WO2024085674A1 - Cas 단백질 및 박테리아 톡신을 포함하는 융합 단백질 및 이의 용도 - Google Patents

Cas 단백질 및 박테리아 톡신을 포함하는 융합 단백질 및 이의 용도 Download PDF

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WO2024085674A1
WO2024085674A1 PCT/KR2023/016230 KR2023016230W WO2024085674A1 WO 2024085674 A1 WO2024085674 A1 WO 2024085674A1 KR 2023016230 W KR2023016230 W KR 2023016230W WO 2024085674 A1 WO2024085674 A1 WO 2024085674A1
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ssda
fusion protein
protein
sequence
crispr
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French (fr)
Korean (ko)
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김용섭
권지연
김대식
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Asan Foundation
Sungkyunkwan University
University of Ulsan Foundation for Industry Cooperation
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Asan Foundation
Sungkyunkwan University
University of Ulsan Foundation for Industry Cooperation
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Priority to JP2025522573A priority patent/JP2025535373A/ja
Priority to CN202380073912.9A priority patent/CN120092083A/zh
Publication of WO2024085674A1 publication Critical patent/WO2024085674A1/ko
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
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Definitions

  • It relates to fusion proteins comprising Cas proteins and bacterial toxins and uses thereof.
  • Genome editing is a technology that freely edits the genetic information of living organisms. Through advances in the field of life sciences and the development of genome sequencing technology, we have gained a broad understanding of various genetic information. For example, understanding of genes for the reproduction of animals and plants, disease and growth, genetic mutations that cause various human genetic diseases, and production of biofuels has already been secured, but this can be directly utilized to improve living things and prevent human diseases. To reach the level of treatment, further technological advancement is essential.
  • Total editing technology can dramatically expand its scope of use by changing the genetic information of animals, plants, and microorganisms, including humans.
  • Genetic scissors are molecular tools designed and manufactured to accurately cut desired genetic information and play a key role in genome editing technology.
  • next generation sequencing technology which has advanced the field of genetic sequencing to the next level, genetic scissors is becoming a core technology that expands the speed and scope of genetic information utilization and creates new industrial fields.
  • APOBEC or AID proteins which are commonly used proteins for cytosine base correction using Cas9, are relatively large in size, making it difficult to manufacture them as vectors for use as cell therapy products. Accordingly, there is a demand for the production of a CRISPR-Cas system that is easy to manufacture as a vector and has excellent base correction efficiency.
  • One aspect is to provide a fusion protein comprising a Cas protein (CRISPR-associated protein) and a bacterial toxin.
  • Another aspect is to provide a polynucleotide encoding the fusion protein.
  • Another aspect is providing a vector containing the polynucleotide.
  • Another aspect includes the fusion protein or a polynucleotide encoding the same; and a CRISPR-Cas system comprising a guide polynucleotide.
  • Another aspect is to provide a method of editing a nucleic acid comprising contacting a nucleic acid molecule with the CRISPR-Cas system.
  • One aspect provides a fusion protein comprising a Cas protein (CRISPR-associated protein) and a bacterial toxin.
  • Cas protein may be a CRISPR-binding endonuclease.
  • the Cas protein can cleave all or part of a specific target polynucleotide sequence.
  • the Cas protein may be a class 2 Cas (Class 2 CRISPR associated system) protein.
  • the class 2 Cas proteins may be included in type II, type V, or type VI systems.
  • the type II system may have the cas1, cas2, and cas9 genes. Type II systems can be further classified into three subtypes: subtypes II-A, II-B, and II-C.
  • Subtype II-A may contain an additional gene, csn2.
  • An organism with a subtype II-A system may include Streptococcus thermophilus.
  • Subtype II-B lacks csn2, but may have cas4.
  • An organism with a subtype II-B system may include Legionella pneumophila.
  • Subtype II-C is the most common type II system found in bacteria and can have only three proteins: Cas1, Cas2, and Cas9.
  • An organism with a subtype II-C system may include Neisseria lactamica .
  • the type V system may have the cas12 gene and the cas1 and cas2 genes.
  • the Cas12 gene may encode the protein Cas12, which has a RuvC-like nuclease domain homologous to the respective region of Cas9 but lacks the HNH nuclease domain present in the Cas9 protein.
  • the type VI system may have the cas13 gene and the cas1 and cas2 genes.
  • the RuvC-like nuclease (RNase H fold domain and HNH (McrA-like) nuclease domain of Cas9 are each capable of cleaving one of the strands of the target nucleic acid.
  • Cas9 cleavage of the type II system Activity may also require hybridization of the crRNA to the tracrRNA to form a duplex that facilitates crRNA and target binding by Cas9.
