Best1 gene editing method using crispr / cas system

Abstract

Disclosed in the present specification are a composition capable of editing an endogenous BEST1 gene in a cell genome by using a CRISPR / Cas system, and a gene editing method using same. The wild-type or mutant BEST1 gene can be edited using the composition and the editing method, and can be used in therapeutic strategies for various diseases associated with the BEST1 gene, such as macular dystrophy.

Description

BEST1 gene editing method using the CRISPR / CAS system

[0001] The invention disclosed in this specification relates to a technology for editing genes using the CRISPR / Cas system. The gene targeted for editing in this specification is the BEST1 gene, and mutations in the BEST1 gene are associated with various ocular diseases.

[0002]

[0003] Mutations in the BEST1 gene are associated with various ocular diseases. Examples include Best Vitelliform Macular Dystrophy (BVMD) and Autosomal Dominant VitReoretinoChoroidopathy (ADVIRC), which are autosomal dominant diseases. Another example is Autosomal Recessive Bestrophinopathy (ARB). According to US Publication 2023-0174974 A1, the CRISPR / Cas system was used to treat diseases such as Best Vitelliform Macular Dystrophy caused by mutations in the BEST1 gene. This article proposed a strategy for treating patients with both normal and mutant BEST1 genes. Specifically, to knockout the above mutant BEST1 gene, 1) a guide RNA targeting a specific mutant BEST1 gene, and 2) a guide RNA targeting an intron region common to both the wild-type BEST1 gene and the mutant BEST1 gene were used. This is a strategy based on the mechanism that both guide RNAs only work on the mutant BEST1 gene, thereby removing part of the mutant BEST1 gene. However, this method is inefficient because it requires delivering more than two types of guide RNAs to cells. In addition, since it only targets a specific mutant BEST1 gene, it does not cover various BEST1 gene mutation patients.

[0004] Therefore, new treatment strategies are needed.

[0005]

[0006] This specification presents a CRISPR / Cas system and editing method capable of editing the endogenous BEST1 gene within a cellular genome. The technical challenge of this specification is to identify a target sequence capable of editing even when the endogenous BEST1 gene sequence contains a single nucleotide polymorphism (SNP).

[0007]

[0008] To address the above technical challenges, this specification presents the CRISPR / Cas system, the effectiveness of which has been experimentally proven, and the BEST1 gene editing method using it.

[0009]

[0010] The CRISPR / Cas system and gene editing methods provided in this specification can be used to edit the endogenous BEST1 gene within a cell genome. For example, by introducing indels into the endogenous BEST1 gene, its function can be knocked out.

[0011]

[0012] Figure 1 shows the experimental results of indel introduction into the human endogenous BEST1 gene of the cellular genome using the CRISPR / SauriCas9 system according to Experimental Example 2.1. Each label on the horizontal axis represents each target sequence listed in Table 4, and the vertical axis is a numerical value representing the frequency of indel introduction as a percentage.

[0013] Figure 2 shows the results of confirming whether the expression level of the endogenous BEST1 gene was actually reduced by inducing indel introduction into the human endogenous BEST1 gene of the cell genome using the CRISPR / SauriCas9 system according to Experimental Example 2.2. A: Each label on the horizontal axis represents each target sequence listed in Table 4, and the vertical axis is a numerical value representing the frequency of indel introduction as a percentage. B: Each label on the horizontal axis represents each target sequence listed in Table 4, and the vertical axis is a numerical value representing the mRNA expression level for the endogenous BEST1 gene relative to the expression level of the control group (NT).

[0014] Figure 3 shows the experimental results of indel introduction into the human endogenous BEST1 gene of the cellular genome using the CRISPR / SpCas9 system according to Experimental Example 3.1. Each label on the horizontal axis represents each target sequence listed in Table 7, and the vertical axis is a numerical value representing the frequency of indel introduction as a percentage.

[0015] Figure 4 shows the results of inducing indel introduction into the human endogenous BEST1 gene of the cell genome using the CRISPR / SpCas9 system according to Experimental Example 3.2, and confirming whether the expression level of the endogenous BEST1 gene was actually reduced. A: Each label on the horizontal axis represents each target sequence listed in Table 4, and the vertical axis is a numerical value representing the frequency of indel introduction as a percentage. B: Each label on the horizontal axis represents each target sequence listed in Table 4, and the vertical axis is a numerical value representing the mRNA expression level for the endogenous BEST1 gene relative to the expression level of the control group (NT).

[0016]

[0017] 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 included within the scope of the claims.

[0018] This specification discloses a method for editing an endogenous BEST1 gene contained in the genome of a cell, comprising:

[0019] The process of introducing the CRISPR / Cas composition into the above cells,

[0020] wherein the CRISPR / Cas composition comprises:

[0021] Cas protein, or a nucleic acid encoding said Cas protein,

[0022] wherein the Cas protein is a Cas9 protein derived from Streptococcus pyogenes (SpCas9) or an engineered Staphylococcus aureus Cas9 protein (SauriCas9); and

[0023] a guide RNA, or a nucleic acid encoding said guide RNA;

[0024] Here, the guide RNA comprises a guide domain and a scaffold,

[0025] The above guide domain targets a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 17 and SEQ ID NO: 18 to SEQ ID NO: 43 within the sequence of the endogenous BEST1 gene,

[0026] The above scaffold interacts with the Cas protein to form an RNA-protein complex,

[0027] The above guide domain and the above scaffold are sequentially connected in the direction from the 5' end to the 3' end of the guide RNA.

[0028] In one embodiment, the guide domain of the guide RNA may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 95, and SEQ ID NO: 96 to SEQ ID NO: 121.

[0029] In one embodiment, the Cas protein and the scaffold may be selected from the following combinations:

[0030] A scaffold comprising an SpCas9 protein comprising an amino acid sequence of SEQ ID NO: 1 and a nucleic acid sequence of SEQ ID NO: 3; or

[0031] A scaffold comprising a SauriCas9 protein comprising an amino acid sequence of SEQ ID NO: 2 and a nucleic acid sequence of SEQ ID NO: 4.

[0032] In one embodiment, the CRISPR / Cas composition comprises the Cas protein and the guide RNA,

[0033] The above Cas protein and the above guide RNA can form an RNA-protein complex.

[0034] In one embodiment, the CRISPR / Cas composition comprises a CRISPR / Cas system component expression vector,

[0035] The above vector may comprise a nucleic acid encoding the Cas protein and a nucleic acid encoding the guide RNA.

[0036] In one embodiment, the CRISPR / Cas composition may comprise an mRNA encoding the Cas protein and the guide RNA.

[0037] In one embodiment, the performance of the above method may result in the generation of an indel in the endogenous BEST1 gene.

[0038]

[0039] 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.

[0040] Definition of Terms

[0041] approximately

[0042] The term "about" as used in this specification 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.

[0043] Amino acid sequence notation

[0044] Unless otherwise stated, amino acid sequences in this specification are written in the N-terminal to C-terminal direction using the amino acid single-letter notation or the three-letter notation. For example, the notation VSKN means a peptide in which valine, serine, lysine, and asparagine are sequentially linked from the N-terminal to the C-terminal. Another example, the notation Thr-Leu-Lys means a peptide in which threonine, leucine, and lysine are sequentially linked from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the above single-letter notation, other letters are used and additional explanations are provided.

[0045] The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Ty, Y); and Valine (Val, V).

[0046] Nucleic acid sequence notation

[0047] The symbols A, T, C, G, and U used in this specification are to be interpreted as having the 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. The sequence listing attached to this application follows the ST26 format proposed by the World Intellectual Property Organization (WIPO). Accordingly, even if this specification presents a nucleic acid sequence consisting of A, T, C, and G, it should be understood that it is designated as an RNA sequence if it is explicitly stated as such or if it is clear from the context that it refers to an RNA sequence. In this case, T in the sequence can be interpreted as U.

