Cas9 protein variant with low immunogenicity

Abstract

The present invention relates to a Cas9 protein variant having low immunogenicity. In addition, the present invention relates to an in vivo genetic manipulation method using a CRISPR / Cas9 composition comprising the Cas9 protein variant.

Description

CAS9 protein variants with low immunogenicity

[0001] The present invention relates to a Cas9 protein variant having low immunogenicity and a gene editing method using the same.

[0002]

[0003] The high efficiency and precision of CRISPR / Cas9 gene editing-based therapies offer the potential to treat numerous human diseases by targeting previously intractable proteins. As evidence of this, CRISPR / Cas9 gene editing technology has recently entered clinical development. As in-vivo clinical trials commence, the risks associated with administering CRISPR / Cas9 gene editing therapies to patients are becoming increasingly important. Since the early days of the development of CRISPR / Cas9 gene editing technology, there has been speculation that immune responses to microbial Cas9 proteins could impact clinical applications (Chew, WL Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol. Med. 10, https: / doi.org / 10.1002 / wsbm.1408 (2018); Chew, WL et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods. 13, 868-874 (2016)). Since 2018, studies have provided experimental evidence for T and B cell responses to Cas9 derived from Staphylococcus aureus and Streptococcus pyogenes. In these studies, anti-Cas9 antibodies were detected in serum samples from some healthy donors. Additionally, both CD4+ and CD8+ T cells from this donor were found to respond to the Cas9 protein (Charlesworth, CT et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249-254 (2019)).Consistent with these results, in silico evaluation of the Cas9 protein showed that several peptides derived from SpCas9 bind to MHC proteins with high affinity (Ferdosi, SR et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat. Commun. 10, 1842 (2019)). Therefore, pre-existing immunity to delivery of therapeutic Cas9 proteins may reduce their in vivo efficacy and pose serious safety concerns.

[0004]

[0005] One object of the present invention is to provide a Cas9 protein variant having low immunogenicity.

[0006] Another object of the present invention is to provide a CRISPR / Cas9 composition comprising a Cas9 protein variant.

[0007] Another object of the present invention is to provide a gene editing method using a CRISPR / Cas9 composition comprising a Cas9 protein variant.

[0008] Another object of the present invention is to provide a therapeutic agent using a CRISPR / Cas9 composition comprising a Cas9 protein variant.

[0009] Another object of the present invention is to provide a method for treating a disease using a CRISPR / Cas9 composition comprising a Cas9 protein variant.

[0010]

[0011] The present invention provides Cas9 protein variants. In particular, it provides Cas9 protein variants (deimmunized Cas9) that have reduced immunogenicity when administered to a subject.

[0012] The above Cas9 protein variant has an amino acid sequence in which at least one amino acid among the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) in the amino acid sequence represented by SEQ ID NO: 1 is replaced with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); serine (S); glycine (G); lysine (K); arginine (R); threonine (T); glutamine (Q); cysteine ​​(C); and alanine (A). At this time, the Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1, and the Cas9 protein variant has lower immunogenicity in vivo compared to the wild-type Cas9 protein having the amino acid sequence shown in SEQ ID NO: 1.

[0013]

[0014] In one embodiment, the Cas9 protein variant has an amino acid sequence in which the 161st methionine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with histidine (H) or aspartic acid (D).

[0015] In another embodiment, the Cas9 protein variant has an amino acid sequence in which valine at position 189 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid (E).

[0016] In another embodiment, the Cas9 protein variant has an amino acid sequence in which alanine at position 337 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid.

[0017] In another embodiment, the Cas9 protein variant has an amino acid sequence in which lysine at position 961 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid or cysteine ​​(C).

[0018] In another embodiment, the Cas9 protein variant has an amino acid sequence in which the 1194th leucine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine (G) or alanine.

[0019] In another embodiment, the Cas9 protein variant has an amino acid sequence in which asparagine at position 1234 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with aspartic acid or glycine.

[0020] In another embodiment, the Cas9 protein variant has an amino acid sequence in which the 161st methionine; the 337th alanine; the 961st lysine; and the 1194th leucine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). In one specific example, the Cas9 protein variant has histidine or aspartic acid instead of the 161st methionine; glutamic acid or aspartic acid instead of the 337th alanine; aspartic acid or cysteine ​​instead of the 961st lysine in the amino acid sequence shown in SEQ ID NO: 1; And the 1194th leucine is replaced with glycine or alanine. In another specific embodiment, the Cas9 protein variant has an amino acid sequence in which the 161st methionine is replaced with histidine; the 337th alanine is replaced with glutamic acid; the 961st lysine is replaced with cysteine; and the 1194th leucine is replaced with glycine in the amino acid sequence shown in SEQ ID NO: 1. In another specific embodiment, the Cas9 protein variant has an amino acid sequence shown in SEQ ID NO: 43.

[0021]

[0022] In another embodiment, the Cas9 protein variant has an amino acid sequence in which the 961st lysine; the 1194th leucine; and the 1234th asparagine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: aspartic acid (D); cysteine ​​(C); glycine (G); and alanine (A). In one specific embodiment, the Cas9 protein variant has an amino acid sequence in which the 961st lysine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid or cysteine; the 1194th leucine is substituted with glycine or alanine; and the 1234th asparagine is substituted with aspartic acid or glycine. In another specific embodiment, the Cas9 protein variant has an amino acid sequence in which the 961st lysine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine; An amino acid sequence in which glycine is substituted for leucine at position 1194 and glycine is substituted for asparagine at position 1234. In another specific embodiment, the Cas9 protein variant has the amino acid sequence represented by SEQ ID NO: 42.

[0023]

[0024] In another embodiment, the Cas9 protein variant has an amino acid sequence in which the 161st methionine; the 189th valine; the 1194th leucine; and the 1234th asparagine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); glycine (G); and alanine (A). In one specific example, the Cas9 protein variant has an amino acid sequence in which the 161st methionine is substituted with histidine or aspartic acid; the 189th valine is substituted with aspartic acid or glutamic acid; the 1194th leucine is substituted with glycine or alanine; and the 1234th asparagine is substituted with aspartic acid or glycine in the amino acid sequence shown in SEQ ID NO: 1. In another specific embodiment, the Cas9 protein variant has an amino acid sequence in which the methionine at position 161 is replaced by histidine; the valine at position 189 is replaced by aspartic acid; the leucine at position 1194 is replaced by alanine; and the asparagine at position 1234 is replaced by glycine in the amino acid sequence shown in SEQ ID NO: 1. In yet another specific embodiment, the Cas9 protein variant has an amino acid sequence shown in SEQ ID NO: 44.

[0025]

[0026] In another embodiment, the Cas9 protein variant further comprises at least one NLS.

[0027] The present invention provides a CRISPR / Cas9 composition comprising: the Cas9 protein variant or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same.

[0028] In one embodiment, the Cas9 protein variant has an amino acid sequence represented by SEQ ID NO: 42, 43, or 44.

[0029] In another embodiment, the CRISPR / Cas9 composition comprises the Cas9 protein variant and the guide RNA in the form of a ribonucleoprotein (RNP).

[0030] In another embodiment, the CRISPR / Cas9 composition comprises a nucleic acid encoding the Cas9 protein variant and a nucleic acid encoding the guide RNA in the form of a vector.

[0031] The present invention provides a method for editing genes in vivo, comprising administering a CRISPR / Cas9 composition into the body. The CRISPR / Cas9 composition comprises the Cas9 protein variant or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same, wherein the guide RNA has a guide sequence that is custom-designed or artificially designed to target a gene to be edited.

[0032] The present invention provides a therapeutic agent for in vivo administration comprising: the Cas9 protein variant or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same. The guide RNA has a guide sequence that is customized or artificially designed according to the target gene of the disease.

[0033] The present invention provides a method for treating a disease, comprising administering a therapeutic agent for in vivo administration to a subject for treating a disease. The therapeutic agent comprises the Cas9 protein or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same, wherein the guide RNA has a guide sequence that is customized or artificially designed according to the target gene of the disease. The disease is caused by abnormal expression and / or abnormal function of a specific gene or protein, and the disease is a disease whose symptoms can be alleviated or treated by artificially editing a specific gene.

[0034]

[0035] The CRISPR / Cas9 composition comprising the Cas9 protein variant of the present invention having low immunogenicity in vivo will enable more effective in vivo gene editing therapy by reducing the immune response through the antigen presentation process compared to the wild-type Cas9 protein.

[0036]

[0037] Figure 1 shows the method for producing a library of SpCas9.

[0038] Figure 2 shows the 6TG screening results.

[0039] Figure 3 shows the results of determining the average indel activity compared to WT using the Artificial 24-target evaluation system. Mutations selected for the mutation combination are marked with an *.

[0040] Figure 4 shows indels for the EMX1 target of 140 mutants.

[0041] Figure 5 shows the number of mutations in 140 mutants.

[0042] Figure 6 shows the results of three-repeat indel analysis for the EMX1 target of the selected mutant.

[0043] Figure 7 shows the results of three-repeat indel analysis for the EMX1 target with the first mutation combination.

[0044] Figure 8 shows the average indel value for 24 artificial targets with the first mutation combination.

[0045] Figure 9 shows the indel evaluation results for 24 artificial targets using the first mutation combination.

[0046] Figure 10 shows the results of indel analysis for 24 artificial targets using secondary mutation combinations.

[0047] Figure 11 shows the results of indel analysis for 24 targets of the first and second selection mutants.

[0048] Figure 12 shows the average results of 3 indel iterations for each of the 24 artificial targets of the final selection candidates.

[0049] Figure 13 briefly shows the sequence and method of the immune response experiment of WT SpCas9 and deimmunized SpCas9 mutants using human peripheral blood PBMC.

[0050] Figures 14 and 15 are graphs showing the percentage of CD25+ and 41BB+ cells in CD4+, CD8+, or CD4+CD8+ (DP) T cells.

[0051] Figure 16 is a graph showing the percentage of CD25+ and 41BB+ cells in CD4+, CD8+, or CD4+CD8+ (DP) T cells, with WT SpCas9 as the reference (100%).

[0052] Figure 17 is a graph showing the percentage of CD25+ and 41BB+ cells in CD4+, CD8+, or CD4+CD8+ (DP) T cells.

[0053] Figure 18 is a graph showing the percentage of TNFα+, IL-2+, and IFNγ+ cells in re-sensitized T cells.

[0054]

[0055] Hereinafter, the present invention will be described in more detail through specific implementation examples and examples with reference to the drawings attached to this application. The attached drawings include some implementation examples of the present invention, but not all implementation examples. The present invention may be implemented in various forms and is not limited to the specific implementation examples described in this specification. That is, these implementation examples may be variously changed or modified within the spirit and scope of the present invention, as will be apparent to those skilled in the art. Therefore, the invention disclosed in this specification is not limited to the specific implementation examples described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0056]

[0057] Definition of terms

[0058] The definitions of terms used in this specification are as follows.

[0059]

[0060] immune response

[0061] In general, when a foreign substance (protein) is introduced into the body, the foreign substance induces an immune response through the following antigen presentation process. Among the foreign substances, proteins are broken down into short lengths by various degradative organelles within the cell. The peptide chains that are broken down to a size that the cell can take up through endocytosis are retained inside the endosome through endocytosis and enter the cytoplasm of the antigen-presenting cell (APC). The endosomes that have entered the cytoplasm through endocytosis fuse with lysosomes, and at this time, the lysosomal membrane contains Major Histocompatibility Complex (MHC) class 1 and / or Major Histocompatibility Complex (MHC) class 2. The proteins are broken down into short peptide fragments by various degradative enzymes within the lysosome. Peptide fragments of about 12 to 20 amino acids bind to MHC class 1 and / or MHC class 2. The lysosome then fuses with the plasma membrane again, and at this time, the peptide fragments bound to MHC class 1 and / or MHC class 2 are released to the outside of the cell. The peptide fragments bound to MHC class 1 and / or MHC class 2, i.e., antigens, can be recognized by numerous T cells in the body. That is, the T cell receptor attempts to bind to MHC class 1 and / or MHC class 2, and if a bond is formed, the T cell is activated and secretes various cytokines, causing an immune response.

[0062]

[0063] immunogenicity

[0064] The term "immunogenicity" refers to the ability of a human or animal body to elicit an immune response to a foreign substance, such as an antigen, or an externally introduced substance. In the present invention, the phrases "low immunogenicity" or "reduced immunogenicity" can be interpreted as indicating that an immune response is minimal or weak. For example, the phrases "Cas9 protein variant with low immunogenicity" or "Cas9 protein variant with reduced immunogenicity" can be interpreted as indicating that the Cas9 protein, when introduced into the body, elicits minimal or low immune responses resulting from antigen presentation and T cell recognition. In this case, the induced immune response is significantly reduced or lower compared to the immune response elicited when the wild-type Cas9 protein is introduced into the body. In this case, the Cas9 protein variant can be understood to have lower or reduced immunogenicity than the wild-type Cas9 protein.

[0065]

[0066] 'low immune response in vivo' or 'decreased immune response in vivo'

[0067] In the present invention, "low immune response in vivo or reduced immune response in vivo" means a decrease in the immune response by T cells that recognize antigens bound to MHC Class 2 and / or MHC Class 1 during the above-described immune response process. A low immune response in vivo or reduced immune response in vivo may be manifested by a decrease in the activity of CD4+ T cells, CD8+ T cells, etc., or a decrease in the expression of immune-related cytokines (IL-2, IFNγ, etc.). In addition, when it is described that a low immune response in vivo or a reduced immune response in vivo is shown to a specific protein, the specific protein can be understood as having "low immunogenicity" or "reduced immunogenicity." That is, in the present invention, a "low immune response in vivo or reduced immune response in vivo" can be understood as a phenomenon caused by a protein having low immunogenicity (e.g., a Cas9 protein variant).

[0068]

[0069] CRISPR / Cas9 system

[0070] The CRISPR / Cas9 system is a type of immune system found in prokaryotic organisms and is a promising approach for performing targeted genetic changes in eukaryotic cells. The CRISPR / Cas9 system comprises the Cas9 protein and a guide RNA. The CRISPR / Cas9 system is a well-known technology and has been described in detail in various publications, including H. Wang, M. La Russa, LS Qi, CRISPR / Cas9 in genome editing and beyond, Annu Rev Biochem, 85 (2016), pp. 227-264; L. Cong, FA Ran, D. Cox, et al., Multiplex genome engineering using CRISPR / Cas systems, Science, 339 (2013), pp. 819-823; and WO2014 / 065596 (International Publication No.). The term "Cas9 protein" as used herein is a general term for nucleases that can be interpreted as being used in the CRISPR / Cas system.

[0071]

[0072] variant (or mutant)

[0073] The term "variant" or "mutant" refers to a protein in which at least one amino acid sequence has been artificially altered from the amino acid sequence of a wild-type protein, and the variant has an amino acid sequence that is different from the amino acid sequence of the wild-type protein. In this case, the wild-type protein refers to a protein that exists in a natural state and is an original protein without any artificial modification. For example, an SpCas9 protein variant is a protein in which at least one amino acid sequence selected from the amino acid sequence of a wild-type SpCas9 protein has been artificially altered, and the SpCas9 protein variant has an amino acid sequence that is different from the amino acid sequence of the wild-type SpCas9 protein. In this case, the artificial modification may be, but is not limited to, deletion of the selected amino acid or substitution with another amino acid.