  • the RuvC-like nuclease domain of Cas12 can cleave both strands of the target nucleic acid in a staggered configuration, creating a 5' overhang. These 5' overhangs can facilitate DNA insertion via non-homologous end joining methods.
  • the Cas12 cleavage activity of the type V system also does not require hybridization of the crRNA to the tracrRNA to form a duplex, and the crRNA of the type V system can use a single crRNA with a stem-loop structure to form an internal duplex.
  • the Type V system can induce single or double strand breaks at the location of the target sequence. Strand breakage may be a staggered cleavage with a 5' overhang.
  • the Cas protein may include Cas9 or Cas12.
  • the Cas12 protein may refer to a protein derived from various bacterial species.
  • the Cas protein is Streptococcus , Campylobacter , Nitratifractor , Staphylococcus , Parvibaculum , Roseburia , Neisseria ), Gluconacetobacter , Azospirillum , Sphaerochaeta , Lactobacillus , Eubacterium , Corynebacter , Carnobacterium ( Carnobacterium , Rhodobacter , Listeria , Paludibacter , Clostridium , Lachnospiraceae , Clostridiaridium , Leptotrichia ( Leptotrichia , Francisella , Legionella , Alicyclobacillus , Methanomethyophilus , Porphyromonas , Prevotella , Bacteroidetes ( Bacteroidetes , Helcococcus , Letospira
  • the Cas12 protein is Francisella tularensis 1, Francisella tularensis subspecies novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio Proteoclasticus , Peregrinibacterium GW2011_GWA2_33_10, Parcubacterium GW2011_GWC2_44_17, Smitella spp. SCADC, Asidaminococcus spp.
  • Lachnospiraceae bacterium MA2020 Lachnospiraceae bacterium MA2020, Candidatus methanoplasma thermitum, Eubacterium elligens, Morag Derived from a bacterial species selected from the group consisting of Sella boboculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas creviolicanis 3, Prevotella deciens and Porphyromonas macacae It could be.
  • the Cas12 protein may be any one selected from the group consisting of Cas12a, mgCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, and Cas12j.
  • the Cas12 protein may include a modification of the Cas12 protein.
  • the Cas12 protein has nuclease activity
  • the Cas12 protein has reduced nuclease activity, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, compared to the wild type enzyme. Or it can be modified to have 100% nuclease inactivation.
  • the Cas9 may be Saccharomyces pneumoniae ( S. pneumoniae ), Streptococcus pyogenes ( S. pyogenes ), Streptococcus thermophilus ( S. thermophilus ), Campylobacter jejuni ( C. jejuni ) Cas9, , may contain mutated Cas9 derived from these organisms.
  • the enzyme may be a Cas9 homolog or ortholog.
  • the CRISPR enzyme can be codon-optimized for expression in eukaryotic cells.
  • the CRISPR enzyme can induce one or two strand breaks at the location of the target sequence.
  • the Cas9 protein may include a modification of the Cas9 protein.
  • the Cas9 protein When the Cas9 protein has nuclease activity, the Cas9 protein has reduced nuclease activity, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, compared to the wild type enzyme. Or it can be modified to have 100% nuclease inactivation.
  • the Cas9 may be Cas9 D10A.
  • the Cas protein may be a Cas9 protein or a Cas12 protein.
  • At least one nuclear localization signal can be attached to a nucleic acid sequence encoding a Cas protein.
  • at least one C-terminal or N-terminal NLS may be attached.
  • the codon optimized Cas protein may comprise an NLS attached to the C-terminus of the protein.
  • Cas is transferred to a specific site within a cell, such as an organelle, such as a mitochondria.
  • localizations include, but are not limited to, localization to plastids, chloroplasts, vesicles, Golgi, (nuclear or cellular) membranes, ribosomes, nucleoli, ER, cytoskeleton, vacuoles, centrosomes, nucleosomes, granules, centrioles, etc.
  • the tag can be fused to the Cas protein.
  • the Cas protein and bacterial toxin can be fused through a linker.
  • the linker may be located at the C-terminus, N-terminus, or both the C-terminus and the N-terminus of the Cas protein, and the bacterial toxin can bind to the Cas protein through the linker.
  • the suitable linker motifs and linker configurations are described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69, the entire contents of which are hereby incorporated by reference.
  • the bacterial toxin may be single-stranded DNA deaminase toxin A (SsdA).
  • the SsdA may be derived from a Pseudomonas SP. strain.
  • the strains of the Pseudomonas genus include Pseudomonas syringae , Pseudomonas congelans, Pseudomonas savastanoi, Pseudomonas viridiflava , and Pseudomonas coronafaciens . ), Pseudomonas Fluorescens ( Pseudomonas fluorescens ), Pseudomonas sp. MPC6, Pseudomonas sp. GL-R-26, or Pseudomonas sp. It could be GL-RE-26.