[0048] CRISPR / Cas system

[0049] generalization

[0050] The CRISPR / Cas system is an immune system found in prokaryotic organisms, comprising Cas proteins and guide RNAs. The detailed structure of Cas proteins and guide RNAs is described in detail in the public document WO2018 / 231018 (International Publication No. 2018 / 231018). The term "Cas protein" as used in this specification is a general term for nucleases that can be interpreted as being utilized in the CRISPR / Cas system. Below, the DNA cleavage process of the most commonly used CRISPR / Cas9 system is briefly described using an example.

[0051] Cas9 protein

[0052] In the CRISPR / Cas9 complex, the protein with nuclease activity that cleaves nucleic acids is called the Cas9 protein. The above Cas9 protein corresponds to Class 2, Type II in the CRISPR / Cas system classification, and includes Cas9 proteins derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, and Streptosporangium roseum. Of these, the most commonly used Cas9 protein for research purposes is the Cas9 protein derived from Streptococcus pyogenes.

[0053] guide RNA

[0054] In the CRISPR / Cas9 complex, the RNA that guides the CRISPR / Cas9 complex to recognize a specific sequence contained in the target nucleic acid is called a guide RNA. The structure of the guide RNA can be broadly divided into 1) a scaffold and 2) a guide domain. The scaffold is the part that enables the guide RNA to bind to the Cas9 protein and form a complex. Typically, the scaffold includes the tracrRNA and crRNA repeat sequences. The specific nucleic acid sequence of the scaffold is determined by the Cas9 protein that functions with it. The guide domain is a part that can complementarily bind to a certain length of nucleotide sequence in the target nucleic acid. The guide domain is a part that is designed according to the intention and is determined by the target nucleotide sequence of interest.

[0055] The process by which the CRISPR / Cas9 complex cuts the target nucleic acid

[0056] In order for the CRISPR / Cas9 complex to cleave the target nucleic acid, 1) the CRISPR / Cas9 complex must contact the target nucleic acid and the Cas9 protein must recognize a nucleotide sequence of a certain length, and 2) a portion of the guide RNA (the guide sequence portion above) must complementarily bind to a portion adjacent to the PAM sequence above.

[0057] At this time, the nucleotide sequence of a certain length recognized by the Cas9 protein is called a protospacer-adjacent motif (PAM). This is a sequence determined according to the type or origin of the Cas9 protein. For example, the Cas9 protein derived from Streptococcus pyogenes can recognize the 5'-NGG-3' sequence in the target nucleic acid. Here, the above N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G).

[0058] For the CRISPR / Cas9 complex to cleave a target nucleic acid, the guide domain of the guide RNA must complementarily bind to a sequence region adjacent to the PAM sequence. Therefore, the guide domain is determined to match the sequence of the target nucleic acid, specifically, the sequence region adjacent to the PAM sequence.

[0059] When the CRISPR / Cas9 complex cleaves the target nucleic acid, any position within the PAM sequence portion of the target nucleic acid and / or the sequence portion that complementarily binds to the guide sequence is cleaved.

[0060] Target strand, non-target strand

[0061] The CRISPR / Cas9 complex has cleavage activity against double-stranded DNA. In the double-stranded DNA, the strand that can bind and hybridize with the guide domain is called the target strand (TS). The strand complementary to the target strand, which contains a protospacer that does not bind to the guide domain, is called the non-target strand (NTS). The guide sequence portion can complementarily bind to a specific sequence contained in the target strand (TS) of the double-stranded DNA. The guide sequence and the protospacer sequence contained in the non-target strand (NTS) of the double-stranded DNA are equivalent sequences. Specifically, the only difference is that the guide sequence is an RNA sequence, and the protospacer sequence contained in the non-target strand (NTS) is the corresponding DNA sequence.

[0062] BEST1 gene and related diseases

[0063] BEST1 protein

[0064] Bestrophin-1 (BEST1) is a membrane protein found primarily in the retinal pigment epithelium (RPE). BEST1 is abundant in the basolateral cell membrane of the RPE. It functions as an intracellular calcium-activated chloride channel in the cell membrane. BEST1 consists of 585 amino acids, and both its N- and C-termini are located intracellularly.

[0065] Disease caused by mutations in the BEST1 gene

[0066] Mutations in the BEST1 gene are associated with various eye diseases. Examples include Best Vitelliform Macular Dystrophy (BVMD) and Autosomal Dominant VitReoretinoChoroidopathy (ADVIRC), which are autosomal dominant disorders. Another example is Autosomal Recessive Bestrophinopathy (ARB).

[0067] Conventional treatment strategies

[0068] As described above, many diseases caused by mutations in the BEST1 gene are autosomal dominant. According to US Publication No. 2023-0174974 A1, the CRISPR / Cas system was used to treat diseases such as Best's vitellogenic macular dystrophy caused by mutations in the BEST1 gene. The above document proposed a strategy for treating patients with both a normal and mutant BEST1 gene. Specifically, to knock out the mutant BEST1 gene, 1) a guide RNA targeting a specific mutant BEST1 gene and 2) a guide RNA targeting an intron region common to both the wild-type BEST1 gene and the mutant BEST1 gene were used. This strategy is based on the mechanism that both guide RNAs work only on the mutant BEST1 gene, thereby removing part of the mutant BEST1 gene. However, this method is inefficient because it requires delivering more than two types of guide RNAs to cells. Additionally, since it targets only a specific mutant BEST1 gene, it does not cover patients with various BEST1 gene mutations.

[0069] BEST1 gene editing method using the CRISPR / Cas system

[0070] Overview of the BEST1 gene editing method using the CRISPR / Cas system

[0071] This specification discloses a method for editing the BEST1 gene using the CRISPR / Cas system. The BEST1 gene editing method comprises delivering the CRISPR / Cas system to a cell. The purpose is to induce the CRISPR / Cas system to contact the genome of the cell. The CRISPR / Cas system, which forms a complex with the Cas protein and guide RNA, targets the endogenous BEST1 gene. Consequently, the CRISPR / Cas system acts on the endogenous BEST1 gene, thereby editing the endogenous BEST1 gene. A representative editing mode is the introduction of indels. This specification describes in detail the CRISPR / Cas system used in the method and a method for introducing the CRISPR / Cas system into a cell.

[0072] CRISPR / Cas system

[0073] The CRISPR / Cas system comprises a Cas protein and a guide RNA. This specification uses a CRISPR / Cas9 system derived from Streptococcus pyogenes (CRISPR / SpCas9 system) or an engineered CRISPR / Cas9 system derived from Staphylococcus aureus (CRISPR / SauriCas9 system). The CRISPR / Cas system comprises a Cas9 protein derived from Streptococcus pyogenes or an engineered Cas9 protein derived from Staphylococcus aureus. The guide RNA interacts with the Cas9 protein to form a complex, targeting a target sequence within the endogenous BEST1 gene. The CRISPR / Cas system can take various forms, such as an RNA-protein complex, a vector, or a composition. Each specific configuration is described in the "Possible Embodiments of the Invention" section.

[0074] How to Edit the BEST1 Gene Using the CRISPR / Cas System

[0075] This specification discloses a method for editing the BEST1 gene using the CRISPR / Cas system. This method involves introducing the CRISPR / Cas system into a cell. The specific introduction process may vary depending on how the CRISPR / Cas system is implemented. Once introduced into a cell, the CRISPR / Cas system contacts the cellular genome and edits the endogenous BEST1 gene. The specific details of this method are described in the "Possible Embodiments of the Invention" section.

[0076] Method for reducing the expression level of the endogenous BEST1 gene in a tissue using the CRISPR / Cas system

[0077] This specification discloses a method for reducing the expression of an endogenous BEST1 gene in a tissue using a CRISPR / Cas system. The endogenous BEST1 gene may be a wild-type BEST1 gene or a mutant BEST1 gene. Specifically, the mutant BEST1 gene may be a single nucleotide polymorphism (SNP) mutation. The method includes administering the CRISPR / Cas system to the tissue. The specific process may vary depending on how the CRISPR / Cas system is implemented. The CRISPR / Cas system administered to the tissue is delivered to each cell in the tissue and edits the endogenous BEST1 gene in each cell. Consequently, expression of the endogenous BEST1 gene is reduced throughout the tissue. The specific details of the method are described in the "Possible Embodiments of the Invention" section.