[0074]

[0075] Amino acid sequence notation

[0076] Unless otherwise stated, when describing amino acid sequences in this specification, amino acid single-letter notation or three-letter notation is used, and is written from the N-terminus to the C-terminus. For example, when written as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected from the N-terminus to the C-terminus. Another example, when written as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminus to the C-terminus. In the case of amino acids that cannot be expressed with the single-letter notation, other letters are used and additional explanations are provided.

[0077] 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).

[0078]

[0079] vector

[0080] As used herein, "vector" refers to any material capable of transporting genetic material into a cell, unless otherwise specified. For example, a vector may be a DNA molecule containing the genetic material of interest, for example, a nucleic acid encoding the Cas9 protein of a CRISPR / Cas9 system, and / or a nucleic acid encoding a guide RNA. In another example, a vector may be an RNA molecule containing the genetic material of interest, for example, a nucleic acid encoding the Cas9 protein of a CRISPR / Cas9 system, and / or a guide RNA. However, the present invention is not limited thereto. The above terms include all meanings that can be recognized by a person skilled in the art and may be appropriately interpreted according to the context.

[0081]

[0082] NLS (Nuclear Localization Sequence)

[0083] The term "NLS (nuclear localization sequence or nuclear localization signal)" refers to a peptide of a certain length, or its sequence, that acts as a kind of "tag" by attaching to a protein that is the target of transport when transporting a substance outside the cell nucleus into the nucleus through nuclear transport. Specifically, the NLS is an NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 45); an NLS from nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 46)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 47) or RQRRNELKRSP (SEQ ID NO: 48); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 49); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 50) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 51) and PPKKARED (SEQ ID NO: 52) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 53) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 54) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 55) and PKQKKRK (SEQ ID NO: 56) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 57) of the hepatitis virus delta antigen; The NLS sequence may be derived from, but is not limited to, the sequence REKKKFLKRR (SEQ ID NO: 58) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 59) of the human poly(ADP-ribose) polymerase; or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 60) of the steroid hormone receptor (human) glucocorticoid.The term “NLS” as used herein has all meanings that can be recognized by a person of ordinary skill in the art and may be interpreted appropriately depending on the context.

[0084]

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0086]

[0087] Challenges or limitations of using gene editing therapies in vivo

[0088] CRISPR / Cas9-mediated genome editing technology offers promising opportunities for the treatment of both single-gene diseases and complex multigenic disorders such as cancer. Compared to existing genome editing technologies such as ZFN and TLAEN, CRISPR / Cas9 technology has proven to be a powerful tool for genome editing strategies due to its ease of use, site-specific activity, and limited off-target effects (L. Cong, FA Ran, D. Cox, et al. Multiplex genome engineering using CRISPR / Cas systems, Science, 339 (2013), pp. 819-823). With less than a decade between its discovery and first clinical application, its enormous potential is evident. The current preclinical cell therapy pipeline is replete with CRISPR / Cas9 genome editing strategies, with promising and effective therapeutic strategies awaiting transplantation of cells into patients after ex vivo editing, in vivo editing of patient cells, or direct delivery of Cas9 and guide RNA to cells of interest or diseased cells. However, recent demonstration that routine prior exposure to Staphylococcus aureus and Streptococcus pyogenes results in pre-existing immunity to Cas9 poses a significant threat to further clinical development.

[0089] Immune responses to proteins used in therapeutic applications can impact the safety and / or efficacy of these products. Consequently, immunogenicity risk assessments are necessary during drug development and approval. According to FDA guidance, Cas proteins are likely to fall into the high-risk category because they were originally isolated from human pathogens (US Food and Drug Administration. Immunogenicity assessment for therapeutic protein products (Guidance for Industry, 2014)). Immunogenicity risk assessments seek to answer two fundamental questions: (i) What is the probability that a therapeutically used protein will elicit an immune response? (ii) What are the clinical consequences of such an immune response? As previously described, several reports have shown that Cas proteins elicit immune responses in vivo, and these studies may provide an answer to the first question. Furthermore, recent mouse studies of liver genome editing using AAV-packaged CRISPR-Cas9 provide an opportunity to glimpse the in vivo consequences of pre-existing immunity to Cas proteins. Notably, genome editing was accompanied by an increase in CD8+ T cells and a cytotoxic T cell response in the liver, which resulted in hepatocyte death, loss of the recombinant AAV genome, and complete elimination of genome-edited cells (Li, A. et al. AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9. Mol. Ther. 28, 1432-1441 (2020)).

[0090] Recognizing the above issues, the present inventors developed SpCas9 protein variants with novel mutations based on the wild-type SpCas9 protein to reduce the immunogenicity of existing Cas proteins, and confirmed the low immunogenicity of the developed SpCas9 protein variants. Below, the SpCas9 protein variants with low immunogenicity in vivo and their applications are described in detail.

[0091]

[0092] Cas9 protein variants

[0093] In one aspect of the present invention, a Cas9 protein variant is disclosed.

[0094] The above Cas9 protein variant is a Streptococcus pyogenes-derived Cas9 (SpCas9) protein variant that has low immunogenicity when introduced into a living body. At this time, when the Cas9 protein variant is introduced into a living body, compared to when the wild-type SpCas9 protein is introduced, an immune response occurring during an antigen presentation process and / or a T cell recognition process is almost absent or reduced. In particular, when the Cas9 protein variant is introduced into a living body, compared to when the wild-type SpCas9 protein is introduced, an antigen bound to MHC Class 2 and / or MHC Class 1 during the antigen presentation process is reduced, thereby reducing an immune response by T cells that recognize the antigen. Alternatively, when the Cas9 protein variant is introduced into a living body, compared to when the wild-type SpCas9 protein is introduced, the activity of CD4+ T cells and / or CD8+ T cells is reduced. Alternatively, when the Cas9 protein variant is introduced into a living body, the expression of immune-related cytokines (IL-2, IFNγ, etc.) is reduced compared to when the wild-type SpCas9 protein is introduced.

[0095] The above Cas9 protein variant has a mutation that minimizes immunogenicity without affecting the nuclease activity of the Cas9 protein. The mutation is a substitution of an amino acid at a position selected by the inventors through bioinformatics analysis in the amino acid sequence constituting the SpCas9 protein with a different specific amino acid.

[0096] In the present specification, the Cas9 protein variant is referred to as a Cas9 protein, SpCas9 protein, SpCas9 protein variant, novel Cas9 protein, novel SpCas9 protein, Cas9 mutation, SpCas9 mutation, Cas9 mutant, SpCas9 mutant, deimmunized Cas9 protein, deimmunized Cas9 protein variant, deimmunized SpCas9 protein or deimmunized SpCas9 protein variant, and the terms may be used interchangeably. However, wild-type Cas9 protein or wild-type SpCas9 protein is used to distinguish it from the terms.

[0097] Hereinafter, the Cas9 protein variant of the present invention is described in various embodiments.

[0098] Example 1 of Deimmunized Cas9 Protein Variants

[0099] In one embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least one amino acid among the following is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 238th phenylalanine (F); 295th asparagine (N); 337th alanine (A); 619th isoleucine (I); 620th valine (V); 647th valine (V); 648th methionine (M); 659th tryptophan (W); 794th glutamine (Q); 884th arginine (R); 956th isoleucine (I); 961st lysine (K); 978th ​​isoleucine (I); 1042nd isoleucine (I); 1144th leucine (L); 1163rd leucine (L); 1194th leucine (L); and 1234th asparagine (N). Here, the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the wild-type SpCas9 protein. The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0100] In one specific example, the Cas9 protein variant comprises amino acid sequences represented by SEQ ID NO: 1, wherein the 161st methionine (M); the 189th valine (V); the 238th phenylalanine (F); the 295th asparagine (N); the 337th alanine (A); the 619th isoleucine (I); the 620th valine (V); the 647th valine (V); the 648th methionine (M); the 659th tryptophan (W); the 794th glutamine (Q); the 884th arginine (R); the 956th isoleucine (I); the 961st lysine (K); the 978th ​​isoleucine (I); the 1042nd isoleucine (I); the 1144th leucine (L); the 1163rd leucine (L); the 1194th leucine (L); And at least one amino acid selected from the 1234th asparagine (N) may be substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); serine (S); glycine (G); lysine (K); arginine (R); threonine (T); glutamine (Q); cysteine ​​(C); and alanine (A). At this time, the Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0101] In another specific embodiment, the Cas9 protein variant may have one or more mutations selected from the following in the amino acid sequence shown in SEQ ID NO: 1: methionine (M) at position 161 is replaced with histidine (H) or aspartic acid (D); valine (V) at position 189 is replaced with aspartic acid (D) or glutamic acid (E); phenylalanine (F) at position 238 is replaced with serine (S) or aspartic acid (D); asparagine (N) at position 295 is replaced with aspartic acid (D) or glycine (G); alanine (A) at position 337 is replaced with glutamic acid (E) or aspartic acid (D); isoleucine (I) at position 619 is replaced with lysine (K) or arginine (R); The valine (V) at position 620 is substituted with serine (S) or threonine (T); the valine (V) at position 647 is substituted with aspartic acid (D) or glycine (G); the methionine (M) at position 648 is substituted with lysine (K) or glutamic acid (E); the tryptophan (W) at position 659 is substituted with glutamine (Q) or histidine (H); the glutamine (Q) at position 794 is substituted with glutamic acid (E) or aspartic acid (D); the arginine (R) at position 884 is substituted with glutamic acid (E) or aspartic acid (D); the isoleucine (I) at position 956 is substituted with threonine (T) or glycine (G); The 961st lysine (K) is substituted with aspartic acid (D) or cysteine ​​(C); the 978th ​​isoleucine (I) is substituted with glutamic acid (E) or glycine (G); the 1042nd isoleucine (I) is substituted with glutamic acid (E) or aspartic acid (D); the 1144th leucine (L) is substituted with glutamine (Q) or asparagine (N); the 1163rd leucine (L) is substituted with glutamine (Q) or glycine (G); the 1194th leucine (L) is substituted with glycine (G) or alanine (A); and the 1234th asparagine (N) is substituted with aspartic acid (D) or glycine (G).

[0102] In one specific example, the Cas9 protein variant may have histidine (H) or aspartic acid (D) instead of methionine (M) at position 161 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 2. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 3.

[0103] In another specific example, the Cas9 protein variant may have aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 4. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 5.

[0104] In another specific example, the Cas9 protein variant may have serine (S) or aspartic acid (D) instead of phenylalanine (F) at position 238 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 6. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 7.

[0105] In another specific example, the Cas9 protein variant may have aspartic acid (D) or glycine (G) instead of asparagine (N) at position 295 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 8. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 9.

[0106] In another specific example, the Cas9 protein variant may have glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 10. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 11.

[0107] In another specific example, the Cas9 protein variant may have lysine (K) or arginine (R) instead of isoleucine (I) at position 619 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 12. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 13.

[0108] In another specific example, the Cas9 protein variant may have serine (S) or threonine (T) instead of valine (V) at position 620 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 14. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 15.

[0109] In another specific example, the Cas9 protein variant may have aspartic acid (D) or glycine (G) instead of valine (V) at position 647 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 16. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 17.

[0110] In another specific example, the Cas9 protein variant may have lysine (K) or glutamic acid (E) instead of methionine (M) at position 648 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 18. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 19.

[0111] In another specific example, the Cas9 protein variant may have glutamine (Q) or histidine (H) instead of tryptophan (W) at position 659 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 20. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 21.

[0112] In another specific example, the Cas9 protein variant may have glutamic acid (E) or aspartic acid (D) instead of glutamine (Q) at position 794 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 22. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 23.

[0113] In another specific example, the Cas9 protein variant may have glutamic acid (E) or aspartic acid (D) instead of arginine (R) at position 884 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 24. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 25.

[0114] In another specific embodiment, the Cas9 protein variant may have a threonine (T) or a glycine (G) instead of the 956th isoleucine (I) in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 26. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 27.

[0115] In another specific example, the Cas9 protein variant may have aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 28. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 29.

[0116] In another specific example, the Cas9 protein variant may have glutamic acid (E) or glycine (G) instead of isoleucine (I) at position 978 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 30. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 31.

[0117] In another specific example, the Cas9 protein variant may have glutamic acid (E) or aspartic acid (D) instead of isoleucine (I) at position 1042 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 32. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 33.

[0118] In another specific embodiment, the Cas9 protein variant may have glutamine (Q) or asparagine (N) instead of leucine (L) at position 1144 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 34. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 35.

[0119] In another specific example, the Cas9 protein variant may have glutamine (Q) or glycine (G) instead of leucine (L) at position 1163 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 36. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 37.

[0120] In another specific embodiment, the Cas9 protein variant may have glycine (G) or alanine (A) instead of leucine (L) at position 1194 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 38. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 39.

[0121] In another specific example, the Cas9 protein variant may have aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234 in the amino acid sequence shown in SEQ ID NO: 1. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 40. In another example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 41.

[0122] Example 2 of Deimmunized Cas9 Protein Variants

[0123] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least one amino acid selected from the following is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0124] In one specific example, the Cas9 protein variant may have at least one amino acid selected from the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N) substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0125] In another specific embodiment, the Cas9 protein variant may have at least one mutation selected from the following in the amino acid sequence shown in SEQ ID NO: 1: a replacement of the 161st methionine (M) with a histidine (H) or an aspartic acid (D); a replacement of the 189th valine (V) with an aspartic acid (D) or a glutamic acid (E); a replacement of the 337th alanine (A) with a glutamic acid (E) or an aspartic acid (D); a replacement of the 961st lysine (K) with an aspartic acid (D) or a cysteine ​​(C); a replacement of the 1194th leucine (L) with a glycine (G) or an alanine (A); and a replacement of the 1234th asparagine (N) with an aspartic acid (D) or a glycine (G). For example, the Cas9 protein variant may have an amino acid sequence selected from among the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

[0126] Example 3 of Deimmunized Cas9 Protein Variants

[0127] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least two amino acids selected from the following are independently substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0128] In one specific example, the Cas9 protein variant may have at least two amino acids selected from the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) in the amino acid sequence shown in SEQ ID NO: 1, each substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0129] In another specific embodiment, the Cas9 protein variant may have at least two mutations selected from the following in the amino acid sequence shown in SEQ ID NO: 1: a substitution of the 161st methionine (M) with a histidine (H) or an aspartic acid (D); a substitution of the 189th valine (V) with an aspartic acid (D) or a glutamic acid (E); a substitution of the 337th alanine (A) with a glutamic acid (E) or an aspartic acid (D); a substitution of the 961st lysine (K) with an aspartic acid (D) or a cysteine ​​(C); a substitution of the 1194th leucine (L) with a glycine (G) or an alanine (A); and a substitution of the 1234th asparagine (N) with an aspartic acid (D) or a glycine (G).