  • the N-terminus of SsdA may have a PAAR domain
  • the C-terminus of SsdA may have a DYW deaminase domain.
  • the SsdA is identical to the deaminase used in existing base corrections in that it has the common amino acid motifs HxE and CxxC motifs, but is different from the deaminase used in existing base corrections in that it additionally has the SGW motif. there is.
  • the SsdA is a deaminase that is structurally and evolutionarily different from other deaminase enzymes previously used in base correction technology and is classified as a DYW-like deaminase.
  • the schematic diagram and structural domain differences between SsdA and other deaminase enzymes used in existing base correction technologies are shown in Figure 1.
  • the SsdA may include the amino acid sequence of SEQ ID NO: 1.
  • the SsdA may include a toxin domain.
  • the toxin domain of SsdA is a part with deaminase activity and has a length of 100 to 200 amino acids, for example, 120 to 180 amino acids, 120 to 170 amino acids, 130 to 160 amino acids, or 140 to 160 amino acids. It can have a length of .
  • the toxin domain of SsdA may include the amino acid sequence of SEQ ID NO: 2.
  • the base sequence of the toxin domain is shown in Table 1 below.
  • the amino acid sequence (toxin domain) of SEQ ID NO: 2 of SsdA may include a catalytic active site.
  • the catalytic active site may include an HxE motif, CxxC motif, or SGW motif.
  • the SGW motif is an additional motif that only the SsdA enzyme has, unlike existing deaminase enzymes such as APOBEC and AID.
  • the HxE motif may include amino acids 301 to 303 of SEQ ID NO: 1 (PAAR domaomn-containing protein), the HxE motif may include amino acids 347 to 349, and the SGW motif may include amino acids 301 to 303. It can be included.
  • the SsdA is at least 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 1 (PAAR domaomn-containing protein). It may have a sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the fusion protein may be a DYW deaminase protein bound to a Cas protein.
  • the SsdA may be inactivated SsdA.
  • the inactivated SsdA may have an amino acid mutation in the catalytic active site of activated SsdA.
  • the inactivated SsdA may have lower cytotoxicity than SsdA.
  • the inactivated SsdA may have amino acid mutations at positions G302 and E349 in the amino acid sequence of SEQ ID NO: 1 (PAAR domaomn-containing protein).
  • the amino acid mutation means that the wild type protein is substituted with an amino acid other than the amino acid at positions G302 and E349.
  • the other amino acids are arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), and glutamine (Q).
  • the inactivated SsdA may have G302D, E349A, or their corresponding amino acid mutation in the amino acid sequence of SEQ ID NO: 1. More specifically, the inactivated SsdA having the G302D mutation in the amino acid sequence of SEQ ID NO: 1 (PAAR domaomn-containing protein) may be SEQ ID NO: 17.
  • the inactivated SsdA is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 17. It may have a sequence identity of more than 96%, more than 97%, more than 98%, or more than 99%.
  • the SsdA can induce deamination of single-stranded DNA, and the SsdA can be a cytidine deaminase.
  • cytidine deaminase refers to an enzyme that has the activity of removing the amino (-NH2) group of cytosine, cytidine, or deoxycytidine. do.
  • cytidine deaminase is used as a concept including cytosine deaminase.
  • the cytidine deaminase may be used interchangeably with the cytosine deaminase.
  • the cytidine deaminase has the activity of converting cytosine, a base present in nucleotides (e.g., cytosine present in DNA or RNA), to uracil (C-to-U conversion or C-to-U editing). It refers to all enzymes that have a . It converts cytosine located on the strand where the PAM sequence of the target site sequence (target nucleic acid sequence) exists to uracil.
  • the bacterial toxin may be bound to the end of the Cas protein.
  • the bacterial toxin may be bound to the C-terminus, N-terminus, or both C-terminus and N-terminus of the Cas protein.
  • the fusion protein may further include a DNA glycosylase inhibitor.
  • the DNA glycosylase inhibitor is a thymine glycosylase inhibitor, a uracil glycosylase inhibitor, an oxoguanine glycosylase inhibitor, or an alkylguanine DNA glycosylase inhibitor ( It may be an alkylguanine glycosylase inhibitor).
  • the uracil DNA glycosylase inhibitor may be a uracil DNA glycosylase inhibitor derived from Bacillus subtilis bacteriophage, PBS1, a uracil DNA glycosylase inhibitor derived from Bacillus subtilis bacteriophage, or PBS2, It is not limited to this.
  • Another aspect provides a polynucleotide encoding the fusion protein.
  • Another aspect provides a vector containing the polynucleotide.
  • vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • a vector is a nucleic acid molecule that is single-stranded, double-stranded, or partially double-stranded; A nucleic acid molecule containing one or more free ends, a nucleic acid molecule lacking free ends (e.g., circular); Nucleic acid molecules including DNA, RNA, or both; and various other polynucleotides known in the art.
  • plasmid which may refer to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques.
  • viral vectors which contain viral-derived DNA or RNA sequences into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). May be present in vector for packaging purposes.
  • a recombinant expression vector may contain a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which may mean that the recombinant expression vector contains one or more regulatory elements, and one or more regulatory elements may be used for expression. It can be selected based on the host cell and can be operably linked to the nucleic acid sequence to be expressed.
  • operably linked means that the nucleotide sequence of interest allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell if the vector is introduced into the host cell). It may mean that it is connected to the control element(s) in a way that
  • regulatory elements refers to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly- U sequence). Regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)]. Regulatory elements may include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences).
  • the vector contains one or more pol III promoters (e.g., 1, 2, 3, 4, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3 , 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof.
  • pol III promoters may include, but are not limited to, the U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, ⁇ -actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • vectors can include lentiviruses and adeno-associated viruses (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9), and these types of vectors can also be used to target certain types of cells. can be selected for targeting.
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 lentiviruses and adeno-associated viruses
  • nucleic acid molecules within the vector system may be located on the same or different vectors.
  • the vector e.g., a plasmid or viral vector
  • the tissue of interest e.g., by intramuscular injection, while in other cases delivery is intravenous, transdermal, intranasal, buccal, mucosal, or otherwise. This can be accomplished through other delivery methods. Such delivery may occur via either a single dose or multiple doses.
  • the actual dosage delivered herein will depend on a variety of factors, such as the choice of vector, target cell, organism, or tissue, general condition of the subject to be treated, degree of transformation/modification sought, route of administration, method of administration, and trait sought. It can vary greatly depending on the type of conversion/transformation, etc.
  • the dosage can be determined by, for example, carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluent, pharmaceutically acceptable carrier (e.g. For example, phosphate buffered saline), pharmaceutically acceptable excipients, and/or other compounds known in the art.
  • the dosage may include one or more pharmaceutically acceptable salts, such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic acid salts such as acetate, propionate, malonate, benzoate, etc.
  • auxiliary components such as wetting or emulsifying agents, pH buffering components, gels or gelling substances, flavoring agents, colorants, microspheres, polymers, suspending agents, etc. may also be presented herein.
  • one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, water retention agents, suspending agents, surfactants, antioxidants, fillers, chelating agents, coating agents, chemical stabilizers, etc.
  • Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, Includes parachlorophenol, gelatin, albumin, and combinations thereof.
  • delivery for disease treatment can be achieved via AAV.
  • a therapeutically effective dosage for in vivo delivery of AAV to a human may be a saline solution ranging from about 20 ml to about 50 ml containing from about 1 1010 to about 1 10100 AAV per ml of solution. Dosages can be adjusted to balance therapeutic benefits against any side effects.
  • fusion protein comprising a Cas protein and a bacterial toxin or a polynucleotide encoding the fusion protein; and a CRISPR-Cas system comprising a guide polynucleotide.
  • the fusion protein and the polynucleotide encoding it are as described above.
  • the guide polynucleotide may include a targeting sequence and/or an activation sequence.
  • targeting sequence can refer to a polynucleotide comprising DNA, or a mixture of DNA and RNA, that is complementary to a sequence in a target nucleic acid.
  • targeting sequences may also include other nucleic acids, or nucleic acid analogs, or combinations thereof.
  • the targeting sequence may consist solely of DNA because such constructs are less likely to degrade within the host cell. In some embodiments, such configurations may increase target sequence recognition specificity and/or reduce the occurrence of off-target binding/hybridization.
  • the targeting sequence may include a guide sequence or a spacer sequence. The length of the domains of the targeting sequence is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. , may be 26, 27, 28, 29 or 30 nucleotides long.
  • the term “activation sequence” may refer to a portion of a polynucleotide containing RNA or DNA, or a mixture of DNA and RNA, that can interact, associate, or bind to a Cas protein.
  • the activation region may also include other nucleic acids, or nucleic acid analogs, or combinations thereof.
  • the activation sequence may be adjacent to or connected to the target sequence.
  • the activation region can be downstream of the targeting region.
  • the activation region can be upstream of the targeting region.
  • the activation sequence may include a direct repeat sequence, CRISPR RNA (crRNA) and/or trans-activating RNA (tracrRNA).
  • the guide polynucleotide, guide RNA, mature crRNA, and immature crRNA may comprise or consist of a direct repeat sequence and a guide sequence or spacer sequence.
