Prime editor for correcting EYS gene and use thereof

Abstract

The present specification provides: a prime editor which is screened through a high-throughput screening method and verified in actual human retinal cells, and can actually correct the c.4957dupA mutation of the EYS gene; and components thereof. The prime editor disclosed in the present specification can act on human retinal cells including the c.4957dupA mutation in the EYS gene, and thereby correct the EYS gene to a normal state. Furthermore, the present specification provides: a prime editor that not only corrects the c.4957dupA mutation of the EYS gene, but also performs co-editing in which silent mutations are introduced near the mutation; and components thereof. The prime editor for performing co-editing can correct the mutation of the EYS gene with greater efficiency. 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

EYS Gene Editing Prime Editor and its Uses

[0001] The invention disclosed herein relates to a technique for treating genetic diseases using a prime editor.

[0002]

[0003] retinitis pigmentosa

[0004] retinitis pigmentosa

[0005] Retinitis pigmentosa (RPE) is a hereditary eye disease that can cause serious complications ranging from night blindness and visual field narrowing to blindness in both eyes. It is a relatively common eye condition, with a high prevalence worldwide, affecting approximately 1 in 3,000 people. However, there are currently few effective treatments for RPE.

[0006] c.4957dupA mutation in the EYS gene

[0007] Recent advancements in Next Generation Sequencing (NGS) technology have led to the discovery of over 50 different genetic mutations that can cause RPE. Among these, mutations in the Eyes Shut Homolog (EYS) gene are known to be a single mutation that can cause RPE. EYS mutations are particularly prevalent in Asian RPE patients.

[0008] Among single-gene mutations that cause RPE, there is the c.4957dupA mutation, which occurs when an adenine nucleotide is duplicated at position 4957 of the EYS gene. Research has shown that a significant number of Asian RPE patients, particularly those in Korea and Japan, are caused by the c.4957dupA mutation in the EYS gene.

[0009] Therefore, it is expected that there will be many patients who can be treated for retinitis pigmentosa by correcting the c.4957dupA mutation in the EYS gene.

[0010] Limitations of existing treatment strategies

[0011] For diseases caused by genetic mutations, strategies were mainly used to deliver the wild-type gene of the mutated gene into cells. The methods for delivering the wild-type gene include 1) a method using adeno-associated virus, 2) a method using lentivirus, and 3) a method of directly delivering mRNA to cells. However, 1) adeno-associated virus can only deliver genes of 4.8 kb or less, but the EYS gene exceeds 10 kb, so it must be divided into multiple viruses and delivered separately, which drastically reduces delivery efficiency, 2) although lentiviruses are free in the size of the gene that can be delivered, there is a risk of the cell randomly inserting it into the genome, and 3) mRNA is unstable, so the gene expression time is limited, so there are disadvantages in that the therapeutic agent must be continuously administered.

[0012] Using the recently spotlighted CRISPR / Cas gene scissors, 1) a treatment strategy that aims for a knockout or knockdown effect by repeatedly cutting the double-stranded DNA of the target gene to create indels in the target gene, and 2) a treatment strategy that uses donor DNA to insert the information of the donor DNA into the gene through the homologous recombination (HDR) mechanism and shows 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 correcting genes using donor DNA has limitations in that its efficiency is too low to see a significant therapeutic effect.

[0013] Prime Editor

[0014] Prime editor refers to a protein-nucleic acid complex used in a gene editing technology called prime editing. The prime editor comprises a prime editor protein including a CRISPR / Cas protein with a mutated double-strand cleavage activity and a reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA). Prime editing is a technology that corrects a gene as intended by 1) binding the prime editor protein to a target gene by the guide of the pegRNA, 2) reverse transcribing the correction sequence included in the 3' end of the pegRNA by the prime editor protein into cDNA, and 3) inserting the reverse transcribed cDNA in place of the mutated sequence of the target gene.

[0015]

[0016] In this specification, we disclose a prime editor capable of correcting the c.4957dupA mutation of the EYS gene.

[0017] Applying the above prime editor to correct only the c.4957dupA mutation in retinitis pigmentosa caused by the c.4957dupA mutation in the EYS gene is a very effective treatment strategy that overcomes the limitations of the existing treatment strategies mentioned above. Theoretically, thousands or tens of thousands of prime editors, especially pegRNAs, that can correct the c.4957dupA mutation in the EYS gene can be designed. However, not all of the thousands or tens of thousands of theoretically designed prime editors actually work to correct the c.4957dupA mutation in the EYS gene. To date, very little has been revealed about which components of the above prime editor affect the prime editing efficiency. Therefore, it is nearly impossible to predict whether the theoretically designed prime editor will actually work based only on its components.

[0018] Therefore, one of the most important technical challenges to be solved in this specification is to select a prime editor that exhibits an effect of correcting the c.4957dupA mutation of the EYS gene in actual human retinal cells from among numerous prime editors that can be theoretically designed, and to specify its composition, thereby providing a prime editor that can exhibit an actual therapeutic effect.

[0019]

[0020] To address the above technical challenges, the present disclosure provides a prime editor and its components that can correct the c.4957dupA mutation of the EYS gene by screening through a high-throughput screening method and validating it in actual human retinal cells.

[0021] In particular, we aim to provide a pegRNA that can correct the c.4957dupA mutation of the EYS gene by working together with a prime editing protein comprising a Nickase mutant of the Cas9 protein derived from Streptococcus pyogenes.

[0022]

[0023] 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.

[0024]

[0025] Figure 1 is a schematic diagram showing the structure of pegRNA and extended pegRNA.

[0026] Figure 2 is a schematic diagram illustrating the relationship between the guide domain of pegRNA, the primer binding site (PBS), the reverse transcriptase template (RTT), and the target nucleic acid. Specifically, the prime editor recognizes the PAM of the non-target strand of the target nucleic acid, and the guide domain complementarily binds to the target sequence of the target strand of the target nucleic acid.

[0027] Figure 3 is a schematic diagram illustrating the relationship between the guide domain of pegRNA, the primer binding site (PBS), the reverse transcriptase template (RTT), and the target nucleic acid. Specifically, it shows the positional relationship of the PBS and RTT sequences after the target nucleic acid is nicked by the prime editor.

[0028]

[0029] Hereinafter, the present invention will be described in more detail through specific implementations and examples with reference to the attached drawings. It should be noted that the attached drawings include some, but not all, implementations of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementations described herein. These implementations should be considered as provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementations of the invention disclosed herein. Therefore, the invention disclosed herein is not limited to the specific implementations described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0030] Disclosed herein is a pegRNA for a prime editor for correcting the c.4957dupA variant of the EYS gene, comprising:

[0031] 5' - [Guide Domain] - [Scaffold] - [Prime Editing Domain] - 3'

[0032] Here, the scaffold can interact with the prime editor to form a complex,

[0033] The above guide domain and the above prime editing domain are composed of a combination selected from the following:

[0034] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 376 to 394 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 8383 to 8484;

[0035] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 395 to 413 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 8485 to 8889;

[0036] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 414 to 432 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 8890 to 9272;

[0037] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 433 to 451 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 9273 to 9523;

[0038] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 452 to 470 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 9524 to 9620;

[0039] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 471 to 489 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 9621 to 9738;

[0040] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 490 to 508 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 9739 to 10131;

[0041] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 509 to 527 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10132 to 10437;

[0042] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 528 to 546 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10438 to 10537;

[0043] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 547 to 565 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10538 to 10754;

[0044] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 566 to 584 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10755 to 10863;

[0045] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 585 to 603 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10864 to 10982;

[0046] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 604 to 622 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 10983 to 11210;

[0047] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 623 to 641 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 11211 to 11539; and

[0048] A guide domain targeting a target sequence of a nucleic acid sequence selected from among SEQ ID NOs: 642 to 660 and a prime editing domain consisting of a nucleic acid sequence selected from among SEQ ID NOs: 11540 to 11958;

[0049] In one embodiment, the scaffold may be a pegRNA comprised of a nucleic acid sequence selected from SEQ ID NO: 31 to SEQ ID NO: 33.

[0050] Disclosed herein is a prime editor composition for correcting the c.4957dupA variant of the EYS gene, comprising:

[0051] a prime editor protein, or a nucleic acid encoding said prime editor protein; and

[0052] The pegRNA, or a nucleic acid encoding the pegRNA,

[0053] Here, the prime editor protein comprises a Cas9 protein derived from Streptococcus pyogenes or a variant thereof, and a reverse transcriptase,

[0054] The scaffold of the above pegRNA interacts with the prime editor protein to form a complex.

[0055] In one embodiment, the prime editor composition may include the prime editor protein and the pegRNA, and the prime editor protein and the pegRNA may be combined to form a complex.

[0056] In one embodiment, the prime editor composition may comprise a nucleic acid encoding the prime editor and a nucleic acid encoding the pegRNA.

[0057] In one embodiment, the prime editor protein may be selected from: NRCH-PE2max; VRQR-PE2max; and PE2max.

[0058] This specification provides pegRNAs for Prime Editor to correct the c.4957dupA variant of the EYS gene, including:

[0059] 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3'

[0060] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0061] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0062] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0063] The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 15572 to SEQ ID NO: 15881,

[0064] The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent,

[0065] The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent,

[0066] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0067] This specification provides pegRNAs for Prime Editor to correct the c.4957dupA variant of the EYS gene, including:

[0068] pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, represented by the following structure:

[0069] 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3'

[0070] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0071] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135,

[0072] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0073] The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 16243 to SEQ ID NO: 16265,

[0074] The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent,

[0075] The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent,

[0076] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0077] This specification provides a prime editing composition for correcting the c.4957dupA variant of the EYS gene, comprising:

[0078] VRQR-PE2max prime editor protein, or a nucleic acid encoding said prime editor protein; and

[0079] A pegRNA selected from any one of claims 1 to 2, or a nucleic acid encoding the pegRNA,

[0080] Here, the scaffold of the pegRNA interacts with the prime editor protein to form a complex.

[0081] As an example,

[0082] The above prime editing composition may include a prime editor complex in which the VRQR-PE2max prime editor protein and the pegRNA are bound.

[0083] As an example,

[0084] The above prime editing composition may include a nucleic acid encoding the VRQR-PE2max prime editor protein and a nucleic acid encoding the pegRNA.

[0085] As an example,

[0086] The nucleic acid encoding the VRQR-PE2max prime editor protein and the nucleic acid encoding the pegRNA are included in an expression vector,

[0087] The above expression vector can be configured to express the VRQR-PE2max prime editor protein and the pegRNA in a cell.

[0088] This specification provides a pharmaceutical composition for treating retinitis pigmentosa caused by the c.c4957dupA mutation of the EYS gene, comprising:

[0089] The above prime editing composition; and

[0090] Pharmaceutically acceptable carrier.

[0091] This specification provides a method for treating retinitis pigmentosa caused by the c.c4957dupA mutation in the EYS gene, comprising:

[0092] A process of administering a therapeutically effective amount of the above prime editing composition to a patient.

[0093]

[0094] Definition of Terms

[0095] approximately

[0096] The term "about" as used herein means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0097] Nucleic acid sequence notation

[0098] The symbols A, T, C, G, and U used herein are to be interpreted as meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively; when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide containing each of the above nucleosides.

[0099] target gene or target nucleic acid

[0100] As used herein, "target gene" or "target nucleic acid" basically refers to a gene or nucleic acid within a cell 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 subject. Unless otherwise specified, the target gene or target nucleic acid may refer to either a gene or nucleic acid inherent to the target cell or an exogenous gene or nucleic acid, and is not particularly limited as long as it can be the target of gene editing. The target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and / or RNA. Furthermore, the terms encompass all meanings recognizable to those skilled in the art and may be appropriately interpreted depending on the context.

[0101] Target strand and non-target strand

[0102] When the terms "target strand" and "nontarget strand" are used herein to refer to a target nucleic acid, they should be understood in the context of the Cas9 protein (and the prime editor protein including it) and the guide RNA (and pegRNA). When the target nucleic acid is a double-stranded nucleic acid and is targeted by the prime editor, the target strand and the nontarget strand are distinguished according to the following criteria. The nontarget strand is a strand that includes a protospacer adjacent motif (PAM) sequence that the Cas9 protein can recognize, and to which the guide RNA (and pegRNA) does not complementarily bind. The target strand refers to a strand to which the guide domain of the guide RNA (and pegRNA) complementarily binds after the Cas9 protein recognizes the PAM sequence of the nontarget strand. In addition, the terms include all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.

[0103] EYS Gene Editing Prime Editor

[0104] EYS Gene Editing Prime Editor Overview

[0105] Disclosed herein are a prime editor capable of correcting the c.4957dupA mutation of the EYS gene and components thereof. The prime editor comprises a prime editor protein and pegRNA. Here, the prime editor protein comprises a Cas9 nickase variant derived from Streptococcus pyogenes and a reverse transcriptase. The pegRNA comprises a guide domain, a scaffold, and a prime editing domain, wherein the guide domain, the scaffold, and the prime editing domain are sequentially linked from the 5' end to the 3' end. Here, 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 around the c.4957dupA mutation of the EYS gene, and 2) provides a normal gene sequence template so that the c.4957dupA mutation of the EYS gene can be corrected through the reverse transcriptase included in the prime editor protein. The prime editor protein 1) generates a nick in the target nucleic acid so that the PBS of the pegRNA can bind to the target nucleic acid and position it appropriately, and 2) reverse transcribes DNA using the RTT as a template so that gene correction can be induced.

[0106] If the sequences of the mutant and normal genes to be corrected are clearly specified, a pegRNA that enables gene correction can theoretically be designed using Prime Editor. When designing the pegRNA, the main design variables are 1) a guide domain capable of targeting the target nucleic acid around the correction target, 2) an RTT sequence and its length according to the sequence of the target nucleic acid and the nick occurrence position, and 3) a PBS sequence length according to the sequence of the target nucleic acid and the nick occurrence position. By changing these, thousands or tens of thousands of pegRNAs can be designed in theory. However, since very little is known about which pegRNA configuration will exhibit the prime editing effect, it is nearly impossible to select a pegRNA sequence that can actually be used for gene correction based only on the theoretically designed sequence information.

[0107] Therefore, the inventors of the present invention selected a method of selecting pegRNAs by confirming through actual experiments whether each pegRNA truly exhibits a prime editing effect. Specifically, the inventors of the present invention synthesized almost all pegRNAs that could be theoretically designed to select a pegRNA capable of correcting the c.4957dupA mutation of the EYS gene, and screened pegRNAs capable of gene editing using a high-throughput screening method. Furthermore, the inventors verified that the pegRNAs showing prime editing efficiency as a result of the screening could correct the c.4957dupA mutation of the EYS gene in actual human retinal cells, thereby completing the present invention by specifying the sequence of the pegRNA that can be used to correct the c.4957dupA mutation of the EYS gene.

[0108] Prime Editor Protein

[0109] 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 having nickase activity and a reverse transcriptase enzyme. In particular, the Cas protein having nickase activity is a mutant of the Cas9 protein derived from Streptococcus pyogenes. The above prime editor protein can cleave only one of the two strands of the target double-stranded nucleic acid (hereinafter referred to as "nicking"). Specifically, the prime editor protein generates a nick in a non-target strand of the target double-stranded nucleic acid. Here, since the position of the target double-stranded nucleic acid is specified, it is also possible to know at which position the prime editor protein will generate a nick, and thus, each configuration (e.g., RTT and PBS sequences) of pegRNA for prime editing correction can be designed based on this.

[0110] The composition of the above prime editor protein is specifically exemplified in the “Prime Editor Protein” section of “Possible Embodiments of the Invention” below.

[0111] pegRNA for EYS gene correction

[0112] The prime editor capable of correcting the c.4957dupA mutation of the EYS gene disclosed herein comprises a pegRNA. The pegRNA comprises a guide domain, a scaffold, a reverse transcriptase template (RTT), and a primer binding site (PBS). Here, the reverse transcriptase template and the primer binding site are collectively referred to as the prime editing domain. Optionally, the pegRNA may additionally comprise an RNA linker and a tmpknot or tevopreQ1 at the 3' end.

[0113] The structures of the above pegRNA and extended pegRNA are schematically shown in Figure 1.

[0114] The above pegRNA can be designed in countless forms by changing the sequence of the guide domain, the reverse transcriptase template sequence, and the primer binding site sequence.

[0115] The composition of the above pegRNA is specifically exemplified in the “pegRNA” section of “Possible Embodiments of the Invention” below.

[0116] Method for designing pegRNA for gene editing

[0117] A pegRNA for gene correction can be designed to 1) specify the target gene site for correction (the c.4957dupA mutation site of the EYS gene) and 2) specify the prime editor protein to be used and the position where the nick is generated in the target nucleic acid.

[0118] Specifically, pegRNA for gene correction can be designed in the following manner: The following description is an example to explain the design method, and although the numbers are given in order, it does not necessarily have to be performed in that order, and it can be designed in other ways as long as the purpose is achieved.

[0119] 1) Specify the sequence before and after correction of the target gene to be corrected.

[0120] 2) Depending on the prime editor protein to be used, the guide domain of pegRNA is designed so that the prime editor can be guided to a predetermined target nucleic acid near the gene site to be corrected.

[0121] 3) Depending on the sequence of the target nucleic acid, the position where a nick is generated by the prime editor protein is specified.

[0122] 4) Based on the position where the above nick occurs, the primer binding site (PBS) of pegRNA is designed so that it can complementarily bind to the 5'-terminal nucleotide of the non-target strand.

[0123] 5) A reverse transcriptase template (RTT) of pegRNA is designed so that the sequence can be reverse transcribed after proofreading by a reverse transcriptase in the 3' end direction of the non-target strand to which the primer binding site binds. The relationship between the guide domain of the pegRNA, the primer binding site (PBS), the reverse transcriptase template (RTT), and the target nucleic acid is schematically shown in Figures 2 and 3.

[0124] Figures 2 and 3 only show a portion of the target nucleic acid and pegRNA, and the portions indicated as 3' and 5' are not the actual nucleic acid ends but are shown to indicate the direction. Figure 2 shows a state in which the prime editor recognizes the PAM of the non-target strand of the target nucleic acid, and the guide domain complementarily binds to the target sequence of the target strand of the target nucleic acid. In this case, a single-stranded cleavage (nick) occurs at a specific position of the non-target strand. Figure 3 shows the positional relationship of the PBS and RTT sequences after a nick occurs in the target nucleic acid by the prime editor. The primer binding site of the pegRNA complementarily binds to the 5' end of the non-target strand of the cleaved target nucleic acid to establish a position, and allows prime editing to occur using the reverse transcriptase template as a template.

[0125] As above, the guide domain, primer binding site, and reverse transcriptase template of the pegRNA can be designed.

[0126] In addition, 1) the guide domain can be designed in various ways by varying the position and length of the target nucleic acid, 2) the PBS can be designed in various ways by varying the length that complementarily binds to the 5'-terminal nucleotide of the non-target strand, and 4) the RTT can be designed in various ways by varying the length of the sequence after proofreading reverse transcribed using a reverse transcriptase. Therefore, theoretically, thousands or tens of thousands of pegRNAs can be designed by varying the guide domain, PBS, and RTT, respectively.

[0127] The difficulty of selecting a functional pegRNA

[0128] As mentioned above, if 1) the target gene site for correction (the c.4957dupA mutation site of the EYS gene) is specified, and 2) the prime editor protein to be used and the position where the nick occurs in the target nucleic acid are specified, a pegRNA for correcting the target gene site can be designed. However, although the number of pegRNAs that can be theoretically designed reaches thousands or tens of thousands, the pegRNA pattern with high gene editing efficiency and the factors that determine the efficiency have not been properly identified yet. Therefore, it is very difficult to select a pegRNA that can be used for actual gene correction based only on the sequence information of the designed pegRNA.

[0129] Approach #1 of the inventors of this application - Selection and validation through high-throughput screening.

[0130] The inventors of the present application employed a method to select pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene through actual experiments. First, the inventors of the present application designed nearly all pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene using prime editor proteins capable of recognizing various PAMs. They then experimentally confirmed whether each pegRNA actually exhibited a prime editing effect and selected pegRNAs capable of correcting the c.4957dupA mutation in the EYS gene. To efficiently perform the above task, a high-throughput screening method was introduced. This high-throughput screening method was based on the method disclosed in 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 pegRNA showing a corrective effect in the high-throughput screening method can correct the endogenous EYS gene of actual human retinal cells (ARPE19), it was shown that the pegRNA selected by the high-throughput screening method can correct the c.4957dupA mutation of the EYS gene in actual retinal cells.

[0131] Approach #2 of the present inventors - Introducing co-editing around the target of pegRNA editing.

[0132] In addition to the above approach, the inventors designed a pegRNA that introduces co-editing, which simultaneously corrects the c.4957dupA mutation site in the EYS gene and other bases surrounding the target base. Researchers have shown that adding co-editing in this way can avoid the inhibition of correction efficiency caused by the DNA mismatch repair system within the cell, thereby increasing the actual correction efficiency. If such co-editing affects gene function, it is not suitable for the purpose of correction. Therefore, co-editing is selected so as not to change the amino acid encoded by the codon at the corresponding position in the gene. The inventors designed and tested a pegRNA with co-editing and confirmed that the pegRNA corrected the c.4957dupA mutation site in the EYS gene without affecting the amino acid sequence encoded by the gene.