[0130] Example 4 of Deimmunized Cas9 Protein Variants

[0131] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least three or more amino acids selected from the following are independently substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0132] In one specific example, the Cas9 protein variant may have at least three amino acids selected from the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N) substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0133] In another specific embodiment, the Cas9 protein variant may have at least three mutations selected from the following in the amino acid sequence shown in SEQ ID NO: 1: a substitution of the 161st methionine (M) with a histidine (H) or an aspartic acid (D); a substitution of the 189th valine (V) with an aspartic acid (D) or a glutamic acid (E); a substitution of the 337th alanine (A) with a glutamic acid (E) or an aspartic acid (D); a substitution of the 961st lysine (K) with an aspartic acid (D) or a cysteine ​​(C); a substitution of the 1194th leucine (L) with a glycine (G) or an alanine (A); and a substitution of the 1234th asparagine (N) with an aspartic acid (D) or a glycine (G).

[0134] In another specific example, the Cas9 protein variant may have aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961 in the amino acid sequence shown in SEQ ID NO: 1; glycine (G) or alanine (A) instead of leucine (L) at position 1194; and aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234. For example, the Cas9 protein variant may have one sequence selected from the amino acid sequences shown in SEQ ID NO: 42 and SEQ ID NOs: 63 to 69.

[0135] In a preferred embodiment of the present invention, the Cas9 protein variant may have cysteine ​​(C) instead of lysine (K) at position 961 in the amino acid sequence shown in SEQ ID NO: 1; glycine (G) instead of leucine (L) at position 1194; and glycine (G) instead of asparagine (N) at position 1234. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 42.

[0136] Example 5 of Deimmunized Cas9 Protein Variants

[0137] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least four amino acids selected from the following are independently substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0138] In one specific example, the Cas9 protein variant may have at least four amino acids selected from the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N) substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0139] In another specific embodiment, the Cas9 protein variant may have at least four mutations selected from the following in the amino acid sequence shown in SEQ ID NO: 1: a substitution of the 161st methionine (M) with a histidine (H) or an aspartic acid (D); a substitution of the 189th valine (V) with an aspartic acid (D) or a glutamic acid (E); a substitution of the 337th alanine (A) with a glutamic acid (E) or an aspartic acid (D); a substitution of the 961st lysine (K) with an aspartic acid (D) or a cysteine ​​(C); a substitution of the 1194th leucine (L) with a glycine (G) or an alanine (A); and a substitution of the 1234th asparagine (N) with an aspartic acid (D) or a glycine (G).

[0140] In another specific example, the Cas9 protein variant may have histidine (H) or aspartic acid (D) instead of methionine (M) at position 161 in the amino acid sequence shown in SEQ ID NO: 1; glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337; aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961; and glycine (G) or alanine (A) instead of leucine (L) at position 1194. For example, the Cas9 protein variant may have one sequence selected from the amino acid sequences shown in SEQ ID NO: 43 and SEQ ID NOs: 70 to 84.

[0141] In a preferred embodiment of the present invention, the Cas9 protein variant may have histidine (H) instead of methionine (M) at position 161 in the amino acid sequence shown in SEQ ID NO: 1; glutamic acid (E) instead of alanine (A) at position 337; cysteine ​​(C) instead of lysine (K) at position 961; and glycine (G) instead of leucine (L) at position 1194. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 43.

[0142] In another specific example, the Cas9 protein variant may have histidine (H) or aspartic acid (D) instead of methionine (M) at position 161 in the amino acid sequence shown in SEQ ID NO: 1; aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189; glycine (G) or alanine (A) instead of leucine (L) at position 1194; and aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234. For example, the Cas9 protein variant may have one sequence selected from the amino acid sequences shown in SEQ ID NO: 44 and SEQ ID NOs: 85 to 99.

[0143] In a preferred embodiment of the present invention, the Cas9 protein variant may have histidine (H) instead of methionine (M) at position 161 in the amino acid sequence shown in SEQ ID NO: 1; aspartic acid (D) instead of valine (V) at position 189; alanine (A) instead of leucine (L) at position 1194; and glycine (G) instead of asparagine (N) at position 1234. For example, the Cas9 protein variant may have the amino acid sequence shown in SEQ ID NO: 44.

[0144] Example 6 of Deimmunized Cas9 Protein Variants

[0145] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which at least five amino acids selected from the following are independently substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0146] In one specific example, the Cas9 protein variant may have at least five amino acids selected from the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) in the amino acid sequence shown in SEQ ID NO: 1, each substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0147] In another specific embodiment, the Cas9 protein variant may have at least five mutations selected from the following in the amino acid sequence shown in SEQ ID NO: 1: a replacement of the 161st methionine (M) with a histidine (H) or an aspartic acid (D); a replacement of the 189th valine (V) with an aspartic acid (D) or a glutamic acid (E); a replacement of the 337th alanine (A) with a glutamic acid (E) or an aspartic acid (D); a replacement of the 961st lysine (K) with an aspartic acid (D) or a cysteine ​​(C); a replacement of the 1194th leucine (L) with a glycine (G) or an alanine (A); and a replacement of the 1234th asparagine (N) with an aspartic acid (D) or a glycine (G).

[0148] Example 7 of Deimmunized Cas9 Protein Variants

[0149] In another embodiment, the Cas9 protein variant of the present invention has an amino acid sequence in which the following amino acids are independently substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 1: 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N). The Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1.

[0150] In one specific example, the Cas9 protein variant may have an amino acid sequence represented by SEQ ID NO: 1 in which the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) are each substituted with one amino acid selected from the following: histidine (H); aspartic acid (D); glutamic acid (E); cysteine ​​(C); glycine (G); and alanine (A). The Cas9 protein variant may have an amino acid sequence different from the amino acid sequence represented by SEQ ID NO: 1.

[0151] In another specific embodiment, the Cas9 protein variant may have the following mutations in the amino acid sequence shown in SEQ ID NO: 1: the 161st methionine (M) is substituted with histidine (H) or aspartic acid (D); the 189th valine (V) is substituted with aspartic acid (D) or glutamic acid (E); the 337th alanine (A) is substituted with glutamic acid (E) or aspartic acid (D); the 961st lysine (K) is substituted with aspartic acid (D) or cysteine ​​(C); the 1194th leucine (L) is substituted with glycine (G) or alanine (A); and the 1234th asparagine (N) is substituted with aspartic acid (D) or glycine (G).

[0152] Additional components of the deimmunized Cas9 protein variant

[0153] The Cas9 protein variant disclosed herein may further comprise at least one nuclear localization signal (NLS) at the internal, N-terminal and / or C-terminal end of the constituent amino acid sequence.

[0154] The above NLS is PKKKRKV (SEQ ID NO: 45), KRPAATKKAGQAKKKK (SEQ ID NO: 46), PAAKRVKLD (SEQ ID NO: 47), RQRRNELKRSP (SEQ ID NO: 48), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 49), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 50), VSRKRPRP (SEQ ID NO: 51), PPKKARED (SEQ ID NO: 52), PQPKKKPL (SEQ ID NO: 53), SALIKKKKKMAP (SEQ ID NO: 54), DRLRR (SEQ ID NO: 55), PKQKKRK (SEQ ID NO: 56), RKLKKKIKKL (SEQ ID NO: 57), REKKKFLKRR (SEQ ID NO: 58), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 59), and RKCLQAGMNLEARKTKK (SEQ ID NO: 60), but is not limited thereto.

[0155] In one specific embodiment, the Cas9 protein variant has a cysteine ​​(C) instead of lysine (K) at position 961; a glycine (G) instead of leucine (L) at position 1194; and a glycine (G) instead of asparagine (N) at position 1234 in the amino acid sequence shown in SEQ ID NO: 1, and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof. In another specific embodiment, the Cas9 protein variant has an amino acid sequence shown in SEQ ID NO: 42, and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof.

[0156] In another specific embodiment, the Cas9 protein variant has a histidine (H) instead of the 161st methionine (M) in the amino acid sequence shown in SEQ ID NO: 1; a glutamic acid (E) instead of the 337th alanine (A) in the amino acid sequence shown in SEQ ID NO: 1; a cysteine ​​(C) instead of the 961st lysine (K) in the amino acid sequence shown in SEQ ID NO: 1; and a glycine (G) instead of the 1194th leucine (L), and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof. In another specific embodiment, the Cas9 protein variant has an amino acid sequence shown in SEQ ID NO: 43, and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof.

[0157] In another specific embodiment, the Cas9 protein variant has histidine (H) instead of methionine (M) at position 161; aspartic acid (D) instead of valine (V) at position 189; alanine (A) instead of leucine (L) at position 1194; and glycine (G) instead of asparagine (N) at position 1234 in the amino acid sequence shown in SEQ ID NO: 1, and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof. In another specific embodiment, the Cas9 protein variant has the amino acid sequence shown in SEQ ID NO: 44, and may further comprise at least one NLS at the N-terminus and / or the C-terminus thereof.

[0158] Hereinafter, a composition comprising the Cas9 protein variant and a gene editing method using the same and its use are described.

[0159]

[0160] Composition comprising a Cas9 protein variant

[0161] In one aspect of the present invention, a composition comprising the Cas9 protein variant described above is disclosed. The composition is a CRISPR / Cas9 composition comprising the Cas9 protein variant described above and a guide RNA.

[0162] The CRISPR / Cas9 composition comprises a Cas9 protein variant or a nucleic acid encoding the Cas9 protein variant; and a guide RNA or a nucleic acid encoding the guide RNA. The Cas9 protein variant is one of the Cas9 protein variants described in the "Cas9 Protein Variant" section. The guide RNA is an RNA molecule capable of forming a complex with the SpCas9 protein to target a predetermined target gene. The guide RNA is described below, but is not limited thereto, and has the same meaning as commonly understood by a person skilled in the art.

[0163] guide RNA

[0164] The guide RNA included in the above CRISPR / Cas9 construct can form a complex with the SpCas9 protein to target a predetermined target gene.

[0165] The above guide RNA includes crRNA and tracrRNA.

[0166] The above crRNA has a guide sequence and a direct repeat sequence sequentially linked in the 5' to 3' direction.

[0167] At this time, the direct repeat sequence is a nucleic acid sequence of 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 61). Or the above direct repeat sequence is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, A nucleic acid sequence having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0168] Here, the "guide sequence" is a sequence that can complementarily bind to a portion of a nucleic acid sequence of a predetermined target gene. The guide sequence may be designed to fit the predetermined target gene or artificially. The guide sequence has a length of 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.

[0169] The above tracrRNA has a nucleic acid sequence of 5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 61). Or the tracrRNA has a nucleic acid sequence of 5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 61) and 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, Has a nucleic acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0170] In one embodiment, the guide RNA may be a dual guide RNA comprising two molecules, the crRNA and the tracrRNA, as separate molecules. In this case, the direct repeat sequence of the crRNA may complementarily bind to a portion of the tracrRNA sequence.

[0171] In another embodiment, the guide RNA may be a single guide RNA in which the 3' end of the crRNA is linked to the 5' end of the tracrRNA. The crRNA may be linked to the tracrRNA via a linker. In this case, the linker may be 5'-GAAA-3'.

[0172] From another perspective, the guide RNA can be described as comprising a guide sequence capable of complementarily binding to a portion of a nucleic acid sequence of a target gene and a scaffold sequence involved in forming a complex with the SpCas9 protein.

[0173] That is, the scaffold of the guide RNA is composed of a portion of the crRNA sequence (direct repeat) and a tracrRNA sequence, and in one specific example, the scaffold sequence of the guide RNA that interacts with the SpCas9 protein can be represented as 5'-GUUUUAGAGCUAGAAA UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 174).

[0174] Depending on the choice of the skilled artisan, the guide RNA may use a wild-type sequence or a modified sequence known in the art, as long as it can interact with the SpCas9 protein.

[0175] Form of CRISPR / Cas9 composition

[0176] The CRISPR / Cas9 composition of the present invention

[0177] A Cas9 protein variant or a nucleic acid encoding said Cas9 protein variant; and

[0178] Guide RNA or a nucleic acid encoding said guide RNA

[0179] The CRISPR / Cas9 composition may have various forms, and examples of suitable forms of the CRISPR / Cas9 composition of the present invention are described.

[0180] Composition form 1

[0181] In one embodiment, the CRISPR / Cas9 composition may comprise the Cas9 protein variant and the guide RNA in the form of a ribonucleoprotein (RNP). In this case, the ribonucleoprotein may be an RNA-protein complex formed by interaction between the direct repeat portion of the guide RNA and the tracrRNA, i.e., a scaffold, with the Cas9 protein variant.

[0182] Composition form 2

[0183] In another embodiment, the CRISPR / Cas9 composition may comprise a nucleic acid encoding the Cas9 protein variant and a nucleic acid encoding the guide RNA in the form of a vector.

[0184] In one specific embodiment, the vector may be composed of DNA or RNA. In another specific embodiment, the vector may be composed of DNA and RNA. In this case, the vector can express the Cas9 protein variant and the guide RNA in vivo.

[0185] At this time, the vector may be a non-viral vector or a viral vector. At this time, the viral vector may be selected from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.

[0186] In one specific embodiment, the CRISPR / Cas9 composition may comprise a nucleic acid encoding the Cas9 protein variant and a nucleic acid encoding the guide RNA in a single vector. In this case, the CRISPR / Cas9 composition may comprise a single vector molecule.

[0187] In another specific embodiment, the CRISPR / Cas9 composition may comprise a nucleic acid comprising the Cas9 protein variant and a nucleic acid encoding the guide RNA, each in a separate vector. In this case, the CRISPR / Cas9 composition may comprise two or more vector molecules.

[0188] In another specific example, the CRISPR / Cas9 composition may comprise a vector comprising an mRNA encoding the Cas9 protein variant and a nucleic acid encoding the guide RNA. In this case, the nucleic acid encoding the guide RNA may be DNA.

[0189] Composition form 3

[0190] In another embodiment, the CRISPR / Cas9 composition may comprise a nucleic acid encoding the Cas9 protein variant and the guide RNA. In this case, the nucleic acid encoding the Cas9 protein variant may be mRNA or DNA, and may be contained in a vector.

[0191] In another embodiment, the CRISPR / Cas9 composition may comprise the Cas9 protein variant and a nucleic acid encoding the guide RNA. In this case, the nucleic acid encoding the guide RNA may be DNA and may be contained in a vector.

[0192]

[0193] Gene editing method using a composition comprising a Cas9 protein variant

[0194] In one aspect of the present invention, a gene editing method using a composition comprising a Cas9 protein variant is disclosed. The gene editing method is a method for editing genes within a cell. In particular, the gene editing method is a method for editing genes within a living body.

[0195] Intracellular gene editing methods

[0196] In one embodiment, the gene editing method is a method of editing a gene present in a cell.

[0197] The method comprises the step of introducing, administering, injecting, and / or delivering a CRISPR / Cas9 composition into a cell, wherein the CRISPR / Cas9 composition is one of the CRISPR / Cas9 compositions described in the "Composition Comprising a Cas9 Protein Variant" section described above.

[0198] The cell may be a human cell, a non-human animal cell, or a plant cell. In one specific example, the cell is a human cell. For example, the cell may be a human-derived somatic cell, a human-derived stem cell, or a human-derived immune cell.