[0078] Feature #1 of the BEST1 gene editing method using the CRISPR / Cas system - Endogenous BEST1 gene targeting

[0079] The BEST1 gene editing method using the CRISPR / Cas system provided in this specification targets the endogenous BEST1 gene. The method is characterized by its ability to edit both wild-type and mutant BEST1 genes.

[0080] Feature #2 of the BEST1 gene editing method using the CRISPR / Cas system - Uses the proven effective CRISPR / Cas system.

[0081] The BEST1 gene editing method using the CRISPR / Cas system provided in this specification has been proven effective through practical experiments. While technological advancements have made it relatively easy to design CRISPR / Cas systems that target specific genes, using the CRISPR / Cas system provided in this specification to perform the above method allows access to the genome of the cell nucleus and editing of the actual BEST1 gene.

[0082] Feature #3 of the BEST1 gene-editing method using the CRISPR / Cas system - It can be used in disease treatment strategies mediated by the BEST1 gene.

[0083] The BEST1 gene editing method provided in this specification is characterized by its ability to edit both wild-type and mutant BEST1 genes without distinction. In other words, since the method is not dependent on a specific BEST1 gene mutation sequence, it can be applied to any patient. If the function of a patient's endogenous BEST1 gene can be disrupted through gene editing, it is easy to treat the related disease by delivering a normal BEST1 gene from outside. Therefore, the BEST1 gene editing method provided in this specification can be utilized to treat BEST1 gene-related diseases with appropriate additions. The characteristic of the method not being dependent on a specific BEST1 gene mutation sequence allows for the formation of a highly versatile treatment strategy.

[0084]

[0085] [Possible embodiments of the invention]

[0086] Cas protein

[0087] Example 1, specific Cas9 proteins

[0088] Cas9 protein, selected from:

[0089] Cas9 protein (SpCas9) derived from Streptococcus pyogenes or a variant thereof,

[0090] The above SpCas9 protein recognizes the 5'-NGG-3' protospacer adjacent motif (PAM); and,

[0091] Engineered Staphylococcus aureus-derived Cas9 protein (SauriCas9) or a variant thereof;

[0092] The above SauriCas9 protein recognizes the PAM of 5'-NNGG-3';

[0093] Here, the above N is A, T, C, or G.

[0094] Example 2, Sequence limitation

[0095] In Example 1,

[0096] If the above Cas9 protein is SpCas9, IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 1) contains the amino acid sequence,

[0097] If the above Cas9 protein is SauriCas9, it contains the amino acid sequence of KTIGKRVVLIEKYTTDILGNLYKTPLPKKPQLIFKRGEL (SEQ ID NO: 2).

[0098] Example 3, including NLS

[0099] In any one of Examples 1 to 2, the Cas9 protein further comprises one or more nuclear localization signals (NLS).

[0100] Example 4, NLS-containing structure

[0101] In Example 3, the above Cas9 protein comprises the following structure:

[0102] [NLS1] - [Cas9] - [NLS2]

[0103] Here, the above NLS1 is the first nuclear localization signal or is absent,

[0104] The above Cas9 is the Cas9 protein,

[0105] The above NLS2 is either a second nuclear localization signal or is absent,

[0106] However, this does not apply to cases where both NLS1 and NLS2 are absent.

[0107] Example 5, NLS sequence limitation

[0108] In either Example 3 or Example 4,

[0109] One or more of the above nuclear location signals are each independently selected from the following groups:

[0110] PKKKRKV (SEQ ID NO: 130) ;KRPAATKKAGQAKKKK (SEQ ID NO: 131) ;PAAKRVKLD (SEQ ID NO: 132) ;RQRRNELKRSP (SEQ ID NO: 133) ;NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 134) ;RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 135) ;VSRKRPRP (SEQ ID NO: 136) ;PPKKARED (SEQ ID NO: 137) ;PQPKKKPL (SEQ ID NO: 138) ;SALIKKKKKMAP (SEQ ID NO: 139) ;DRLRR (SEQ ID NO: 140) ;PKQKKRK (SEQ ID NO: 141) ;RKLKKKIKKL (SEQ ID NO: 142);REKKKFLKRR (SEQ ID NO: 143);KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144);and RKCLQAGMNLEARKTKK (SEQ ID NO: 145).

[0111] Target nucleic acid

[0112] Example 6, target nucleic acid in human BEST1 gene

[0113] target nucleic acid,

[0114] Here, the above target nucleic acid is all or part of the human BEST1 gene,

[0115] The above target nucleic acid is double-stranded DNA, comprising a first strand and a second strand,

[0116] The first and second strands above are complementary sequences,

[0117] The first strand above contains the nucleic acid sequence 5'-[protospacer]-[PAM]-3',

[0118] The second strand above contains the target sequence,

[0119] The above target sequence is a nucleic acid sequence complementary to the above protospacer.

[0120] Example 7, Exon Specificity

[0121] In Example 6,

[0122] The above target nucleic acid is all or part of exon 1, exon 2, or exon 3 of the human BEST1 gene.

[0123] Example 8, Protospacer and PAM Specificity

[0124] In any one of Examples 6 to 7,

[0125] The above protospacer sequence and PAM sequence are selected from the following group of combinations:

[0126] A nucleic acid sequence selected from among GCTTGTGTAAGTGATGGTCA (SEQ ID NO: 5), CACTTGGCTTGTGTAAGTGA (SEQ ID NO: 6), CATCACTTACACAAGCCAAG (SEQ ID NO: 7), CTAAGCGGGCATTAGCCACT (SEQ ID NO: 8), CAAGTGGCTAATGCCCGCTT (SEQ ID NO: 9), GCGGGAGAAGGAGCCTAAGC (SEQ ID NO: 10), AGCATCTACAAGCTGCTATA (SEQ ID NO: 11), GGATGATGTAGTAGCAGAGC (SEQ ID NO: 12), ACATCATCCGCTTTATTTAT (SEQ ID NO: 13), CCCAGGCTTCTACGTGACGC (SEQ ID NO: 14), ACCAGCGTCACGTAGAAGCC (SEQ ID NO: 15), CTCGTACTGGTTCCACCAGC (SEQ ID NO: 16), and ACCAGTACGAGAACCTGCCG (SEQ ID NO: 17). A protospacer, and a PAM of 5'-NGG-3'; and

[0127] ACCATCACTTACACAAGCCAA (SEQ ID NO: 18), GCCACTTGGCTTGTGTAAGTG (SEQ ID NO: 19), GCCAAGTGGCTAATGCCCGCT (SEQ ID NO: 20), GCCTAAGCGGGCATTAGCCAC (SEQ ID NO: 21), AGGCGGGAGAAGGAGCCTAAG (SEQ ID NO: 22), CAGCACAGCAGCAGGCGGGAG (SEQ ID NO: 23), CTTCTCCCGCCTGCTGCTGTG (SEQ ID NO: 24), TCCCGCCTGCTGCTGTGCTGG (SEQ ID NO: 25), CCCGCCTGCTGCTGTGCTGGC (SEQ ID NO: 26), CCCCGCCAGCACAGCAGCAGG (SEQ ID NO: 27), GCAGCATCTACAAGCTGCTAT (SEQ ID NO: 28), GCGGATGATGTAGTAGCAGAG (SEQ ID NO: 29), CGTGACGCTGGTCGTGACCCG (SEQ ID NO: 30), TTCTCGTACTGGTTCCACCAG (SEQ ID NO: 31), GTTCTCGTACTGGTTCCACCA (SEQ ID NO: 32), GAACCAGTACGAGAACCTGCC (SEQ ID NO: 33), GGGCCACGGCAGGTTCTCGTA (SEQ ID NO: 34), GGCTCATGAGGCGGTCGGGCC (SEQ ID NO: 35), TGGCCCGACCGCCTCATGAGC (SEQ ID NO: 36), ACACCAGGCTCATGAGGCGGT (SEQ ID NO: 37), GACACCAGGCTCATGAGGCGG (SEQ ID NO: 38), GACCGCCTCATGAGCCTGGTG (SEQ ID NO: 39), ACCGCCTCATGAGCCTGGTGT (SEQ ID NO: 40), GCCCGACACCAGGCTCATGAG (SEQ ID NO: 41), GAAGCCCGACACCAGGCTCAT (SEQ ID NO: 42), and a protospacer of a nucleic acid sequence selected from GCCTGGTGTCGGGCTTCGTCG (SEQ ID NO: 43), and a PAM of 5'-NNGG-3';

[0128] Here, the above N is A, T, C, or G.