[0133] The following examples and experimental examples describe in detail the prime editor and its configuration that can correct the c.4957dupA mutation of the EYS gene.

[0134] [Possible embodiments of the invention]

[0135] Below, possible embodiments of the invention are disclosed. The description below provides examples of embodiments that can be derived from the technical concepts described in this specification. It should be understood that the invention disclosed in this specification is not limited to the specific embodiments described below, and that variations and other implementations thereof are also included within the scope of the invention disclosed in this specification.

[0136] Prime Editor Protein

[0137] Example 1: SpCas9-based prime editor protein

[0138] Prime Editor proteins including:

[0139] A Cas9 protein derived from Streptococcus pyogenes, a variant of the Cas9 protein derived from Streptococcus pyogenes, or a Cas9 protein exhibiting nickase activity among the variants of the Cas9 protein derived from Streptococcus pyogenes; and

[0140] Reverse transcriptase enzyme.

[0141] Example 2: Specificity of the Nickeyase mutation

[0142] In Example 1, the Cas9 protein exhibiting the nickase activity includes the H840A mutation based on SEQ ID NO: 1.

[0143] Example 3, PE2max-induced mutation

[0144] In any one of Examples 1 to 2, the variant of the Cas9 protein, or the Cas9 protein exhibiting the nickase activity, further comprises one or more mutations selected from the following, based on SEQ ID NO: 1:

[0145] R221K; and N394K.

[0146] Example 4: Specific location of nick occurrence

[0147] In any one of Examples 1 to 3, the Cas9 protein exhibiting the nickase activity can recognize a PAM (protospacer adjacent motif) sequence of a non-target strand of a target double-stranded nucleic acid, and can cleave between the 3rd nucleotide and the 4th nucleotide in the 5'-terminal direction from the 5'-terminal nucleotide of the PAM sequence of the non-target strand.

[0148] Example 5, PAM mutant

[0149] In any one of Examples 1 to 4, the variant of the Cas9 protein, or the Cas9 protein exhibiting the nickase activity, can recognize a PAM sequence different from 5'-NGG-3'.

[0150] Example 6, PAM specific

[0151] In any one of Examples 1 to 5, the variant of the Cas9 protein, or the Cas9 protein exhibiting the nickase activity, can recognize one or more PAM sequences selected from the following:

[0152] 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'.

[0153] Example 7, PAM mutation specificity

[0154] In any one of Examples 1 to 6, the variant of the Cas9 protein, or the Cas9 protein exhibiting the nickase activity, comprises at least one mutation selected from the following, based on SEQ ID NO: 1:

[0155] I322V, S409I, E427G, R654L, R753G, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, and H1349R;

[0156] L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, and T1337R; and

[0157] D1135V, G1218R, R1335Q, and T1337R.

[0158] Example 8, Cas9 protein sequence limitation

[0159] In any one of Examples 1 to 7, the Cas9 protein, the variant of the Cas9 protein, or the Cas9 protein exhibiting nickase activity comprises an amino acid sequence selected from the following:

[0160] Sequence numbers 1 to 3; and Sequence numbers 19 to 24.

[0161] Example 9, Reverse Transcriptase Limited

[0162] In any one of Examples 1 to 8, the reverse transcriptase is selected from the following:

[0163] Sequence number 12 to sequence number 13.

[0164] Example 10, Addition of LA Protein

[0165] In any one of Examples 1 to 9, the prime editor protein comprises La Protein.

[0166] Example 11, LA Protein Limited

[0167] In any one of Examples 1 to 10, the La Protein is

[0168] Example 12, Other Domains

[0169] In any one of Examples 1 to 11, the prime editor protein further comprises one or more domains selected from the following:

[0170] One or more Nuclear Localization Signals (NLS); and a linker.

[0171] Example 13, NLS sequence limitation

[0172] In Example 12, each of the one or more NLSs independently comprises a sequence selected from the following:

[0173] Sequence number 4 to sequence number 7.

[0174] Example 14, linker-specific

[0175] In any one of Examples 12 to 13, the linker comprises the sequence of SEQ ID NO: 14.

[0176] Example 15, Prime Editor Protein Limited

[0177] In any one of Examples 1 to 14, the prime editor protein is selected from the group consisting of:

[0178] PE2; PE2max; PE7max; NRCH-PE2; NRCH-PE2max; NRCH-PE7max; VRQR-PE2; VRQR-PE2max; and VRQR-PE7max.

[0179] Example 16, Prime Editor Sequence Limitation

[0180] In any one of Examples 1 to 15, the prime editor protein comprises an amino acid sequence selected from SEQ ID NO: 16 to SEQ ID NO: 18 and SEQ ID NO: 25 to SEQ ID NO: 27.

[0181] Example 17, Prime Editor, Structure Limited

[0182] Prime Editor proteins including:

[0183] [NLS1] - [Cas9 protein] - [1st linker] - [reverse transcriptase] - [2nd linker] - [La Protein] - [NLS2]

[0184] Here, the NLS1 is the first NLS (Nuclear Localization Signal) or is absent,

[0185] The above NLS2 is a second NLS or is absent,

[0186] The above Cas9 protein comprises a sequence selected from among SEQ ID NO: 19 and SEQ ID NO: 22 to SEQ ID NO: 24,

[0187] The above first linker is an amino acid linker or is absent,

[0188] The above reverse transcriptase comprises a sequence selected from SEQ ID NO: 12 to SEQ ID NO: 13.

[0189] The above second linker is an amino acid linker or is absent,

[0190] The above La Protein is La Protein or is absent.

[0191] Example 18, NLS and linker sequence definition

[0192] In Example 17,

[0193] The above NLS1 and NLS2 each independently comprise an amino acid sequence selected from SEQ ID NO: 4 to SEQ ID NO: 7.

[0194] Example 19, linker-specific

[0195] In Example 17,

[0196] The first linker and the second linker are each independently an amino acid sequence of SEQ ID NO: 16313.

[0197] Example 20, Prime Editor Full-Length Sequence Limited

[0198] In Example 17,

[0199] The above prime editor protein comprises an amino acid sequence selected from SEQ ID NO: 28 to SEQ ID NO: 30 and SEQ ID NO: 16314.

[0200] pegRNA

[0201] Example 21, pegRNA

[0202] pegRNA for Prime Editor to correct the c.4957dupA variant in the EYS gene.

[0203] Example 22, Meaning of c.4957dupA correction

[0204] In Example 21, correcting the c.4957dupA variant means correcting the sequence ATCAAAGTAAT (SEQ ID NO: 15539), which is a sequence including the c.4957dupA variant, of the EYS gene including the c.4957dupA variant, to ATCAAGTAAT (SEQ ID NO: 15540).

[0205] Example 23, Prime Editor Protein Limited

[0206] In Example 21, the prime editor comprises a prime editor protein selected from Examples 1 to 20.

[0207] Example 24, pegRNA structure #1

[0208] In any one of Examples 21 to 23, the pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0209] The above prime editing domain comprises a reverse transcriptase template (RTT) and a primer binding site (PBS),

[0210] The above pegRNA has a guide domain, a scaffold, and a prime editing domain sequentially connected from the 5' end to the 3' end.

[0211] The above guide domain can target a target nucleic acid contained in the EYS gene,

[0212] The above scaffold can interact with the prime editor protein of the above prime editor to form a complex,

[0213] The above prime editing domain is sequentially connected to a reverse transcriptase template (RTT) and a primer binding site (PBS) in the 5'-to-3'-end direction.

[0214] Example 25, pegRNA structure #2

[0215] In any one of Examples 21 to 24, the pegRNA has the following structure:

[0216] 5' - [Guide Domain] - [Scaffold] - [Prime Editing Domain] - 3'

[0217] Here, the guide domain can target a target nucleic acid contained in the EYS gene,

[0218] The above scaffold can interact with the prime editor protein of the above prime editor to form a complex,

[0219] The above prime editing domain includes a reverse transcriptase template (RTT) and a primer binding site (PBS).

[0220] Example 26, Meaning of Target Nucleic Acid Target

[0221] In any one of Examples 21 to 25,

[0222] The above target nucleic acid refers to double-stranded DNA of all or part of the EYS gene,

[0223] The above target nucleic acid comprises a target strand and a nontarget strand,

[0224] Since the sequence of the target strand and the sequence of the non-target strand are complementary sequences to each other, the sequence of the target nucleic acid can be specified only by the sequence of the target strand or the sequence of the non-target strand.

[0225] The description that the above guide domain targets the above target sequence contained in the above EYS gene has the following selected meanings:

[0226] The guide domain can complementarily bind to and / or hybridize with the target strand of the target nucleic acid;

[0227] The sequence of the above guide domain includes a sequence complementary to all or part of the sequence of the target strand of the above target nucleic acid;

[0228] The sequence of the above guide domain is identical to, matches, homologous to, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid; or

[0229] Any suitable combination of the above descriptions, within the scope recognizable by a person skilled in the art.

[0230] Example 27, Non-target strand sequence limitation

[0231] In Example 26,

[0232] The sequence of the non-target strand of the target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 376 to SEQ ID NO: 660 and SEQ ID NO: 946 to SEQ ID NO: 1230,

[0233] The sequence of the guide domain of the above pegRNA comprises a sequence that is identical, matched, homologous, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid.

[0234] Example 28, Sequence limitation similar to non-target strand

[0235] In Example 26,

[0236] The sequence of the non-target strand of the target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 376 to SEQ ID NO: 660 and SEQ ID NO: 946 to SEQ ID NO: 1230,

[0237] The sequence of the guide domain of the pegRNA comprises a sequence that is identical, matched, homologous, and / or equivalent to all or part of the sequence of the non-target strand of the target nucleic acid by at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0238] Example 29, same as the non-target strand, but with mismatches

[0239] In Example 26,

[0240] The sequence of the non-target strand of the target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 376 to SEQ ID NO: 660 and SEQ ID NO: 946 to SEQ ID NO: 1230,

[0241] The sequence of the guide domain of the above pegRNA comprises a sequence that is identical, matched, homologous, and / or equivalent to the entire or a portion of the non-target strand of the target nucleic acid except for 1, 2, 3, 4, or 5 nucleotide bases.

[0242] Example 30, target strand sequence limitation, complementary sequence

[0243] In Example 26,

[0244] The sequence of the target strand of the above target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 661 to SEQ ID NO: 945,

[0245] The sequence of the guide domain of the above pegRNA includes a sequence complementary to all or part of the sequence of the target strand.

[0246] Example 31, restriction of sequences similar to sequences complementary to the target strand sequence

[0247] In Example 26,

[0248] The sequence of the target strand of the above target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 661 to SEQ ID NO: 945,

[0249] The sequence of the guide domain of the pegRNA comprises a sequence that is at least 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to all or part of the sequence of the target strand.

[0250] Example 32, complementary to the target strand, but containing a mismatch

[0251] In Example 24,

[0252] The sequence of the target strand of the above target nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 661 to SEQ ID NO: 945,

[0253] The sequence of the guide domain of the above pegRNA includes a sequence in which all or part of the sequence of the target strand is complementary to the remaining bases except for 1, 2, 3, 4, or 5 nucleotide bases.

[0254] Example 33, target strand sequence limitation, binding relationship limitation

[0255] In any one of Examples 21 to 32,

[0256] The guide domain of the pegRNA can complementarily bind and / or hybridize with the target strand.

[0257] Example 34, limiting the length of the guide domain

[0258] In any one of Examples 21 to 33, the nucleotide length of the guide domain is 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt, or has a length within a range comprised of the above numerical ranges (e.g., a length between 12 nt and 30 nt).

[0259] Example 35, Guide Domain Limited

[0260] In any one of Examples 21 to 34, the sequence of the guide domain comprises a nucleic acid sequence selected from the group consisting of:

[0261] A nucleic acid sequence selected from SEQ ID NO: 76 to SEQ ID NO: 375; and

[0262] A sequence that is identical, matches, homologous, and / or equivalent to a nucleic acid sequence selected from SEQ ID NO: 76 to SEQ ID NO: 375 by at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0263] Example 36, including G or GG at the 5' end

[0264] In Example 35, the sequence of the guide domain further includes G or GG at the 5' end.

[0265] Example 37, Scaffold Sequence Limitation

[0266] In any one of Examples 21 to 36, the sequence of the scaffold comprises a nucleic acid sequence selected from the group consisting of:

[0267] A nucleic acid sequence selected from SEQ ID NO: 31 to SEQ ID NO: 33; and

[0268] A sequence that is identical or identical to a nucleic acid sequence selected from SEQ ID NOs: 31 to 33 by at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or is identical or matched or homologous to a nucleic acid sequence selected from SEQ ID NOs: 31 to 33, and is capable of interacting with the prime editor protein to form a complex.

[0269] Example 38, Prime Editing Domain Sequence Limitation

[0270] In any one of Examples 21 to 37,

[0271] The above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8383 to SEQ ID NO: 11957.

[0272] Example 39, RTT and PBS each limited

[0273] In any one of Examples 21 to 38,

[0274] The sequence of the above prime editing domain is the following sequence sequentially connected from the 5' end to the 3' end:

[0275] A reverse transcriptase template sequence selected from SEQ ID NO: 1231 to SEQ ID NO: 4806 and SEQ ID NO: 16195 to SEQ ID NO: 16217; and

[0276] A primer binding site sequence selected from among SEQ ID NO: 4807 to SEQ ID NO: 8382 and SEQ ID NO: 16218.

[0277] Example 40, Guide Domain and Prime Editing Domain Matching

[0278] In any one of Examples 21 to 39, the sequence of the guide domain of the pegRNA and the sequence of the prime editing domain are expressed in a combination selected from the group consisting of:

[0279] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 95, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8383 to SEQ ID NO: 8484;

[0280] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 115, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8485 to SEQ ID NO: 8889;

[0281] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8890 to SEQ ID NO: 9272;

[0282] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 136 to SEQ ID NO: 155, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9273 to SEQ ID NO: 9523;

[0283] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 156 to SEQ ID NO: 175, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9524 to SEQ ID NO: 9620;

[0284] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO: 195, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9621 to SEQ ID NO: 9738;

[0285] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 196 to SEQ ID NO: 215, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9739 to SEQ ID NO: 10131;

[0286] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10132 to SEQ ID NO: 10437;

[0287] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 236 to SEQ ID NO: 255, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10438 to SEQ ID NO: 10537;

[0288] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 256 to SEQ ID NO: 275, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10538 to SEQ ID NO: 10754;

[0289] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 276 to SEQ ID NO: 295, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10755 to SEQ ID NO: 10863;

[0290] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 296 to SEQ ID NO: 315, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10864 to SEQ ID NO: 10982;

[0291] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 316 to SEQ ID NO: 335, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10983 to SEQ ID NO: 11210;

[0292] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 336 to SEQ ID NO: 355, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11211 to SEQ ID NO: 11539;

[0293] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 375, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11540 to SEQ ID NO: 11958; and

[0294] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the sequence of the above prime editing domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16243 to SEQ ID NO: 16265.

[0295] Example 41, Guide domain, RTT, and PBS sequence matching

[0296] In any one of Examples 21 to 40, the sequence of the guide domain of the pegRNA and the sequence of the prime editing domain are expressed in a combination selected from the group consisting of:

[0297] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 95, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 1231 to SEQ ID NO: 1332 and a primer binding site sequence selected from SEQ ID NO: 4807 to SEQ ID NO: 4908 are sequentially connected from the 5' end to the 3' end;

[0298] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 115, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 1333 to SEQ ID NO: 1737 and a primer binding site sequence selected from SEQ ID NO: 4909 to SEQ ID NO: 5313 are sequentially connected from the 5' end to the 3' end;

[0299] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 1738 to SEQ ID NO: 2120 and a primer binding site sequence selected from SEQ ID NO: 5314 to SEQ ID NO: 5696 are sequentially connected from the 5' end to the 3' end;

[0300] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 136 to SEQ ID NO: 155, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 2121 to SEQ ID NO: 2371 and a primer binding site sequence selected from SEQ ID NO: 5697 to SEQ ID NO: 5947 are sequentially connected from the 5' end to the 3' end;

[0301] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 156 to SEQ ID NO: 175, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 2372 to SEQ ID NO: 2468 and a primer binding site sequence selected from SEQ ID NO: 5948 to SEQ ID NO: 6044 are sequentially connected from the 5' end to the 3' end;

[0302] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO: 195, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 2469 to SEQ ID NO: 2586 and a primer binding site sequence selected from SEQ ID NO: 6045 to SEQ ID NO: 6162 are sequentially connected from the 5' end to the 3' end;

[0303] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 196 to SEQ ID NO: 215, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 2587 to SEQ ID NO: 2979 and a primer binding site sequence selected from SEQ ID NO: 6163 to SEQ ID NO: 6555 are sequentially connected from the 5' end to the 3' end;

[0304] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 2980 to SEQ ID NO: 3285 and a primer binding site sequence selected from SEQ ID NO: 6556 to SEQ ID NO: 6861 are sequentially connected from the 5' end to the 3' end;

[0305] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 236 to SEQ ID NO: 255, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 3286 to SEQ ID NO: 3385 and a primer binding site sequence selected from SEQ ID NO: 6862 to SEQ ID NO: 6961 are sequentially connected from the 5' end to the 3' end;

[0306] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 256 to SEQ ID NO: 275, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 3386 to SEQ ID NO: 3602 and a primer binding site sequence selected from SEQ ID NO: 6962 to SEQ ID NO: 7178 are sequentially connected from the 5' end to the 3' end;

[0307] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 276 to SEQ ID NO: 295, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 3603 to SEQ ID NO: 3711 and a primer binding site sequence selected from SEQ ID NO: 7179 to SEQ ID NO: 7287 are sequentially connected from the 5' end to the 3' end;

[0308] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 296 to SEQ ID NO: 315, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 3712 to SEQ ID NO: 3830 and a primer binding site sequence selected from SEQ ID NO: 7288 to SEQ ID NO: 7406 are sequentially connected from the 5' end to the 3' end;

[0309] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 316 to SEQ ID NO: 335, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 3831 to SEQ ID NO: 4058 and a primer binding site sequence selected from SEQ ID NO: 7407 to SEQ ID NO: 7634 are sequentially connected from the 5' end to the 3' end;

[0310] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 336 to SEQ ID NO: 355, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 4059 to SEQ ID NO: 4387 and a primer binding site sequence selected from SEQ ID NO: 7635 to SEQ ID NO: 7963 are sequentially connected from the 5' end to the 3' end;

[0311] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 375, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 4388 to SEQ ID NO: 4806 and a primer binding site sequence selected from SEQ ID NO: 7964 to SEQ ID NO: 8382 are sequentially connected from the 5' end to the 3' end; and

[0312] The sequence of the above guide domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the sequence of the above prime editing domain comprises a nucleic acid sequence in which a reverse transcriptase template sequence selected from SEQ ID NO: 16195 to SEQ ID NO: 16217 and a primer binding site sequence of SEQ ID NO: 16218 are sequentially connected from the 5' end to the 3' end.

[0313] Example 42, pegRNA full-length sequence limitation

[0314] In any one of Examples 21 to 41, the sequence of the pegRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11959 to SEQ ID NO: 15534.

[0315] Example 43, adding tmpknot

[0316] In any one of Examples 21 to 42, the pegRNA additionally comprises tmpknot, tevopreQ1, or both at the 3' end.

[0317] Example 44, tmpknot sequence limitation

[0318] In Example 43, the sequence of tmpknot includes the nucleic acid sequence of SEQ ID NO: 34, and the sequence of tevopreQ1 includes the nucleic acid sequence of SEQ ID NO: 16242.

[0319] Example 45, tmpknot connection linker

[0320] In any one of Examples 43 to 44, the pegRNA further comprises an RNA linker, and the tmpknot is connected to the 3' end of the pegRNA via the RNA linker.