[0199] At this time, the introduction, administration, injection, and / or delivery may utilize microinjection, electroporation, gene gun, sonoporation, magnetofection, and / or temporary cell compression or squeezing. Alternatively, the introduction, administration, injection, and / or delivery may utilize nanoparticles. For example, the introduction, administration, injection, and / or delivery may utilize lipid nanoparticles (LNPs) and / or PEG.

[0200] The cells into which the CRISPR / Cas9 composition has been introduced, administered, injected, and / or delivered may be transplanted and / or delivered to a subject, as needed. The subject is an organism with an immune system. In one specific example, the subject may be a human. In another specific example, the subject may be a non-human animal.

[0201] In one example, cells into which the CRISPR / Cas9 composition is introduced, administered, injected, and / or delivered, such as stem cells or immune cells, can be administered (transplanted and / or delivered) to a subject for the treatment of a desired disease, and the present specification also discloses methods of treatment and / or uses comprising the administration of such cells.

[0202] In another example, a tissue comprising cells into which the CRISPR / Cas9 composition has been introduced, administered, injected, and / or delivered, for example, liver tissue (comprising liver cells into which the CRISPR / Cas9 composition has been introduced, administered, injected, and / or delivered), can be transplanted (or delivered) into a subject for the treatment of a desired disease, and the present specification also discloses methods of treatment and / or uses involving transplantation of such tissue (comprising cells into which the CRISPR / Cas9 composition has been introduced, administered, injected, and / or delivered).

[0203] In vivo gene editing methods

[0204] In another embodiment, the gene editing method is a method of editing a gene in vivo.

[0205] The method comprises administering a CRISPR / Cas9 composition to a subject. The CRISPR / Cas9 composition is one of the CRISPR / Cas9 compositions described in the "Composition Comprising a Cas9 Protein Variant" section described above. The subject is an organism having an immune system. In one embodiment, the subject may be a human. In another embodiment, the subject may be a non-human animal.

[0206] Hereinafter, the use of a composition comprising the Cas9 protein variant and a gene editing method using the same will be described.

[0207]

[0208] A therapeutic agent using a composition comprising a Cas9 protein variant

[0209] In one aspect of the present invention, a disease treatment using a composition comprising a Cas9 protein variant is disclosed. The Cas9 protein variant of the present invention exhibits low immunogenicity in vivo, resulting in a reduced immune response compared to the wild-type Cas9 protein. Therefore, utilizing the Cas9 protein variant with these characteristics, it is possible to develop a therapeutic agent for in vivo administration that enables effective gene editing without inducing an immune response.

[0210] The therapeutic agent comprises a CRISPR / Cas9 composition. The CRISPR / Cas9 composition is one of the CRISPR / Cas9 compositions described in the "Composition Comprising a Cas9 Protein Variant" section described above. The guide RNA of the CRISPR / Cas9 composition has a guide sequence that is customized or artificially designed according to a target gene. The target gene is a gene associated with a disease. For example, the target gene is a gene that causes the disease. For another example, the target gene is a gene involved in causing the disease. For another example, the target gene is a gene involved in exacerbating the symptoms of the disease.

[0211] The above therapeutic agent is used for alleviating symptoms or treating diseases by artificially editing or manipulating specific genes.

[0212]

[0213] Method for treating a disease using a composition comprising a Cas9 protein variant

[0214] In one aspect of the present invention, a method for treating a disease using a composition comprising a Cas9 protein variant is disclosed. By administering a therapeutic agent comprising a Cas9 protein variant with low immunogenicity in vivo to a patient, the target gene of the disease can be artificially edited or manipulated without a significant immune response, thereby alleviating or treating the symptoms of the disease.

[0215] The above-described method for treating a disease comprises administering a therapeutic agent to a subject. The therapeutic agent is one of the therapeutic agents described in the "Treatment agent using a composition comprising a Cas9 protein variant" described above.

[0216] In this case, the subject is a human or non-human animal for which treatment is sought. For example, the subject is a human or non-human animal that has developed a disease. For another example, the subject is a human or non-human animal diagnosed as having a disease.

[0217] In this case, the disease is caused by a mutation in a specific gene. Alternatively, the disease is caused by the abnormal expression and / or abnormal function of a specific protein. In this case, the specific gene or the gene expressing the specific protein can be identified as a target gene. By artificially editing or manipulating this target gene, the symptoms of the disease can be alleviated or treated, or the onset of the disease can be inhibited.

[0218] At this time, the administration is administered in various ways depending on the disease. For example, the administration can be performed by injection, transfusion, implantation, or transplantation. In addition, the administration is administered by various routes depending on the disease. For example, the administration can be administered intraneurally, subcutaneously, intradermally, intraocularly, intravitreally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, intralymphatic, or intraperitoneally.

[0219]

[0220] Possible embodiments of the invention

[0221] Below, we list possible embodiments of the invention provided in this specification. The following embodiments provided in this paragraph are merely illustrative of the invention. Therefore, the invention provided in this specification should not be construed as limited to these embodiments. Furthermore, the brief descriptions provided with the embodiment numbers are merely for convenience in distinguishing between the embodiments and should not be construed as limitations on the invention disclosed in this specification.

[0222]

[0223] Cas9 protein

[0224] Example 1. SpCas9 protein variants

[0225] A Cas9 protein variant from Streptococcus pyogenes with low immunogenicity in vivo.

[0226] Example 2. Meaning of low immunogenicity 1

[0227] In Example 1, having low immunogenicity in vivo means that when the SpCas9 protein variant is introduced in vivo, compared to when the wild-type SpCas9 protein is introduced, there is little or no immune response occurring in the antigen presentation process and / or T cell recognition process.

[0228] Example 3. Meaning of low immunogenicity 2

[0229] In Example 1, the low immunogenicity in vivo means that when the SpCas9 protein variant is introduced in vivo, the antigen bound to MHC Class 2 and / or MHC Class 1 during the antigen presentation process is reduced compared to when the wild-type SpCas9 protein is introduced.

[0230] Example 4. Meaning of low immunogenicity 3

[0231] In Example 1, the low immunogenicity in vivo means that when the SpCas9 protein variant is introduced in vivo compared to when the wild-type SpCas9 protein is introduced, the antigen bound to MHC Class 2 and / or MHC Class 1 during the antigen presentation process is reduced, thereby reducing the immune response by T cells recognizing it.

[0232] Example 5. Meaning of low immunogenicity 4

[0233] In Example 1, having low immunogenicity in vivo means that when the SpCas9 protein variant is introduced in vivo, the activity of CD4+ T cells and / or CD8+ T cells is reduced compared to when the wild-type SpCas9 protein is introduced.

[0234] Example 6. Meaning of low immunogenicity 5

[0235] In Example 1, the low immunogenicity in vivo means that when the SpCas9 protein variant is introduced into the body, the expression of immune-related cytokines (IL-2, IFNγ, etc.) is reduced compared to when the wild-type SpCas9 protein is introduced.

[0236] Example 7. Mutation sites of SpCas9 protein variants (20 sites)

[0237] In any one of Examples 1 to 6, the SpCas9 protein variant In the amino acid sequence shown in , the 161st methionine (M); the 189th valine (V); the 238th phenylalanine (F); the 295th asparagine (N); the 337th alanine (A); the 619th isoleucine (I); the 620th valine (V); the 647th valine (V); the 648th methionine (M); the 659th tryptophan (W); the 794th glutamine (Q); the 884th arginine (R); the 956th isoleucine (I); the 961st lysine (K); An amino acid sequence in which at least one amino acid among the 978th ​​isoleucine (I); the 1042nd isoleucine (I); the 1144th leucine (L); the 1163rd leucine (L); the 1194th leucine (L); and the 1234th asparagine (N)) is substituted with another amino acid.

[0238] Example 8. Mutation sites of SpCas9 protein variants (6 locations)

[0239] In any one of Examples 1 to 7, the SpCas9 protein variant has an amino acid sequence in which at least one amino acid among the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) in the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid.

[0240] Example 9. SpCas9 protein variant 1 (deimmunized SpCas9 protein variant 1)

[0241] In Example 7, the SpCas9 protein variant has an amino acid sequence as shown in SEQ ID NO: 1, including the 161st methionine (M); the 189th valine (V); the 238th phenylalanine (F); the 295th asparagine (N); the 337th alanine (A); the 619th isoleucine (I); the 620th valine (V); the 647th valine (V); the 648th methionine (M); the 659th tryptophan (W); the 794th glutamine (Q); the 884th arginine (R); the 956th isoleucine (I); the 961st lysine (K); the 978th ​​isoleucine (I); the 1042nd isoleucine (I); the 1144th leucine (L); the 1163rd leucine (L); the 1194th leucine (L); and at least one amino acid selected from the 1234th asparagine (N) is substituted with one amino acid selected from the following:

[0242] Histidine (H); Aspartic acid (D); Glutamic acid (E); Serine (S); Glycine (G); Lysine (K); Arginine (R); Threonine (T); Glutamine (Q); Cysteine ​​(C); and Alanine (A),

[0243] At this time, the SpCas9 protein variant has an amino acid sequence different from the amino acid sequence shown in sequence number 1.

[0244] Example 10. SpCas9 protein variant 2 (deimmunized SpCas9 protein variant 2)

[0245] In Example 7 or Example 9, the SpCas9 protein variant has at least one mutation selected from the amino acid sequence shown in SEQ ID NO: 1:

[0246] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0247] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0248] Having serine (S) or aspartic acid (D) instead of phenylalanine (F) at position 238;

[0249] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 295;

[0250] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0251] Having lysine (K) or arginine (R) instead of isoleucine (I) at position 619;

[0252] Having serine (S) or threonine (T) instead of valine (V) at position 620;

[0253] Having aspartic acid (D) or glycine (G) instead of valine (V) at position 647;

[0254] Having lysine (K) or glutamic acid (E) instead of methionine (M) at position 648;

[0255] Having glutamine (Q) or histidine (H) instead of tryptophan (W) at position 659;

[0256] Having glutamic acid (E) or aspartic acid (D) instead of glutamine (Q) at position 794;

[0257] Having glutamic acid (E) or aspartic acid (D) instead of arginine (R) at position 884;

[0258] Having threonine (T) or glycine (G) instead of isoleucine (I) at position 956;

[0259] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0260] Having glutamic acid (E) or glycine (G) instead of isoleucine (I) at position 978;

[0261] Having glutamic acid (E) or aspartic acid (D) instead of isoleucine (I) at position 1042;

[0262] Having Glutamine (Q) or Asparagine (N) instead of Leucine (L) at position 1144;

[0263] Having Glutamine (Q) or Glycine (G) instead of Leucine (L) at position 1163;

[0264] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0265] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0266] Example 11. SpCas9 protein variant 3 (deimmunized SpCas9 protein variant 3)

[0267] In Example 10, the SpCas9 protein variant has one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 2 to SEQ ID NO: 41.

[0268] ;

[0269] ;

[0270] ;

[0271] ;

[0272] ;

[0273] ;

[0274] ;

[0275] ;

[0276] ;

[0277] ;

[0278] ;

[0279] ;

[0280] ;

[0281] ;

[0282] ;

[0283] ;

[0284] ;

[0285] ;

[0286] ;

[0287] ;

[0288] ;

[0289] ;

[0290] ;

[0291] ;

[0292] ;

[0293] ;

[0294] ;

[0295] ;

[0296] ;

[0297] ;

[0298] ;

[0299] ;

[0300] ;

[0301] ;

[0302] ;

[0303] ;

[0304] ;

[0305] ;

[0306] ; and

[0307]

[0308] Example 12. SpCas9 protein variant 4 (deimmunized SpCas9 protein variant 4)

[0309] In Example 8, the SpCas9 protein variant has at least one amino acid selected from the 161st methionine (M); the 189th valine (V); the 337th alanine (A); the 961st lysine (K); the 1194th leucine (L); and the 1234th asparagine (N) in the amino acid sequence shown in SEQ ID NO: 1 substituted with one amino acid selected from the following:

[0310] Histidine (H); Aspartic acid (D); Glutamic acid (E); Cysteine ​​(C); Glycine (G); and Alanine (A),

[0311] At this time, the SpCas9 protein variant has an amino acid sequence different from the amino acid sequence shown in sequence number 1.

[0312] Example 13. SpCas9 protein variant 5 (deimmunized SpCas9 protein variant 5)

[0313] In Example 8 or Example 12, the SpCas9 protein variant has at least one mutation selected from the amino acid sequence shown in SEQ ID NO: 1:

[0314] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0315] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0316] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0317] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0318] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0319] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0320] Example 14. SpCas9 protein variant 6 (deimmunized SpCas9 protein variant 6)

[0321] In Example 13, the SpCas9 protein variant has an amino acid sequence selected from among the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

[0322] Example 15. SpCas9 protein variant 7 (deimmunized SpCas9 protein variant 7)

[0323] In Example 8 or Example 12, the SpCas9 protein variant has at least two mutations selected from the amino acid sequence shown in SEQ ID NO: 1:

[0324] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0325] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0326] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0327] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0328] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0329] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0330] Example 16. SpCas9 protein variant 8 (deimmunized SpCas9 protein variant 8)

[0331] In Example 8 or Example 12, the SpCas9 protein variant has at least three mutations selected from the amino acid sequence shown in SEQ ID NO: 1:

[0332] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0333] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0334] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0335] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0336] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0337] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0338] Example 17. SpCas9 protein variant 9 (deimmunized SpCas9 protein variant 9)

[0339] In Example 16, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0340] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0341] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0342] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0343] Example 18. SpCas9 protein variant 10 (deimmunized SpCas9 protein variant 10)

[0344] In Example 17, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0345] Having cysteine ​​(C) instead of lysine (K) at position 961;

[0346] Having glycine (G) instead of leucine (L) at position 1194; and

[0347] Having glycine (G) instead of asparagine (N) at position 1234.

[0348] Example 19. SpCas9 protein variant 11 (deimmunized SpCas9 protein variant 11)

[0349] In Example 18, the SpCas9 protein variant has the amino acid sequence shown in SEQ ID NO: 42.

[0350]

[0351] Example 20. SpCas9 protein variant 12 (deimmunized SpCas9 protein variant 12)

[0352] In Example 8 or Example 12, the SpCas9 protein variant has at least four mutations selected from the amino acid sequence shown in SEQ ID NO: 1:

[0353] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0354] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0355] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0356] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0357] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0358] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0359] Example 21. SpCas9 protein variant 13 (deimmunized SpCas9 protein variant 13)

[0360] In Example 20, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0361] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0362] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0363] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961; and

[0364] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194.

[0365] Example 22. SpCas9 protein variant 14 (deimmunized SpCas9 protein variant 14)

[0366] In Example 21, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0367] Having histidine (H) instead of methionine (M) at position 161;

[0368] Having glutamic acid (E) instead of alanine (A) at position 337;

[0369] Having cysteine ​​(C) instead of lysine (K) at position 961; and

[0370] Having glycine (G) instead of leucine (L) at position 1194.

[0371] Example 23. SpCas9 protein variant 15 (deimmunized SpCas9 protein variant 15)

[0372] In Example 22, the SpCas9 protein variant has the amino acid sequence shown in SEQ ID NO: 43.