  • the guide RNA or mature crRNA may comprise or consist of a direct repeat sequence linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5') from the guide sequence or spacer sequence.
  • the guide polynucleotide may include crRNA and tracrRNA.
  • the guide polynucleotide may be dual guide RNA or single-chain guide RNA (sgRNA).
  • sgRNA single-chain guide RNA
  • the system can form a deletion, insertion, substitution, or indel of at least one nucleotide in the nucleotide sequence of a target nucleic acid molecule. .
  • the nucleic acid may be RNA or DNA.
  • the system is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 in the nucleotide sequence of the target nucleic acid molecule.
  • the system is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 bp to 35 bp, 1 bp to 30 bp, 1 bp to 25 bp, 1 bp to 20 bp, 1 bp to 15 bp, 1 bp to 10 bp, 1 bp to 5 bp, 5 bp to 60 bp, 5 bp to 55 bp, 5 bp to 50 bp, 5 bp to 45 bp, 5 bp to 40 bp, 5 bp to 35 bp, 5 bp to 30 bp, 5 bp to 25 bp, 5 bp to 20 bp, 5 bp to 15 bp, 5 bp to 10 bp, 10 bp to 60 bp, 10 bp, bp
  • the system is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 bp to 35 bp, 1 bp to 30 bp, 1 bp to 25 bp, 1 bp to 20 bp, 1 bp to 15 bp, 1 bp to 10 bp, 1 bp to 5 bp, 5 bp to 60 bp, 5 bp to 55 bp, 5 bp to 50 bp, 5 bp to 45 bp, 5 bp to 40 bp, 5 bp to 35 bp, 5 bp to 30 bp, 5 bp to 25 bp, 5 bp to 20 bp, 5 bp to 15 bp, 5 bp to 10 bp, 10 bp to 60 bp, 10 bp, bp
  • the system has an efficiency of forming indels of nucleotides of 5% to 50%, for example, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%. , 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10 % to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 15% 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25% , 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 50%, 30% to 45%, 30% to 40%, 30 % to 35%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 50%, 40% to 45%, or 45% to 50%.
  • the system has an efficiency of forming substitutions in the sequence of nucleotides of 1% to 20%, for example, 1% to 18%, 1% to 16%, 1% to 14%, 1% to 1%. 12%, 1% to 10%, 1% to 8%, 1% to 6%, 1% to 4%, 1% to 2%, 2% to 20%, 2% to 18%, 2% to 16% , 2% to 14%, 2% to 12%, 2% to 10%, 2% to 8%, 2% to 6%, 2% to 4%, 4% to 20%, 4% to 18%, 4 % to 16%, 4% to 14%, 4% to 12%, 4% to 10%, 4% to 8%, 4% to 6%, 6% to 20%, 6% to 18%, 6% to 6% 16%, 6% to 14%, 6% to 12%, 6% to 10%, 6% to 8%, 8% to 20%, 8% to 18%, 8% to 16%, 8% to 14% ,
  • the system can form an editing window of at least 4 nucleotides in the nucleotide sequence of a target nucleic acid molecule.
  • the system has at least 50 nucleotides, such as at least 49 nucleotides, at least 48 nucleotides, at least 47 nucleotides, at least 46 nucleotides, at least 45 nucleotides, at least 44 nucleotides, at least 43 nucleotides, at least 42 nucleotides, at least 41 nucleotides, at least 40 nucleotides, at least 39 nucleotides, at least 38 nucleotides, at least 37 nucleotides, at least 36 nucleotides, at least 35 nucleotides, at least 34 nucleotides, at least 33 nucleotides, at least 32 nucleotides, at least 31 nucleotides, at least 30 nucleotides, at least 29 nucleotides , at least 28 nucleotides, at least
  • the system is 1 bp to 20 bp, 1 bp to 19 bp, 1 bp to 18 bp, 1 bp to 17 bp, 1 bp to 16 bp, 1 bp from the 5' end of the gRNA target sequence.
  • the nucleotide editing window is the first cytosine (C 1 ) to the 20th cytosine (C 20 ), C 2 to C 20 , and C 3 from the 5' end of the gRNA target sequence.
  • Another aspect provides a method of editing a nucleic acid comprising contacting a nucleic acid molecule with the CRISPR-Cas system.
  • the nucleic acid and CRISPR-Cas system are as described above.
  • the editing may involve forming a deletion, insertion, substitution, or indel of at least one nucleotide sequence among the nucleotide sequences of the nucleic acid molecule.