[0321] Example 46, linker sequence limitation

[0322] In Example 45, the sequence of the RNA linker is TACAACAA (SEQ ID NO: 35), AATAATGC (SEQ ID NO: 36), CCCAGCTT (SEQ ID NO: 37), AGATAAGC (SEQ ID NO: 38), GGGCAAAT (SEQ ID NO: 39), AAGTGAGA (SEQ ID NO: 40), CACAGAAA (SEQ ID NO: 41), AATAACAA (SEQ ID NO: 42), AACGACGC (SEQ ID NO: 43), AAAGAAGA (SEQ ID NO: 44), CACAGCAT (SEQ ID NO: 45), AAGTAGAC (SEQ ID NO: 46), AAGAAAGA (SEQ ID NO: 47), AGCATAGA (SEQ ID NO: 48), ACCGCATA (SEQ ID NO: 49), CCAGCCTC (SEQ ID NO: 50), CCCATATA (SEQ ID NO: 51), CTCCACGC (SEQ ID NO: 52), CTAAATAA (SEQ ID NO: 53), AATATATA (SEQ ID NO: 54), CCAAACAC (SEQ ID NO: 55), TCGCTACT (SEQ ID NO: 56), ATATACGT (SEQ ID NO: 57), CAATTTAT (SEQ ID NO: 58), CTTCATAA (SEQ ID NO: 59), ATATACTT (SEQ ID NO: 60), TTACAAAT (SEQ ID NO: 61), ATAAATAA (SEQ ID NO: 62), CACATATA (SEQ ID NO: 63), ATTATTAC (SEQ ID NO: 64), ACAAATTA (SEQ ID NO: 65), CAAATAGC (SEQ ID NO: 66), CCATATTA (SEQ ID NO: 67), ATCCCTAA (SEQ ID NO: 68), TAAGGTAC (SEQ ID NO: 69), TTATAAGA (SEQ ID NO: 70), TAAAGTGA (SEQ ID NO: 71), CTCCAGGA (SEQ ID NO: 72), AGGACAGC (SEQ ID NO: 73), AGATAGCT (SEQ ID NO: 74), AGAACGGA (SEQ ID NO: 75), GGATAACT (SEQ ID NO: 16219), GAAGGAAG (SEQ ID NO: 16220), AAGAAAGC (SEQ ID NO: 16221), GGAAGAGA (SEQ ID NO: 16222), AATATAAA (SEQ ID NO: 16223), GAAAGAGA (SEQ ID NO: 16224), GGAAGAAG (SEQ ID NO: 16225), AATGATAA (SEQ ID NO: 16226),A nucleic acid selected from among GGAGGAAA (SEQ ID NO: 16227), AAAGAGAA (SEQ ID NO: 16228), GAGAAGGC (SEQ ID NO: 16229), AATAAGGA (SEQ ID NO: 16230), GAGGGAAC (SEQ ID NO: 16231), AGAGAAAC (SEQ ID NO: 16232), GAAAGAAA (SEQ ID NO: 16233), AAATAGAG (SEQ ID NO: 16234), AATAATAG (SEQ ID NO: 16235), AGGAAGAC (SEQ ID NO: 16236), AGGGATAC (SEQ ID NO: 16237), AGAAAGCA (SEQ ID NO: 16238), AGGAACAA (SEQ ID NO: 16239), GGATAGAA (SEQ ID NO: 16240), and AGGAGGAA (SEQ ID NO: 16241). Contains sequences.,

[0323] Example 47, including Poly-U

[0324] In any one of Examples 21 to 46, the pegRNA additionally comprises a Poly-U sequence at the 3' end.

[0325] Example 48, poly-U sequence limitation

[0326] In Example 47, the Poly-U sequence is 5'-UUUUUU-3'.

[0327] Example 49, Markushi-type claim

[0328] pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, represented by the following structure:

[0329] 5'-[5'-end appendage]-[guide domain]-[scaffold]-[reverse transcriptase template (RTT)]-[primer binding site (PBS)]-[linker]-[stabilizing motif]-[Poly-U]-3'

[0330] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0331] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 375,

[0332] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0333] The sequence of the above reverse transcriptase template comprises a sequence selected from the group consisting of SEQ ID NO: 1231 to SEQ ID NO: 4806 and SEQ ID NO: 16195 to SEQ ID NO: 16217,

[0334] The sequence of the above primer binding site includes a sequence selected from the group consisting of SEQ ID NO: 4807 to SEQ ID NO: 8382 and SEQ ID NO: 16218,

[0335] The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent,

[0336] The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, or the above stabilizing motif is absent,

[0337] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0338] Example 50, VRQR-Validation

[0339] In any one of Examples 21 to 49,

[0340] The above guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0341] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8966, SEQ ID NO: 9105, SEQ ID NO: 9109, SEQ ID NO: 9111, SEQ ID NO: 9139 to SEQ ID NO: 9140, SEQ ID NO: 9179, SEQ ID NO: 9185, SEQ ID NO: 9210, and SEQ ID NO: 9214.

[0342] Example 51, DNA encoding pegRNA

[0343] DNA encoding the pegRNA of any one of Examples 21 to 50.

[0344] Co-editing pegRNA

[0345] Example 52, Co-editing target

[0346] In Example 21, the pegRNA comprises a sequence ATCAAAGTAAT (SEQ ID NO: 15539) including the c.4957dupA position of the mutant EYS gene, ATCAAGCAAT (SEQ ID NO: 15541), ATCATCAAAT (SEQ ID NO: 15542), ATCATCTAAT (SEQ ID NO: 15543), ATCATCGAAT (SEQ ID NO: 15544), ATCATCCAAT (SEQ ID NO: 15545), ATCTAGTAAT (SEQ ID NO: 15546), ATCGAGTAAT (SEQ ID NO: 15547), ATCCAGTAAT (SEQ ID NO: 15548), ATCTTCAAAT (SEQ ID NO: 15549), ATCGTCAAAT (SEQ ID NO: 15550), ATCCTCAAAT (SEQ ID NO: 15551), ATCTTCTAAT (SEQ ID NO: 15552), ATCGTCTAAT (SEQ ID NO: 15553), ATCCTCTAAT (SEQ ID NO: 15554), ATCTTCGAAT (SEQ ID NO: 15555), ATCGTCGAAT (SEQ ID NO: 15556), ATCCTCGAAT (SEQ ID NO: 15557), ATCTTCCAAT (SEQ ID NO: 15558), ATCGTCCAAT (SEQ ID NO: 15559), ATCCTCCAAT (SEQ ID NO: 15560), AAGCAGTAAT (SEQ ID NO: 15561), AAGTAGTAAT (SEQ ID NO: 15562), AAGCTCAAAT (SEQ ID NO: 15563), AAGCTCTAAT (SEQ ID NO: 15564), AAGCTCGAAT (SEQ ID NO: 15565), AAGCTCCAAT (SEQ ID NO: 15566), AAGTTCAAAT (SEQ ID NO: 15567), AAGTTCTAAT (SEQ ID NO: 15568), AAGTTCGAAT (SEQ ID NO: 15569), AAGTTCCAAT (SEQ ID NO: 15570), ATCAAGTAAC (SEQ ID NO: 15571), ATCAAGCAATT (SEQ ID NO: 16289), ATCATCAAATT (SEQ ID NO: 16290), ATCATCCAATT (SEQ ID NO: 16291), ATCATCTAATT (SEQ ID NO: 16292), ATCATCGAATT (SEQ ID NO: 16293), ATCCAGTAATT (SEQ ID NO: 16294),ATCTAGTAATT (SEQ ID NO: 16295), ATCGAGTAATT (SEQ ID NO: 16296), ATCCTCAAATT (SEQ ID NO: 16297), ATCCTCCAATT (SEQ ID NO: 16298), ATCCTCTAATT (SEQ ID NO: 16299), ATCCTCGAATT (SEQ ID NO: 16300), ATCTTCAAATT (SEQ ID NO: 16301), ATCTTCCAATT (SEQ ID NO: 16302), ATCTTCTAATT (SEQ ID NO: 16303), ATCTTCGAATT (SEQ ID NO: 16304), ATCGTCAAATT (SEQ ID NO: 16305), ATCGTCCAATT (SEQ ID NO: 16306), ATCGTCTAATT (SEQ ID NO: 16307), ATCGTCGAATT (SEQ ID NO: 16308), To correct one selected from among AAGTAGTAATT (SEQ ID NO: 16309), AAGCAGTAATT (SEQ ID NO: 16310), and ATCAAGTAATT (SEQ ID NO: 16311).

[0347] Example 53, Prime Editing Domain Limited

[0348] In Example 52, the pegRNA has a guide domain, a scaffold, and a prime editing domain sequentially connected in the 5'-end to 3'-end direction,

[0349] The above scaffold can interact with the above prime editor protein to form a complex,

[0350] The above guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0351] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15572 to SEQ ID NO: 15881.

[0352] Example 54, pegRNA sequence definition

[0353] In Example 53, the pegRNA consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15882 to SEQ ID NO: 16191 and SEQ ID NO: 16266 to SEQ ID NO: 16288.

[0354] Example 55, Markushi-type claim

[0355] pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, represented by the following structure:

[0356] 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3'

[0357] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0358] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0359] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0360] The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 15572 to SEQ ID NO: 15881,

[0361] The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent,

[0362] The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent,

[0363] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0364] Example 56, Markushi Form Claim #2

[0365] pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, represented by the following structure:

[0366] 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3'

[0367] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0368] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135,

[0369] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0370] The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 16243 to SEQ ID NO: 16265,

[0371] The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent,

[0372] The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent,

[0373] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0374] Prime Editor Complex

[0375] Example 57, Prime Editor Complex

[0376] Prime Editor Complex including:

[0377] A prime editor protein of any one of Examples 1 to 20; and

[0378] Any one of the pegRNAs of Examples 21 to 51,

[0379] Here, the scaffold of the pegRNA interacts with the prime editor protein to form a complex.

[0380] Example 58, VRQR-Validation

[0381] In Example 57,

[0382] The above prime editor protein is VRQR-PE2max,

[0383] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0384] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0385] The above scaffold can interact with the above prime editor protein to form a complex,

[0386] The above guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0387] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8966, SEQ ID NO: 9105, SEQ ID NO: 9109, SEQ ID NO: 9111, SEQ ID NO: 9139 to SEQ ID NO: 9140, SEQ ID NO: 9179, SEQ ID NO: 9185, SEQ ID NO: 9210, and SEQ ID NO: 9214.

[0388] Example 59, VRQR-Validation co-editing #1

[0389] In Example 57,

[0390] The above prime editor protein is VRQR-PE2max,

[0391] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0392] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0393] The above scaffold can interact with the above prime editor protein to form a complex,

[0394] The above guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0395] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15572 to SEQ ID NO: 15881.

[0396] Example 60, VRQR-Validation co-editing #2

[0397] In Example 57,

[0398] The above prime editor protein is VRQR-PE2max,

[0399] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0400] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0401] The above scaffold can interact with the above prime editor protein to form a complex,

[0402] The above guide domain consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, or comprises the nucleic acid sequence,

[0403] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16243 to SEQ ID NO: 16265.

[0404] Example 61, NRCH-Validation

[0405] In Example 57,

[0406] The above prime editor protein is NRCH-PE2max,

[0407] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0408] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0409] The above scaffold can interact with the above prime editor protein to form a complex,

[0410] The above guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235,

[0411] The above prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10248, SEQ ID NO: 10275 to SEQ ID NO: 10276, SEQ ID NO: 10304, SEQ ID NO: 10331 to SEQ ID NO: 10332, SEQ ID NO: 10336, SEQ ID NO: 10359, and SEQ ID NO: 10387 to SEQ ID NO: 10388.

[0412] Example 62, VRQR-All

[0413] In Example 57,

[0414] The above prime editor protein is VRQR-PE2max,

[0415] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0416] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0417] The above scaffold can interact with the above prime editor protein to form a complex,

[0418] The above guide domain and the above prime editing domain are a combination selected from the group consisting of:

[0419] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 95, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11959 to SEQ ID NO: 11978, SEQ ID NO: 11980 to SEQ ID NO: 11998, SEQ ID NO: 12000 to SEQ ID NO: 12024, and SEQ ID NO: 12026 to SEQ ID NO: 12060;

[0420] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 115, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12061 to SEQ ID NO: 12097, and SEQ ID NO: 12099 to SEQ ID NO: 12465;

[0421] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12466 to SEQ ID NO: 12476, and SEQ ID NO: 12478 to SEQ ID NO: 12848;

[0422] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 136 to SEQ ID NO: 155, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12849 to SEQ ID NO: 13099;

[0423] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 156 to SEQ ID NO: 175, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13100 to SEQ ID NO: 13117, SEQ ID NO: 13119 to SEQ ID NO: 13164, and SEQ ID NO: 13166 to SEQ ID NO: 13196;

[0424] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO: 195, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13197 to SEQ ID NO: 13314;

[0425] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 196 to SEQ ID NO: 215, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13315 to SEQ ID NO: 13707;

[0426] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13708 to SEQ ID NO: 13728, SEQ ID NO: 13730 to SEQ ID NO: 13810, and SEQ ID NO: 13812 to SEQ ID NO: 14013;

[0427] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 236 to SEQ ID NO: 255, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14014 to SEQ ID NO: 14021, SEQ ID NO: 14023 to SEQ ID NO: 14030, SEQ ID NO: 14032 to SEQ ID NO: 14052, SEQ ID NO: 14054 to SEQ ID NO: 14070, SEQ ID NO: 14072 to SEQ ID NO: 14092, and SEQ ID NO: 14094 to SEQ ID NO: 14113;

[0428] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 256 to SEQ ID NO: 275, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14114 to SEQ ID NO: 14330;

[0429] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 276 to SEQ ID NO: 295, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14331 to SEQ ID NO: 14351, SEQ ID NO: 14353 to SEQ ID NO: 14362, and SEQ ID NO: 14364 to SEQ ID NO: 14439;

[0430] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 296 to SEQ ID NO: 315, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14440 to SEQ ID NO: 14558;

[0431] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 316 to SEQ ID NO: 335, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14559 to SEQ ID NO: 14695, and SEQ ID NO: 14697 to SEQ ID NO: 14786;

[0432] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 336 to SEQ ID NO: 355, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14787 to SEQ ID NO: 15115; and

[0433] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 375, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15116 to SEQ ID NO: 15381, and SEQ ID NO: 15383 to SEQ ID NO: 15534.

[0434] Example 63, NRCH-All

[0435] In Example 57,

[0436] The above prime editor protein is NRCH-PE2max,

[0437] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0438] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0439] The above scaffold can interact with the above prime editor protein to form a complex,

[0440] The above guide domain and the above prime editing domain are a combination selected from the group consisting of:

[0441] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 95, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11959 to SEQ ID NO: 12060;

[0442] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 115, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12061 to SEQ ID NO: 12465;

[0443] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12466 to SEQ ID NO: 12848;

[0444] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 136 to SEQ ID NO: 155, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12849 to SEQ ID NO: 13099;

[0445] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 156 to SEQ ID NO: 175, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13100 to SEQ ID NO: 13164, and SEQ ID NO: 13166 to SEQ ID NO: 13196;

[0446] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO: 195, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13197 to SEQ ID NO: 13239, SEQ ID NO: 13241 to SEQ ID NO: 13302, and SEQ ID NO: 13304 to SEQ ID NO: 13314;

[0447] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 196 to SEQ ID NO: 215, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13315 to SEQ ID NO: 13707;

[0448] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13708 to SEQ ID NO: 14013;

[0449] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 236 to SEQ ID NO: 255, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14014 to SEQ ID NO: 14018, SEQ ID NO: 14020 to SEQ ID NO: 14032, SEQ ID NO: 14034 to SEQ ID NO: 14100, and SEQ ID NO: 14102 to SEQ ID NO: 14113;

[0450] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 256 to SEQ ID NO: 275, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14114 to SEQ ID NO: 14330;

[0451] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 276 to SEQ ID NO: 295, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14331 to SEQ ID NO: 14439;

[0452] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 296 to SEQ ID NO: 315, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14440 to SEQ ID NO: 14556, and SEQ ID NO: 14558;

[0453] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 316 to SEQ ID NO: 335, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14559 to SEQ ID NO: 14695, and SEQ ID NO: 14697 to SEQ ID NO: 14786;

[0454] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 336 to SEQ ID NO: 355, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14787 to SEQ ID NO: 15115; and

[0455] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 375, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15116 to SEQ ID NO: 15381, and SEQ ID NO: 15383 to SEQ ID NO: 15534.

[0456] Example 64, PE2max-All

[0457] In Example 57,

[0458] The above prime editor protein is PE2max,

[0459] The above pegRNA comprises a guide domain, a scaffold, and a prime editing domain,

[0460] The guide domain, scaffold, and prime editing domain of the above pegRNA are sequentially connected from the 5' end to the 3' end,

[0461] The above scaffold can interact with the above prime editor protein to form a complex,

[0462] The above guide domain and the above prime editing domain are a combination selected from the group consisting of:

[0463] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 76 to SEQ ID NO: 95, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11959 to SEQ ID NO: 11998, SEQ ID NO: 12000 to SEQ ID NO: 12024, SEQ ID NO: 12026 to SEQ ID NO: 12042, and SEQ ID NO: 12044 to SEQ ID NO: 12060;

[0464] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 115, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12061 to SEQ ID NO: 12097, and SEQ ID NO: 12099 to SEQ ID NO: 12465;

[0465] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12466 to SEQ ID NO: 12476, and SEQ ID NO: 12478 to SEQ ID NO: 12848;

[0466] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 136 to SEQ ID NO: 155, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12849 to SEQ ID NO: 13099;

[0467] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 156 to SEQ ID NO: 175, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13100 to SEQ ID NO: 13162, SEQ ID NO: 13164 to SEQ ID NO: 13180, SEQ ID NO: 13182 to SEQ ID NO: 13185, and SEQ ID NO: 13187 to SEQ ID NO: 13196;

[0468] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO: 195, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13197 to SEQ ID NO: 13239, and SEQ ID NO: 13241 to SEQ ID NO: 13314;

[0469] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 196 to SEQ ID NO: 215, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13315 to SEQ ID NO: 13549, and SEQ ID NO: 13551 to SEQ ID NO: 13707;

[0470] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13708 to SEQ ID NO: 13728, SEQ ID NO: 13730 to SEQ ID NO: 13810, and SEQ ID NO: 13812 to SEQ ID NO: 14013;

[0471] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 236 to SEQ ID NO: 255, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14014 to SEQ ID NO: 14018, SEQ ID NO: 14020 to SEQ ID NO: 14021, SEQ ID NO: 14023 to SEQ ID NO: 14034, SEQ ID NO: 14036 to SEQ ID NO: 14038, SEQ ID NO: 14040 to SEQ ID NO: 14048, SEQ ID NO: 14050 to SEQ ID NO: 14073, SEQ ID NO: 14075 to SEQ ID NO: 14100, SEQ ID NO: 14102 to SEQ ID NO: 14105, and SEQ ID NO: 14107 to SEQ ID NO: 14113;

[0472] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 256 to SEQ ID NO: 275, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14114 to SEQ ID NO: 14330;

[0473] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 276 to SEQ ID NO: 295, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14331 to SEQ ID NO: 14351, SEQ ID NO: 14353 to SEQ ID NO: 14362, SEQ ID NO: 14364 to SEQ ID NO: 14415, and SEQ ID NO: 14417 to SEQ ID NO: 14439;

[0474] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 296 to SEQ ID NO: 315, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14440 to SEQ ID NO: 14556, and SEQ ID NO: 14558;

[0475] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 316 to SEQ ID NO: 335, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14559 to SEQ ID NO: 14786;

[0476] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 336 to SEQ ID NO: 355, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14787 to SEQ ID NO: 15115; and

[0477] The guide domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 375, and the prime editing domain consists of or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15116 to SEQ ID NO: 15261, SEQ ID NO: 15263 to SEQ ID NO: 15523, and SEQ ID NO: 15525 to SEQ ID NO: 15534.

[0478] Prime Editor Expression Vector

[0479] Example 65, Prime Editor Expression Vector

[0480] Prime Editor expression vectors containing:

[0481] A nucleic acid encoding a prime editor protein of any one of Examples 1 to 20; and

[0482] A nucleic acid encoding a pegRNA of any one of Examples 21 to 51,

[0483] Here, the scaffold of the pegRNA can interact with the prime editor protein to form a complex,

[0484] The above prime editor expression vector is capable of expressing the prime editor protein and the pegRNA respectively in a cell.

[0485] Example 66, Validation

[0486] In Example 65, the prime editor expression vector is capable of expressing the prime editor protein and pegRNA included in the prime editor complex of Example 58 or Example 61, respectively.

[0487] Example 67, Co-editing #1

[0488] Prime Editor expression vectors containing:

[0489] A nucleic acid encoding the VRQR-PE2max prime editor protein; and

[0490] A nucleic acid encoding a pegRNA of any one of Examples 52 to 56,

[0491] Here, the scaffold of the pegRNA can interact with the prime editor protein to form a complex,

[0492] The above prime editor expression vector is capable of expressing the prime editor protein and the pegRNA respectively in a cell.

[0493] Example 68, vector of two or more molecules

[0494] In any one of Examples 65 to 67, the prime editor expression vector includes the prime editor protein encoding nucleic acid sequence information and the pegRNA encoding nucleic acid sequence information divided into two or more vector molecules.

[0495] Example 69, Prime Editor Codon Limited

[0496] In any one of Examples 65 to 68, the nucleic acid encoding the prime editor protein comprises a nucleic acid sequence selected from SEQ ID NO: 16192 to SEQ ID NO: 16194.

[0497] Example 70, nucleic acid limitation

[0498] In any one of Examples 65 to 69, the nucleic acid means DNA or RNA.