[0129] Example 9, First-strand sequence specification

[0130] In any one of Examples 6 to 8,

[0131] The first strand above comprises a nucleic acid sequence selected from the group consisting of:

[0132] GCTTGTGTAAGTGATGGTCATGG (SEQ ID NO: 44), CACTTGGCTTGTGTAAGTGATGG (SEQ ID NO: 45), CATCACTTACACAAGCCAAGTGG (SEQ ID NO: 46), CTAAGCGGGCATTAGCCACTTGG (SEQ ID NO: 47), CAAGTGGCTAATGCCCGCTTAGG (SEQ ID NO: 48), GCGGGAGAAGGAGCCTAAGCGGG (SEQ ID NO: 49), AGCATCTACAAGCTGCTATATGG (SEQ ID NO: 50), GGATGATGTAGTAGCAGAGCAGG (SEQ ID NO: 51), ACATCATCCGCTTTATTTATAGG (SEQ ID NO: 52), CCCAGGCTTCTACGTGACGCTGG (SEQ ID NO: 53), ACCAGCGTCACGTAGAAGCCTGG (SEQ ID NO: 54), CTCGTACTGGTTCCACCAGCGGG (SEQ ID NO: 55), and A nucleic acid sequence selected from ACCAGTACGAGAACCTGCCGTGG (SEQ ID NO: 56); and

[0133] ACCATCACTTACACAAGCCAAGTGG (SEQ ID NO: 57), GCCACTTGGCTTGTGTAAGTGATGG (SEQ ID NO: 58), GCCAAGTGGCTAATGCCCGCTTAGG (SEQ ID NO: 59), GCCTAAGCGGGCATTAGCCACTTGG (SEQ ID NO: 60), AGGCGGGAGAAGGAGCCTAAGCGGG (SEQ ID NO: 61), CAGCACAGCAGCAGGCGGGAGAAGG (SEQ ID NO: 62), CTTCTCCCGCCTGCTGCTGTGCTGG (SEQ ID NO: 63), TCCCGCCTGCTGCTGTGCTGGCGGG (SEQ ID NO: 64), CCCGCCTGCTGCTGTGCTGGCGGGG (SEQ ID NO: 65), CCCCGCCAGCACAGCAGCAGGCGGG (SEQ ID NO: 66), GCAGCATCTACAAGCTGCTATATGG (SEQ ID NO: 67), GCGGATGATGTAGTAGCAGAGCAGG (SEQ ID NO: 68), CGTGACGCTGGTCGTGACCCGCTGG (SEQ ID NO: 69), TTCTCGTACTGGTTCCACCAGCGGG (SEQ ID NO: 70), GTTCTCGTACTGGTTCCACCAGCGG (SEQ ID NO: 71), GAACCAGTACGAGAACCTGCCGTGG (SEQ ID NO: 72), GGGCCACGGCAGGTTCTCGTACTGG (SEQ ID NO: 73), GGCTCATGAGGCGGTCGGGCCACGG (SEQ ID NO: 74), TGGCCCGACCGCCTCATGAGCCTGG (SEQ ID NO: 75), ACACCAGGCTCATGAGGCGGTCGGG (SEQ ID NO: 76), GACACCAGGCTCATGAGGCGGTCGG (SEQ ID NO: 77), GACCGCCTCATGAGCCTGGTGTCGG (SEQ ID NO: 78), A nucleic acid sequence selected from among ACCGCCTCATGAGCCTGGTGTCGGG (SEQ ID NO: 79), GCCCGACACCAGGCTCATGAGGCGG (SEQ ID NO: 80), GAAGCCCGACACCAGGCTCATGAGG (SEQ ID NO: 81), and GCCTGGTGTCGGGCTTCGTCGAAGG (SEQ ID NO: 82);

[0134] A contiguous nucleic acid sequence of 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, or 20nt within a nucleic acid sequence selected from SEQ ID NO: 44 to SEQ ID NO: 56; and

[0135] A contiguous nucleic acid sequence of 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, or 20nt within a nucleic acid sequence selected from SEQ ID NO: 57 to SEQ ID NO: 82.

[0136] Guide RNA targeting the BEST1 gene

[0137] Example 10, Guide RNA

[0138] Guide RNA containing a guide domain and a scaffold,

[0139] Here, the above guide domain targets the target nucleic acid of any one of Examples 6 to 9,

[0140] The above scaffold can interact with any one of the Cas9 proteins of Examples 1 to 5 to form a complex.

[0141] Example 11, Guide Domain Sequence Limitation

[0142] In Example 10, the above guide domain is a nucleic acid sequence selected from the following:

[0143] GCUUGUGUAAGUGAUGGUCA (SEQ ID NO: 83), CACUUGGCUUGUGUAAGUGA (SEQ ID NO: 84), CAUCACUUACACAAGCCAAG (SEQ ID NO: 85), CUAAGCGGGCAUUAGCCACU (SEQ ID NO: 86), CAAGUGGCUAAUGCCCGCUU (SEQ ID NO: 87), GCGGGAGAAGGAGCCUAAGC (SEQ ID NO: 88), AGCAUCUACAAGCUGCUAUA (SEQ ID NO: 89), GGAUGAUGUAGUAGCAGAGC (SEQ ID NO: 90), ACAUCAUCCGCUUUAUUUAU (SEQ ID NO: 91), CCCAGGCUUCUACGUGACGC (SEQ ID NO: 92), ACCAGCGUCACGUAGAAGCC (SEQ ID NO: 93), CUCGUACUGGUUCCACCAGC (SEQ ID NO: 94), and ACCAGUACGAGAACCUGCCG (SEQ ID NO: 95), and ACCAUCACUUACACAAGCCAA (SEQ ID NO: 96), GCCACUUGGCUUGUGUAAGUG (SEQ ID NO: 97), GCCAAGUGGCUAAUGCCCGCU (SEQ ID NO: 98), GCCUAAGCGGGCAUUAGCCAC (SEQ ID NO: 99), AGGCGGGAGAAGGAGCCUAAG (SEQ ID NO: 100), CAGCACAGCAGCAGGCGGGAG (SEQ ID NO: 101), CUUCUCCCGCCUGCUGCUGUG (SEQ ID NO: 102), UCCCGCCUGCUGCUGUGCUGG (SEQ ID NO: 103), CCCGCCUGCUGCUGUGCUGGC (SEQ ID NO: 104), CCCCGCCAGCACAGCAGCAGG (SEQ ID NO: 105), GCAGCAUCUACAAGCUGCUAU (SEQ ID NO: 106), GCGGAUGAUGUAGUAGCAGAG (SEQ ID NO: 107), CGUGACGCUGGUCGUGACCCG (SEQ ID NO: 108), UUCUCGUACUGGUUCCACCAG (SEQ ID NO: 109), GUUCUCGUACUGGUUCCACCA (SEQ ID NO: 110), GAACCAGUACGAGAACCUGCC (SEQ ID NO: 111), GGGCCACGGCAGGUUCUCGUA (SEQ ID NO: 112), GGCUCAUGAGGCGGUCGGGCC (SEQ ID NO: 113),A nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of UGGCCCGACCGCCUCAUGAGC (SEQ ID NO: 114), ACACCAGGCUCAUGAGGCGGU (SEQ ID NO: 115), GACACCAGGCUCAUGAGGCGG (SEQ ID NO: 116), GACCGCCUCAUGAGCCUGGUG (SEQ ID NO: 117), ACCGCCUCAUGAGCCUGGUGU (SEQ ID NO: 118), GCCCGACACCAGGCUCAUGAG (SEQ ID NO: 119), GAAGCCCGACACCAGGCUCAU (SEQ ID NO: 120), and GCCUGGUGUCGGGCUUCGUCG (SEQ ID NO: 121);

[0144] A nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 95, and SEQ ID NO: 96 to SEQ ID NO: 121; and

[0145] A contiguous nucleic acid sequence of 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, or 20nt selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 95, and SEQ ID NO: 96 to SEQ ID NO: 121.