[0373]

[0374] Example 24. SpCas9 protein variant 16 (deimmunized SpCas9 protein variant 16)

[0375] In Example 20, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0376] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0377] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0378] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0379] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0380] Example 25. SpCas9 protein variant 17 (deimmunized SpCas9 protein variant 17)

[0381] In Example 24, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0382] Having histidine (H) instead of methionine (M) at position 161;

[0383] Having aspartic acid (D) instead of valine (V) at position 189;

[0384] Having Alanine (A) instead of Leucine (L) at position 1194; and

[0385] Having glycine (G) instead of asparagine (N) at position 1234.

[0386] Example 26. SpCas9 protein variant 18 (deimmunized SpCas9 protein variant 18)

[0387] In Example 25, the SpCas9 protein variant has the amino acid sequence shown in SEQ ID NO: 44.

[0388]

[0389] Example 27. SpCas9 protein variant 19 (deimmunized SpCas9 protein variant 19)

[0390] In Example 8 or Example 12, the SpCas9 protein variant has at least five mutations selected from the amino acid sequence shown in SEQ ID NO: 1:

[0391] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0392] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0393] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0394] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0395] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0396] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0397] Example 28. SpCas9 protein variant 20 (deimmunized SpCas9 protein variant 20)

[0398] In Example 8 or Example 12, the SpCas9 protein variant has the following mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0399] Having histidine (H) or aspartic acid (D) instead of methionine (M) at position 161;

[0400] Having aspartic acid (D) or glutamic acid (E) instead of valine (V) at position 189;

[0401] Having glutamic acid (E) or aspartic acid (D) instead of alanine (A) at position 337;

[0402] Having aspartic acid (D) or cysteine ​​(C) instead of lysine (K) at position 961;

[0403] Having glycine (G) or alanine (A) instead of leucine (L) at position 1194; and

[0404] Having aspartic acid (D) or glycine (G) instead of asparagine (N) at position 1234.

[0405] Example 29. SpCas9 protein variant 21 (deimmunized SpCas9 protein variant 21)

[0406] In Example 7, Example 8, Example 9, Example 10, Example 12, Example 13, Example 15, Example 16, or Example 20, the SpCas9 protein variant has the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

[0407] Example 30. Including additional NLS

[0408] In any one of Examples 1 to 29, the SpCas9 protein variant further comprises at least one NLS at the N-terminus and / or the C-terminus.

[0409] Example 31. Inclusion of additional NLS

[0410] In Example 30, the NLS is each independently PKKKRKV (SEQ ID NO: 45), KRPAATKKAGQAKKKK (SEQ ID NO: 46), PAAKRVKLD (SEQ ID NO: 47), RQRRNELKRSP (SEQ ID NO: 48), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 49), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 50), VSRKRPRP (SEQ ID NO: 51), PPKKARED (SEQ ID NO: 52), PQPKKKPL (SEQ ID NO: 53), SALIKKKKKMAP (SEQ ID NO: 54), DRLRR (SEQ ID NO: 55), PKQKKRK (SEQ ID NO: 56), RKLKKKIKKL (SEQ ID NO: 57), Having an amino acid sequence selected from among REKKKFLKRR (SEQ ID NO: 58), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 59), and RKCLQAGMNLEARKTKK (SEQ ID NO: 60).

[0411] Example 32. Cas9 nomenclature

[0412] In any one of Examples 1 to 31, the SpCas9 protein variant may be referred to as a Cas9 protein, a novel Cas9 protein, a novel SpCas9 protein, a Cas9 protein variant, a Cas9 mutation, a SpCas9 mutation, a Cas9 mutant, a SpCas9 mutant, a deimmunized Cas9 protein, a deimmunized Cas9 protein variant, a deimmunized SpCas9 protein, or a deimmunized SpCas9 protein variant.

[0413] guide RNA

[0414] Example 33. SpCas9 guide RNA

[0415] Guide RNA that can target a predetermined target gene by forming a complex with the Cas9 protein derived from Streptococcus pyogenes.

[0416] Example 34. Guide RNA sequence

[0417] In Example 33, the guide RNA comprises crRNA and tracrRNA,

[0418] The above crRNA has a guide sequence and a direct repeat sequence sequentially linked in the 5' to 3' direction.

[0419] At this time, the direct repeat sequence is a nucleic acid sequence of 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 61), or a nucleic acid sequence shown in the above SEQ ID NO: 61 and 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, represented by a nucleic acid sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical;

[0420] The above tracrRNA has a nucleic acid sequence of 5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 61), or 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, Expressed as a nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0421] Example 35. Guide sequence of guide RNA

[0422] In Example 34, the guide sequence is a sequence capable of complementarily binding to a portion of a nucleic acid sequence of a predetermined target gene.

[0423] Example 36. Guide sequence of guide RNA

[0424] In Example 34 or Example 35, the guide sequence can be designed (or artificially) to fit a predetermined target gene.

[0425] Example 37. Guide sequence of guide RNA

[0426] In any one of Examples 34 to 36, the guide sequence is 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 in length.

[0427] Example 38. Dual guide RNA

[0428] In any one of Examples 33 to 37, the guide RNA is a dual guide RNA of two molecules,

[0429] At this time, the direct repeat sequence and a part of the tracrRNA can complementarily bind.

[0430] Example 39. Single guide RNA

[0431] In any one of Examples 33 to 37, the guide RNA is connected to the 3' end of the crRNA and the 5' end of the tracrRNA via a linker.

[0432] Example 40. Linker of a single guide RNA

[0433] In Example 39, the linker is 5'-GAAA-3'.

[0434] CRISPR / Cas9 composition

[0435] Example 41. CRISPR / Cas9 composition comprising SpCas9 protein variant

[0436] Any one of the SpCas9 protein variants of Examples 1 to 31 or a nucleic acid encoding the SpCas9 protein variant; and

[0437] The guide RNA of any one of Examples 33 to 40 or a nucleic acid encoding the guide RNA

[0438] A CRISPR / Cas9 composition comprising:

[0439] Example 42. Form 1 of CRISPR / Cas9 composition

[0440] In Example 41, the CRISPR / Cas9 composition comprises the SpCas9 protein variant and the guide RNA in the form of ribonucleoprotein (RNP).

[0441] Example 43. Ribonucleoprotein (RNP) form

[0442] In Example 42, the ribonucleoprotein is formed by the direct repeat portion of the guide RNA and the tracrRNA interacting with the SpCas9 protein variant.

[0443] Example 44. Form 2 of CRISPR / Cas9 composition

[0444] In Example 41, the CRISPR / Cas9 composition comprises a nucleic acid encoding the SpCas9 protein variant and a nucleic acid encoding the guide RNA in the form of a vector.

[0445] Example 45. Vector

[0446] In Example 44, the vector can express the SpCas9 protein variant and the guide RNA in vivo.

[0447] Example 46. Single vector

[0448] In Example 44 or Example 45, the vector is a single vector of one molecule, wherein the nucleic acid encoding the SpCas9 protein variant and the nucleic acid encoding the guide RNA are contained in a single vector.

[0449] Example 47. Multiple vectors

[0450] In Example 44 or Example 45, the vector is a vector of two or more molecules, wherein the nucleic acid encoding the SpCas9 protein variant and the nucleic acid encoding the guide RNA are each contained in separate vectors.

[0451] Example 48. mRNA

[0452] In any one of Examples 44 to 47, the nucleic acid encoding the SpCas9 protein variant is mRNA, and the nucleic acid encoding the guide RNA is DNA.

[0453] Example 49. Types of vectors

[0454] In any one of Examples 44 to 48, the vector is a non-viral vector or a viral vector.

[0455] Example 50. Types of viral vectors

[0456] In Example 49, the viral vector is selected from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.

[0457] Example 51. Form 3 of CRISPR / Cas9 composition

[0458] In Example 41, the CRISPR / Cas9 composition comprises a nucleic acid encoding the SpCas9 protein variant and the guide RNA, wherein the nucleic acid encoding the SpCas9 protein variant is mRNA or DNA and is included in a vector.

[0459] Example 52. Form 4 of CRISPR / Cas9 composition

[0460] In Example 41, the CRISPR / Cas9 composition comprises the SpCas9 protein variant and a nucleic acid encoding the guide RNA, wherein the nucleic acid encoding the guide RNA is DNA and includes a vector.

[0461] Gene editing methods

[0462] Example 53. Gene editing method 1

[0463] A method for editing genes within a cell, comprising:

[0464] A step of introducing, administering, injecting and / or delivering the CRISPR / Cas9 composition of any one of Examples 41 to 52 into a cell;

[0465] At this time, the guide RNA has a guide sequence that is custom-designed (or artificially) to target the gene to be edited.

[0466] Example 54. Gene editing method 2

[0467] A method for editing a gene in vivo, comprising:

[0468] A step of administering to a target a CRISPR / Cas9 composition of any one of Examples 41 to 52;

[0469] At this time, the guide RNA has a guide sequence that is custom-designed (or artificially) to target the gene to be edited.

[0470] Example 55. Subject 1

[0471] In Example 54, the subject is an organism having an immune system.

[0472] Example 56. Subject 2

[0473] In Example 54 or Example 55, the subject is a human or a non-human animal.

[0474] Disease treatments and methods of treatment

[0475] Example 57. Therapeutic agent for in vivo administration

[0476] A therapeutic agent for in vivo administration comprising a CRISPR / Cas9 composition of any one of Examples 41 to 52,

[0477] At this time, the guide RNA has a guide sequence that is custom-designed (or artificially) according to the target gene of the disease.

[0478] Example 58. Method for treating disease

[0479] A method for treating a disease, comprising administering the therapeutic agent of Example 57 to a subject for whom treatment of the disease is desired.

[0480] Example 59. Disease

[0481] In Example 57 or Example 58, the disease is caused by abnormal expression and / or abnormal function performance of a specific gene or a specific protein, and the disease refers to a disease whose symptoms can be alleviated or treated by artificially editing (or manipulating) a specific gene.

[0482] Example 60. Subject

[0483] In Example 58, the subject is a human or non-human animal diagnosed as having or being capable of developing a disease.

[0484] Example 61. Administration method 1

[0485] In Example 58, the administration is administered in various ways depending on the disease.

[0486] Example 62. Administration method 2

[0487] In Example 58 or Example 61, the administration is performed by injection, transfusion, implantation or transplantation.

[0488] Example 63. Route of administration

[0489] In Example 58, Example 61 or Example 62, the administration is intraneural, subcutaneously, intradermally, intraocularly, intravitreally, intratumorally, intranodally, intramedullary, intramuscularly, intravenous, intralymphatic, or intraperitoneally.

[0490]

[0491] 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.

[0492]

[0493] Experimental Example 1: Screening of Deimmunized SpCas9

[0494] The aim is to obtain a deimmunized SpCas9 mutant with a reduced immune response by interfering with the antigen presentation process of the SpCas9 protein.

[0495] The SpCas9 protein is cleaved into short peptide fragments via intracellular proteolytic mechanisms. These peptide fragments, each with a specific sequence, bind to MHC-I and MHC-II proteins and are used for antigen presentation. Bioinformatics analysis can predict which SpCas9 protein fragments are most likely to bind to MHC-I and MHC-II proteins and be used for antigen presentation. Furthermore, specific amino acid substitution mutations can be used to weaken antigen presentation.

[0496] Through antigen presentation, MHC-I activates cytotoxic T cells, while MHC-II activates antibody-producing B cells. Weakening antigen presentation through substitution mutations in SpCas9 is expected to reduce the responses of T and B cells, which are classified as adaptive immune cells.

[0497] Experimental process and results

[0498] Experimental Example 1-1. Selection of Deimmunized SpCas9 Mutants through Bioinformatics Analysis

[0499] Bioinformatics analysis was used to select substitution mutations that maximize the deimmune effect without significantly reducing SpCas9 nuclease activity. A total of 40 first- and second-rank mutations were identified at 20 locations.

[0500] Selected Mutation LocationMutationA.BCDEFGHIJKLMNOPQRSTNameD1M161 (A)1st rankH1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0M161H, A1D22nd rankD2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0M161D, A2D3V189 (B)1st rankD0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0V189D, B1D42RankE0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0V189E, B2D5F238 (C)1RankS0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0F238S, C1D62RankD0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0F238D, C2D7N295 (D)1RankD0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0N295D, D1D82rankG0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0N295G, D2D9A337 (E)1rankE0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0A337E, E1D102rankD0.0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0A337D, E2D11I619 (F)1rankK0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0I619K, F1D122RankingR0.0.0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0I619R, F2D13V620 (G)1SankingS0.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0V620S, G1D142TankingT0.0.0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0V620T, G2D15V647 (H)1DankingD0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0V647D, H1D162RankG0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0V647G, H2D17M648 (I)1st rank K0.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0M648K, I1D182nd rank E0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0M648E, I2D19W659 (J)1st rank Q0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0W659Q, J1D202nd rank H0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0.0.0W659H, J2D21Q794 (K)1st rank E0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0Q794E, K1D222 rank D0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0.0Q794D, K2D23R884 (L)1st rank E0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.0R884E, L1D242 rank D0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0R884D, L2D25I956 (M)1stT0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0I956T, M1D262TrongG0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.0I956G, M2D27K961 (N)1stD0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.0K961D, N1D282TrongC0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0K961C, N2D29I978 (O)1st rankE0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0I978E, O1D302rankG0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0I978G, O2D31I1042 (P)1st rankE0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0I1042E, P1D322rankD0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0I1042D, P2D33L1144 (Q)1st place Q0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0L1144Q, Q1D342Rank N0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0L1144N, Q2D35L1163 (R)1Rank Q0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0L1163Q, R1D362Rank G0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0L1163G, R2D37L1194 (S)1st rank G0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0L1194G, S1D382nd rank A0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.0L1194A, S2D39N1234 (T)1st rank D0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1N1234D, T1D402nd rank G0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2N1234G, T20 : WT / 1 : 1st rank mutation / 2 : 2nd rank mutation.

[0501] Experimental Example 1-2. Construction of a Screening Library Plasmid

[0502] At 20 locations, library 1 was constructed with a combination of wild type (WT) and first-rank mutations, and library 2 was constructed with a combination of first-rank and second-rank mutations, resulting in 220 types of libraries (approximately 1 million). Primers containing mutations were ordered, and a short first-round PCR was performed. The fragments were assembled to produce PCR products containing all mutations, which were then cloned into a piggybac vector (CMV promoter, 2NLS) through a Gibson assembly experiment (Fig. 1).

[0503] Experimental Example 1-3. Creation of Piggybac stable cell lines and 6TG screening.

[0504] The stable cell line was transfected into 2*10^6 HeLa cells in a 150-pi culture plate using 2μg of Cas9 plasmid, 2μg of transposase vector, and 12μl of Lipofectamine 2000. Puromycin selection was performed at 2μg / ml from the next day. After 2-3 weeks of puromycin selection, 6*10^6 HeLa cells in 150-pi were transfected using 20μg of HPRT sgRNA plasmid and 40μl of Lipofectamine 2000. After 2-3 days, 30μM 6TG screening was performed for 2 weeks on 2*10^6 HeLa cells in 150-pi. Due to the large number of cell colonies, stable cell production and screening were repeated once more (Fig. 2). The HPRT target sequences used in the experiments are as follows: TCGAGATGTGATGAAGGAGA (SEQ ID NO: 175) in the first screening and GTAGCCCTCTGTGTGCTCAA (SEQ ID NO: 176) in the second screening.