[0499] Prime Editor Composition

[0500] Example 71, Prime Editor Composition

[0501] Prime Editor composition comprising:

[0502] A prime editor protein of any one of Examples 1 to 20, or a nucleic acid encoding said prime editor protein; and

[0503] Any one of the pegRNAs of Examples 21 to 51, or a nucleic acid encoding the pegRNA,

[0504] Here, the scaffold of the pegRNA can interact with the prime editor protein to form a complex.

[0505] Example 72, Co-editing

[0506] Prime Editor composition comprising:

[0507] VRQR-PE2max prime editor protein, or a nucleic acid encoding said prime editor protein; and

[0508] Any one of the pegRNAs of Examples 52 to 56, or a nucleic acid encoding the pegRNA,

[0509] Here, the scaffold of the pegRNA can interact with the prime editor protein to form a complex.

[0510] Example 73, nucleic acid limitation

[0511] In any one of Examples 71 to 72, the nucleic acid encoding the prime editor protein and the nucleic acid encoding the pegRNA are each independently selected from among DNA and RNA (or mRNA).

[0512] Example 74, VRQR-PE2max only

[0513] In Example 72, the VRQR-PE2max prime editor protein comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 27.

[0514] Example 75, composition comprising a complex

[0515] In any one of Examples 71 to 74, the prime editor composition comprises the prime editor protein and the pegRNA in the form of a complex of any one of Examples 57 to 64.

[0516] Example 76, Composition Comprising a Vector

[0517] In any one of Examples 71 to 75, the prime editor composition comprises a nucleic acid encoding the prime editor protein and a nucleic acid encoding the pegRNA in the form of a vector of any one of Examples 65 to 70.

[0518] Example 77, Composition containing mRNA

[0519] In any one of Examples 71 to 76, the prime editor composition comprises an mRNA encoding the prime editor protein and the pegRNA or a nucleic acid encoding the pegRNA.

[0520] Prime Editor 3

[0521] Example 78, Prime Editor 3

[0522] Prime Editor composition comprising:

[0523] A prime editor protein of any one of Examples 1 to 20, or a nucleic acid encoding said prime editor protein;

[0524] pegRNA of Example 56, or a nucleic acid encoding said pegRNA; and

[0525] ngRNA, or a nucleic acid encoding said ngRNA,

[0526] Here, the ngRNA is represented by the following structure:

[0527] 5'-[5' terminal appendage]-[guide domain]-[scaffold]-[Poly-U]-3'

[0528] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0529] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 16315 to SEQ ID NO: 16348,

[0530] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0531] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0532] Here, the prime editor protein and the pegRNA can bind to form a prime editing complex,

[0533] The above prime editor protein and the above ngRNA can combine to form a nickase complex.

[0534] Example 79, containing protein and RNA

[0535] In Example 78,

[0536] The above prime editor composition comprises the above prime editor protein, the above pegRNA, and the above ngRNA.

[0537] Example 80, containing an encoding nucleic acid

[0538] In Example 78,

[0539] The above prime editor composition comprises a nucleic acid encoding the prime editor protein, a nucleic acid encoding the pegRNA, and a nucleic acid encoding the ngRNA.

[0540] Example 81, including vectors

[0541] In Example 80,

[0542] The above Prime Editor composition comprises one or more vectors,

[0543] Each of the above vectors is independently configured to express the prime editor protein, the pegRNA, the ngRNA, or any combination thereof.

[0544] Example 82, ngRNA limitation

[0545] In any one of Examples 78 to 81,

[0546] The above ngRNA comprises any one nucleic acid sequence selected from the group consisting of SEQ ID NO: 16349 to SEQ ID NO: 16382.

[0547] Dead-sgRNA

[0548] Example 83, Dead-sgRNA

[0549] Prime Editor composition comprising:

[0550] A prime editor protein of any one of Examples 1 to 20, or a nucleic acid encoding said prime editor protein;

[0551] pegRNA of Example 56, or a nucleic acid encoding said pegRNA; and

[0552] dsgRNA, or a nucleic acid encoding said dsgRNA,

[0553] Here, the dsgRNA is represented by the following structure:

[0554] 5'-[5' terminal appendage]-[guide domain]-[scaffold]-[Poly-U]-3'

[0555] Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent,

[0556] The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 16383 to SEQ ID NO: 16403,

[0557] The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33,

[0558] The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

[0559] Here, the prime editor protein and the pegRNA can bind to form a prime editing complex,

[0560] The above prime editor protein and the above dsgRNA can combine to form a proximal CRISPR complex (proxy-CRISPR complex).

[0561] Example 84, dd

[0562] In Example 83,

[0563] The above prime editor composition comprises the above prime editor protein, the above pegRNA, and the above dsgRNA.

[0564] Example 85, containing an encoding nucleic acid

[0565] In Example 83,

[0566] The above prime editor composition comprises a nucleic acid encoding the prime editor protein, a nucleic acid encoding the pegRNA, and a nucleic acid encoding the dsgRNA.

[0567] Example 86, including vectors

[0568] In Example 85,

[0569] The above Prime Editor composition comprises one or more vectors,

[0570] Each of the above vectors is independently configured to express the prime editor protein, the pegRNA, the dsgRNA, or any combination thereof.

[0571] Example 87, dsgRNA limitation

[0572] In any one of Examples 83 to 86,

[0573] The above ngRNA comprises any one nucleic acid sequence selected from the group consisting of SEQ ID NO: 16404 to SEQ ID NO: 16424.

[0574] Pharmaceutical composition comprising a prime editor

[0575] Example 88, Pharmaceutical Composition

[0576] A pharmaceutical composition comprising:

[0577] A therapeutically effective amount of a prime editor complex selected from any one of Examples 57 to 64, a prime editor expression vector selected from any one of Examples 65 to 70, a prime editor composition selected from any one of Examples 71 to 77, a prime editor composition selected from any one of Examples 78 to 82, or a prime editor composition selected from any one of Examples 83 to 87; and

[0578] Pharmaceutically acceptable carrier.

[0579] Prime Editor's therapeutic uses and treatment methods for retinitis pigmentosa

[0580] Example 89, Use for the treatment of retinitis pigmentosa

[0581] Use of a prime editor complex selected from any one of Examples 57 to 64, a prime editor expression vector selected from any one of Examples 65 to 70, a prime editor composition selected from any one of Examples 71 to 77, a prime editor composition selected from any one of Examples 78 to 82, or a prime editor composition selected from any one of Examples 83 to 87, or a pharmaceutical composition selected from any one of Examples 88 for treating retinitis pigmentosa.

[0582] Example 90, Preparation of a drug for treating retinitis pigmentosa

[0583] A use for manufacturing a drug for treating retinitis pigmentosa, comprising a prime editor complex selected from any one of Examples 57 to 64, a prime editor expression vector selected from any one of Examples 65 to 70, a prime editor composition selected from any one of Examples 71 to 77, a prime editor composition selected from any one of Examples 78 to 82, or a prime editor composition selected from any one of Examples 83 to 87, or a pharmaceutical composition selected from any one of Example 88.

[0584] Example 91, Method for Treating Retinitis Pigmentosa

[0585] 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 57 to 64, a prime editor expression vector selected from any one of Examples 65 to 70, a prime editor composition selected from any one of Examples 71 to 77, a prime editor composition selected from any one of Examples 78 to 82, or a prime editor composition selected from any one of Examples 83 to 87, or a pharmaceutical composition selected from any one of Examples 88.

[0586] Example 92, Retinitis Pigmentosa Limited

[0587] In any one of Examples 89 to 91, the retinitis pigmentosa is caused by a c.4957dupA mutation in the EYS gene.

[0588] Method for correcting EYS gene mutation

[0589] Example 93, Method for Correcting EYS Gene Mutation

[0590] Methods for correcting the c.4957dupA mutation in the EYS gene, including:

[0591] A process of administering, introducing, injecting, and / or delivering to a cell having a c.4957dupA mutation in an EYS gene a prime editor complex selected from any one of Examples 57 to 64, a prime editor expression vector selected from any one of Examples 65 to 70, a prime editor composition selected from any one of Examples 71 to 77, a prime editor composition selected from any one of Examples 78 to 82, or a prime editor composition selected from any one of Examples 83 to 87, or a pharmaceutical composition selected from any one of Examples 88.

[0592] Example 94, PE3 related

[0593] In Example 93,

[0594] The method comprises administering, introducing, injecting, and / or delivering the prime editor composition of any one of Examples 78 to 82 to a cell having a c.4957dupA mutation in the EYS gene,

[0595] As a result of performing the above process, the prime editing complex of the prime editor composition contacts the EYS gene of the cell genome to correct the c.4957dupA mutation portion,

[0596] The nickase complex of the above prime editor composition causes a nick near the portion of the EYS gene of the cell genome that is corrected by the prime editing complex, thereby increasing the correction efficiency of the prime editing complex.

[0597] Example 95, Dead sgRNA related

[0598] In Example 93,

[0599] The method comprises administering, introducing, injecting, and / or delivering the prime editor composition of any one of Examples 83 to 87 to a cell in which the c.4957dupA mutation in the EYS gene has occurred,

[0600] As a result of performing the above process, the prime editing complex of the prime editor composition contacts the EYS gene of the cell genome to correct the c.4957dupA mutation portion,

[0601] The proximal CRISPR complex of the above prime editor composition binds near the portion of the EYS gene of the cell genome that the prime editing complex corrects, thereby increasing chromatin accessibility and increasing the correction efficiency of the prime editing complex.

[0602] The above proximal CRISPR complex does not cleave nucleic acids.

[0603] [Experimental Example]

[0604] Hereinafter, the invention provided by this specification will be described in more detail through experimental examples and examples. These examples are intended solely to illustrate the subject matter disclosed by this specification, and it will be apparent to those skilled in the art that the scope of the subject matter disclosed by this specification is not limited by these examples.

[0605] Experimental Example 1. Experimental Method and Materials

[0606] Experimental Example 1.1. Plasmid Library Preparation Method

[0607] A plasmid library containing the pegRNA coding sequence and its corresponding target sequence was constructed through the following two-step cloning process:

[0608] (Step I) Gibson assembly process; and

[0609] (Step II) Restriction enzyme-induced cutting (cut) and ligation (lysate) process.

[0610] The two-step cloning process described above effectively prevents uncoupling of the pegRNA and the target sequence corresponding to each pegRNA during PCR amplification. The two-step process was implemented using a previously reported method, modified appropriately.

[0611] Step I: Construction of an initial plasmid library containing pegRNA-encoding and target sequence pairs.

[0612] Oligonucleotide pools containing each pegRNA coding sequence and its corresponding target sequence were amplified by PCR for 20 cycles using Phusion Polymerase (NEB), and the amplicons were then gel-purified. The Lenti_gRNA-Puro vector (Addgene #84752) was digested with BsmBI enzyme (NEB) 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 a MicroPulser (Bio-Rad). Luria-Bertani (LB) medium (1 ml) was added to the transformation mixture and incubated at 37°C for 1 hour. The cells were then plated on LB agar plates containing 100 μg / ml carbenicillin and cultured. Aliquots (10, 1, and 0.1 μl) of each culture were plated separately to determine the library coverage. Plasmids were extracted from the harvested colonies. The initial plasmid library coverage was calculated to be 65X.

[0613] Step II: Insertion of the sgRNA scaffold

[0614] The initial plasmid library generated in Step I was digested with BsmBI for 9 h. The digested product was size-selected on a 1% agarose gel and gel-purified. The sgRNA-optimized scaffold sequence (Dang et al., 2015) was chemically synthesized as an oligonucleotide (IDT) and PCR-amplified for 20 cycles using Pfu polymerase and primer pairs containing BsmBI restriction sites in each pair. The PCR-amplified product was inserted into the PCR-Blunt II-TOPO vector (Solgent), digested with BsmBI for 4 h, and gel-purified on a 4% agarose gel. The purified inserts were ligated with the digested initial plasmid library vector at a molar ratio of 20:1 using T4 ligase (Enzynomics) overnight at room temperature (25°C). The ligation products were purified by isopropanol precipitation and electroporated into Endura electrocompetent cells (Lucigen). Colonies were harvested, and the final plasmid library was extracted using the QIAGEN Plasmid Maxi kit. The coverage of the final plasmid library was calculated to be 70X.

[0615] Experimental Example 1.4. Construction of a Prime Editor Protein Expression Vector

[0616] BSD (blasticidin) Selection vector

[0617] The pLenti-PE2max-BSD plasmid (Addgene #191102) was used for the PE2max prime editor protein, the pLenti-NRCH-PE2max-BSD (Addgene #191103) plasmid was used for the NRCH-PE2max prime editor protein, and the pLenti-VRQR-PE2max-BSD plasmid was used for additional cloning.

[0618] The above vector construction method was referenced from the method described in Yu et al., Prediction of efficiencies for diverse prime editing systems in multiple cell types, 2023, Cell 186, 2256-2272. In addition, for the cloned pLenti-VRQR-PE2max-BSD plasmid, the PAM variant mutation information described in Kim et al., Prediction of the sequence-specific cleavage activity of Cas9 variants, 2020, Nat Biotechnol 38, 1328-1336 was used.

[0619] Puromycin Selection Vector

[0620] 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-VRQR-PE2max-puro plasmid was used for the VRQR-PE2max prime editor protein.

[0621] The construction method for each plasmid is as follows:

[0622] The BSD antibiotic resistance gene region of the pLenti-PE2max-BSD plasmid (Addgene #191102), the pLenti-NRCH-PE2max-BSD (Addgene #191103) plasmid for the NRCH-PE2max prime editor protein, and the pLenti-VRQR-PE2max -BSD plasmid for the VRQR-PE2max prime editor were used after converting them to the puromycin (puro) antibiotic resistance gene sequence. The puro antibiotic resistance gene was obtained by PCR amplification for 20 cycles in lentiGuide-puro (Addgene #52963) and the puro antibiotic resistance gene sequence was inserted into the plasmid from which the BSD antibiotic resistance gene region was cut out using the Gibson assembly method.

[0623] Experimental Example 1.5. Lentivirus Production

[0624] HEK293T cells (1.0 Х 10 7) were seeded in 150 mm cell culture dishes containing DMEM (Dulbecco's Modified Eagle Medium). After 21 h, the library plasmid and psPAX2 (Addgene #12260) were mixed with pMD2.G (Addgene #12259) at a mass ratio of 4:3:1 and co-transfected into HEK293T cells using polyethylenimine. After 20 h of transfection, the medium was replaced with maintenance medium. After 48 h of transfection, the supernatant containing 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 ultra-pure 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 the virus titer, serial dilutions of the aliquoted virus were transduced into HEK293T cells in the presence of polybrene (10 μg / ml). Both untransduced cells and cells treated with serially diluted viruses were cultured in the presence of 2 μg / ml puromycin (Invitrogen). When most of the untransduced cells were dead, the number of viable cells in the virus-treated population was counted to estimate the virus titer. The production of lentiviruses containing the prime editor expressing plasmids (PE2max, NRCH-PE2max, VRQR-PE2max) followed the same protocol as above.

[0625] Experimental Example 1.6. Generation of Prime Editor Protein-Expressing Cell Lines

[0626] HEK293T cells were plated in 150 mm cell culture dishes at a density of 1.0 х 10 per plate. 7Cells were seeded at a cell density of 10 and cultured overnight. Lentiviruses expressing the prime editor (PE2max, NRCH-PE2max, VRQR-PE2max) prepared in Experimental Example 1.5 were transduced into cells at a multiplicity of infection (MOI) of 0.1 to 0.3. After transduction, cells were cultured for 19 hours and maintained in 20 μg / ml BSD (blasticidin) for the next 7 days to remove untransduced cells. After BSD selection, each cell line was continuously maintained in 10 μg / ml BSD.

[0627] Experimental Example 1.7. Library Transduction

[0628] A lentivirus containing the library prepared according to Experimental Example 1.5 was transduced into a prime editor protein-expressing cell line according to Experimental Example 1.6. The specific process is as follows:

[0629] 1) Cell lines according to Experimental Example 1.6 were seeded into 6 150 mm dishes per cell line, 1.5 Х 10 per dish 7 The cells were seeded at a density of 10 cells and cultured for 21 hours.

[0630] 2) The lentiviral library prepared according to Experimental Example 1.5 was transduced into the cells of 1) at a multiplicity of infection (MOI) of 0.5 together with 10 μg / ml of polybrene, so that the library coverage was > 500x in each of two repeated experiments.

[0631] 3) The cells in 2) were cultured overnight and cultured for 6 days in the presence of 2 μg / ml of puromycin to remove non-transduced cells.

[0632] 4) Cells were harvested and used 8 days after transduction.

[0633] Experimental Example 1.8: Deep Sequencing

[0634] For Experimental Example 2, genomic DNA was extracted from harvested cells using the Wizard Genomic DNA Purification Kit (Promega). For high-throughput experiments, integrated barcode and target sequences were PCR amplified using 2X Taq PCR Smart mix (SolGent). For each cell library, the first PCR contained a total of 240 μg of genomic DNA per cell line screen. This corresponds to 10 6 Assuming 10 μg of genomic DNA per cell, this represents a 1,000-fold increase in library size. Forty-eight independent 50-μl PCR reactions were performed with an initial genomic DNA concentration of 5 μg per reaction, and the products were pooled and gel-purified using the MEGAquick-spin Whole Fragment DNA Purification Kit (iNtRON Biotechnology).

[0635] The first PCR was performed under the following conditions: 95°C for 1 min; followed by 25 cycles of 95°C for 1 min, 55°C for 30 s, and 72°C for 30 s; followed by a final extension at 72°C for 2 min.

[0636] Then, 50 ng of purified DNA was amplified by PCR using primers containing both Illumina adapter and index sequences.

[0637] Secondary PCR was performed under the following conditions: 95°C for 1 min; followed by 10 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; followed by a final extension at 72°C for 30 s.

[0638] The products of the second PCR were gel-purified and deep sequenced.

[0639] For Experimental Example 3, to measure individual prime editing efficiencies at endogenous sites in ARPE19 and HEK293T cell lines harboring the EYS c.4957dupA mutation, independent first PCRs were performed in 50 μL reaction volumes containing 50–100 ng of initial genomic DNA template per sample. A second PCR to attach Illumina adapters and index sequences was performed in a 30 μL reaction volume containing 1 μL of template from the first PCR product. After gel purification, the resulting amplicons were analyzed using iSeq (Illumina, San Diego, CA).

[0640] Experimental Example 1.9. Prime Editing Efficiency Analysis

[0641] Each pegRNA and target sequence pair was verified by barcode sequence. Reads containing the designated edits, without unintended mutations within the broad target sequence, were considered to represent prime editor-induced mutations. To exclude background prime editing frequency arising from array synthesis and PCR amplification procedures, the observed prime editing frequency was subtracted from the background prime editing frequency derived in the absence of the prime editor, as follows.

[0642]

[0643] Deep sequencing data was filtered to improve analysis accuracy. Deep sequencing reads with fewer than 100 reads or a background prime editing frequency of 3% or higher were excluded from analysis.

[0644] Experimental Example 1.10. Guide RNA Preparation

[0645] The ngRNA of Experimental Example 5 or the dsgRNA of Experimental Example 6 was prepared by the following method:

[0646] 1) A plasmid was prepared by changing the scaffold sequence to the sequence of SEQ ID NO: 31 through Hifi gibson assembly in pRG2 (addgene#104174).

[0647] 2) After cutting the above plasmid with BsaI restriction enzyme, a fragment annealed with the guide sequence of the desired guide RNA (ngRNA or dsgRNA) was inserted through ligation.

[0648] Scaffold of the existing pRG2 plasmid: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 33).

[0649] Optimized scaffold of newly cloned plasmid: GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 31).

[0650] Experimental Example 2. Screening for pegRNAs capable of correcting the EYS c.4957dupA mutation.

[0651] Experimental Example 2.1. Preparation of a pegRNA Screening Library

[0652] To identify the most efficient pegRNA and target sequence for correcting the EYS c.4957dupA target, we designed all possible pegRNAs for a total of 95 guide sequences and conducted pegRNA screening. Specifically, for each target sequence (guide sequence), pegRNAs were designed with all possible combinations of reverse transcriptase template sequence length (minimum editable length ~ 50 nt) and primer binding site sequence length (7 to 17 nt) and included in the library.

[0653] According to the above design, an oligonucleotide pool containing 24,000 pairs of pegRNA and target sequences prepared by Experimental Example 1.1 was synthesized by Twist Bioscience (San Francisco, CA). Specifically, each oligonucleotide contained a 19-nt guide sequence, a BsmBI restriction site #1, a 15-nt barcode stuffer sequence, a BsmBI restriction site #2, a reverse transcriptase template sequence, a primer binding site sequence, a poly T sequence, a 20-nt barcode sequence (identification barcode) sequence, and a corresponding 74-nt target sequence including a PAM and a reverse transcriptase template binding region. Here, the barcode stuffer sequence was later removed by cleavage with BsmBI, and the identification barcode sequence was located upstream of the target sequence, so that individual pegRNA and target sequence pairs could be identified after deep sequencing. Oligonucleotides containing unintended BsmBI restriction sites within the sequence were excluded from the screening. To identify the most efficient pegRNA for the target, pegRNAs were generated with all possible combinations of reverse transcriptase template sequence length (minimum editable length ~ 50 nt) and primer binding site sequence length (7 to 17 nt) for a total of 95 guide sequence pairs.