[0146] Example 12: Meaning of targeting

[0147] In any one of Examples 10 to 11,

[0148] The meaning of targeting the above target nucleic acid is selected from the group consisting of:

[0149] Can be hybridized with the second strand of the above target nucleic acid;

[0150] Having a sequence that is equivalent to, identical to, matched with, homologous to, or identical to the protospacer of the target nucleic acid by at least N%;

[0151] Having a sequence complementary to the target sequence of the above target nucleic acid by at least M%; and

[0152] The target nucleic acid above has L or fewer mismatches with the target sequence,

[0153] Here, the above N is about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100,

[0154] The above M is about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100,

[0155] The above L is 0, 1, 2, 3, 4, or 5.

[0156] Example 13, Scaffold

[0157] In any one of Examples 10 to 12,

[0158] The above scaffold contains a nucleic acid sequence selected from the group consisting of FALSE.

[0159] Example 14, Scaffold-like sequence

[0160] In Example 13,

[0161] The above scaffold is a sequence that is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of No entry found.

[0162] CRISPR / Cas complex

[0163] Example 15, CRISPR / Cas complex

[0164] CRISPR / Cas complexes containing:

[0165] The Cas9 protein of any one of Examples 1 to 5; and

[0166] The guide RNA of any one of Examples 10 to 14.

[0167] Here, the scaffold of the above guide RNA interacts with the above Cas9 protein to form a complex.

[0168] Example 16, SpCas9

[0169] In Example 15,

[0170] The above Cas9 protein is a Cas9 protein (SpCas9) derived from Streptococcus pyogenes or a variant thereof,

[0171] The scaffold of the above guide RNA comprises a nucleic acid sequence selected from among SEQ ID NO: 3,

[0172] The first strand of the target nucleic acid of the above guide RNA is GCTTGTGTAAGTGATGGTCA (SEQ ID NO: 5), CACTTGGCTTGTGTAAGTGA (SEQ ID NO: 6), CATCACTTACACAAGCCAAG (SEQ ID NO: 7), CTAAGCGGGCATTAGCCACT (SEQ ID NO: 8), CAAGTGGCTAATGCCCGCTT (SEQ ID NO: 9), GCGGGAGAAGGAGCCTAAGC (SEQ ID NO: 10), AGCATCTACAAGCTGCTATA (SEQ ID NO: 11), GGATGATGTAGTAGCAGAGC (SEQ ID NO: 12), ACATCATCCGCTTTATTTAT (SEQ ID NO: 13), CCCAGGCTTCTACGTGACGC (SEQ ID NO: 14), ACCAGCGTCACGTAGAAGCC (SEQ ID NO: 15), CTCGTACTGGTTCCACCAGC (SEQ ID NO: 16), and A protospacer comprising a nucleic acid sequence selected from among ACCAGTACGAGAACCTGCCG (SEQ ID NO: 17) and a PAM of 5'-NGG-3'.

[0173] Example 17, SpCas9, guide domain limited

[0174] In Example 16, the guide domain of the above guide RNA comprises a nucleic acid sequence selected from SEQ ID NO: 83 to SEQ ID NO: 95.

[0175] Example 18, SauriCas9

[0176] In Example 15,

[0177] The above Cas9 protein is an engineered Staphylococcus aureus-derived Cas9 protein (SauriCas9) or a variant thereof,

[0178] The scaffold of the above guide RNA comprises a nucleic acid sequence selected from among SEQ ID NO: 4,

[0179] The first strand of the target nucleic acid of the above guide RNA is ACCATCACTTACACAAGCCAA (SEQ ID NO: 18), GCCACTTGGCTTGTGTAAGTG (SEQ ID NO: 19), GCCAAGTGGCTAATGCCCGCT (SEQ ID NO: 20), GCCTAAGCGGGCATTAGCCAC (SEQ ID NO: 21), AGGCGGGAGAAGGAGCCTAAG (SEQ ID NO: 22), CAGCACAGCAGCAGGCGGGAG (SEQ ID NO: 23), CTTCTCCCGCCTGCTGCTGTG (SEQ ID NO: 24), TCCCGCCTGCTGCTGTGCTGG (SEQ ID NO: 25), CCCGCCTGCTGCTGTGCTGGC (SEQ ID NO: 26), CCCCGCCAGCACAGCAGCAGG (SEQ ID NO: 27), GCAGCATCTACAAGCTGCTAT (SEQ ID NO: 28), GCGGATGATGTAGTAGCAGAG (SEQ ID NO: 29), CGTGACGCTGGTCGTGACCCG (SEQ ID NO: 30), TTCTCGTACTGGTTCCACCAG (SEQ ID NO: 31), GTTCTCGTACTGGTTCCACCA (SEQ ID NO: 32), GAACCAGTACGAGAACCTGCC (SEQ ID NO: 33), GGGCCACGGCAGGTTCTCGTA (SEQ ID NO: 34), GGCTCATGAGGCGGTCGGGCC (SEQ ID NO: 35), TGGCCCGACCGCCTCATGAGC (SEQ ID NO: 36), ACACCAGGCTCATGAGGCGGT (SEQ ID NO: 37), GACACCAGGCTCATGAGGCGG (SEQ ID NO: 38), GACCGCCTCATGAGCCTGGTG (SEQ ID NO: 39), ACCGCCTCATGAGCCTGGTGT (SEQ ID NO: 40), GCCCGACACCAGGCTCATGAG (SEQ ID NO: 41), GAAGCCCGACACCAGGCTCAT (SEQ ID NO: 42), and a protospacer of a nucleic acid sequence selected from GCCTGGTGTCGGGCTTCGTCG (SEQ ID NO: 43), and a PAM of 5'-NNGG-3'.

[0180] Example 19, SauriCas9, Guide Domain Limited

[0181] In Example 18, the guide domain of the above guide RNA comprises a nucleic acid sequence selected from SEQ ID NO: 96 to SEQ ID NO: 121.

[0182] Example 20, Complex Alternative Expression

[0183] In any one of Examples 15 to 19, the CRISPR / Cas complex is replaced with a selected expression from the following:

[0184] RNA-protein complex; ribonucleoprotein (RNP); and RNA-guided endonuclease.

[0185] CRISPR / Cas component expression vector

[0186] Example 21, component expression vector

[0187] CRISPR / Cas component expression vectors containing:

[0188] A nucleic acid encoding the Cas9 protein of any one of Examples 1 to 5; and

[0189] A nucleic acid encoding the guide RNA of any one of Examples 10 to 14.

[0190] Example 22, vector types

[0191] In Example 21, the above CRISPR / Cas component expression vector is a non-viral vector or a viral vector.

[0192] Example 23, nonviral vector type

[0193] In Example 22, the above non-viral vector is selected from among a plasmid, a phage, naked DNA, a DNA complex, mRNA, and a PCR amplicon.

[0194] Example 24, Type of Viral Vector

[0195] In Example 22, the viral vector is selected from among retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus.

[0196] Example 25, adeno-associated virus

[0197] In Example 24, the above viral vector is an adeno-associated virus, and is a serotype selected from the following:

[0198] Wild type serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10;

[0199] Synthetic (variant) serotypes such as AAV-DJ, AAV-DJ8, AAV-DJ9, and AAV6.2; and

[0200] Other AAV variant serotypes.

[0201] Example 26, single vector

[0202] In any one of Examples 21 to 25, the nucleic acid encoding the above Cas9 protein and the nucleic acid encoding the above guide RNA are contained in one nucleic acid molecule.

[0203] Example 27, Two or more vectors

[0204] In any one of Examples 21 to 26, the nucleic acid encoding the Cas9 protein and the nucleic acid encoding the guide RNA are each included in the nucleic acid of the molecule.

[0205] Example 28, including promoter

[0206] In any one of Examples 21 to 27, the vector comprises one or more promoters.