  • the modification is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 bp to 35 bp, 1 bp to 30 bp, 1 bp to 25 bp, 1 bp to 20 bp, 1 bp to 15 bp, 1 bp to 10 bp, 1 bp to 5 bp, 5 bp to 60 bp, 5 bp to 55 bp, 5 bp to 50 bp, 5 bp to 45 bp, 5 bp to 40 bp, 5 bp to 35 bp, 5 bp to 30 bp, 5 bp to 25 bp, 5 bp to 20 bp , 5 bp to 15 bp, 5 bp to 10 bp, 10
  • the modification is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 bp to 35 bp, 1 bp to 30 bp, 1 bp to 25 bp, 1 bp to 20 bp, 1 bp to 15 bp, 1 bp to 10 bp, 1 bp to 5 bp, 5 bp to 60 bp, 5 bp to 55 bp, 5 bp to 50 bp, 5 bp to 45 bp, 5 bp to 40 bp, 5 bp to 35 bp, 5 bp to 30 bp, 5 bp to 25 bp, 5 bp to 20 bp , 5 bp to 15 bp, 5 bp to 10 bp, 10
  • the modification is 1 bp to 60 bp, for example, 1 bp to 55 bp, 1 bp to 50 bp, 1 bp to 45 bp, 1 bp to 40 bp, 1 bp to 35 bp, 1 bp to 30 bp, 1 bp to 25 bp, 1 bp to 20 bp, 1 bp to 15 bp, 1 bp to 10 bp, 1 bp to 5 bp, 5 bp to 60 bp, 5 bp to 55 bp, 5 bp to 50 bp, 5 bp to 45 bp, 5 bp to 40 bp, 5 bp to 35 bp, 5 bp to 30 bp, 5 bp to 25 bp, 5 bp to 20 bp , 5 bp to 15 bp, 5 bp to 10 bp, 10
  • the method of modifying the nucleic acid has an efficiency of forming indels of the nucleotide sequence of 5% to 50%, for example, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35 %, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 50%, 30% to 45%, 30% It may be from 40% to 40%, 30% to 35%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 50%, 40% to 45%, or 45% to 50%.
  • the method of modifying the nucleic acid has an efficiency of forming a nucleotide sequence substitution of 1% to 20%, for example, 1% to 18%, 1% to 16%, 1% to 14%, 1% to 12%, 1% to 10%, 1% to 8%, 1% to 6%, 1% to 4%, 1% to 2%, 2% to 20%, 2% to 18%, 2% to 16%, 2% to 14%, 2% to 12%, 2% to 10%, 2% to 8%, 2% to 6%, 2% to 4%, 4% to 20%, 4% to 18 %, 4% to 16%, 4% to 14%, 4% to 12%, 4% to 10%, 4% to 8%, 4% to 6%, 6% to 20%, 6% to 18%, 6% to 16%, 6% to 14%, 6% to 14%, 6% to 12%, 6% to 10%, 6% to 8%, 8% to 20%, 8% to 18%, 8% to 16%, 8% to 14%
  • the method of modifying a nucleic acid can form an editing window of at least 4 nucleotides in the nucleotide sequence of a target nucleic acid molecule.
  • the method of modifying the nucleic acid includes at least 50 nucleotides, such as at least 49 nucleotides, at least 48 nucleotides, at least 47 nucleotides, at least 46 nucleotides, at least 45 nucleotides, at least 44 nucleotides, at least 43 nucleotides, at least 42 nucleotides, at least 41 nucleotides, at least 40 nucleotides, at least 39 nucleotides, at least 38 nucleotides, at least 37 nucleotides, at least 36 nucleotides, at least 35 nucleotides, at least 34 nucleotides, at least 33 nucleotides, at least 32 nucleotides, at least 31 nucleotides, at least 30 nucleotides , at least 29
  • the method of modifying the nucleic acid includes 1 bp to 20 bp, 1 bp to 19 bp, 1 bp to 18 bp, 1 bp to 17 bp, and 1 bp to 16 bp from the 5' end of the gRNA target base sequence.
  • the editing window of the method for modifying the nucleic acid is the cytosine located first from the 5' end of the gRNA target base sequence (C 1 ) to the cytosine located 20th (C 20 ), C 2 to C 20 , C 3 to C 20 , C 4 to C 20 , C 1 to C 19 , C 2 to C 19 , C 3 to C 19 , C 4 to C 19 , C 1 to C 18 , C 2 to C 18 , C 3 to C 18 , C 4 to C 18 , C 1 to C 17 , C 2 to C 17 , C 3 to C 17 , C 4 to C 17 , C 1 to C 16 , C 2 to C 16 , C 3 to C 16 , C 4 to C 16 , C 1 to C 15 , C 2 to C 15 , C 3 to C 15 , C 4 to C 15 , C 1 to C 14 , C 2 to C 14 , C 3 to C 14 , C 4 to C 14 , C 1 to C 13 ,
  • a fusion protein, a polypeptide thereof, and a CRISPR-Cas system including the same can perform effective base correction.