[0505] Experimental Example 1-4. Activity Test of 6TG-Screened Deimmunized Cas9 Candidates

[0506] After gDNA purification and cloning into a plasmid, full sequencing was performed on 100 E. coli colonies per library. The EMX1 indel test was used to initially select those with less activity compared to the WT. The EMX1 target sequence of the sgRNA used here is GAGTCCGAGCAGAAGAAGAA (SEQ ID NO: 100). For the 24 targets, those with less activity compared to the WT were then subjected to a secondary selection.

[0507] For this purpose, an artificial 24-target indel evaluation system was secured.

[0508] Using the Piggybac system, we integrated artificial sequences containing sgRNA and target sequences into the genome of HEK293T cells. Key sequences integrated via the Piggybac system are listed below. Sequence information corresponding to each NNN is shown in the table and was used to prepare a total of 24 artificial targets.

[0509] U6 promoter sgRNA GX20 spacer sgRNA scaffold 10bp Barcode Target NGG PAM

[0510] TTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC TTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTNNNNNNNNNNNNGANNNNNNNNNNNNNNNNNNNNNNNGGAGCTTGGCGTAACGGCTTAACTAGA (SEQ ID NO: 101)

[0511] Sequence information #sgRNA G20 spacer 10bp BarcodeTargetNGG PAM Note 1 GAGAAGGAGTAACATCC (SEQ ID NO: 102) AGGGAGAACC (SEQ ID NO: 126) AAGAAGAAGGAGTAACATCC (SEQ ID NO: 150) TGG 2 GTTGTGCTCTTTGCTCTCTCA (SEQ ID NO: 103) GTCCGGTGGC (SEQ ID NO: 127) CTGTGCTCTTTGCTCTCTCA (SEQ ID NO: 151) TGG 3 GTTTCATTACAATCGCGTGGC (SEQ ID NO: 104) TAGATAGATA (SEQ ID NO: 128) TTTCATTACAATCGCGTGGC (SEQ ID NO: 152) TGG 4 GTACCACCGGATGGGACTGG (SEQ ID NO: 105) CCCACTTGGT (SEQ ID NO: 129) GTACCACCGGATGGGACTGG (SEQ ID NO: 153) AGG 5GTACGGGTGGCTCTCAAGCG (SEQ ID NO: 106) TCCTGGAAGGC (SEQ ID NO: 130) GTACGGTGGCTCTCAAGCG (SEQ ID NO: 154) CGGBarcode 11bp 6GGCTGACCGCGATGCCTACC (SEQ ID NO: 107) AAGTATACCA (SEQ ID NO: 131) TGCTGACCGCGATGCCTACC (SEQ ID NO: 155) AGG 7GCCGTCTGTGGATAGGAGAG (SEQ ID NO: 108) GACCGCCTAT (SEQ ID NO: 132) ACCGTCTGTGGATAGGAGAG (SEQ ID NO: 156) CGG 8GCCTCGGGGCTGAGCGTGCG (SEQ ID NO: 109) TGCTATGTTT (SEQ ID NO: 133)GCCTCGGGGCTGAGCGTGCG (SEQ ID NO: 157)CGG 9GTCTGTTGAAAAAGAGAACT (SEQ ID NO: 110)TAAGTCCACG (SEQ ID NO: 134)CTCTGTTGAAAAAGAGAACT (SEQ ID NO: 158)TGG 10GCTGCCGACTCCGGTGCCGT (SEQ ID NO: 111)ATACTCACTG (SEQ ID NO: 135)GCTGCCGACTCCGGTGCCGT (SEQ ID NO: 159)CGG 11GATCACTGTCAACTACGGCT (SEQ ID NO: 112)TAGCCGATCT (SEQ ID NO: 136)GATCACTGTCAACTACGGCT (SEQ ID NO:160)TGG 12GTGCTGCGGTTGTCCCATTG(SEQ ID NO: 113)CCCTTTGACT(SEQ ID NO: 137)GTGCTGCGGTTGTCCCATTG(SEQ ID NO: 161)GGG 13GAGCCTGTACAGCGGCCTGC(SEQ ID NO: 114)TGCATTGGAA(SEQ ID NO: 138)CAGCCTGTACAGCGGCCTGC(SEQ ID NO: 162)TGG 14GGGGAGGAAACCCTACAACC(SEQ ID NO: 115)CATGCTTACT(SEQ ID NO: 139)CGGGAGGAAACCCTACAACC(SEQ ID NO: 163)CGG 15GCACCCTTTTCCTCTTGGGG(SEQ ID NO: 116)GCACTAATTT(SEQ ID NO: 140)TCACCCTTTTCCTCTTGGGG(SEQ ID NO: 164)CGG 16GCAGATGCCCAGCGGGTAGC(SEQ ID NO: 117)CGCCGAGGGC(SEQ ID NO: 141)ACAGATGCCCAGCGGGTAGC(SEQ ID NO: 165)AGG 17GTGTGAAGTAATCTTAGGGT(SEQ ID NO: 118)GGAACTAATT(SEQ ID NO: 142)TTGTGAAGTAATCTTAGGGT(SEQ ID NO: 166)TGG 18GGAACCCGAGTGCTGCTTGG(SEQ ID NO: 119)CTGTATCACA(SEQ ID NO: 143)AGAACCCGAGTGCTGCTTGG(SEQ ID NO: 167)CGG 19GCGGAAGCCCGACGGTTGCT(SEQ ID NO: 120)CTGCCCCGAC(SEQ ID NO: 144)GCGGAAGCCCGACGGTTGCT(SEQ ID NO: 168)CGG 20GCCTGCGAGGCGGCCCACTT(SEQ ID NO: 121)AACCGAAGTT(SEQ ID NO: 145)ACCTGCGAGGCGGCCCACTT(SEQ ID NO: 169)CGG 21GGTAGGTCACCAGAAAGCAG(SEQ ID NO: 122)TTCTATCTGT(SEQ ID NO: 146)CGTAGGTCACCAGAAAGCAG(SEQ ID NO: 170)AGG 22GCGGTCTTGGGCGCGCTCTG(SEQ ID NO: 123)ACGTATAGCT(SEQ ID NO: 147)TCGGTCTTGGGCGCGCTCTG(SEQ ID NO: 171)CGG 23GTGTAGCTGGCGTAATCTGT(SEQ ID NO:124)TAGCTGTCGA(SEQ ID NO: 148)GTGTAGCTGGCGTAATCTGT(SEQ ID NO: 172)AGG 24GCACCCTAAGGGGGGAGAAA(SEQ ID NO: 125)GGTGTGGTAC(SEQ ID NO: 149)GCACCCTAAGGGGGGAGAAA(SEQ ID NO: 173)TGG

[0512] Integration experiments were conducted using Transposase vector and Transposone vector at a molar ratio of 1:2.5 according to the Piggybac system manual, and a total of 1 μg of plasmid was prepared. The prepared plasmid was transfected into 24-well HEK293T 1*10^5 cells using 2 μl of Lipofectamin 2000. The transposon vector containing the artificial sequence was transfected into each well so that one artificial sequence was inserted into each well. After transfection, selection was performed for one week with puromycin at a concentration of 2 μg / ml, and finally, equal amounts of cells were pooled to create a cell library with 24 artificial targets. When using the created artificial 24-target indel evaluation system, the plasmid containing the Cas9 to be evaluated was delivered to the cell library, and the indel activity value for each target was averaged and calculated.

[0513] Experimental Example 1-5. Activity Changes According to 40 Substitution Mutations

[0514] After securing 40 types of single mutations through site-directed mutagenesis, the average indel activity compared to the WT was confirmed using the artificial 24-target evaluation system obtained above (Fig. 3). Five mutations (Q794D, R884D, K961C, L1194G, N1234G) were selected based on their activity rank within the top 10 and non-overlapping positions.

[0515] 24 targets relative activity spCas9 domain abbreviation position information WT 100% vs. Rank Mutation added REC-1, 2D1M161H 79.80% 15 - D2M161D 65.38% 30 - D3V189D 85.12% 8 - D4V189E 77.60% 17 - D5F238S 79.90% 14 - D6F238D 25.51% 37 - D7N295D 80.26% 13 - D8N295G 64.92% 31 - D9A337E 75.70% 20 - D10A337D 84.27% 9 - Rec-3D1 1I619K28.73%36-D12I619R20.01%38-D13V620S75.67%21-D14V620T74.69%23-D15V647D66.93%28-D16V6 47G78.48%16-D17M648K76.58%18-D18M648E80.65%12-D19W659Q60.79%33-D20W659H11.27%39-HNHD21Q7 94E81.63%11-D22Q794D91.68%2SelectedD23R884E87.17%5-D24R884D90.79%3SelectedRuvC-3D25I956T84.22%10-D26I956G66.03%29-D27K961D68.07%27-D28K961C90.35%4SelectedD29I978E72.12%26-D30I978G43.91%34-D31I1 042E31.21%35-D32I1042D5.65%40-CTDD33L1144Q73.43%25-D34L1144N76.30%19-D35L1163Q64.17%32-D36L1163G73.54%24-D37L1194G85.22%7SelectedD38L1194A74.77%22-D39N1234D86.21%6-D40N1234G93.48%1Selected

[0516] Experimental Example 1-6. Selection of Three Representative Deimmunized SpCas9 Candidates

[0517] The mutations selected in Experiments 4 and 5 above were mixed and cloned into various deimmunized spCas9s. Mutants showing an activity of 70% or more compared to the WT were selected through an average indel activity test using the Artificial 24 target analysis system. Subsequently, three deimmunized spCas9 candidates (Mutants 1-3) were selected through bioinformatics analysis to have a high degree of deimmunity and no problems in the actual protein production process. The final three candidates are expected to weaken the antigen presentation of MHC-II. The three selected deimmunized spCas9 candidates have the amino acid sequences represented by SEQ ID NO: 43 (Mutant 1), SEQ ID NO: 42 (Mutant 2), and SEQ ID NO: 44 (Mutant 3), respectively.

[0518] deimmunized spCas9 candidate 3 types 24 targets (WT 100%) (N=3) RNP EMX1WT = 100% (N=3)ABCDEFGHIJKLMNOPQRSTMutation numberMutation typeCc(Mutant 1)76.07%38.06%1.0.0.0.1.0.0.0.0.0.0.0.0.2.0.0.0.0.1.04M161H, A337E, K961C, L1194GDcde (Mutant 2)88.84%33.62%0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.1.23K961C, L1194G, N1234G2_64(Mutant 3)84.57%94.09%1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.24M161H, V189D, L1194A, N1234G

[0519] Predicted immune scores of three deimmunized spCas9 candidatesMHC-IIMHC-IsumIEDB(26 alleles)IEDB(8 alleles)ProPred(8 alleles)IEDB(26 alleles)IEDB(8 alleles)WT107732946211883663422Mutant 198828044311783643253Mutant 2101228945911803643304Mutant 3104431844411723643342

[0520] MHCI, MHC II Binding peptide Prediction tool

[0521] 1. IEDB - https: / / www.iedb.org /

[0522] 2. ProPred - https: / webs.iiitd.edu.in / raghava / propred /

[0523] Experimental Example 1-7. Deimmunized SpCas9 Screening Experiment and Data Analysis

[0524] The mutant screening process prior to immunological experiments aims to screen for mutants with the highest number of mutations that minimize the immune response without significantly reducing Cas9 editing activity. The experimental process, briefly described above, is described in detail, along with the results of analysis of the acquired data.

[0525] 1) Analysis of the results of EMX1 indel activity analysis experiments on 140 deimmunized Cas9 mutant candidates obtained after two 6TG screenings.

[0526] Transfection was performed on 1x10^5 HEK239T cells in 24 wells using 500 ng of Cas9 plasmid, 500 ng of EMX1 sgRNA plasmid, and 2 μl of Lipofectamine2000. Two days later, gDNA was prepped and Indel analysis was performed.

[0527] Through a single repeated experiment, mutants with appropriate Indel activity against the EMX1 target were first screened, and the selected candidates were marked with * in front of the sample name in the table and circled on the graph. (Table 6, Fig. 4) The selection criteria were different depending on the number of mutations. In the case of M>5 (number of mutations exceeding 5), most of the Indel activities were absent, so the mutant with the highest indel was selected, and in the case of M=5, no selection was made, and in the cases of M=4 and M=3, two mutants with the highest indels were selected each. In the cases of M=2 and M=1, it was determined that the number of mutations needed to be added to reduce immunity, so the selection criterion was set to be an Indel activity of 70% or more compared to the WT.

