim 3, wherein the prime editor composition comprises the nucleic acid encoding the prime editor protein and the nucleic acid encoding the pegRNA.

6. The prime editor composition according to any one of claims 3 to 5, wherein the prime editor protein is selected from NRCH-hyPE2max;NRCH-PE2max;NRCH-PE2; and PE2max.

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