[0207] Example 29, Promoter, Operably Linked

[0208] In Example 28, the above vector comprises two or more promoters,

[0209] One of which is operably linked to a nucleic acid encoding the above Cas9 protein,

[0210] The other is operably linked to a nucleic acid encoding the above guide RNA.

[0211] Example 30, Promoter Only

[0212] In Example 28 or Example 29, the above promoters are each independently selected from the following groups:

[0213] SV40 early promoter; mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE); rous sarcoma virus (RSV) promoter; human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)); enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1;31(17)); and human H1 promoter (H1).

[0214] Example 31, Other Vector Configurations

[0215] In any one of Examples 21 to 30, the above vector further comprises a selected configuration from the following:

[0216] An enhancer; an intron; a polyadenylation signal; a Kozak consensus sequence; an internal ribosome entry site (IRES); a splice acceptor; a 2A sequence; an origin of replication; or a suitable combination of the aforementioned components.

[0217] Example 32, nucleic acid type

[0218] In any one of Examples 21 to 31, the above nucleic acids are each independently DNA or RNA.

[0219] CRISPR / Cas composition

[0220] Example 33, composition

[0221] CRISPR / Cas composition comprising:

[0222] The Cas9 protein of any one of Examples 1 to 5, or a nucleic acid encoding the Cas9 protein; and

[0223] The guide RNA of any one of Examples 10 to 14, or a nucleic acid encoding the guide RNA,

[0224] Here, the scaffold of the above Cas9 protein and the above guide RNA can interact to form a complex.

[0225] Example 34, including complex

[0226] In Example 33, the CRISPR / Cas composition comprises a CRISPR / Cas complex of any one of Examples 15 to 20.

[0227] Example 35, including vectors

[0228] In Example 33, the above CRISPR / Cas composition is included in the form of any one of the vectors of Examples 21 to 32.

[0229] Example 36, Cas9 encoding nucleic acid and guide RNA

[0230] In Example 33, the above CRISPR / Cas composition comprises a nucleic acid encoding the above Cas9 protein and the above guide RNA.

[0231] Example 37, nucleic acid limitation

[0232] In any one of Examples 33 to 36, the above nucleic acids are each independently DNA or RNA.

[0233] How to Edit the BEST1 Gene

[0234] Example 38, BEST1 gene editing method

[0235] Methods for editing the BEST1 gene in cells, including:

[0236] The process of introducing the CRISPR / Cas system into the cells above,

[0237] Here, the above CRISPR / Cas system is a CRISPR / Cas complex of any one of Examples 15 to 20, a vector of any one of Examples 21 to 32, or a composition of any one of Examples 33 to 37.

[0238] Example 39, Cell-limited

[0239] In Example 38, the above cells are human cells.

[0240] Example 40, Isolated Cells

[0241] In any one of Examples 38 to 39, the above cells are isolated human cells.

[0242] Example 41, BEST1 gene type limitation

[0243] In any one of Examples 38 to 40, the BEST1 gene is a wild-type BEST1 gene or a mutant BEST1 gene.

[0244] Example 42, mutant BEST1 gene

[0245] In Example 41, the above mutant BEST1 gene includes a single nucleotide polymorphism (SNP).

[0246] Example 43, SNP-limited

[0247] In Example 42, the single nucleotide polymorphism included in the BEST1 gene of the above mutation is selected from the group consisting of:

[0248] rs1800009; rs2524294; rs909268; rs2668898; rs972355; rs972353; rs2736597; rs1800007; rs760306; rs974121; rs168991; rs195161; rs149698; rs1534842; rs3758976; rs1800008; rs195158; rs195157; rs195156; rs2009875; rs2955684; rs2955683; rs17185413; rs972354; rs195163; rs2668897; rs1109748; rs195160; rs183176; rs195167; rs195165; rs195164; rs2736594; rs195162; rs113492158; rs195166; rs741886; rs2736596; rs1801621; rs17156609; rs1534843; rs73491300; rs74754540; rs112769638; rs74369809; rs78054615; rs144630276; rs141507235; rs114944671; rs1801327; rs78012644; rs112665957; rs139745332; rs77543508; rs78545127; rs116516743; rs1805140; rs73493205; rs77651946; rs195159; rs195155; rs2727272; rs2668899; rs2736595; rs56215258; rs174481; rs168990; rs111509315; rs1735379; and rs112720784;

[0249] Here, each of the above lists the RSIDs from NCBI's 2018 database of Single Nucleotide Polymorphisms (dbSNP).

[0250] Example 44, Gene Editing Environment

[0251] In any one of Examples 38 to 43, the gene editing method is performed in vitro, in vivo, or ex vivo.

[0252] Example 45, Delivery Method

[0253] In any one of Examples 38 to 44, the process of introducing the CRISPR / Cas system into the above cell is performed by one or more methods selected from the group consisting of:

[0254] Electroporation; gene gun; sonoporation; magnetofection; nanoparticle method; transient cell compression or squeezing method; microinjection; cationic liposome method; lithium acetate-DMSO; lipid-mediated transfection; calcium phosphate precipitation; lipofection; PEI (polyethyleneimine)-mediated transfection; DEAE-dextran-mediated transfection; and nanoparticle-mediated nucleic acid delivery (see Panyam et al., Adv Drμg Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023).

[0255] Example 46, Gene Editing Alternative Expression

[0256] In any one of Examples 38 to 45, "BEST1 gene editing" is replaced with an expression selected from the group consisting of:

[0257] Artificial manipulation of the BEST1 gene; introduction of indels into the BEST1 gene; knockout of the BEST1 gene; and disruption of the BEST1 gene.

[0258] Example 47, Alternative Expression for "Introduce"

[0259] In any one of Examples 38 to 46, the verb "introduce" is replaced with an expression selected from the group consisting of:

[0260] Treat; administer; inject; insert; implant; deliver; and transfect.

[0261] Methods for reducing the expression of the endogenous BEST1 gene in tissues

[0262] Example 48, reduction of endogenous BEST1 gene expression

[0263] A method for reducing the expression level of an endogenous BEST1 gene in a tissue, comprising:

[0264] The process of treating the above tissues with the CRISPR / Cas system,

[0265] Here, the above CRISPR / Cas system is a CRISPR / Cas complex of any one of Examples 15 to 20, a vector of any one of Examples 21 to 32, or a composition of any one of Examples 33 to 37.

[0266] Example 49, Organizational Limitation

[0267] In Example 48, the above tissue is ocular tissue.

[0268] Example 50, Endogenous BEST1 Gene Limitation

[0269] In any one of Examples 48 to 49,

[0270] The endogenous BEST1 gene above is either a wild-type BEST1 gene or a mutant BEST1 gene.

[0271] Example 51, limited to mutant BEST1 gene

[0272] In Example 50,

[0273] The above mutant BEST1 gene contains a single nucleotide polymorphism (SNP).

[0274] Example 52, single nucleotide polymorphism limitation

[0275] In Example 51, the single nucleotide polymorphism included in the BEST1 gene of the above mutation is selected from the group consisting of:

[0276] rs1800009; rs2524294; rs909268; rs2668898; rs972355; rs972353; rs2736597; rs1800007; rs760306; rs974121; rs168991; rs195161; rs149698; rs1534842; rs3758976; rs1800008; rs195158; rs195157; rs195156; rs2009875; rs2955684; rs2955683; rs17185413; rs972354; rs195163; rs2668897; rs1109748; rs195160; rs183176; rs195167; rs195165; rs195164; rs2736594; rs195162; rs113492158; rs195166; rs741886; rs2736596; rs1801621; rs17156609; rs1534843; rs73491300; rs74754540; rs112769638; rs74369809; rs78054615; rs144630276; rs141507235; rs114944671; rs1801327; rs78012644; rs112665957; rs139745332; rs77543508; rs78545127; rs116516743; rs1805140; rs73493205; rs77651946; rs195159; rs195155; rs2727272; rs2668899; rs2736595; rs56215258; rs174481; rs168990; rs111509315; rs1735379; and rs112720784;

[0277] Here, each of the above lists the RSIDs from NCBI's 2018 database of Single Nucleotide Polymorphisms (dbSNP).