  • the small size of the polypeptide makes it easy to combine with the Cas protein, and when used as a cell therapy agent, it has the effect of facilitating delivery through a vector.
  • Figure 1 is a schematic diagram showing the structural/evolutionary differences between SsdA and other deaminase enzymes used in existing base correction technologies.
  • Figure 2 is a diagram showing the process of deamidating single-stranded DNA with the base sequence of SEQ ID NO: 4 by treating it with SsdA and cutting the single-stranded DNA by treating it with UDG and NaOH, and a Western blot confirming whether SsdA was deaminated. This is a photo showing the results.
  • Figure 3 is a graph showing the base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of target DNA:
  • Figure 3a is a graph showing base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of RNF2 DNA It is a graph showing the base correction efficiency of the CRISPR-Cas system including, and
  • Figure 3b is a graph showing the base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of HEK2 DNA.
  • Figure 4 is a graph showing the cytotoxicity of Cas9(D10A)-SsdA fusion protein, Cas9(D10A)-SsdA-UGI fusion protein, dCas9-SsdA fusion protein, and dCas9-SsdA-UGI fusion protein.
  • Figure 5 is a graph showing the base correction efficiency and indel formation efficiency of the CRISPR-Cas system containing Cas9 and SsdA fusion proteins:
  • Figure 5A shows the CRISPR-Cas system comprising Cas9(D10A)-SsdA fusion protein, Cas9(D10A)-SsdA-UGI fusion protein, dCas9-SsdA fusion protein, or dCas9-SsdA-UGI fusion protein for HEK2, HEK3, and It is a graph showing the base correction efficiency and indel formation efficiency in HEK4 and RNF2, and Figure 5b is a graph showing the cjCas9(D8A)-SsdA fusion protein, cjCas9(D8A)-SsdA-UGI fusion protein, or cjCas9(L58Y/D900K)(D8A).
  • -CRISPR-Cas system containing the SsdA-UGI fusion protein is a graph showing the base correction efficiency in each of EPAS1_e2, EPAS1_e5, HIF_e8, HIF_e9, and TFPi, and Figure 5c shows cjCas9 in a cell line suppressing Uracil-DNA glycosylase (UDG) expression.
  • UDG Uracil-DNA glycosylase
  • Figure 6 is a schematic diagram showing a plasmid for producing a fusion protein in which SsdA and uracil glycosylase inhibitor (UGI) are bound to the C terminus, N terminus, and both N and C termini of the Cas protein.
  • SsdA and uracil glycosylase inhibitor URI
  • Figure 7 is a graph confirming the base correction efficiency according to the binding position of SsdA and Cas proteins:
  • Figure 7A shows Cas9(D10A)-SsdA fusion protein (SsdA-C), SsdA-Cas9(D10A) fusion protein (SsdA-N, SsCBE), or SsdA-Cas9(D10A)-SsdA(SsdA-NC) fusion protein.
  • FIG. 7b is a graph showing the cytosine base correction efficiency in each of the CRISPR-Cas systems including HEK2, HEK3, HEK4, RNF2, FANCF, TYRO3, CCR5, or EMX1
  • Figure 7b is a graph showing the SsdA-Cas9(D10A) fusion protein (SsdA-N, CRISPR-Cas system containing a fusion protein combining one or two UGIs at the C terminus, N terminus, and both C and N termini of (SsCBE) HEK2, HEK3, HEK4, RNF2, FANCF, TYRO3, CCR5, or
  • SsdA-N SsdA-N
  • Figure 8 shows UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2), BE3 at the target site (HEK2, HEK3, HEK2-2, HEK3-8, HEK4-2, or HEK4-7 site). This is a graph showing the substitution frequency (%) of BE4max.
  • Figure 9 includes Cas9(D10A)-SsdA(G54D)-UGI fusion protein, Cas9(D10A)-SsdA(G54D) fusion protein, dCas9-SsdA(G54D)-UGI fusion protein, or dCas9-SsdA(G54D) fusion protein.
  • This is a graph showing the base correction efficiency and indel formation efficiency in HEK2, HEK3, and HEK4 for each CRISPR-Cas system.
  • the coding sequences of Cas9 and UGI were obtained through PCR amplification, the SsdA sequence was amplified from gBlock using Gibson Assembly Master Mix (New England Biolabs), and subcloned with pCMV plasmid. did.
  • PAAR-domin-containing protein amino acid sequence of SEQ ID NO: 1 and the SsdAI amino acid sequence of SEQ ID NO: 3 are shown in Table 2.
  • SsdA protein PAAR-domin-containing protein
  • the SsdA protein cloned in Example 1 was purified using E. coli BL21.