[0528] 140 Mutant EMX1 indelsample name (Library_Colony)Mutation typeNumber of mutationsWT 100% ContrastABCDEFFGGGHIJJKLMNOP1_10.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.0.1.0244.95%*1_20.0.0.0.0.0.1.0.0.0.1.0.0.0.1.0.0.0.0.0.0.0277.94%1_30.0.0.1.0.0.1.0.0.0.0.0.0.0.1.1.0.0.1.1610.39%1_50.0.0.1.0.1.1.0.0.0.0.0.1.0.0.0.0.1.0.0.0.0.150.45%1_70.0.0. 0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.1248.96%1_100.0.0.0.0.0.1.1.0.0.0.1.1.0.0.0.1.1.0.0.0.0.0.0.0434.75%1_110.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.010.45%1_121.0.0.0.0.0.0.0.0.0.0.0.1.1.0.1.0.1.0.1.0.1.0.1.0.162.76%*1_130.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0. 0.0.0195.65%1_150.0.0.0.0.1.1.1.1.1.0.0.1.1.0.0.0.0.180.08%1_171.0.0.0.0.0.0.1.0.0.0.1.1.0.0.0.1.1.0.0.0.1.1.0.170.09%*1_180.0.0.1.0.0.0.0.0.0.0.1.1.0.0.0.0.0.0350.79%1_210.0.0.0.0.0.1.0.0.0.0.1.0.0.0.1.0.1.1.1.0.1.170.09%1_220.1.1.0.0.0.0 .0.0.0.1.1.1.1.0.0.0.0.0.170.31%1_230.0.0.0.0.1.1.0.0.0.0.0.0.0.0.0.0.1.1.150.52%1_240.0.1.1.0.0.1.0.0.0.1.1.1.0.1.0.0.0.1.1.1.0.1.0.0.0.1.190.15%1_290.1.0.0.0.1.1.1.1.1.1.1.0.1.1.1.1.0.1.1.1.0.0.11321.86%1_310.0.0.0.0.0.1.0.0.0.0.0.0.0.0.0.1.1.1.0425.16%1_320.0.0.0.0.0.0.0.0.1.1.1.0.0.0.0.0.1.0.040.48%1_340.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.033.79%1_351.1.0.0.0.0.1.0.1.0.0.0.0.0.0.0.1.1.1.070.34%1_380.0.0.0.0.1.1.1.1.0.0.1.0.1.1.0.0.1.1.090.17%1_390.0.0.0.0.1.0.0.1.0.0.0.0.0.0.0.0.0.0.1333.33%*1_420.0.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0361.12%1_431.1.1.1.0.1.1.1.0.0.0.0.1.0.1.0.0.1.0.0100.13%*1_440.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0195.05%1_450.0.0.0.0.0.1.0.0.0.1.0.1.0.0.0.1.1.0.160.09%1_460.0.0.0.0.0.1.0.1.1.0.1.1.0.1.1.1.0.0.190.41%1_490.0.0.0.0.0.0.0.1.0.0.1.0.0.1.0.0.0.0.031.46%1_510.1.0.0.0.0.1.0.0.0.0.0.1.0.0.1.0.1.0.160.00%1_541.1.1.1.0.1.1.1.0.0.1.0.0.1.0.1.1.1.1.0140.17%1_550.0.0.0.0.0.0.1.1.0.1.1.0.0.0.0.1.0.1.170.31%1_570.1.1.0.0.0.0.0.1.1.1.1.1.0.0.1.1.0.0.090.00%1_591.0.0.0.0.1.0.1.0.0.0.1.1.0.0.0.0.0.0.050.65%1_600.0.3.1.0.0.1.0.0.0.0.1.0.0.0.1.1.0.1.182.56%1_610.0.0.0.0.0.1.1.1.1.1.0.0.0.0.0.0.0.0.160.16%*1_631.0.0.0.0.0.1.1.0.0.0.1.0.0.0.0.0.0.0.0468.85%1_641.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0186.03%1_650.0.1.1.0.0.1.0.0.1.0.0.0.0.1.1.1.1.1.090.28%1_680.0.0.0.0.1.1.1.0.0.0.0.0.1.1.0.0.1.1.070.23%1_690.1.0.1.0.0.0.1.0.1.0.0.1.0.1.0.0.1.0.180.20%*1_730.0.3.1.0.0.0.1.0.0.1.1.1.0.0.0.0.0.0.0641.21%1_740.0.0.0.0.0.1.0.0.1.0.1.0.0.1.0.0.1.0.050.56%1_750.0.0.1.0.1.0.1.1.0.0.1.1.1.0.0.0.1.1.090.80%1_780.1.0.0.0.1.1.1.0.0.0.0.1.0.1.0.0.0.0.060.00%1_790.0.0.1.0.0.1.1.0.0.1.1.0.0.0.0.1.1.0.076.80%1_800.0.0.0.0.0.0.0.0.0.0.1.0.1.0.0.1.0.0.130.56%1_810.1.0.0.0.0.0.0.0.0.0.0.1.1.1.0.0.0.0.158.78%1_830.0.1.0.0.1.0.0.1.0.0.1.1.0.0.0.0.0.0.050.31%1_851.0.0.0.0.1.0.0.0.1.0.0.1.0.1.0.0.1.1.187.69%1_880.1.1.1.0.0.0.0.0.0.0.1.0.0.0.0.0.0.0.040.39%1_890.0.0.0.0.1.1.1.0.1.1.1.1.0.0.0.0.1.0.190.38%1_900.0.1.0.0.0.1.0.0.0.0.1.0.0.0.0.0.0.0.145.28%1_910.0.0.0.0.0.1.0.0.0.1.0.1.1.0.0.0.0.0.0439.37%1_920.1.0.0.0.0.1.1.1.0.0.1.0.1.0.0.0.0.0.060.26%1_940.0.0.1.0.1.0.1.0.0.0.0.0.0.0.0.0.0.0.030.40%1_951.1.0.1.0.1.1.1.0.0.0.0.1.1.0.0.0.0.0.190.28%1_970.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0.010.51%1_991.0.0.1.0.0.1.0.1.0.1.1.1.1.0.1.0.1.1.0110.16%1_1020.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.010.64%1_1030.0.0.1.0.0.1.0.1.0.1.1.1.0.1.1.1.1.0.1110.13%1_1040.0.0.0.0.0.0.0.0.0.0.0.0.0.1.1.0.0.1.141.77%1_1050.1.1.0.0.0.0.0.0.0.0.0.0.0.1.1.0.0.0.040.49%1_1060.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.048.15%1_1070.0.0.1.0.1.1.0.0.1.1.0.0.0.0.0.0.0.0.051.40%1_1081.1.0.1.0.1.1.1.0.0.1.1.0.1.0.1.1.1.1.0130.00%1_1100.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.020.63%1_1110.0.0.0.0.1.1.1.0.1.0.0.0.0.0.1.0.1.0.050.57%1_1120.1.0.1.0.0.1.0.0.0.0.0.1.0.1.0.0.1.0.170.84%1_1130.0.0.0.0.1.0.1.0.0.1.1.0.0.1.0.1.1.0.180.50%1_1140.0.0.0.0.1.1.1.0.0.0.0.1.1.0.1.0.1.0.073.28%1_1150.0.0.0.0.0.0.1.1.0.0.0.1.0.0.1.1.1.0.060.42%1_1170.0.0.0.0.0.1.0.0.0.1.0.0.1.1.0.1.0.1.061.49%1_1180.0.1.0.0.1.0.1.0.1.0.1.1.0.1.0.1.1.1.1110.17%1_1190.0.0.1.0.0.1.1.0.0.0.1.1.1.1.0.1.1.1.1110.00%1_1220.0.1.0.0.0.0.1.1.1.1.0.1.1.1.0.0.1.0.1100.52%1_1240.0.0.1.0.0.1.1.1.1.0.1.0.0.0.0.0.0.0.170.10%1_1250.0.0.1.0.0.0.0.0.0.0.1.0.0.0.0.0.1.0.140.29%1_1260.0.0.0.0.0.1.1.0.0.0.1.1.0.1.0.1.0.0.060.10%1_1270.0.0.0.0.0.1.0.0.1.1.1.0.1.1.0.1.0.0.180.22%1_1280.0.0.0.0.0.0.1.1.0.0.0.0.0.1.0.0.0.0.0319.43%1_1290.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.1.1.0420.53%1_1300.0.1.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0227.18%1_1351.0.1.1.0.1.0.1.0.0.0.0.1.0.0.1.1.0.1.1100.13%2_11.1.1.2.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.053.82%2_20.0.0.0.0.1.1.2.2.1.1.2.2.1.1.1.1.1.2.1150.13%2_30.2.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0228.78%2_52.1.2.2.2.1.2.1.2.1.1.1.2.2.2.1.2.2.2.1200.32%2_71.0.0.0.1.0.0.1.1.1.1.2.2.1.2.1.2.2.2.1150.18%*2_100.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0182.13%*2_120.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0185.90%2_140.0.0.0.1.2.1.2.2.2.2.2.1.2.1.1.1.1.1.1160.45%2_172.2.1.1.0.0.0.0.0.0.0.0.0.0.0.1.1.1.2.290.21%2_192.1.2.1.2.1.2.2.1.1.2.1.1.2.1.2.2.2.2.2200.62%2_232.2.1.2.2.2.2.2.2.2.1.1.2.1.2.2.2.1.2.2200.13%2_262.2.0.0.0.0.0.0.0.0.0.1.1.2.2.1.1.2.1.2110.25%*2_290.0.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0182.62%2_300.0.0.0.0.1.2.1.1.2.1.1.2.2.2.2.2.1.1.11516.66%2_341.2.2.1.2.2.2.2.1.2.2.1.1.1.1.2.0.0.0.0160.12%2_352.1.1.2.2.2.2.2.1.2.2.1.1.1.1.1.2.2.1.2200.22%2_392.1.0.0.1.0.0.0.0.0.2.2.1.1.1.1.1.1.0.0110.29%2_401.1.2.2.2.1.2.2.2.2.2.2.2.1.1.0.0.0.0.0150.28%2_410.0.0.1.1.2.2.2.1.1.1.2.2.2.2.2.2.1.1.2170.09%2_420.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.1.2.140.40%2_431.0.0.2.1.0.0.0.0.0.0.0.0.0.0.0.2.2.2.2711.16%2_522.1.2.1.2.2.1.1.1.1.1.2.2.1.2.1.2.2.1.2200.33%2_591.1.2.1.1.1.2.1.2.1.2.1.1.1.2.2.1.2.2.2200.20%2_630.0.0.1.1.2.1.1.2.1.2.1.2.2.2.1.0.0.0.0130.39%*2_641.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.2445.22%2_720.0.2.2.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0317.20%2_731.2.2.1.1.2.2.2.2.1.1.2.1.2.1.2.1.1.1.2200.19%2_740.0.0.0.0.0.0.0.0.0.0.2.1.1.2.2.2.1.2.290.22%2_761.2.2.2.2.2.2.2.2.2.2.1.2.2.2.2.2.1.1.1200.08%2_801.2.1.1.1.2.1.1.2.1.2.1.1.2.2.0.1.0.2.1180.22%2_810.0.0.0.0.2.2.2.1.2.1.1.1.1.2.2.1.0.1.2140.44%2_820.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0.0.0.0122.32%2_920.0.0.2.1.2.1.2.2.1.1.2.1.1.2.0.0.0.0.0120.42%2_930.0.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0152.49%2_961.1.2.2.2.1.1.2.1.2.2.1.2.2.2.1.2.2.1.2200.17%2_970.1.1.1.0.0.1.0.0.1.1.1.0.0.0.1.2.1.2.1120.27%2_990.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.1.2.2429.51%*2_1011.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0185.94%2_1031.1.2.2.1.2.2.1.2.0.0.0.0.0.0.0.0.0.0.0913.52%2_1101.2.2.1.1.2.1.1.2.1.1.1.2.1.2.1.2.1.2.2200.00%2_1142.1.1.2.1.1.2.2.1.1.1.2.1.1.2.1.2.1.1.2200.23%2_1160.0.0.0.0.2.0.2.1.2.1.2.2.d.1.1.1.2.1.2140.14%2_1181.0.2.1.2.2.2.2.1.1.2.2.1.1.1.1.1.2.1.2191.54%2_1202.1.2.2.1.2.2.2.1.1.2.2.2.1.2.1.2.1.2.2200.17%2_1292.1.0.0.0.0.0.0.0.0.0.1.2.1.1.1.2.2.2.2110.43%2_1300.0.2.1.2.1.2.1.2.1.1.1.2.1.1.1.2.1.2.2180.20%2_1331.1.1.1.1.1.1.2.2.2.2.2.1.2.2.2.0.0.0.0160.16%2_1342.2.2.2.1.2.2.2.2.2.2.1.2.2.2.2.2.2.0.0180.29%2_1352.0.0.0.2.2.1.1.2.1.2.1.2.1.1.1.1.2.2.0160.23%2_1372.1.1.1.1.2.2.1.2.2.2.2.1.3.2.0.0.0.0.0150.42%2_1411.1.1.1.1.2.1.1.1.2.1.2.2.2.2.2.2.1.1.2200.24%2_1421.2.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0514.48%2_1432.1.0.0.2.1.2.1.2.2.1.1.2.1.3.2.1.2.1.2180.18%2_1472.2.1.2.2.2.1.2.2.2.2.1.2.1.2.2.2.2.2.2200.38%2_1492.2.1.1.0.2.2.1.1.2.1.1.1.2.2.2.2.2.2.2.2.2200.21%2_1501.2.1.2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.260.09%.

[0529] When looking at the mutation number composition of the 140 mutants used in the experiment, it can be seen that the proportion of mutants with a small number of mutations is high (Figure 5).

[0530] To ensure accurate experiments, the selected mutant candidates were re-prepared with Plsamid Miniprep to ensure good sample quality. Then, indel analysis experiments against the EMX1 target were performed in triplicate. WT spCas9 indel activity was considered 100%, and the indel data are summarized in Table 7 and Figure 6.

[0531] EMX1 3-repeat indel analysis of selected mutantsEMX1(N=3)1_73(M=6)1_63(M=4)2_64(M=4)1_18(M=3)1_42(M=3)1_2(M=2)1_13(M=1)1_44(M=1)2_10(M=1)2_12(M=1)2_29(M=1)2_101(M=1)143.68%94.33%97.01%71.32%109.16%82.70%90.06%99.47%103.3 7% 100.80% 87.12% 105.20% 248.42% 94.00% 96.75% 72.47% 95.49% 86.68% 93.07% 98.60% 103.78% 105.02% 89.91% 108.16% 350.68% 88.17% 95.74% 73.86% 96.38% 88.10% 92.75% 95.30% 106.18% 106.22% 89.79% 101.64%

[0532] 2) Mutant selection and first mutation combination experiment

[0533] Mutants 1_63, 2_64, 1_42, 2_10, 2_12, and 2_101 were selected through EMX1 indel 3-repeat analysis, and the following mutants were obtained through the first mutation combination cloning experiment (Table 8).

[0534] Mutant obtained by 1st mutation combination cloning Sample name (Library_Colony) Mutation type Number of mutations ABCDEFFGGGHIJJKLMNOP1_420.0.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.031_42a(1_42 + 1_63)0.0.1.1.1.0.1.1.0.0.0.1.0.0.0.0.0.0.0.0.061_42b(1_42 + 2_10)0.0.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.041_42c(1_42 + 2_10 + 2_101)1.0.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.1.052_641.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2.242_64a(2_64 + 1_63)1.1.0.0.0.0.1.1.0.0.0.1.0.0.0.0.0.0.2.272_64c(2_64 + 2_12)1.1.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.2.25M=2(2_10 + 2_101)1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1.02C(2_10 + 2_12 + 2_101)1.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.1.03

[0535] The combined mutants were subjected to three-repeat indel analysis for the EMX1 target (Table 9, Fig. 7). The values ​​were analyzed based on the WT indel activity set at 100%.

[0536] 1st mutation combination EMX1 3-repeat indel analysis EMX1 (N=3) 1_42 (M=3) 1_42a (M=6) 1_42b (M=4) 1_42c (M=5) 2_64 (M=4) 2_64a (M=7) 2_64c (M=5) M=2C (M=3) 181.55% 57.85% 76.53% 83.69% 90.13% 64.46% 84.3 1%101.99%103.83%276.97%57.80%75.20%84.07%90.07%65.54%87.06%108.45%101.64%384.44%54.88%77.18%81.78%87.97%61.84%89.71%101.42%96.30%

[0537] As the number of mutations increased, a tendency for Indel activity to decrease was confirmed. However, since the activity may specifically decrease for the EMX1 target, an additional two-repeat indel analysis was performed for 24 artificial targets for precise analysis. First, the average indel for each repeat for the 24 targets was calculated, and the average was calculated again using the average for each repeat. When considering SpCas9 as 100%, the indel activity of all mutants decreased, and six mutants maintained an average indel of 70% compared to the WT: 1_42, 1_42b, 2_64, 2_64c, M=2, and C (Fig. 8). Next, the average indel of 48 indel data obtained from two repeats of 24 artificial targets was calculated. Similar to the previously confirmed Fig. 8, a tendency for indels to decrease compared to the WT was confirmed (Fig. 9).

[0538] 3) Secondary mutation combination and indel performance evaluation

[0539] We performed a secondary mutation combination experiment and indel performance evaluation by adding the mutations (Q794D, R884D, K961C, L1194G, N1234G) selected in the performance evaluation of the M=1 mutant to the 1_42 (A), 2_64 (B), and C mutants, as well as a combination of the five selected mutations. One experiment was repeated using 24 artificial targets, and the average indel was calculated and quantified based on WT = 100%. Mutants showing indels of 70% or more compared to WT were marked with an * in front of the sample name (Table 10). When graphed as Indel %, the same results as in the table were confirmed (Fig. 10).