[0278] Example 53, Alternative expression of reduced expression level

[0279] In any one of Examples 38 to 47, "reducing the expression level of the endogenous BEST1 gene" is replaced with an expression selected from the group consisting of:

[0280] Suppresses the expression of the endogenous BEST1 gene; reduces the activity of the endogenous BEST1 gene; prohibits the expression of the endogenous BEST1 gene; attenuates the expression of the endogenous BEST1 gene; restricts the expression of the endogenous BEST1 gene; blocks the expression of the endogenous BEST1 gene; downregulates the expression of the endogenous BEST1 gene; and silences the expression of the endogenous BEST1 gene.

[0281] Example 54, alternative expression for processing

[0282] In any one of Examples 48 to 53, the verb "process" is replaced with an expression selected from the group consisting of:

[0283] introduce; administer; inject; insert; implant; and deliver.

[0284] Uses of the CRISPR / Cas System

[0285] Example 55, Use of the CRISPR / Cas System #1 - BEST1 Gene Editing Use

[0286] Use of the CRISPR / Cas system in any one of the methods of Examples 38 to 47,

[0287] Here, the above CRISPR / Cas system is a CRISPR / Cas complex of any one of Examples 15 to 20, a vector of any one of Examples 21 to 32, or a composition of any one of Examples 33 to 37.

[0288] Example 56, Use of the CRISPR / Cas System #2 - Use to Reduce Endogenous BEST1 Gene Expression

[0289] Use of the CRISPR / Cas system in any one of the methods of Examples 48 to 54,

[0290] Here, the above CRISPR / Cas system is a CRISPR / Cas complex of any one of Examples 15 to 20, a vector of any one of Examples 21 to 32, or a composition of any one of Examples 33 to 37.

[0291]

[0292] [Experimental Example]

[0293] The invention provided by this specification is described in more detail below 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 these examples are not intended to limit the scope of the subject matter disclosed by this specification.

[0294] Experimental Example 1. Experimental Method and Materials

[0295] Experimental Example 1.1. Production of a Vector Expressing CRISPR / Cas Components

[0296] An adeno-associated virus vector capable of expressing the SauriCas9 protein and guide RNA was prepared using the following method:

[0297] 1) The codon-optimized SauriCas9 protein sequence and the EFS promoter sequence capable of expressing it were inserted between the inverted tandem repeats (ITRs) of the AAV2 vector using Gibson assembly.

[0298] 2) Guide RNA was inserted through oligo cloning using the BspQ1 site.

[0299] Experimental Example 1.2. Preparation of CRISPR / Cas Complex

[0300] CRISPR / Cas complex was prepared by mixing 4 μg of SpCas9 protein (enzynomics' spCas9 nuclease M058H) and single guide RNA (synthego, 2.8 μg) for 5 minutes.

[0301] Experimental Example 1.3. Cell Culture

[0302] HEK293 (CRL-1573, ATCC) was maintained in 2 subcultures / 1W using Dulbecco's Modified Eagle Medium (DMEM) (WelGene) supplemented with 10% fetal calf serum (WelGene) and 1H penicillin / streptomycin (WelGene).

[0303] Experimental Example 1.4. Transfection #1 - Vector Transfection

[0304] Cells cultured according to Experimental Example 1.3 were cultured in DMEM containing 10% FBS and 1% P / S at 37°C and 5% CO2. The above cells were seeded at 1X10 per well in a 24-well plate. 5 The cells were cultured for 16 hours after inoculation. Afterwards, the cells were treated with a mixture of 2 μL of Lipofectamine® 2000, 1 μg of the vector according to Experimental Example 1.1, and 100 μL of Opti-MEM medium. After 6 hours, additional medium was added to reduce cytotoxicity. Genomic DNA was isolated from the cells 48 hours later.

[0305] Experimental Example 1.5. Transfection #2 - RNP Transfection

[0306] Cells cultured according to Experimental Example 1.3 were cultured in DMEM containing 10% FBS and 1% P / S at 37°C and 5% CO2. The CRISPR / Cas complex according to Experimental Example 1.2 was transfected into the above cells using Neon TM Electroporation was performed using the Transfection System 10 μL kit. 1.2 x 10 cells per well in a 24-well plate per transfection. 5 Electroporation was performed under cell conditions. The electroporation conditions were set at 1150 V / 20 ms / 2 pulses. The transfection process was performed according to the manufacturer's protocol. Genomic DNA was isolated from the cells after 48 hours.

[0307] Experimental Example 1.6. qRT-PCR

[0308] A total of 100–1,000 ng of extracted RNA was used to produce cDNA using a cDNA Reverse-transcription kit (ThermoFisher). qRT-PCR was performed with 10–15 ng of cDNA using SYBR Green Master Mix in QuantStudio 5 according to the manufacturer's protocol (ThermoFisher). BEST1 expression levels were calculated using CT values, and hGAPDH was used as an internal control. The primer sequences used are shown in the table below.

[0309] Primer nameSequence (5'-3')SEQ ID NOBEST1_FGGCAGAACACAAGCAGTTGG122BEST1_RGTGTCCACACTGAGTACGCA123hGAPDH_FGTCTCCTCTGACTTCAACAGCG124hGAPDH_RACCACCCTGTTGCTGTAGCCAA125

[0310] Experimental Example 1.7. Indel Analysis (Targeted Deep Sequencing)

[0311] HEK293 cells to be sequenced were centrifuged (2500 rpm, 5 minutes) and genomic DNA was isolated using a genomic DNA extraction kit (Favorgene). Genomic DNA isolated according to Experimental Example 1.4 or Experimental Example 1.5 was amplified and labeled to read only the sequences through two PCRs, and the PCR products were analyzed using an illumina mini-seq instrument. The PCR reaction mixture consisted of 10 μL of SUN-PCR Blend (SUN GENETICS, SG-PT02), 0.5 μL of forward primer (10 pmole), 0.5 μL of reverse primer (10 pmole), 2 μL of genomic DNA, and 7 μL of distilled water, making a total volume of 20 μL. PCR was performed at 95°C for 2 minutes to allow the complementary bonds of the entire DNA to be broken, followed by 35 cycles of 95°C for 30 seconds, 58°C for 20 seconds, and 72°C for 20 seconds, and then 72°C for 5 minutes. The resulting reads were analyzed using Cas9-Analyzer of CRISPR / RGEN Tools (www.rgenome.net) to quantitatively count insertion or deletion (Indels) reads at the on-target genomic location, and the gene correction efficiency by the vector was compared and evaluated.

[0312] The primer sequences used for targeted deep sequencing are listed in the table below:

[0313] namesequence (5' to 3')SEQ ID NOhBEST1_E1_DF1ACACTCTTTTCCCTACACGACGCTCTTCCGATCTCTCTCTACCAGGACCCAAGC126hBEST1_E1_DR1GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCACATCCTTCCCAGG12 7hBEST1_E3_DF1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCCGCTCGCAGCAGAAAGC128hBEST1_E3_DR1GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAGCGTGCCGCCGCAGCAGC129

[0314] Experimental Example 2. BEST1 Gene Editing Using CRISPR / SauriCas9

[0315] Experimental Example 2.1. Confirming the Efficiency of BEST1 Gene Editing Using CRISPR / SauriCas9

[0316] The CRISPR / SauriCas9 component expression vector manufactured according to Experimental Example 1.1 was transfected into cells according to Experimental Example 1.3 according to Experimental Example 1.4 to induce editing of the BEST1 gene. The indel introduction efficiency was analyzed according to Experimental Example 1.7.

[0317] The composition of CRISPR / SauriCas9 used in the experiment is shown in the following table:

[0318] Cas ProteinAmino acid sequenceSauriCas9KKTIGKRVVLIEKYTTDILGNLYKTPLPKKPQLIFKRGEL(SEQ ID NO: 2)

[0319]

[0320] The underlined portion of the above target sequence represents the PAM sequence. The experimental results are shown in Fig. 1. The experimental results confirmed that a certain level of indel introduction effect was observed for a significant number of target sequences.