[0654] Experimental Example 2.2. High-throughput evaluation of cell libraries

[0655] A lentivirus containing the library of Experimental Example 2.1 was prepared according to Experimental Example 1.5, and the lentivirus was transfected into the prime editor protein expression cell line of Experimental Example 1.6 according to Experimental Example 1.7.

[0656] The resulting DNA was then transduced. Deep sequencing was performed according to Experimental Example 1.7, and the results were analyzed for prime editing efficiency according to Experimental Example 1.8. Prime editing efficiency was measured twice and the average value was used.

[0657] The pegRNA screening results are presented in the table below:

[0658] NRCH-PE2max 실험 결과LabelPrime EditorpegRNA(SEQ ID NO)Adjusted Prime editing efficiency average (%)Readcount averageBackground efficiency (error)peg-0007NRCH-PE2max119650.08%641.50.00%peg-0027NRCH-PE2max119850.11%4310.00%peg-0030NRCH-PE2max119870.21%447.50.00%peg-0044NRCH-PE2max119980.21%1203.50.00%peg-0061NRCH-PE2max120150.83%628.50.00%peg-0063NRCH-PE2max120170.07%1481.50.00%peg-0072NRCH-PE2max120260.03%15100.00%peg-0075NRCH-PE2max120290.24%419.50.00%peg-0080NRCH-PE2max120340.14%808.50.00%peg-0081NRCH-PE2max120350.32%5900.00%peg-0082NRCH-PE2max120360.58%5190.00%peg-0083NRCH-PE2max120370.10%495.50.00%peg-0084NRCH-PE2max120380.06%8150.00%peg-0085NRCH-PE2max120390.11%8370.00%peg-0090NRCH-PE2max120440.62%2610.00%peg-0091NRCH-PE2max120451.06%371.50.00%peg-0093NRCH-PE2max120470.27%7430.07%peg-0097NRCH-PE2max120510.45%2830.00%peg-0098NRCH-PE2max120521.27%2880.00%peg-0105NRCH-PE2max120590.34%407.50.00%peg-0106NRCH-PE2max120600.35%1300.00%peg-0110NRCH-PE2max120640.07%20750.00%peg-0111NRCH-PE2max120650.71%7050.00%peg-0112NRCH-PE2max120660.63%2134.50.00%peg-0113NRCH-PE2max120670.91%4317.50.05%peg-0114NRCH-PE2max120680.37%817.50.00%peg-0115NRCH-PE2max120690.49%7090.07%peg-0116NRCH-PE2max120700.31%49430.01%peg-0117NRCH-PE2max120710.47%12360.00%peg-0119NRCH-PE2max120730.08%495.50.12%peg-0120NRCH-PE2max120740.20%1463.50.07%peg-0121NRCH-PE2max120750.12%13270.04%peg-0122NRCH-PE2max120760.67%16440.03%peg-0123NRCH-PE2max120770.30%18500.00%peg-0124NRCH-PE2max120780.11%31500.00%peg-0125NRCH-PE2max120790.06%41620.00%peg-0126NRCH-PE2max120800.09%2167.50.02%peg-0127NRCH-PE2max120810.14%52850.00%peg-0129NRCH-PE2max120830.19%18090.00%peg-0130NRCH-PE2max120840.04%23000.00%peg-0134NRCH-PE2max120880.03%19690.00%peg-0136NRCH-PE2max120900.06%1688.50.00%peg-0138NRCH-PE2max120920.03%15510.00%peg-0148NRCH-PE2max121020.29%2061.50.00%peg-0149NRCH-PE2max121031.99%18600.00%peg-0150NRCH-PE2max121041.56%3091.50.02%peg-0152NRCH-PE2max121060.26%5680.00%peg-0153NRCH-PE2max121071.19%16150.00%peg-0154NRCH-PE2max121080.22%28280.00%peg-0155NRCH-PE2max121090.14%3620.00%peg-0156NRCH-PE2max121101.02%11270.00%peg-0157NRCH-PE2max121110.18%1128.50.00%peg-0158NRCH-PE2max121120.83%4960.50.00%peg-0159NRCH-PE2max121130.66%4153.50.01%peg-0160NRCH-PE2max121141.20%20940.00%peg-0161NRCH-PE2max121151.22%1509.50.00%peg-0162NRCH-PE2max121160.47%2339.50.00%peg-0163NRCH-PE2max121170.18%5360.50.00%peg-0164NRCH-PE2max121180.16%15930.00%peg-0166NRCH-PE2max121200.11%26080.00%peg-0167NRCH-PE2max121210.19%836.50.00%peg-0168NRCH-PE2max121220.03%1934.50.00%peg-0171NRCH-PE2max121250.03%3121.50.00%peg-0172NRCH-PE2max121260.22%15110.00%peg-0176NRCH-PE2max121300.13%3920.00%peg-0178NRCH-PE2max121320.30%336.50.00%peg-0184NRCH-PE2max121380.07%13680.00%peg-0185NRCH-PE2max121390.51%1423.50.00%peg-0186NRCH-PE2max121401.05%1342.50.03%peg-0187NRCH-PE2max121413.40%1690.50.03%peg-0188NRCH-PE2max121421.13%8810.00%peg-0189NRCH-PE2max121430.40%369.50.00%peg-0191NRCH-PE2max121440.48%626.50.00%peg-0192NRCH-PE2max121451.66%4790.00%peg-0193NRCH-PE2max121461.38%3013.50.00%peg-0194NRCH-PE2max121471.07%28681.09%peg-0195NRCH-PE2max121480.66%20940.00%peg-0196NRCH-PE2max121490.34%1312.50.00%peg-0197NRCH-PE2max121501.06%6470.00%peg-0198NRCH-PE2max121510.67%29710.03%peg-0199NRCH-PE2max121520.45%2912.50.00%peg-0200NRCH-PE2max121531.28%1063.50.00%peg-0201NRCH-PE2max121540.62%1180.50.00%peg-0202NRCH-PE2max121550.16%6340.00%peg-0203NRCH-PE2max121560.21%3140.50.00%peg-0204NRCH-PE2max121570.25%23690.00%peg-0205NRCH-PE2max121580.02%27700.00%peg-0210NRCH-PE2max121630.14%21170.00%peg-0212NRCH-PE2max121650.26%337.50.00%peg-0213NRCH-PE2max121660.14%1727.50.00%peg-0215NRCH-PE2max121680.08%18470.00%peg-0221NRCH-PE2max121740.10%22220.00%peg-0222NRCH-PE2max121750.24%15060.00%peg-0223NRCH-PE2max121760.76%8980.05%peg-0224NRCH-PE2max121772.34%2586.50.00%peg-0225NRCH-PE2max121781.73%10250.00%peg-0226NRCH-PE2max121790.68%10850.05%peg-0227NRCH-PE2max121801.55%30960.02%peg-0228NRCH-PE2max121811.28%26820.04%peg-0229NRCH-PE2max121820.62%3960.00%peg-0230NRCH-PE2max121832.12%36210.01%peg-0231NRCH-PE2max121840.96%11330.00%peg-0232NRCH-PE2max121850.11%35950.58%peg-0233NRCH-PE2max121862.12%20250.00%peg-0234NRCH-PE2max121871.81%3727.50.01%peg-0235NRCH-PE2max121881.67%18220.06%peg-0236NRCH-PE2max121890.61%3881.50.00%peg-0237NRCH-PE2max121900.93%4296.50.00%peg-0238NRCH-PE2max121910.60%6877.50.00%peg-0239NRCH-PE2max121920.59%18600.00%peg-0240NRCH-PE2max121930.12%25040.00%peg-0241NRCH-PE2max121940.17%27320.00%peg-0242NRCH-PE2max121950.14%13890.00%peg-0244NRCH-PE2max121970.10%19680.00%peg-0245NRCH-PE2max121980.33%1667.50.00%peg-0246NRCH-PE2max121990.19%23380.02%peg-0247NRCH-PE2max122000.13%1927.50.00%peg-0249NRCH-PE2max122020.62%12680.00%peg-0250NRCH-PE2max122030.10%1099.50.00%peg-0251NRCH-PE2max122040.20%779.50.00%peg-0256NRCH-PE2max122090.03%3831.50.00%peg-0257NRCH-PE2max122100.00%22310.02%peg-0258NRCH-PE2max122110.02%2090.50.00%peg-0259NRCH-PE2max122120.31%2529.50.02%peg-0260NRCH-PE2max122131.18%8990.00%peg-0261NRCH-PE2max122143.64%3036.50.02%peg-0262NRCH-PE2max122151.19%3320.15%peg-0263NRCH-PE2max122160.25%405.50.11%peg-0264NRCH-PE2max122171.41%22700.00%peg-0265NRCH-PE2max122182.14%2141.50.00%peg-0266NRCH-PE2max122191.19%375.50.00%peg-0267NRCH-PE2max122201.37%1175.50.00%peg-0268NRCH-PE2max122211.52%4202.50.00%peg-0269NRCH-PE2max122220.62%17070.00%peg-0270NRCH-PE2max122230.98%4527.50.01%peg-0271NRCH-PE2max122241.02%47720.01%peg-0272NRCH-PE2max122251.80%11720.00%peg-0273NRCH-PE2max122260.88%22130.00%peg-0274NRCH-PE2max122270.83%17170.00%peg-0275NRCH-PE2max122281.01%60860.02%peg-0276NRCH-PE2max122290.09%573.50.00%peg-0277NRCH-PE2max122300.17%26230.00%peg-0279NRCH-PE2max122310.24%4936.50.00%peg-0280NRCH-PE2max122320.22%12120.00%peg-0281NRCH-PE2max122330.28%878.50.00%peg-0282NRCH-PE2max122340.33%3794.50.05%peg-0283NRCH-PE2max122350.10%2623.50.00%peg-0284NRCH-PE2max122360.08%12480.00%peg-0286NRCH-PE2max122380.12%822.50.00%peg-0287NRCH-PE2max122390.20%26940.00%peg-0288NRCH-PE2max122400.04%11880.00%peg-0289NRCH-PE2max122410.28%5220.00%peg-0290NRCH-PE2max122420.50%4050.00%peg-0295NRCH-PE2max122470.13%1554.50.00%peg-0297NRCH-PE2max122491.58%836.50.00%peg-0298NRCH-PE2max122500.55%2226.50.02%peg-0299NRCH-PE2max122511.29%1412.50.00%peg-0300NRCH-PE2max122521.61%590.50.00%peg-0301NRCH-PE2max122531.17%1199.50.00%peg-0302NRCH-PE2max122542.33%2381.50.00%peg-0303NRCH-PE2max122550.89%1233.50.00%peg-0304NRCH-PE2max122562.12%30970.00%peg-0305NRCH-PE2max122570.49%2095.50.00%peg-0306NRCH-PE2max122581.03%38860.00%peg-0307NRCH-PE2max122591.12%3286.50.02%peg-0308NRCH-PE2max122600.66%17830.06%peg-0309NRCH-PE2max122611.10%27690.02%peg-0310NRCH-PE2max122621.30%1173.50.11%peg-0311NRCH-PE2max122630.33%2405.52.19%peg-0312NRCH-PE2max122641.36%1895.50.00%peg-0313NRCH-PE2max122650.16%622.50.00%peg-0314NRCH-PE2max122660.33%22680.20%peg-0315NRCH-PE2max122670.23%1603.50.00%peg-0316NRCH-PE2max122680.28%5027.50.01%peg-0317NRCH-PE2max122690.32%912.50.00%peg-0318NRCH-PE2max122700.15%20950.00%peg-0319NRCH-PE2max122710.28%14240.00%peg-0320NRCH-PE2max122720.43%9350.00%peg-0321NRCH-PE2max122730.59%1115.50.00%peg-0322NRCH-PE2max122740.09%1687.50.00%peg-0323NRCH-PE2max122750.25%19940.03%peg-0324NRCH-PE2max122760.21%1180.50.00%peg-0326NRCH-PE2max122780.18%1394.50.00%peg-0327NRCH-PE2max122790.09%578.50.00%peg-0329NRCH-PE2max122810.05%969.50.00%peg-0331NRCH-PE2max122830.03%27280.05%peg-0332NRCH-PE2max122840.02%2861.50.00%peg-0333NRCH-PE2max122850.94%16800.00%peg-0334NRCH-PE2max122861.76%2798.50.00%peg-0335NRCH-PE2max122873.09%2534.50.00%peg-0336NRCH-PE2max122881.29%342.50.00%peg-0337NRCH-PE2max122893.65%210.50.00%peg-0338NRCH-PE2max122900.85%23600.04%peg-0339NRCH-PE2max122911.77%1829.50.00%peg-0340NRCH-PE2max122920.24%636.50.00%peg-0341NRCH-PE2max122931.92%31100.00%peg-0342NRCH-PE2max122940.49%2430.50.02%peg-0343NRCH-PE2max122951.36%25970.02%peg-0344NRCH-PE2max122960.34%2868.50.00%peg-0345NRCH-PE2max122972.01%882.50.00%peg-0346NRCH-PE2max122981.55%11310.00%peg-0347NRCH-PE2max122991.17%54130.00%peg-0348NRCH-PE2max123001.80%29930.00%peg-0349NRCH-PE2max123010.85%32000.00%peg-0351NRCH-PE2max123030.22%19410.00%peg-0352NRCH-PE2max123040.00%21080.26%peg-0353NRCH-PE2max123050.29%1710.50.00%peg-0354NRCH-PE2max123060.34%2185.50.02%peg-0355NRCH-PE2max123070.47%321.50.00%peg-0357NRCH-PE2max123090.42%2899.50.00%peg-0358NRCH-PE2max123100.33%1055.50.00%peg-0359NRCH-PE2max123110.20%15060.00%peg-0360NRCH-PE2max123120.34%14610.03%peg-0361NRCH-PE2max123130.13%1394.50.11%peg-0364NRCH-PE2max123160.23%427.50.00%peg-0365NRCH-PE2max123170.85%1140.00%peg-0368NRCH-PE2max123200.00%1367.50.04%peg-0369NRCH-PE2max123210.04%23260.00%peg-0370NRCH-PE2max123220.64%1769.50.00%peg-0371NRCH-PE2max123232.14%45910.18%peg-0372NRCH-PE2max123240.90%1611.50.00%peg-0373NRCH-PE2max123250.35%4430.00%peg-0374NRCH-PE2max123262.08%5660.00%peg-0375NRCH-PE2max123270.62%29370.00%peg-0376NRCH-PE2max123281.70%58610.02%peg-0377NRCH-PE2max123290.35%852.50.00%peg-0378NRCH-PE2max123301.19%17160.03%peg-0379NRCH-PE2max123311.73%2279.50.00%peg-0380NRCH-PE2max123321.21%2696.50.00%peg-0381NRCH-PE2max123331.41%1134.50.00%peg-0382NRCH-PE2max123342.20%24750.02%peg-0383NRCH-PE2max123351.92%3030.50.03%peg-0384NRCH-PE2max123362.58%2157.50.03%peg-0385NRCH-PE2max123371.18%1681.50.00%peg-0386NRCH-PE2max123380.65%5188.50.15%peg-0387NRCH-PE2max123390.37%408.50.00%peg-0388NRCH-PE2max123400.63%24640.16%peg-0389NRCH-PE2max123410.75%1021.50.00%peg-0390NRCH-PE2max123420.45%4415.50.06%peg-0391NRCH-PE2max123430.39%25960.02%peg-0393NRCH-PE2max123450.29%4320.50.08%peg-0394NRCH-PE2max123460.72%3153.50.12%peg-0395NRCH-PE2max123470.15%23950.00%peg-0396NRCH-PE2max123480.75%1277.50.23%peg-0397NRCH-PE2max123490.88%1138.50.00%peg-0398NRCH-PE2max123500.85%410.50.00%peg-0400NRCH-PE2max123520.29%15470.00%peg-0401NRCH-PE2max123530.10%1417.50.00%peg-0405NRCH-PE2max123570.02%28560.00%peg-0406NRCH-PE2max123580.25%3234.50.00%peg-0407NRCH-PE2max123590.16%1708.50.00%peg-0408NRCH-PE2max123600.86%31640.03%peg-0409NRCH-PE2max123611.68%29470.02%peg-0410NRCH-PE2max123620.80%746.50.00%peg-0411NRCH-PE2max123