[0540] Average of 24 artificial target indels of the second mutation combination. Sample name. Mutation type. Number of mutations. WT 100%. Average. indelA.BCDEFFGGGHIJJKLMNOPAa0.0.1.1.1.0.0.0.0.0.2.0.0.0.0.0.0.0.0.0443.25%Ab0.0.1.1.1.0.0.0.0.0.0.2.0.0.0.0.0.0. 0.0441.72%Ac0.0.1.1.1.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0437.55%Ad0.0.1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.1.0439.30%Ae0.0. 1.1.1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.2434.06%Aab0.0.1.1.1.0.0.0.0.0.2.2.0.0.0.0.0.0.0.0540.98%Ace0.0.1.1.1.0.0.0.0. 0.0.0.0.2.0.0.0.0.0.2528.47%Aabce0.0.1.1.1.0.0.0.0.0.2.2.0.2.0.0.0.0.0.2627.60%Aabcde0.0.1.1.1.0.0.0.0.0.2.2.0.2 .0.0.0.0.1.2824.02%Ba1.1.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.0.2.2543.66%Bb1.1.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.0.2.2541. 19%Bc1.1.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.2.2533.49%Bab1.1.0.0.0.0.0.0.0.0.2.2.0.0.0.0.0.0.2.2653.02%Bbc1.1.0.0.0 .0.0.0.0.0.0.2.0.2.0.0.0.0.2.2638.43%Babc1.1.0.0.0.0.0.0.0.0.2.2.0.2.0.0.0.0.2.2737.04%Ca1.0.0.0.1.0.0.0.0.0.2.0 .0.0.0.0.0.0.1.0464.57%Cb1.0.0.0.1.0.0.0.0.0.0.2.0.0.0.0.0.0.1.0463.36%*Cc1.0.0.0.1.0.0.0.0.0.0.0.0.2.0.0.0.0.1.0472.86%Ce1.0.0.0.1.0.0.0.0.0.0.0.0.0.0.0.0.0.1.2453.94%Cab1.0.0.0.1.0.0.0.0.0.2.2.0.0.0.0.0.0.1.0563. 29%Cabce1.0.0.0.1.0.0.0.0.0.0.0.0.2.0.0.0.0.1.2541.90%Cabce1.0.0.0.1.0.0.0.0.0.2.2.0.2.0.0.0.0.1.2644.21 %*Dab0.0.0.0.0.0.0.0.0.0.2.2.0.0.0.0.0.0.0.0282.91%*Dce0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.0.2284.60%* Dcde0.0.0.0.0.0.0.0.0.0.0.0.0.2.0.0.0.0.1.2380.62% Dabcde0.0.0.0.0.0.0.0.0.0.2.2.0.2.0.0.0.0.1.2559.41%.

[0541] 4) Selection of deimmunized SpCas9 candidates

[0542] We selected nine mutants that maintained at least 70% indel activity compared to WT SpCas9 and were predicted to exhibit reduced immune responses based on bioinformatics. The number of potential epitopes likely to elicit an immune response based on bioinformatics analysis was expressed numerically as the average of 24 artificial target indels using the corresponding mutant plasmids, with the WT indel average set to 100% (Table 11).

[0543] Immune response prediction and indel average for 9 Deimmune spCas9 candidates IEDB MHC-II (26 alleles) IEDB MHC-II (8 alleles) ProPred (8 alleles) IEDB MHC-I (27 alleles) IEDB MHC-I (8 alleles) sumWT 100% Average indel Mutation number 1st selection 2nd additional selection WT_SpCas9 107732946211883663422100.000--Cc (Mutant 1) 9882804431178364325372.864 O-Dcde (Mutant 2)10122894591180364330480.623O-2_64c10263134371172364331283.895O-C10323054441182365332895.063--2_64 (Mutant 3)10443184441172364334272.274-ODce10303024621183364334184.602--1_42104531444011 87366335274.533-OM210503104511182365335898.602--Dab10483164521183365336482.912--

[0544] Among the nine mutants, three mutants, Cc, Dcde, and 2_64c, were selected through the first selection. Cc and Dcde produced protein well, but 2_64c failed to produce protein. Therefore, two additional mutants, 2_64 and 1_42, were selected for the second selection and protein production. Although both 2_64 and 1_42 produced protein, 2_64 was prioritized. Therefore, Cc, Dcde, and 2_64, which had no abnormalities in protein production, were selected as the final deimmunized spCas9 mutant candidates for the immune response experiment (see Tables 4 and 5).

[0545] 5) Reconfirmation of Indels in the three final candidate plasmids of deimmunized SpCas9

[0546] We performed three rounds of indel analysis using 24 artificial targets. Although the indel counts are lower than those of the WT, as previously confirmed, when the average WT indel is considered 100%, the average indels of both the first and second selected mutants are over 70% of the WT (Figure 11).

[0547] For the three final deimmunized SpCas9 candidates to be tested in immunological experiments, we analyzed the average number of indels per 24 artificial targets (Figure 12). When the WT target was set at 100%, indel levels sometimes approached 100% or dropped significantly, as in target 3, confirming a target-dependent tendency. In other words, if a well-selected target for therapeutics is chosen, it is expected to exhibit editing performance similar to SpCas9 and reduce immune responses.

[0548]

[0549] Experimental Example 2: Immunogenicity Analysis Using PBMCs

[0550] Experimental Example 2-1. Sequence and method of immune response experiments using WT SpCas9 and deimmunized SpCas9 mutants using human peripheral blood mononuclear cells (PBMCs).

[0551] 1) Among 27 samples, samples with an immune response to WT SpCas9 were selected.

[0552] Since only samples activated by WT SpCas9 could be compared to deimmunized SpCas9s, a process of selecting only samples in which lymphocytes were activated by Wild-Type (WT) SpCas9 was performed first. PBMCs were isolated from the peripheral blood of 27 people and seeded in 96-well plates at 1x10^6 cells / well. After treatment with WT SpCas9 and deimmunized SpCas9 mutants at 20 μg / ml each, they were cultured in a CO2 incubator at 37°C for 16 hours. T cell activation markers 41BB and CD25 were stained and analyzed by flow cytometry.

[0553] 2) Analysis of the immune response when immune cells of selected samples were re-sensitized to WT SpCas9 and deimmunized SpCas9 mutants.

[0554] PBMCs were seeded in 96-well plates, treated with WT and deimmunized SpCas9, and cultured for 16 hours. 41BB+, CD25+, 41BB+CD25+(DP) T cells, and CD5- cells were sorted. Sorted CD5- cells were cultured at a ratio of 1:50 after irradiation (60 Gy) and cultured with IL-2 (50 ng / ml) for 4 weeks. After 4 weeks, CD3- cells were sorted from frozen PBMCs from the same donor and labeled with CFSE. Cultured cells were mixed with CFSE-labeled CD3-depleted cells and stimulated with WT and deimmunized SpCas9, followed by intracellular cytokine staining (ICS).

[0555] Experimental Example 2-2. Selection of WT SpCas9-activated samples and comparison of immune responses between deimmunized SpCas9 samples.

[0556] The activation rate by WT SpCas9 was confirmed to be 61% on average, and a high activation response was observed in CD4+ and CD8+ T cells (Fig. 14). CD4+CD25+ (18 people), CD4+41BB+ (16 people), CD8+CD25+ (10 people), CD8+41BB+ (18 people).

[0557] T cells activated by WT showed low reactivity to deimmunized SpCas9 (Fig. 15). When WT SpCas9 was used as the standard (100%), the reactivity to deimmunized SpCas9 was reduced to approximately 20–30% (Fig. 16).

[0558] Experimental Example 2-3. Comparison of immune responses when immune cells activated with WT SpCas9 were isolated and then re-primed with WT SpCas9 or deimmunized SpCas9.

[0559] Samples 8, 19, and 20, which showed significant activation responses with WT SpCas9, were used in resensitization experiments (Fig. 17).

[0560] We confirmed differences in immune responses to WT and deimmunized SpCas9. In sample 8, IFNγ significantly increased, and in samples 19 and 20, IL-2 significantly increased. While WT SpCas9 elicited higher responses to specific cytokines (IL-2, IFNγ), deimmunized SpCas9 exhibited reduced responses (Fig. 18). These results confirmed the high immunogenicity of WT SpCas9 and the immunogenicity-suppressing properties of deimmunized SpCas9.

[0561] In conclusion, deimmunized SpCas9 effectively suppressed immunogenicity. In contrast, WT SpCas9 induced a strong immune response, and its specificity was demonstrated by a specific cytokine increase pattern. The differences in immune responses between WT and deimmunized SpCas9 support the design effectiveness of deimmunized SpCas9 to reduce immunogenicity.

[0562]

[0563] The present invention can be used in therapeutic applications by enabling gene editing in vivo without immune response problems using a Cas9 protein variant with low immunogenicity.

[0564]

[0565] Amino acid sequence of wild type Cas9 protein and amino acid sequence of Cas9 protein variants, etc.

Claims

1. A Cas9 protein variant having an amino acid sequence in which at least one of the 161st methionine (M); 189th valine (V); 337th alanine (A); 961st lysine (K); 1194th leucine (L); and 1234th asparagine (N) in the amino acid sequence represented by sequence number 1 is substituted with one amino acid selected from the following: Histidine (H); Aspartic acid (D); Glutamic acid (E); Serine (S); Glycine (G); Lysine (K); Arginine (R); Threonine (T); Glutamine (Q); Cysteine ​​(C); and Alanine (A), At this time, the Cas9 protein variant has an amino acid sequence different from the amino acid sequence shown in sequence number 1, The above Cas9 protein variant has lower immunogenicity in vivo compared to the wild-type Cas9 protein having the amino acid sequence shown in SEQ ID NO:

1.

2. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine in the amino acid sequence shown in sequence number 1 is substituted with histidine (H) or aspartic acid (D).

3. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which valine at position 189 in the amino acid sequence shown in sequence number 1 is substituted with aspartic acid or glutamic acid (E).

4. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which alanine at position 337 in the amino acid sequence shown in sequence number 1 is substituted with aspartic acid or glutamic acid.

5. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which lysine at position 961 in the amino acid sequence shown in sequence number 1 is substituted with aspartic acid or cysteine ​​(C).

6. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 1194th leucine in the amino acid sequence shown in sequence number 1 is substituted with glycine (G) or alanine.

7. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which asparagine at position 1234 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid or glycine.

8. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine; the 337th alanine; the 961st lysine; and the 1194th leucine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: Histidine (H); Aspartic acid (D); Glutamic acid (E); Cysteine ​​(C); Glycine (G); and Alanine (A).

9. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 961st lysine; the 1194th leucine; and the 1234th asparagine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: Aspartic acid (D); Cysteine ​​(C); Glycine (G); and Alanine (A).

10. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine; the 189th valine; the 1194th leucine; and the 1234th asparagine in the amino acid sequence shown in SEQ ID NO: 1 are each independently substituted with one amino acid selected from the following: Histidine (H); Aspartic acid (D); Glutamic acid (E); Glycine (G); and Alanine (A).

11. In paragraph 8, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine is replaced with histidine or aspartic acid; the 337th alanine is replaced with glutamic acid or aspartic acid; the 961st lysine is replaced with aspartic acid or cysteine; and the 1194th leucine is replaced with glycine or alanine in the amino acid sequence shown in SEQ ID NO:

1.

12. In paragraph 11, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine is replaced by histidine; the 337th alanine is replaced by glutamic acid; the 961st lysine is replaced by cysteine; and the 1194th leucine is replaced by glycine in the amino acid sequence shown in SEQ ID NO:

1.

13. In paragraph 12, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence represented by SEQ ID NO:

43.

14. In paragraph 9, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which aspartic acid or cysteine ​​is substituted for the 961st lysine, glycine or alanine is substituted for the 1194th leucine, and aspartic acid or glycine is substituted for the 1234th asparagine in the amino acid sequence shown in SEQ ID NO:

1.

15. In paragraph 14, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which cysteine ​​is substituted for lysine at position 961; glycine is substituted for leucine at position 1194; and glycine is substituted for asparagine at position 1234 in the amino acid sequence shown in SEQ ID NO:

1.

16. In paragraph 15, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence represented by SEQ ID NO:

42.

17. In paragraph 10, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the methionine at position 161 is replaced with histidine or aspartic acid; the valine at position 189 is replaced with aspartic acid or glutamic acid; the leucine at position 1194 is replaced with glycine or alanine; and the asparagine at position 1234 is replaced with aspartic acid or glycine in the amino acid sequence shown in SEQ ID NO:

1.

18. In paragraph 17, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence in which the 161st methionine is replaced by histidine; the 189th valine is replaced by aspartic acid; the 1194th leucine is replaced by alanine; and the 1234th asparagine is replaced by glycine in the amino acid sequence shown in SEQ ID NO:

1.

19. In paragraph 18, The above Cas9 protein variant is a Cas9 protein variant having an amino acid sequence represented by SEQ ID NO:

44.

20. In paragraph 1, The above Cas9 protein variant is a Cas9 protein variant further comprising at least one NLS.

21. A CRISPR / Cas9 composition comprising: A Cas9 protein variant of claim 1 or a nucleic acid encoding the same; and Guide RNA or a nucleic acid encoding it.

22. In paragraph 21, A CRISPR / Cas9 composition wherein the Cas9 protein variant is a Cas9 protein variant having an amino acid sequence represented by SEQ ID NO: 42, 43 or 44.

23. In paragraph 21, The above CRISPR / Cas9 composition is a CRISPR / Cas9 composition comprising the Cas9 protein variant and the guide RNA in the form of ribonucleoprotein (RNP).

24. In paragraph 21, The above CRISPR / Cas9 composition is a CRISPR / Cas9 composition comprising a nucleic acid encoding the Cas9 protein variant and a nucleic acid encoding the guide RNA in the form of a vector.

25. A method for editing a gene in a living organism, comprising: A step of administering a CRISPR / Cas9 composition into a living body; The above CRISPR / Cas9 composition comprises the Cas9 protein variant of claim 1 or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same, wherein the guide RNA has a guide sequence that is custom-designed or artificially designed to target a gene to be edited.

26. Therapeutic agents for in vivo administration, including: A Cas9 protein variant of claim 1 or a nucleic acid encoding the same; and Guide RNA or a nucleic acid encoding it; At this time, the guide RNA has a guide sequence that is customized or artificially designed according to the target gene of the disease.

27. A method for treating a disease, comprising a step of administering a therapeutic agent for in vivo administration to a subject to be treated with a disease, wherein the therapeutic agent comprises the Cas9 protein of claim 1 or a nucleic acid encoding the same; and a guide RNA or a nucleic acid encoding the same, wherein the guide RNA has a guide sequence designed either customized or artificially according to a target gene of the disease.

28. In paragraph 27, The above disease is caused by abnormal expression and / or abnormal function performance of a specific gene or a specific protein, and the disease is a disease that can be alleviated or treated by artificially editing a specific gene.

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