[0321] Experimental Example 2.2. Confirmation of BEST1 gene editing efficiency and reduced RNA expression using CRISPR / SauriCas9.

[0322] In Experimental Example 2.1, guide RNAs showing high indel introduction efficiency were selected and transfected into cells using the same method to confirm gene editing efficiency and reduction in BEST1 mRNA expression.

[0323] The composition of CRISPR / SauriCas9 used in the experiment is shown in the following table:

[0324] SauriCas9 protein: Proteins listed in Table 3

[0325] Guide RNA used in Experimental Example 2.2 NameTarget siteTarget Sequence with PAM (5'-3')SEQ ID NOSg14Sauri-hBEST1-Exon3-#02TTCTCGTACTGGTTCCACCAGCGGG70Sg15Sauri-hBEST1-Exon3-#03GTTCTCGTACTGGTTCCACCAGCGG71Sg16Sauri-hBEST1-Exon3-#04GAACCAGTACGAGAACCTGCCGTGG72Sg17Sauri-hBEST1-Exon3-#05GGGCCACGGCAGGTTCTCGTACTGG73

[0326] The underlined portion of the target sequence above represents the PAM sequence. The experimental results are shown in Figure 2. The experimental results showed that when an indel was introduced into the BEST1 gene, mRNA for the BEST1 gene decreased. This indicates that an indel was introduced into the BEST1 gene, and thus, its expression level decreased.

[0327] Experimental Example 3. BEST1 Gene Editing Using CRISPR / SpCas9

[0328] Experimental Example 3.1. Confirming the Efficiency of BEST1 Gene Editing Using CRISPR / SpCas9

[0329] The CRISPR / SpCas9 complex prepared according to Experimental Example 1.2 was transfected into cells according to Experimental Example 1.3 according to Experimental Example 1.5 to induce editing of the BEST1 gene. The indel introduction efficiency was analyzed according to Experimental Example 1.7.

[0330] The composition of CRISPR / SpCas9 used in the experimental example is shown in the following table:

[0331] Cas ProteinAmino acid sequenceSpCas9IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO: 1)

[0332] Guide RNA sequence used in Experimental Example 3.1 NameTarget siteTarget Sequence with PAM (5'-3')SEQ ID NOSg1Sp_hBEST1_Exon1_#01GCTTGTGTAAGTGATGGTCATGG44Sg2Sp_hBEST1_Exon1_#02CACTTGGCTTGTGTAAGTGATGG45Sg3Sp_hBEST1_Exon1_#03CATCACTTACACAAGCCAAGTGG46Sg4Sp_hBEST1_Exon1_#04CTAAGCGGGCATTAGCCACTTGG47Sg5Sp_hBEST1_Exon1_#05CAAGTGGCTAATGCCCGCTTAGG48Sg6Sp_hBEST1_Exon1_#06GCGGGAGAAGGAGCCTAAGCGGG49Sg7Sp_hBEST1_Exon1_#07AG CATCTACAAGCTGCTATATGG50Sg8Sp_hBEST1_Exon1_#08GGATGATGTAGTAGCAGAGCAGG51Sg9Sp_hBEST1_Exon1_#09ACATCATCCGCTTTATTTATAGG52Sg10Sp_hBEST1_Exon3_#17CCCAGGCTTCTACG TGACGCTGG53Sg11Sp_hBEST1_Exon3_#18ACCAGCGTCACGTAGAAGCCTGG54Sg12Sp_hBEST1_Exon3_#19CTCGTACTGGTTCCACCAGCGGG55Sg13Sp_hBEST1_Exon3_#20ACCAGTACGAGAACCTGCCGTGG56

[0333] The underlined portion of the above target sequence represents the PAM sequence. The experimental results are shown in Fig. 3. The experimental results confirmed that a certain level of indel introduction effect was observed for a significant number of target sequences.

[0334] Experimental Example 3.2. Confirmation of BEST1 gene editing efficiency and reduced RNA expression using CRISPR / SpCas9.

[0335] In Experimental Example 3.1, guide RNAs showing high indel introduction efficiency were selected and transfected into cells using the same method to confirm gene editing efficiency and reduction in BEST1 mRNA expression.

[0336] The composition of CRISPR / SpCas9 used in the experiment is shown in the following table:

[0337] SpCas9 protein: Proteins listed in Table 6

[0338] Guide RNA used in Experimental Example 3.2 NameTarget siteTarget Sequence with PAM (5'-3')SEQ ID NOSg2Sp_hBEST1_Exon1_#02CACTTGGCTTGTGTAAGTGATGG45Sg7Sp_hBEST1_Exon1_#07AGCATCTACAAGCTGCTATATGG50Sg9Sp_hBEST1_Exon1_#09ACATCATCCGCTTTATTTATAGG52

[0339] The underlined portion of the target sequence above represents the PAM sequence. The experimental results are shown in Figure 4. The experimental results showed that when an indel was introduced into the BEST1 gene, mRNA for the BEST1 gene decreased. This indicates that an indel was introduced into the BEST1 gene, and thus, its expression level decreased.

[0340]

[0341] This specification discloses a composition capable of editing the endogenous BEST1 gene within a cellular genome using the CRISPR / Cas system, and a gene editing method using the same. The composition and editing method can be used to edit a wild-type or mutant BEST1 gene, and this can be utilized in therapeutic strategies for various diseases associated with the BEST1 gene, such as macular dystrophy. For example, it can be utilized in a therapeutic strategy in which the endogenous BEST1 gene, regardless of whether it is mutant or wild-type, is knocked out to lose its function, and then a normal BEST1 gene is separately introduced.

Claims

1. A method for editing an endogenous BEST1 gene contained in the genome of a cell, comprising: The process of introducing the CRISPR / Cas composition into the above cells, wherein the CRISPR / Cas composition comprises: Cas protein, or a nucleic acid encoding said Cas protein, wherein the Cas protein is a Cas9 protein derived from Streptococcus pyogenes (SpCas9) or an engineered Staphylococcus aureus Cas9 protein (SauriCas9); and a guide RNA, or a nucleic acid encoding said guide RNA; Here, the guide RNA comprises a guide domain and a scaffold, The above guide domain targets a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 17 and SEQ ID NO: 18 to SEQ ID NO: 43 within the sequence of the endogenous BEST1 gene, The above scaffold interacts with the Cas protein to form an RNA-protein complex, The above guide domain and the above scaffold are sequentially connected in the direction from the 5' end to the 3' end of the guide RNA.

2. In a method for editing an endogenous BEST1 gene included in the genome of a cell of paragraph 1, The guide domain of the above guide RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 95, and SEQ ID NO: 96 to SEQ ID NO:

121.

3. A method for editing an endogenous BEST1 gene contained in the genome of any one of the cells selected from clauses 1 and 2, The above Cas protein and the above scaffold are selected from the following combinations: A scaffold comprising an SpCas9 protein comprising an amino acid sequence of SEQ ID NO: 1 and a nucleic acid sequence of SEQ ID NO: 3; or A scaffold comprising a SauriCas9 protein comprising an amino acid sequence of SEQ ID NO: 2 and a nucleic acid sequence of SEQ ID NO:

4.

4. A method for editing an endogenous BEST1 gene contained in the genome of any one of the cells selected from clauses 1 to 3, The CRISPR / Cas composition comprises the Cas protein and the guide RNA, The above Cas protein and the above guide RNA form an RNA-protein complex.

5. A method for editing an endogenous BEST1 gene contained in the genome of any one of the cells selected from clauses 1 to 3, The above CRISPR / Cas composition comprises a CRISPR / Cas system component expression vector, The above vector comprises a nucleic acid encoding the Cas protein and a nucleic acid encoding the guide RNA.

6. A method for editing an endogenous BEST1 gene contained in the genome of any one of the cells selected from clauses 1 to 3, The above CRISPR / Cas composition comprises an mRNA encoding the Cas protein and the guide RNA.

7. A method for editing an endogenous BEST1 gene contained in the genome of any one of the cells selected from clauses 1 to 6, As a result of performing the above method, an indel occurs in the endogenous BEST1 gene.

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