630.68%1247.50.00%peg-0412NRCH-PE2max123640.64%24070.20%peg-0413NRCH-PE2max123652.04%12920.00%peg-0414NRCH-PE2max123660.66%15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50120.37%20830.00%peg-3098NRCH-PE2max150140.10%583.50.08%peg-3100NRCH-PE2max150160.21%1034.50.13%peg-3101NRCH-PE2max150170.25%3013.50.32%peg-3102NRCH-PE2max150180.33%6190.00%peg-3103NRCH-PE2max150190.41%1418.50.04%peg-3104NRCH-PE2max150200.00%11400.04%peg-3105NRCH-PE2max150210.13%1683.50.34%peg-3106NRCH-PE2max150220.36%12120.00%peg-3107NRCH-PE2max150230.09%842.50.74%peg-3112NRCH-PE2max150280.08%3939.50.03%peg-3115NRCH-PE2max150310.12%30470.00%peg-3116NRCH-PE2max150320.82%37770.00%peg-3117NRCH-PE2max150331.09%3287.50.02%peg-3118NRCH-PE2max150340.80%500.50.00%peg-3119NRCH-PE2max150350.68%422.50.00%peg-3120NRCH-PE2max150360.95%732.50.00%peg-3121NRCH-PE2max150370.49%279.50.00%peg-3122NRCH-PE2max150380.12%7190.30%peg-3123NRCH-PE2max150390.61%23500.09%peg-3124NRCH-PE2max150400.10%1545.50.00%peg-3125NRCH-PE2max150410.53%13150.00%peg-3126NRCH-PE2max150420.43%20790.07%peg-3127NRCH-PE2max150430.53%15140.00%peg-3128NRCH-PE2max150441.49%9220.29%peg-3129NRCH-PE2max150450.59%929.50.12%peg-3130NRCH-PE2max150460.57%28560.52%peg-3132NRCH-PE2max150480.19%587.50.00%peg-3133NRCH-PE2max150490.12%2619.50.47%peg-3134NRCH-PE2max150500.67%8940.00%peg-3135NRCH-PE2max150510.34%17900.00%peg-3136NRCH-PE2max150520.40%416.50.00%peg-3137NRCH-PE2max150530.30%835.50.94%peg-3138NRCH-PE2max150540.15%756.50.00%peg-3139NRCH-PE2max150550.20%1550.50.64%peg-3140NRCH-PE2max150560.87%9650.43%peg-3141NRCH-PE2max150570.18%1356.50.00%peg-3143NRCH-PE2max150590.25%2187.50.00%peg-3144NRCH-PE2max150600.19%27920.00%peg-3145NRCH-PE2max150610.67%3581.50.01%peg-3146NRCH-PE2max150620.52%12540.00%peg-3147NRCH-PE2max150631.34%1385.50.00%peg-3148NRCH-PE2max150640.45%14620.10%peg-3149NRCH-PE2max150650.54%1026.50.00%peg-3150NRCH-PE2max150660.51%13960.00%peg-3151NRCH-PE2max150670.37%25650.00%peg-3152NRCH-PE2max150680.17%5830.00%peg-3153NRCH-PE2max150690.15%1683.50.00%peg-3154NRCH-PE2max150700.22%2200.50.00%peg-3155NRCH-PE2max150711.23%1086.50.00%peg-3156NRCH-PE2max150720.75%7950.00%peg-3157NRCH-PE2max150730.31%24470.00%peg-3158NRCH-PE2max150741.10%302.50.00%peg-3159NRCH-PE2max150750.16%22950.00%peg-3160NRCH-PE2max150760.45%12610.10%peg-3161NRCH-PE2max150770.58%3430.00%peg-3162NRCH-PE2max150780.58%3480.00%peg-3164NRCH-PE2max150801.98%407.50.00%peg-3165NRCH-PE2max150810.17%1143.50.00%peg-3168NRCH-PE2max150840.41%872.50.11%peg-3170NRCH-PE2max150860.59%429.50.59%peg-3171NRCH-PE2max150870.06%775.50.00%peg-3172NRCH-PE2max150880.15%13470.00%peg-3173NRCH-PE2max150890.10%1613.50.00%peg-3174NRCH-PE2max150900.13%28450.04%peg-3175NRCH-PE2max150910.35%3059.50.02%peg-3176NRCH-PE2max150920.65%8430.00%peg-3177NRCH-PE2max150932.16%2408.50.00%peg-3178NRCH-PE2max150941.81%3570.00%peg-3179NRCH-PE2max150951.01%1739.50.00%peg-3180NRCH-PE2max150961.51%1356.50.00%peg-3181NRCH-PE2max150970.35%14310.00%peg-3182NRCH-PE2max150980.29%3660.00%peg-3183NRCH-PE2max150990.95%14790.04%peg-3184NRCH-PE2max151000.27%1495.50.00%peg-3185NRCH-PE2max151011.17%3060.50.07%peg-3186NRCH-PE2max151021.43%1508.50.00%peg-3187NRCH-PE2max151030.29%6740.00%peg-3188NRCH-PE2max151040.90%293.50.00%peg-3189NRCH-PE2max151050.90%12600.26%peg-3190NRCH-PE2max151060.92%1585.50.00%peg-3191NRCH-PE2max151070.30%18400.16%peg-3193NRCH-PE2max151090.82%903.50.00%peg-3197NRCH-PE2max151120.27%5680.00%peg-3198NRCH-PE2max151130.07%5090.32%peg-3201NRCH-PE2max151160.23%8660.00%peg-3203NRCH-PE2max151180.08%7321.50.01%peg-3204NRCH-PE2max151190.09%45280.00%peg-3205NRCH-PE2max151200.03%17480.00%peg-3207NRCH-PE2max151220.00%17480.03%peg-3208NRCH-PE2max151230.04%59450.14%peg-3209NRCH-PE2max151240.03%3068.50.00%peg-3210NRCH-PE2max151250.39%2575.50.02%peg-3212NRCH-PE2max151270.03%1591.50.09%peg-3213NRCH-PE2max151280.09%1661.50.00%peg-3215NRCH-PE2max151300.02%2704.50.00%peg-3217NRCH-PE2max151320.03%18600.03%peg-3219NRCH-PE2max151340.03%29830.02%peg-3220NRCH-PE2max151350.04%4437.50.02%peg-3222NRCH-PE2max151370.05%24230.00%peg-3223NRCH-PE2max151380.18%16480.00%peg-3227NRCH-PE2max151420.06%960.50.00%peg-3233NRCH-PE2max151480.08%6270.00%peg-3239NRCH-PE2max151540.04%12350.00%peg-3243NRCH-PE2max151580.04%34660.03%peg-3247NRCH-PE2max151610.05%21000.00%peg-3248NRCH-PE2max151620.09%29500.00%peg-3249NRCH-PE2max151630.02%31850.00%peg-3250NRCH-PE2max151640.03%70650.01%peg-3253NRCH-PE2max151670.09%4822.50.00%peg-3254NRCH-PE2max151680.07%3825.50.01%peg-3256NRCH-PE2max151700.05%45910.00%peg-3257NRCH-PE2max151710.17%1211.50.00%peg-3258NRCH-PE2max151720.06%18310.03%peg-3261NRCH-PE2max151750.03%19460.00%peg-3264NRCH-PE2max151780.17%2102.50.00%peg-3265NRCH-PE2max151790.02%22170.00%peg-3266NRCH-PE2max151800.27%17980.00%peg-3275NRCH-PE2max151890.18%8180.06%peg-3278NRCH-PE2max151920.13%1189.50.00%peg-3279NRCH-PE2max151930.04%15110.06%peg-3281NRCH-PE2max151950.00%34920.01%peg-3285NRCH-PE2max151990.04%1328.50.00%peg-3287NRCH-PE2max152010.02%4227.50.02%peg-3289NRCH-PE2max152030.01%4221.50.00%peg-3292NRCH-PE2max152060.10%33470.02%peg-3293NRCH-PE2max152070.04%3821.50.00%peg-3296NRCH-PE2max152100.10%2101.50.00%peg-3297NRCH-PE2max152110.22%13660.00%peg-3299NRCH-PE2max152130.23%1442.50.00%peg-3308NRCH-PE2max152220.12%4620.00%peg-3313NRCH-PE2max152270.04%13400.00%peg-3319NRCH-PE2max152330.06%32100.02%peg-3325NRCH-PE2max152390.02%15680.03%peg-3326NRCH-PE2max152400.15%1658.50.03%peg-3328NRCH-PE2max152420.02%2702.50.00%peg-3331NRCH-PE2max152450.02%2292.50.02%peg-3332NRCH-PE2max152460.08%29270.03%peg-3334NRCH-PE2max152480.03%58060.00%peg-3335NRCH-PE2max152490.04%4017.50.00%peg-3337NRCH-PE2max152500.12%3766.50.00%peg-3339NRCH-PE2max152520.05%29570.02%peg-3344NRCH-PE2max152570.17%310.50.00%peg-3345NRCH-PE2max152580.29%1580.00%peg-3348NRCH-PE2max152610.06%793.50.00%peg-3355NRCH-PE2max152680.30%457.50.00%peg-3358NRCH-PE2max152710.13%20510.05%peg-3359NRCH-PE2max152720.04%44740.00%peg-3361NRCH-PE2max152740.00%1337.50.04%peg-3362NRCH-PE2max152750.14%11160.00%peg-3365NRCH-PE2max152780.09%33600.00%peg-3366NRCH-PE2max152790.18%1621.50.03%peg-3368NRCH-PE2max152810.06%8340.00%peg-3369NRCH-PE2max152820.15%332.50.00%peg-3375NRCH-PE2max152880.10%36850.00%peg-3377NRCH-PE2max152900.16%1525.50.00%peg-3383NRCH-PE2max152960.08%1835.50.00%peg-3386NRCH-PE2max152990.05%10880.00%peg-3389NRCH-PE2max153010.13%784.50.00%peg-3392NRCH-PE2max153040.47%2120.00%peg-3396NRCH-PE2max153070.04%13830.03%peg-3397NRCH-PE2max153080.08%2396.50.00%peg-3398NRCH-PE2max153090.02%4767.50.02%peg-3400NRCH-PE2max153110.52%7780.00%peg-3401NRCH-PE2max153120.04%1303.50.04%peg-3403NRCH-PE2max153140.04%10970.00%peg-3405NRCH-PE2max153160.00%4943.50.02%peg-3406NRCH-PE2max153170.03%27850.02%peg-3407NRCH-PE2max153180.06%825.50.00%peg-3408NRCH-PE2max153190.59%748.50.00%peg-3410NRCH-PE2max153210.07%4139.50.00%peg-3412NRCH-PE2max153230.07%1394.50.00%peg-3416NRCH-PE2max153270.20%1297.50.00%peg-3417NRCH-PE2max153280.20%1261.50.00%peg-3421NRCH-PE2max153320.08%6640.00%peg-3423NRCH-PE2max153340.30%315.50.00%peg-3426NRCH-PE2max153360.22%268.50.00%peg-3431NRCH-PE2max153410.03%1749.50.00%peg-3434NRCH-PE2max153440.51%500.50.00%peg-3435NRCH-PE2max153450.04%14380.00%peg-3436NRCH-PE2max153460.07%1379.50.04%peg-3440NRCH-PE2max153500.15%7010.06%peg-3443NRCH-PE2max153530.00%52770.02%peg-3444NRCH-PE2max153540.08%2417.50.04%peg-3445NRCH-PE2max153550.22%51410.02%peg-3446NRCH-PE2max153560.03%1591.50.00%peg-3448NRCH-PE2max153580.07%1467.50.03%peg-3449NRCH-PE2max153590.06%2453.50.00%peg-3453NRCH-PE2max153630.00%20560.02%peg-3454NRCH-PE2max153640.10%2008.50.00%peg-3456NRCH-PE2max153660.86%1253.51.79%peg-3457NRCH-PE2max153670.07%22780.00%peg-3458NRCH-PE2max153680.21%7300.00%peg-3460NRCH-PE2max153700.19%7840.00%peg-3467NRCH-PE2max153770.30%636.50.00%peg-3475NRCH-PE2max153850.12%3427.50.01%peg-3477NRCH-PE2max153870.24%19500.00%peg-3479NRCH-PE2max153890.12%1300.50.08%peg-3481NRCH-PE2max153910.04%1279.50.00%peg-3482NRCH-PE2max153920.24%2075.50.00%peg-3483NRCH-PE2max153930.43%3039.52.41%peg-3485NRCH-PE2max153950.09%1133.50.00%peg-3486NRCH-PE2max153960.02%26910.03%peg-3488NRCH-PE2max153980.02%9441.50.02%peg-3489NRCH-PE2max153990.10%2859.50.02%peg-3490NRCH-PE2max154000.01%3666.50.01%peg-3495NRCH-PE2max154050.03%1647.50.00%peg-3496NRCH-PE2max154060.03%16570.00%peg-3499NRCH-PE2max154090.11%8730.00%peg-3500NRCH-PE2max154100.18%19340.03%peg-3502NRCH-PE2max154120.55%999.50.00%peg-3507NRCH-PE2max154160.31%1430.00%peg-3514NRCH-PE2max154230.12%2599.50.00%peg-3515NRCH-PE2max154240.01%2900.50.06%peg-3516NRCH-PE2max154250.04%3901.50.03%peg-3517NRCH-PE2max154260.09%638.50.07%peg-3518NRCH-PE2max154270.07%40600.02%peg-3520NRCH-PE2max154290.13%21400.00%peg-3522NRCH-PE2max154310.02%22670.00%peg-3523NRCH-PE2max154320.09%27760.11%peg-3524NRCH-PE2max154330.02%24990.00%peg-3526NRCH-PE2max154350.10%4889.50.01%peg-3529NRCH-PE2max154380.11%72830.02%peg-3531NRCH-PE2max154400.16%3954.50.00%peg-3532NRCH-PE2max154410.00%10810.04%peg-3533NRCH-PE2max154420.55%933.50.27%peg-3535NRCH-PE2max154440.05%39090.00%peg-3539NRCH-PE2max154480.17%8110.00%peg-3541NRCH-PE2max154490.03%1543.50.04%peg-3542NRCH-PE2max154500.20%5620.00%peg-3543NRCH-PE2max154510.35%3000.00%peg-3546NRCH-PE2max154540.78%293.50.00%peg-3549NRCH-PE2max154570.36%6000.00%peg-3552NRCH-PE2max154600.04%1145.50.04%peg-3553NRCH-PE2max154610.00%1586.50.03%peg-3554NRCH-PE2max154620.09%55150.01%peg-3555NRCH-PE2max154630.06%2345.50.00%peg-3557NRCH-PE2max154650.20%5180.08%peg-3558NRCH-PE2max154660.18%5830.00%peg-3559NRCH-PE2max154670.00%55590.02%peg-3560NRCH-PE2max154680.07%35910.01%peg-3561NRCH-PE2max154690.24%25200.00%peg-3565NRCH-PE2max154730.06%43170.01%peg-3566NRCH-PE2max154740.04%2721.50.04%peg-3568NRCH-PE2max154760.00%33280.03%peg-3569NRCH-PE2max154770.04%41920.01%peg-3570NRCH-PE2max154780.05%3263.50.00%peg-3572NRCH-PE2max154800.09%1545.50.00%peg-3573NRCH-PE2max154810.00%20460.02%peg-3574NRCH-PE2max154820.33%19400.00%peg-3576NRCH-PE2max154840.06%8930.00%peg-3577NRCH-PE2max154850.65%7170.00%peg-3589NRCH-PE2max154960.16%1138.50.00%peg-3593NRCH-PE2max155000.21%42160.01%peg-3595NRCH-PE2max155020.29%22320.00%peg-3597NRCH-PE2max155030.08%648.50.08%peg-3598NRCH-PE2max155040.22%68160.03%peg-3600NRCH-PE2max155060.06%39510.02%peg-3601NRCH-PE2max155070.16%26080.06%peg-3603NRCH-PE2max155090.10%3467.50.00%peg-3604NRCH-PE2max155100.06%44920.03%peg-3606NRCH-PE2max155120.21%18260.00%peg-3608NRCH-PE2max155140.14%2876.50.02%peg-3610NRCH-PE2max155160.06%1683.50.00%peg-3611NRCH-PE2max155170.05%17780.00%peg-3613NRCH-PE2max155190.30%1589.50.00%peg-3614NRCH-PE2max155200.11%13760.00%peg-3615NRCH-PE2max155210.88%7510.00%peg-3617NRCH-PE2max155230.15%20040.02%peg-3619NRCH-PE2max155250.24%2157.50.00%peg-3621NRCH-PE2max155270.13%215.52.79%peg-3626NRCH-PE2max155320.84%335.50.00%peg-3628NRCH-PE2max155330.22%693.50.00%.

[0659]

[0660] VRQR-PE2max 실험 결과LabelPrime EditorpegRNA(SEQ ID NO)Adjusted Prime editing efficiency average (%)Readcount averageBackground efficiency (error)peg-0028VRQR-PE2max119860.07%7940.00%peg-0043VRQR-PE2max119970.04%11700.00%peg-0047VRQR-PE2max120010.31%2880.00%peg-0051VRQR-PE2max120050.08%602.50.00%peg-0054VRQR-PE2max120080.09%496.50.00%peg-0061VRQR-PE2max120150.20%636.50.00%peg-0063VRQR-PE2max120170.04%12870.00%peg-0085VRQR-PE2max120390.13%807.50.00%peg-0088VRQR-PE2max120420.12%484.50.30%peg-0104VRQR-PE2max120580.53%290.50.00%peg-0108VRQR-PE2max120620.02%25950.00%peg-0111VRQR-PE2max120650.20%748.50.00%peg-0112VRQR-PE2max120660.14%1897.50.00%peg-0113VRQR-PE2max120670.24%40290.05%peg-0116VRQR-PE2max120700.09%47110.01%peg-0119VRQR-PE2max120730.09%473.50.12%peg-0124VRQR-PE2max120780.05%2764.50.00%peg-0125VRQR-PE2max120790.03%3892.50.00%peg-0126VRQR-PE2max120800.03%2135.50.02%peg-0127VRQR-PE2max120810.01%4971.50.00%peg-0128VRQR-PE2max120820.06%9890.00%peg-0129VRQR-PE2max120830.03%17440.00%peg-0134VRQR-PE2max120880.51%1744.50.00%peg-0136VRQR-PE2max120900.04%15400.00%peg-0138VRQR-PE2max120920.14%1383.50.00%peg-0142VRQR-PE2max120960.25%7240.00%peg-0145VRQR-PE2max120990.07%1559.50.03%peg-0146VRQR-PE2max121000.11%1437.50.00%peg-0148VRQR-PE2max121020.15%19510.00%peg-0149VRQR-PE2max121030.12%1722.50.00%peg-0150VRQR-PE2max121040.08%31080.02%peg-0154VRQR-PE2max121080.06%26990.00%peg-0155VRQR-PE2max121090.50%3100.00%peg-0158VRQR-PE2max121120.07%3675.50.00%peg-0159VRQR-PE2max121130.14%40870.01%peg-0160VRQR-PE2max121140.03%1874.50.00%peg-0162VRQR-PE2max121160.05%20420.00%peg-0163VRQR-PE2max121170.05%4991.50.00%peg-0164VRQR-PE2max121180.06%1561.50.00%peg-0168VRQR-PE2max121220.03%1937.50.00%peg-0171VRQR-PE2max121250.02%3033.50.00%peg-0174VRQR-PE2max121280.21%15080.00%peg-0175VRQR-PE2max121290.32%725.50.00%peg-0180VRQR-PE2max121340.10%5650.00%peg-0182VRQR-PE2max121360.10%1156.50.04%peg-0184VRQR-PE2max121380.04%1222.50.00%peg-0186VRQR-PE2max121400.10%12270.03%peg-0187VRQR-PE2max121410.00%1624.50.03%peg-0188VRQR-PE2max121420.17%8530.00%peg-0191VRQR-PE2max121440.68%636.50.00%peg-0192VRQR-PE2max121450.14%389.50.00%peg-0193VRQR-PE2max121460.19%2870.50.00%peg-0195VRQR-PE2max121480.05%19050.00%peg-0197VRQR-PE2max121500.71%4950.00%peg-0198VRQR-PE2max121510.02%29550.03%peg-0199VRQR-PE2max121520.29%27840.00%peg-0201VRQR-PE2max121540.29%11260.00%peg-0204VRQR-PE2max121570.35%21300.00%peg-0205VRQR-PE2max121580.05%27720.00%peg-0207VRQR-PE2max121600.18%5070.00%peg-0210VRQR-PE2max121630.20%1962.50.00%peg-0214VRQR-PE2max121670.14%14190.00%peg-0220VRQR-PE2max121730.05%1270.50.04%peg-0221VRQR-PE2max121740.12%2077.50.00%peg-0222VRQR-PE2max121750.11%1413.50.00%peg-0223VRQR-PE2max121760.21%9310.05%peg-0224VRQR-PE2max121770.28%22500.00%peg-0225VRQR-PE2max121780.10%1216.50.00%peg-0227VRQR-PE2max121800.08%2478.50.02%peg-0228VRQR-PE2max121810.07%2380.50.04%peg-0230VRQR-PE2max121830.12%34750.01%peg-0233VRQR-PE2max121860.29%1876.50.00%peg-0234VRQR-PE2max121870.01%35810.01%peg-0236VRQR-PE2max121890.06%37270.00%peg-0237VRQR-PE2max121900.10%4094.50.00%peg-0238VRQR-PE2max121910.01%6479.50.00%peg-0239VRQR-PE2max121920.03%1674.50.00%peg-0240VRQR-PE2max121930.09%22970.00%peg-0241VRQR-PE2max121940.16%27420.00%peg-0244VRQR-PE2max121970.20%1681.50.00%peg-0245VRQR-PE2max121980.03%15230.00%peg-0246VRQR-PE2max121990.00%2195.50.02%peg-0249VRQR-PE2max122020.05%11310.00%peg-0250VRQR-PE2max122030.30%1055.50.00%peg-0251VRQR-PE2max122040.27%7480.00%peg-0252VRQR-PE2max122050.08%17440.00%peg-0256VRQR-PE2max122090.04%35710.00%peg-0259VRQR-PE2max122120.02%2445.50.02%peg-0261VRQR-PE2max122140.36%28550.02%peg-0263VRQR-PE2max122160.00%386.50.11%peg-0264VRQR-PE2max122170.22%20700.00%peg-0265VRQR-PE2max122180.03%1899.50.00%peg-0268VRQR-PE2max122210.12%3947.50.00%peg-0269VRQR-PE2max122220.43%1642.50.00%peg-0270VRQR-PE2max122230.09%4296.50.01%peg-0271VRQR-PE2max122240.13%42940.01%peg-0272VRQR-PE2max122250.41%1099.50.00%peg-0273VRQR-PE2max122260.02%2059.50.00%peg-0274VRQR-PE2max122270.06%15570.00%peg-0279VRQR-PE2max122310.04%4830.50.00%peg-0280VRQR-PE2max122320.24%11350.00%peg-0283VRQR-PE2max122350.12%2316.50.00%peg-0284VRQR-PE2max122360.34%1035.50.00%peg-0285VRQR-PE2max122370.03%1736.50.00%peg-0286VRQR-PE2max122380.14%7550.00%peg-0287VRQR-PE2max122390.08%24700.00%peg-0291VRQR-PE2max122430.18%2990.00%peg-0292VRQR-PE2max122440.04%11070.04%peg-0293VRQR-PE2max122450.06%8740.00%peg-0294VRQR-PE2max122460.00%1591.50.03%peg-0296VRQR-PE2max122480.11%4510.00%peg-0298VRQR-PE2max122500.09%22040.02%peg-0299VRQR-PE2max122510.04%1308.50.00%peg-0301VRQR-PE2max122530.09%11250.00%peg-0302VRQR-PE2max122540.56%2334.50.00%peg-0303VRQR-PE2max122550.36%10390.00%peg-0304VRQR-PE2max122560.14%2965.50.00%peg-0305VRQR-PE2max122570.30%1884.50.00%peg-0306VRQR-PE2max122580.10%3759.50.00%peg-0307VRQR-PE2max122590.36%3038.50.02%peg-0309VRQR-PE2max122610.17%2598.50.02%peg-0314VRQR-PE2max122660.04%21070.20%peg-0316VRQR-PE2max122680.06%48020.01%peg-0318VRQR-PE2max122700.08%18550.00%peg-0320VRQR-PE2max122720.32%918.50.00%peg-0322VRQR-PE2max122740.11%1745.50.00%peg-0323VRQR-PE2max122750.00%1817.50.03%peg-0324VRQR-PE2max122760.10%1099.50.00%peg-0325VRQR-PE2max122770.24%4050.00%peg-0326VRQR-PE2max122780.04%12580.00%peg-0327VRQR-PE2max122790.43%5170.00%peg-0329VRQR-PE2max122810.07%8340.00%peg-0331VRQR-PE2max122830.09%2449.50.05%peg-0332VRQR-PE2max122840.15%2661.50.00%peg-0334VRQR-PE2max122860.41%23320.00%peg-0335VRQR-PE2max122870.11%23210.00%peg-0339VRQR-PE2max122910.11%16470.00%peg-0341VRQR-PE2max122930.24%2829.50.00%peg-0342VRQR-PE2max122940.06%2092.50.02%peg-0343VRQR-PE2max122950.54%23300.02%peg-0344VRQR-PE2max122960.13%28370.00%peg-0345VRQR-PE2max122970.06%931.50.00%peg-0347VRQR-PE2max122990.07%51070.00%peg-0348VRQR-PE2max123000.11%29350.00%peg-0349VRQR-PE2max123010.14%31860.00%peg-0351VRQR-PE2max123030.03%20410.00%peg-0353VRQR-PE2max123050.13%17270.00%peg-0354VRQR-PE2max123060.05%20000.02%peg-0357VRQR-PE2max123090.08%2654.50.00%peg-0358VRQR-PE2max123100.30%10120.00%peg-0359VRQR-PE2max123110.07%1493.50.00%peg-0360VRQR-PE2max123120.00%13440.03%peg-0364VRQR-PE2max123160.27%375.50.00%peg-0367VRQR-PE2max123190.07%1421.50.00%peg-0368VRQR-PE2max123200.24%12460.04%peg-0369VRQR-PE2max123210.04%22380.00%peg-0372VRQR-PE2max123240.04%1405.50.00%peg-0374VRQR-PE2max123260.19%5230.00%peg-0375VRQR-PE2max123270.07%27880.00%peg-0376VRQR-PE2max123280.11%5474.50.02%peg-0378VRQR-PE2max123300.13%1504.50.03%peg-0379VRQR-PE2max123310.07%21360.00%peg-0380VRQR-PE2max123320.57%2479.50.00%peg-0381VRQR-PE2max123330.42%9310.00%peg-0382VRQR-PE2max123341.01%2191.50.02%peg-0383VRQR-PE2max123350.12%2959.50.03%peg-0384VRQR-PE2max123360.65%19790.03%peg-0385VRQR-PE2max123370.15%16540.00%peg-0387VRQR-PE2max123390.13%4100.00%peg-0388VRQR-PE2max123400.05%23550.16%peg-0390VRQR-PE2max123420.06%39600.06%peg-0391VRQR-PE2max123430.05%2378.50.02%peg-0393VRQR-PE2max123450.10%4320.50.08%peg-0394VRQR-PE2max123460.22%2734.50.12%peg-0395VRQR-PE2max123470.09%2217.50.00%peg-0396VRQR-PE2max123480.09%1227.50.23%peg-0397VRQR-PE2max123490.06%10060.00%peg-0398VRQR-PE2max123500.11%4250.00%peg-0399VRQR-PE2max123510.58%6800.00%peg-0400VRQR-PE2max123520.10%1451.50.00%peg-0401VRQR-PE2max123530.16%1340.50.00%peg-0405VRQR-PE2max123570.04%27910.00%peg-0406VRQR-PE2max123580.05%30900.00%peg-0407VRQR-PE2max123590.03%15640.00%peg-0408VRQR-PE2max123600.17%29450.03%peg-0409VRQR-PE2max123610.26%2724.50.02%peg-0410VRQR-PE2max123620.22%658.50.00%peg-0411VRQR-PE2max123630.14%11900.00%peg-0413VRQR-PE2max123650.30%10690.00%peg-0414VRQR-PE2max123660.35%1389.50.00%peg-0415VRQR-PE2max123670.42%45350.00%peg-0416VRQR-PE2max123680.10%25470.02%peg-0417VRQR-PE2max123690.01%35410.00%peg-0418VRQR-PE2max123700.09%6468.50.01%peg-0419VRQR-PE2max123710.15%400.50.00%peg-0421VRQR-PE2max123730.18%22030.00%peg-0422VRQR-PE2max123740.08%30640.03%peg-0423VRQR-PE2max123750.26%21110.18%peg-0426VRQR-PE2max123780.94%3634.50.18%peg-0427VRQR-PE2max123790.00%44120.03%peg-0429VRQR-PE2max123810.02%2915.50.03%peg-0430VRQR-PE2max123820.28%1775.50.00%peg-0431VRQR-PE2max123830.11%1664.50.00%peg-0432VRQR-PE2max123840.14%1789.50.11%peg-0433VRQR-PE2max123850.00%30140.05%peg-0434VRQR-PE2max123860.47%331.50.00%peg-0435VRQR-PE2max123870.11%18440.00%peg-0436VRQR-PE2max123880.40%16890.03%peg-0437VRQR-PE2max123890.12%12370.00%peg-0441VRQR-PE2max123930.13%3661.50.00%peg-0442VRQR-PE2max123940.02%30650.02%peg-0443VRQR-PE2max123950.04%3941.50.02%peg-0444VRQR-PE2max123960.13%23040.00%peg-0445VRQR-PE2max123970.16%2799.50.04%peg-0448VRQR-PE2max124000.15%919.50.05%peg-0451VRQR-PE2max124030.02%2300.50.00%peg-0453VRQR-PE2max124050.18%30430.01%peg-0455VRQR-PE2max124070.07%13480.04%peg-0459VRQR-PE2max124110.09%1215.50.00%peg-0460VRQR-PE2max124120.01%3879.50.14%peg-0464VRQR-PE2max124160.39%18310.03%peg-0467VRQR-PE2max124190.06%39610.23%peg-0470VRQR-PE2max124220.09%536.50.00%peg-0472VRQR-PE2max124240.07%773.51.42%peg-0473VRQR-PE2max124250.37%16790.03%peg-0474VRQR-PE2max124260.25%5800.00%peg-0475VRQR-PE2max124270.22%649.50.00%peg-0479VRQR-PE2max124310.24%9520.00%peg-0480VRQR-PE2max124320.05%4043.50.00%peg-0483VRQR-PE2max124350.10%48280.02%peg-0485VRQR-PE2max124370.21%961.50.00%peg-0486VRQR-PE2max124380.04%31190.03%peg-0487VRQR-PE2max124390.06%5988.50.00%peg-0489VRQR-PE2max124410.09%1179.50.04%peg-0490VRQR-PE2max124420.21%3071.50.01%peg-0491VRQR-PE2max124430.13%26050.02%peg-0492VRQR-PE2max124440.22%22840.00%peg-0493VRQR-PE2max124450.12%21510.00%peg-0494VRQR-PE2max124460.26%14950.00%peg-0495VRQR-PE2max124470.33%15130.06%peg-0497VRQR-PE2max124490.00%11430.19%peg-0499VRQR-PE2max124510.52%14400.03%peg-0500VRQR-PE2max124520.27%6170.46%peg-0501VRQR-PE2max124530.36%5297.50.41%peg-0502VRQR-PE2max124540.07%8250.05%peg-0503VRQR-PE2max124550.49%922.50.00%peg-0504VRQR-PE2max124560.22%18240.49%peg-0505VRQR-PE2max124570.32%2610.50.78%peg-0506VRQR-PE2max124580.46%7660.06%peg-0508VRQR-PE2max124600.22%1902.50.73%peg-0511VRQR-PE2max124630.34%398.50.12%peg-0515VRQR-PE2max124670.06%1509.50.00%peg-0516VRQR-PE2max124680.16%1400.50.00%peg-0517VRQR-PE2max124690.33%15480.00%peg-0518VRQR-PE2max124703.73%34800.07%peg-0519VRQR-PE2max124716.92%3082.50.03%peg-0520VRQR-PE2max124727.90%1360.50.04%peg-0521VRQR-PE2max124735.05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-3214VRQR-PE2max151290.03%41850.01%peg-3216VRQR-PE2max151310.22%908.50.00%peg-3217VRQR-PE2max151320.03%17760.03%peg-3218VRQR-PE2max151330.02%2543.50.11%peg-3219VRQR-PE2max151340.05%2857.50.02%peg-3221VRQR-PE2max151360.30%1106.50.00%peg-3222VRQR-PE2max151370.05%2311.50.00%peg-3227VRQR-PE2max151420.05%10090.00%peg-3233VRQR-PE2max151480.19%577.50.00%peg-3236VRQR-PE2max151510.21%4370.00%peg-3238VRQR-PE2max151530.41%972.50.00%peg-3240VRQR-PE2max151550.06%814.50.00%peg-3241VRQR-PE2max151560.04%1356.50.00%peg-3243VRQR-PE2max151580.15%33080.03%peg-3245VRQR-PE2max151600.24%382.50.00%peg-3247VRQR-PE2max151610.05%1863.50.00%peg-3248VRQR-PE2max151620.11%27020.00%peg-3249VRQR-PE2max151630.10%29740.00%peg-3250VRQR-PE2max151640.09%6608.50.01%peg-3251VRQR-PE2max151650.22%731.50.00%peg-3252VRQR-PE2max151660.51%16831.41%peg-3253VRQR-PE2max151670.14%4578.50.00%peg-3254VRQR-PE2max151680.14%36370.01%peg-3256VRQR-PE2max151700.08%40770.00%peg-3258VRQR-PE2max151720.13%16330.03%peg-3259VRQR-PE2max151730.05%937.50.00%peg-3261VRQR-PE2max151750.03%1761.50.00%peg-3264VRQR-PE2max151780.07%18860.00%peg-3272VRQR-PE2max151860.26%827.50.00%peg-3275VRQR-PE2max151890.54%772.50.06%peg-3276VRQR-PE2max151900.18%495.50.00%peg-3277VRQR-PE2max151910.07%737.50.06%peg-3278VRQR-PE2max151920.20%1127.50.00%peg-3280VRQR-PE2max151940.20%1762.50.09%peg-3281VRQR-PE2max151950.05%34030.01%peg-3282VRQR-PE2max151960.09%21760.02%peg-3285VRQR-PE2max151990.14%1197.50.00%peg-3287VRQR-PE2max152010.09%42060.02%peg-3289VRQR-PE2max152030.09%38960.00%peg-3290VRQR-PE2max152040.05%10690.09%peg-3291VRQR-PE2max152050.27%6250.08%peg-3292VRQR-PE2max152060.00%29460.02%peg-3293VRQR-PE2max152070.10%3594.50.00%peg-3294VRQR-PE2max152080.18%2878.50.00%peg-3295VRQR-PE2max152090.03%56400.01%peg-3296VRQR-PE2max152100.04%2091.50.00%peg-3297VRQR-PE2max152110.04%12470.00%peg-3298VRQR-PE2max152120.02%2358.50.02%peg-3302VRQR-PE2max152160.23%9090.00%peg-3303VRQR-PE2max152170.13%3770.00%peg-3305VRQR-PE2max152190.10%1567.50.00%peg-3306VRQR-PE2max152200.20%416.50.00%peg-3307VRQR-PE2max152210.67%7500.00%peg-3310VRQR-PE2max152240.19%967.50.00%peg-3311VRQR-PE2max152250.29%1237.50.00%peg-3315VRQR-PE2max152290.57%2590.00%peg-3317VRQR-PE2max152310.99%197.50.00%peg-3318VRQR-PE2max152320.65%421.50.00%peg-3319VRQR-PE2max152330.03%3051.50.02%peg-3320VRQR-PE2max152340.07%3768.50.01%peg-3322VRQR-PE2max152360.06%822.50.00%peg-3323VRQR-PE2max152370.11%15780.03%peg-3325VRQR-PE2max152390.07%1434.50.03%peg-3326VRQR-PE2max152400.00%1556.50.03%peg-3327VRQR-PE2max152410.27%3864.50.04%peg-3328VRQR-PE2max152420.06%25820.00%peg-3329VRQR-PE2max152430.12%8300.00%peg-3330VRQR-PE2max152440.42%1236.50.03%peg-3332VRQR-PE2max152460.18%28300.03%peg-3333VRQR-PE2max152470.03%2808.50.00%peg-3334VRQR-PE2max152480.14%5396.50.00%peg-3335VRQR-PE2max152490.14%3918.50.00%peg-3337VRQR-PE2max152500.21%35350.00%peg-3338VRQR-PE2max152510.16%22140.00%peg-3339VRQR-PE2max152520.09%2778.50.02%peg-3340VRQR-PE2max152530.15%22580.00%peg-3341VRQR-PE2max152540.06%8040.00%peg-3343VRQR-PE2max152560.14%1541.50.04%peg-3347VRQR-PE2max152600.66%424.50.00%peg-3348VRQR-PE2max152610.15%6840.00%peg-3351VRQR-PE2max152640.12%7720.00%peg-3354VRQR-PE2max152670.14%640.50.00%peg-3355VRQR-PE2max152680.57%349.50.00%peg-3357VRQR-PE2max152700.28%8350.00%peg-3358VRQR-PE2max152710.00%18760.05%peg-3359VRQR-PE2max152720.08%4298.50.00%peg-3360VRQR-PE2max152730.06%58760.03%peg-3364VRQR-PE2max152770.20%704.50.00%peg-3365VRQR-PE2max152780.14%31240.00%peg-3366VRQR-PE2max152790.12%15920.03%peg-3367VRQR-PE2max152800.48%9940.00%peg-3369VRQR-PE2max152820.16%296.50.00%peg-3370VRQR-PE2max152830.10%1927.50.00%peg-3371VRQR-PE2max152840.09%3384.50.00%peg-3372VRQR-PE2max152850.19%34440.00%peg-3374VRQR-PE2max152870.08%26190.02%peg-3375VRQR-PE2max152880.07%3339.50.00%peg-3381VRQR-PE2max152940.05%11920.00%peg-3382VRQR-PE2max152950.36%503.50.00%peg-3383VRQR-PE2max152960.09%1641.50.00%peg-3384VRQR-PE2max152970.19%262.50.00%peg-3385VRQR-PE2max152980.24%621.50.00%peg-3386VRQR-PE2max152990.27%1129.50.00%peg-3391VRQR-PE2max153030.19%20340.00%peg-3396VRQR-PE2max153070.20%12830.03%peg-3397VRQR-PE2max153080.06%2253.50.00%peg-3398VRQR-PE2max153090.11%4485.50.02%peg-3400VRQR-PE2max153110.08%648.50.00%peg-3401VRQR-PE2max153120.12%1244.50.04%peg-3403VRQR-PE2max153140.10%988.50.00%peg-3404VRQR-PE2max153150.06%26830.03%peg-3405VRQR-PE2max153160.09%45190.02%peg-3406VRQR-PE2max153170.13%2369.50.02%peg-3408VRQR-PE2max153190.70%6750.00%peg-3409VRQR-PE2max153200.23%830.50.07%peg-3410VRQR-PE2max153210.16%3778.50.00%peg-3412VRQR-PE2max153230.04%1243.50.00%peg-3413VRQR-PE2max153240.46%8700.00%peg-3416VRQR-PE2max153270.14%1332.50.00%peg-3418VRQR-PE2max153290.12%1666.50.03%peg-3420VRQR-PE2max153310.09%5060.00%peg-3427VRQR-PE2max153370.85%574.52.43%peg-3431VRQR-PE2max153410.24%15720.00%peg-3432VRQR-PE2max153421.12%274.50.00%peg-3435VRQR-PE2max153450.04%13480.00%peg-3436VRQR-PE2max153460.19%13050.04%peg-3438VRQR-PE2max153480.08%1041.50.00%peg-3439VRQR-PE2max153490.20%9840.04%peg-3440VRQR-PE2max153501.09%7080.06%peg-3441VRQR-PE2max153510.40%4610.00%peg-3443VRQR-PE2max153530.11%4867.50.02%peg-3445VRQR-PE2max153550.11%47840.02%peg-3446VRQR-PE2max153560.03%1410.50.00%peg-3447VRQR-PE2max153570.11%546.50.08%peg-3449VRQR-PE2max153590.11%22770.00%peg-3450VRQR-PE2max153600.27%18670.00%peg-3452VRQR-PE2max153620.15%2401.50.02%peg-3454VRQR-PE2max153640.20%19380.00%peg-3455VRQR-PE2max153650.07%14330.00%peg-3456VRQR-PE2max153660.10%1257.51.79%peg-3457VRQR-PE2max153670.23%21970.00%peg-3461VRQR-PE2max153710.25%151.50.00%peg-3463VRQR-PE2max153730.18%806.50.00%peg-3467VRQR-PE2max153770.09%622.50.00%peg-3475VRQR-PE2max153850.13%3215.50.01%peg-3477VRQR-PE2max153870.22%1843.50.00%peg-3478VRQR-PE2max153880.06%19520.02%peg-3479VRQR-PE2max153890.11%10790.08%peg-3481VRQR-PE2max153910.46%1204.50.00%peg-3482VRQR-PE2max153920.13%1935.50.00%peg-3485VRQR-PE2max153950.06%9990.00%peg-3486VRQR-PE2max153960.02%2419.50.03%peg-3487VRQR-PE2max153970.10%2121.50.00%peg-3488VRQR-PE2max153980.08%8626.50.02%peg-3489VRQR-PE2max153990.10%2460.50.02%peg-3490VRQR-PE2max154000.13%3415.50.01%peg-3491VRQR-PE2max154010.13%1896.50.00%peg-3493VRQR-PE2max154030.26%13730.00%peg-3495VRQR-PE2max154050.06%16020.00%peg-3500VRQR-PE2max154100.00%19450.03%peg-3506VRQR-PE2max154150.15%331.50.00%peg-3507VRQR-PE2max154160.72%130.50.00%peg-3509VRQR-PE2max154180.94%415.50.00%peg-3511VRQR-PE2max154201.60%416.50.00%peg-3514VRQR-PE2max154230.12%24060.00%peg-3515VRQR-PE2max154240.14%25960.06%peg-3516VRQR-PE2max154250.13%3474.50.03%peg-3518VRQR-PE2max154270.14%3710.50.02%peg-3520VRQR-PE2max154290.11%19030.00%peg-3521VRQR-PE2max154300.23%1685.50.00%peg-3522VRQR-PE2max154310.02%22040.00%peg-3524VRQR-PE2max154330.44%20950.00%peg-3526VRQR-PE2max154350.08%4694.50.01%peg-3530VRQR-PE2max154390.14%21860.02%peg-3531VRQR-PE2max154400.07%37380.00%peg-3532VRQR-PE2max154410.14%1122.50.04%peg-3534VRQR-PE2max154430.07%6700.00%peg-3535VRQR-PE2max154440.01%3765.50.00%peg-3536VRQR-PE2max154450.32%3500.00%peg-3538VRQR-PE2max154470.56%11800.00%peg-3539VRQR-PE2max154480.26%841.50.00%peg-3541VRQR-PE2max154490.06%15270.04%peg-3542VRQR-PE2max154500.10%5110.00%peg-3547VRQR-PE2max154550.09%5780.00%peg-3548VRQR-PE2max154560.09%5290.00%peg-3549VRQR-PE2max154570.08%648.50.00%peg-3551VRQR-PE2max154590.07%711.50.00%peg-3552VRQR-PE2max154600.20%1082.50.04%peg-3554VRQR-PE2max154620.10%53990.01%peg-3555VRQR-PE2max154630.09%2319.50.00%peg-3556VRQR-PE2max154640.06%9540.18%peg-3557VRQR-PE2max154650.87%460.50.08%peg-3558VRQR-PE2max154660.20%498.50.00%peg-3559VRQR-PE2max154670.13%5348.50.02%peg-3560VRQR-PE2max154680.16%35120.01%peg-3561VRQR-PE2max154690.20%2335.50.00%peg-3563VRQR-PE2max154710.26%16630.00%peg-3565VRQR-PE2max154730.17%41740.01%peg-3566VRQR-PE2max154740.17%2545.50.04%peg-3567VRQR-PE2max154750.10%4751.50.06%peg-3568VRQR-PE2max154760.20%34930.03%peg-3569VRQR-PE2max154770.06%3675.50.01%peg-3570VRQR-PE2max154780.17%31440.00%peg-3572VRQR-PE2max154800.11%12900.00%peg-3573VRQR-PE2max154810.07%1957.50.02%peg-3575VRQR-PE2max154830.13%4530.00%peg-3579VRQR-PE2max154870.33%7660.00%peg-3580VRQR-PE2max154880.03%1709.50.00%peg-3587VRQR-PE2max154940.20%267.50.00%peg-3592VRQR-PE2max154990.09%21850.00%peg-3593VRQR-PE2max155000.09%40760.01%peg-3594VRQR-PE2max155010.08%18190.00%peg-3595VRQR-PE2max155020.19%2093.50.00%peg-3597VRQR-PE2max155030.19%5820.08%peg-3598VRQR-PE2max155040.02%6242.50.03%peg-3599VRQR-PE2max155050.23%43290.03%peg-3600VRQR-PE2max155060.06%3615.50.02%peg-3601VRQR-PE2max155070.18%23180.06%peg-3602VRQR-PE2max155080.04%24340.00%peg-3603VRQR-PE2max155090.13%2961.50.00%peg-3604VRQR-PE2max155100.00%40010.03%peg-3605VRQR-PE2max155110.16%5279.50.08%peg-3606VRQR-PE2max155120.24%18620.00%peg-3608VRQR-PE2max155140.10%2798.50.02%peg-3609VRQR-PE2max155150.11%25830.00%peg-3610VRQR-PE2max155160.10%1583.50.00%peg-3611VRQR-PE2max155170.28%16050.00%peg-3613VRQR-PE2max155190.03%15830.00%peg-3614VRQR-PE2max155200.07%1274.50.00%peg-3615VRQR-PE2max155210.67%600.50.00%peg-3619VRQR-PE2max155250.18%21770.00%peg-3623VRQR-PE2max155290.22%6610.00%peg-3624VRQR-PE2max155300.09%6280.00%peg-3628VRQR-PE2max155330.17%637.50.00%peg-3629VRQR-PE2max155340.29%340.50.00%.

[0661]

[0662] PE2max 실험 결과LabelPrime EditorpegRNA(SEQ ID NO)Adjusted Prime editing efficiency average (%)Readcount averageBackground efficiency (error)peg-0030PE2max119870.23%3950.00%peg-0044PE2max119980.05%9580.00%peg-0046PE2max120000.04%1144.50.00%peg-0065PE2max120190.10%1017.50.00%peg-0076PE2max120300.36%276.50.00%peg-0079PE2max120330.08%5950.00%peg-0090PE2max120440.38%2660.00%peg-0104PE2max120580.17%2530.00%peg-0111PE2max120650.55%681.50.00%peg-0116PE2max120700.01%4276.50.01%peg-0126PE2max120800.00%2048.50.02%peg-0134PE2max120880.06%1585.50.00%peg-0149PE2max121030.07%1620.50.00%peg-0150PE2max121040.13%27670.02%peg-0159PE2max121130.00%3581.50.01%peg-0162PE2max121160.03%1912.50.00%peg-0163PE2max121170.07%4682.50.00%peg-0167PE2max121210.27%736.50.00%peg-0171PE2max121250.02%2586.50.00%peg-0176PE2max121300.17%344.50.00%peg-0181PE2max121350.04%1270.50.00%peg-0184PE2max121380.40%1089.50.00%peg-0186PE2max121400.01%11710.03%peg-0187PE2max121410.00%15430.03%peg-0188PE2max121420.07%7710.00%peg-0193PE2max121460.05%28960.00%peg-0194PE2max121470.69%25111.09%peg-0197PE2max121500.10%4870.00%peg-0199PE2max121520.02%24690.00%peg-0215PE2max121680.03%1603.50.00%peg-0224PE2max121770.02%21470.00%peg-0226PE2max121790.11%904.50.05%peg-0230PE2max121830.08%3287.50.01%peg-0231PE2max121840.05%9480.00%peg-0233PE2max121860.15%1678.50.00%peg-0234PE2max121870.00%3165.50.01%peg-0236PE2max121890.01%34280.00%peg-0252PE2max122050.03%15390.00%peg-0257PE2max122100.03%20060.02%peg-0259PE2max122120.08%2346.50.02%peg-0261PE2max122140.15%2669.50.02%peg-0267PE2max122200.05%10420.00%peg-0268PE2max122210.03%35920.00%peg-0270PE2max122230.05%38890.01%peg-0279PE2max122310.02%4427.50.00%peg-0283PE2max122350.02%2135.50.00%peg-0298PE2max122500.17%2248.50.02%peg-0301PE2max122530.37%1028.50.00%peg-0302PE2max122540.17%20640.00%peg-0303PE2max122550.05%10810.00%peg-0304PE2max122560.04%26760.00%peg-0306PE2max122580.03%3419.50.00%peg-0309PE2max122610.02%24880.02%peg-0311PE2max122631.53%1921.52.19%peg-0314PE2max122660.03%1975.50.20%peg-0315PE2max122670.05%12290.00%peg-0316PE2max122680.00%44930.01%peg-0323PE2max122750.04%1767.50.03%peg-0326PE2max122780.12%11960.00%peg-0329PE2max122810.13%820.50.00%peg-0334PE2max122860.05%22370.00%peg-0335PE2max122870.02%21650.00%peg-0339PE2max122910.03%1530.50.00%peg-0341PE2max122930.04%26240.00%peg-0343PE2max122950.00%2479.50.02%peg-0348PE2max123000.06%26520.00%peg-0349PE2max123010.04%2851.50.00%peg-0351PE2max123030.36%1815.50.00%peg-0353PE2max123050.03%15410.00%peg-0354PE2max123060.06%1888.50.02%peg-0366PE2max123180.05%943.50.00%peg-0368PE2max123200.01%13410.04%peg-0370PE2max123220.03%1533.50.00%peg-0375PE2max123270.06%2471.50.00%peg-0376PE2max123280.00%50430.02%peg-0378PE2max123300.07%1461.50.03%peg-0380PE2max123320.11%2294.50.00%peg-0381PE2max123330.27%949.50.00%peg-0382PE2max123340.09%21620.02%peg-0383PE2max123350.05%26690.03%peg-0391PE2max123430.05%20700.02%peg-0392PE2max123440.19%7130.45%peg-0395PE2max123470.10%1984.50.00%peg-0398PE2max123500.16%338.50.00%peg-0400PE2max123520.33%1310.50.00%peg-0401PE2max123530.15%1078.50.00%peg-0403PE2max123550.05%11040.00%peg-0405PE2max123570.02%25290.00%peg-0408PE2max123600.01%27360.03%peg-0409PE2max123610.00%2710.50.02%peg-0410PE2max123620.15%6700.00%peg-0415PE2max123670.01%4260.50.00%peg-0417PE2max123690.01%3296.50.00%peg-0421PE2max123730.02%2131.50.00%peg-0422PE2max123740.02%27330.03%peg-0424PE2max123760.16%9110.00%peg-0426PE2max123780.25%31950.18%peg-0427PE2max123790.11%38520.03%peg-0433PE2max123850.27%26860.05%peg-0434PE2max123860.35%295.50.00%peg-0435PE2max123870.22%17340.00%peg-0436PE2max123880.04%1623.50.03%peg-0438PE2max123900.25%7650.52%peg-0439PE2max123910.34%157.50.00%peg-0446PE2max123980.05%18960.00%peg-0450PE2max124020.09%2815.50.00%peg-0453PE2max124050.11%2774.50.01%peg-0455PE2max124070.09%1165.50.04%peg-0457PE2max124090.02%13160.06%peg-0460PE2max124120.06%35630.14%peg-0464PE2max124160.29%1734.50.03%peg-0466PE2max124180.08%623.50.00%peg-0472PE2max124241.14%7901.42%peg-0473PE2max124250.11%1402.50.03%peg-0474PE2max124261.55%460.50.00%peg-0476PE2max124280.14%669.50.43%peg-0477PE2max124290.05%10880.04%peg-0481PE2max124330.15%6060.00%peg-0485PE2max124370.06%8460.00%peg-0492PE2max124440.02%2077.50.00%peg-0495PE2max124470.15%14090.06%peg-0496PE2max124480.19%17480.11%peg-0500PE2max124520.15%538.50.46%peg-0502PE2max124540.21%820.50.05%peg-0503PE2max124550.39%907.50.00%peg-0506PE2max124580.40%6280.06%peg-0508PE2max124601.18%17450.73%peg-0511PE2max124630.32%3310.12%peg-0515PE2max124670.03%1554.50.00%peg-0516PE2max124680.04%12360.00%peg-0519PE2max124710.01%28190.03%peg-0520PE2max124720.16%12890.04%peg-0522PE2max124740.16%6440.00%peg-0524PE2max124760.05%1866.50.00%peg-0527PE2max124790.04%1085.50.00%peg-0532PE2max124840.03%22730.02%peg-0533PE2max124850.04%33600.01%peg-0534PE2max124860.12%24440.00%peg-0535PE2max124870.05%9470.00%peg-0536PE2max124880.05%11230.00%peg-0541PE2max124930.03%1459.50.00%peg-0550PE2max125020.05%2202.50.00%peg-0551PE2max125030.76%940.50.00%peg-0553PE2max125050.05%921.50.00%peg-0555PE2max125070.26%20120.02%peg-0558PE2max125100.21%1541.50.14%peg-0559PE2max125110.04%12780.03%peg-0561PE2max125130.09%16250.00%peg-0564PE2max125160.05%10740.00%peg-0566PE2max125180.03%3852.50.01%peg-0569PE2max125210.00%29320.01%peg-0572PE2max125240.13%759.50.00%peg-0577PE2max125290.12%1700.50.00%peg-0582PE2max125340.09%17610.07%peg-0587PE2max125390.06%3034.50.02%peg-0588PE2max125400.12%21940.02%peg-0591PE2max125430.15%649.50.00%peg-0592PE2max125440.25%5820.00%peg-0593PE2max125450.11%889.50.00%peg-0594PE2max125460.26%21690.02%peg-0595PE2max125470.01%323.50.14%peg-0596PE2max125480.03%1981.50.02%peg-0597PE2max125490.14%3265.50.13%peg-0599PE2max125510.07%29740.03%peg-0601PE2max125530.01%50360.01%peg-0602PE2max125540.00%3168.50.01%peg-0603PE2max125550.07%30990.01%peg-0612PE2max125640.08%16800.02%peg-0614PE2max125660.24%2510.50.00%peg-0622PE2max125740.01%2174.50.02%peg-0623PE2max125750.03%3418.50.00%peg-0624PE2max125760.33%24770.00%peg-0625PE2max125770.28%15210.00%peg-0626PE2max125780.35%771.50.00%peg-0627PE2max125790.27%539.50.00%peg-0628PE2max125800.25%1158.50.09%peg-0632PE2max125840.02%24270.02%peg-0633PE2max125850.04%1408.50.00%peg-0636PE2max125880.08%3055.50.00%peg-0638PE2max125900.07%1483.50.00%peg-0640PE2max125920.10%547.50.00%peg-0643PE2max125950.23%1982.50.00%peg-0651PE2max126030.04%1200.50.11%peg-0655PE2max126070.10%1887.50.00%peg-0658PE2max126100.17%1267.50.03%peg-0659PE2max126110.25%813.50.00%peg-0663PE2max126150.21%983.50.04%peg-0664PE2max126160.01%828.50.05%peg-0666PE2max126180.09%22350.02%peg-0667PE2max126190.01%1202.50.03%peg-0668PE2max126200.03%1665.50.03%peg-0669PE2max126210.10%3852.50.09%peg-0670PE2max126220.00%17240.02%peg-0671PE2max126230.04%12260.00%peg-0672PE2max126240.29%29870.01%peg-0673PE2max126250.05%29970.00%peg-0674PE2max126260.00%27580.02%peg-0677PE2max126290.17%15310.00%peg-0679PE2max126310.04%1019.50.00%peg-0681PE2max126330.10%5400.00%peg-0682PE2max126340.08%13880.03%peg-0685PE2max126370.13%1084.50.00%peg-0690PE2max126420.16%19330.00%peg-0693PE2max126450.09%10070.00%peg-0694PE2max126460.06%33030.00%peg-0695PE2max126470.69%12680.03%peg-0696PE2max126480.10%494.50.00%peg-0698PE2max126490.04%26660.03%peg-0702PE2max126530.00%16880.03%peg-0703PE2max126540.10%21060.02%peg-0705PE2max126560.24%23190.00%peg-0706PE2max126570.07%30410.01%peg-0707PE2max126580.00%1280.50.03%peg-0708PE2max126590.25%1903.50.11%peg-0710PE2max126610.02%6220.14%peg-0711PE2max126620.05%8190.00%peg-0713PE2max126640.01%4115.50.04%peg-0717PE2max126680.33%992.50.00%peg-0718PE2max126690.07%1247.50.00%peg-0726PE2max126770.03%1660.50.00%peg-0727PE2max126780.03%15140.00%peg-0728PE2max126790.13%3927.50.01%peg-0729PE2max126800.50%1047.50.00%peg-0730PE2max126810.33%11810.06%peg-0731PE2max126820.01%10050.04%peg-0732PE2max126830.11%4750.00%peg-0733PE2max126840.08%2244.50.08%peg-0734PE2max126850.55%33000.04%peg-0736PE2max126870.07%15440.00%peg-0737PE2max126880.17%8530.00%peg-0738PE2max126890.28%13390.00%peg-0740PE2max126910.01%19610.02%peg-0743PE2max126940.23%1712.50.00%peg-0744PE2max126950.05%20160.00%peg-0747PE2max126980.10%25550.03%peg-0752PE2max127030.04%1680.50.02%peg-0753PE2max127040.08%778.50.05%peg-0754PE2max127050.20%7680.00%peg-0757PE2max127080.06%811.50.00%peg-0758PE2max127090.09%11540.00%peg-0760PE2max127110.07%2069.50.00%peg-0762PE2max127130.03%32440.00%peg-0765PE2max127160.15%2739.50.05%peg-0766PE2max127170.05%12700.03%peg-0767PE2max127180.03%15200.00%peg-0768PE2max127190.20%34950.04%peg-0772PE2max127230.06%2907.50.05%peg-0775PE2max127260.12%12950.00%peg-0778PE2max127290.31%1322.50.00%peg-0779PE2max127300.19%21060.02%peg-0780PE2max127310.01%6960.06%peg-0781PE2max127320.00%37810.05%peg-0782PE2max127330.17%30770.21%peg-0787PE2max127380.12%1277.50.07%peg-0789PE2max127400.04%11460.00%peg-0793PE2max127440.26%963.50.00%peg-0799PE2max127500.32%18070.04%peg-0800PE2max127510.10%4174.50.02%peg-0801PE2max127520.14%7090.00%peg-0802PE2max127530.05%960.50.00%peg-0803PE2max127540.26%38290.04%peg-0804PE2max127550.57%15050.33%peg-0805PE2max127560.09%961.50.00%peg-0806PE2max127570.03%1746.50.00%peg-0807PE2max127580.21%1269.50.00%peg-0808PE2max127590.18%2039.50.02%peg-0811PE2max127620.15%1766.50.02%peg-0816PE2max127670.11%1401.50.00%peg-0817PE2max127680.25%6630.07%peg-0819PE2max127700.31%1011.50.08%peg-0820PE2max127710.12%20780.04%peg-0822PE2max127730.05%1006.50.04%peg-0823PE2max127740.38%662.50.00%peg-0831PE2max127820.02%23310.00%peg-0832PE2max127830.03%17680.00%peg-0834PE2max127850.01%1720.50.02%peg-0835PE2max127860.18%3430.50.01%peg-0837PE2max127880.05%21400.00%peg-0838PE2max127890.60%2589.50.02%peg-0839PE2max127900.15%13840.03%peg-0840PE2max127910.09%1516.50.00%peg-0841PE2max127920.45%2471.50.03%peg-0842PE2max127930.08%1651.50.08%peg-0843PE2max127940.96%476.50.00%peg-0844PE2max127950.10%5330.00%peg-0845PE2max127960.13%15420.00%peg-0849PE2max128000.04%2567.50.02%peg-0851PE2max128020.78%12050.99%peg-0852PE2max128030.19%1896.50.00%peg-0853PE2max128040.27%1650.50.00%peg-0854PE2max128050.01%16240.51%peg-0857PE2max128080.30%729.50.05%peg-0858PE2max128090.74%7830.22%peg-0861PE2max128120.00%1511.50.03%peg-0867PE2max128180.12%21940.00%peg-0868PE2max128190.07%9260.05%peg-0869PE2max128200.07%31580.01%peg-0870PE2max128210.06%27160.03%peg-0873PE2max128240.21%2123.50.02%peg-0874PE2max128250.23%18600.15%peg-0875PE2max128260.08%1344.50.06%peg-0876PE2max128270.34%2004.50.04%peg-0879PE2max128300.19%37500.09%peg-0881PE2max128320.25%2377.50.00%peg-0882PE2max128330.03%7100.11%peg-0884PE2max128350.27%12000.00%peg-0885PE2max128360.17%2630.00%peg-0888PE2max128390.28%3142.50.14%peg-0889PE2max128400.36%1234.50.00%peg-0890PE2max128410.15%652.50.00%peg-0894PE2max128450.09%1623.50.02%peg-0895PE2max128460.57%4300.09%peg-0900PE2max128500.02%3139.50.01%peg-0909PE2max128590.08%2786.50.03%peg-0910PE2max128600.18%5250.00%peg-0912PE2max128620.04%1296.50.00%peg-0920PE2max128700.06%9160.00%peg-0923PE2max128730.02%2192.50.00%peg-0924PE2max128740.03%19110.09%peg-0928PE2max128780.14%3240.00%peg-0929PE2max128790.00%2386.50.02%peg-0930PE2max128800.02%24710.00%peg-0932PE2max128820.02%29920.00%peg-0939PE2max128890.14%37100.01%peg-0947PE2max128970.08%12910.00%peg-0955PE2max129050.02%20380.00%peg-0959PE2max129090.07%13800.00%peg-0963PE2max129130.07%706.50.00%peg-0975PE2max129250.00%3610.50.01%peg-0976PE2max129260.04%1179.50.00%peg-0980PE2max129300.12%2466.50.00%peg-0981PE2max129310.00%3285.50.01%peg-0982PE2max129320.07%14630.00%peg-0985PE2max129350.02%15620.11%peg-0987PE2max129370.03%1414.50.00%peg-0998PE2max129480.03%16320.00%peg-0999PE2max129490.05%1067.50.00%peg-1001PE2max129510.03%15950.00%peg-1007PE2max129560.05%20330.02%peg-1010PE2max129590.81%1175.50.65%peg-1015PE2max129630.04%1247.50.00%peg-1016PE2max129640.00%35480.03%peg-1019PE2max129670.27%537.50.00%peg-1024PE2max129720.06%1705.50.00%peg-1029PE2max129770.00%23790.02%peg-1032PE2max129800.27%1018.50.00%peg-1033PE2max129810.10%4700.00%peg-1041PE2max129890.20%1336.50.00%peg-1043PE2max129910.12%423.50.00%peg-1050PE2max129980.84%6720.00%peg-1051PE2max129990.02%21670.00%peg-1057PE2max130050.22%874.50.00%peg-1061PE2max130090.06%16690.00%peg-1069PE2max130170.07%4403.50.01%peg-1071PE2max130190.03%15220.00%peg-1073PE2max130210.11%13030.00%peg-1074PE2max130220.00%1624.50.03%peg-1077PE2max130250.13%1184.50.00%peg-1086PE2max130340.00%27870.02%peg-1091PE2max130390.00%2552.50.02%peg-1093PE2max130410.10%35580.02%peg-1095PE2max130430.07%846.50.00%peg-1096PE2max130440.07%1586.50.00%peg-1099PE2max130470.05%10060.00%peg-1103PE2max130510.03%2004.50.00%peg-1105PE2max130530.08%5920.00%peg-1108PE2max130560.00%40490.01%peg-1113PE2max130610.01%44990.00%peg-1114PE2max130620.02%59570.01%peg-1120PE2max130680.05%35340.03%peg-1121PE2max130690.01%1249.50.03...

Claims

1. pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, including: 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3' Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent, The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 216 to SEQ ID NO: 235, The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33, The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 15572 to SEQ ID NO: 15881, The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent, The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent, The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

2. pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, including: pegRNA for Prime Editor to correct the c.4957dupA variant of the EYS gene, represented by the following structure: 5'-[5'-end appendage]-[guide domain]-[scaffold]-[prime editor domain]-[linker]-[stabilizing motif]-[Poly-U]-3' Here, the sequence of the 5' terminal addition is 5'-G-3' or 5'-GG-3', or the 5' terminal addition is absent, The sequence of the above guide domain comprises a sequence selected from the group consisting of SEQ ID NO: 116 to SEQ ID NO: 135, The sequence of the above scaffold comprises a sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 33, The sequence of the above prime editor domain comprises a sequence selected from the group consisting of SEQ ID NO: 16243 to SEQ ID NO: 16265, The sequence of the above linker comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 75 and SEQ ID NO: 16219 to SEQ ID NO: 16241, or the linker is absent, The sequence of the above stabilizing motif comprises a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 16242, but the stabilizing motif is absent, The sequence of the above Poly-U is SEQ ID NO: 15535 to SEQ ID NO: 15538, or the sequence of Poly-U is absent.

3. Prime editing composition for correcting the c.4957dupA variant of the EYS gene, comprising: VRQR-PE2max prime editor protein, or a nucleic acid encoding said prime editor protein; and A pegRNA selected from any one of claims 1 to 2, or a nucleic acid encoding the pegRNA, Here, the scaffold of the pegRNA interacts with the prime editor protein to form a complex.

4. In the prime editing composition of paragraph 3, The above prime editing composition comprises a prime editor complex in which the VRQR-PE2max prime editor protein and the pegRNA are bound.

5. In the prime editing composition of paragraph 3, The above prime editing composition comprises a nucleic acid encoding the VRQR-PE2max prime editor protein and a nucleic acid encoding the pegRNA.

6. In the prime editing composition of paragraph 5, The nucleic acid encoding the VRQR-PE2max prime editor protein and the nucleic acid encoding the pegRNA are included in an expression vector, The above expression vector is configured to express the VRQR-PE2max prime editor protein and the pegRNA in a cell.

7. A pharmaceutical composition for treating retinitis pigmentosa caused by the c.c4957dupA mutation of the EYS gene, comprising: A prime editing composition selected from any one of claims 3 to 6; and Pharmaceutically acceptable carrier.

8. Treatment of retinitis pigmentosa caused by the c.c4957dupA mutation in the EYS gene, including: A process of administering to a patient a therapeutically effective amount of a prime editing composition selected from any one of claims 3 to 6.

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