  • pET-28b-His6-SsdA-SsdAI was administered to E. coli BL21 using 0.5mM IPTG, and His6-SsdA and SsdAI complex proteins were purified using Ni-NTA agarose beads (Qiagen). And to separate His6-SsdA from SsdAI, His6-SsdA and SsdAI complex proteins were denatured with denaturation buffer (8M urea, 50mM Tris-HCl pH 7.5, 500mM NaCl, and 1mM DTT) and then incubated at 4°C for 16 hours. did.
  • denaturation buffer 8M urea, 50mM Tris-HCl pH 7.5, 500mM NaCl, and 1mM DTT
  • the suspension buffer containing the denatured protein complex was mixed with Ni-NTA agarose beads (Qiagen) and loaded on a gravity-flow column to remove unbound SsdAI. Afterwards, denaturation buffer with decreasing concentrations of urea (6 M, 4 M, 2 M, 1 M, and 0 M) was treated for refolding of SsdA. Refolded proteins bound to Ni-NTA agarose beads were eluted with an elution buffer containing 300 mM imidazole, and then the eluted proteins were incubated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM DTT, and 40 ⁇ m. It was dialyzed using % (w/v) glycerol and concentrated using Amicon Ultra-15 Centrifugal Filter Unit (Millipore). And the concentration of His6-SsdA protein was analyzed by SDS-PAGE.
  • HEK293T cells (ATCC CRL-11268) were preserved in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene), and then TC-treated 48-well plates (Corning Life Sciences) were seeded at 6 x 10 4 cells per well. Twenty-four hours after seeding, at approximately 60% cell confluency, 500 ng of plasmid (250 ng of Cas9-SsdA expression plasmid and 250 ng of gRNA expression plasmid) and 1.5 uL of Lipofectamine 2000 (Thermo Fisher Scientific) Transfection was performed using .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Welgene penicillin/streptomycin
  • the transfected cells were incubated at 37°C for 3 days, and the cells were directly lysed using lysis buffer (10 mM Tris-HCl at pH 7.5, 0.05% SDS, 100 mg/mL proteinase K; QIAGEN). Genomic DNA was prepared. The cell lysate was incubated at 56°C for 30 minutes and further incubated at 99°C for 15 minutes to inactivate Proteinase K.
  • the target region was amplified by PCR (2 or 3 times in total) and sequenced using an Illumina MiniSeq or iSeq 100 sequencing system.
  • single-stranded DNA having the nucleotide sequence of SEQ ID NO: 4 (5'- Aaaaaaaaaaaaaaaagcgaaaaaaaaaaaaaaaaa-3') was treated with 1 to 200 nM of SsdA at 37°C for 1 hour, then uracil DNA Glycosylase: UDG) was treated at 37°C for 30 minutes to remove DNA bases changed to uracil to create an abasic site. Afterwards, the abasic site was cleaved by treating with 100 mM NaOH and incubating for 2 minutes at 95°C. Then, the cleavage was confirmed through Western blot, and the results are shown in Figure 2.
  • Figure 2 is a diagram showing the process of deamidating single-stranded DNA with the base sequence of SEQ ID NO: 4 by treating it with SsdA and cutting the single-stranded DNA by treating it with UDG and NaOH, and a Western blot confirming whether SsdA was deaminated. This is a photo showing the results.
  • target DNA was treated with the CRISPR-Cas system containing SsdA, dCas9, and gRNA to remove cytosine. The formation of amination was confirmed.
  • the base sequences of the target DNA are shown in Table 3.
  • target dna target site sequence number RNF2 GTC3ATC6TTAGTC12ATTACCTGAGG SEQ ID NO: 5
  • HEK2 GAAC4AC6AAAGC11ATAGACTGCGGG SEQ ID NO: 6
  • RNF2 DNA and HEK2 DNA were treated with 100 nM Cas9, 300 nM sgRNA, and 40 nM SsdA, respectively, and then incubated at 37°C for 8 hours to induce conversion of cytosine to uracil at the target position of the target DNA. did. Then, 8 hours later, sgRNA, Cas9, and SsdA were removed by treatment with RNase and Protease K. Then, DNA purification was performed using the qiagen DNA extraction kit. Afterwards, PCR was performed using a primer containing the target site, and the base correction efficiency was measured through deep sequencing, and the results are shown in Figure 3.
  • Figure 3 is a graph showing the base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of target DNA:
  • Figure 3a is a graph showing the base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of RNF2 DNA
  • Figure 3b is a graph showing base correction efficiency of the CRISPR-Cas system including Cas protein, SsdA, and guide polynucleotide at the target site of HEK2 DNA. This is a graph showing the base correction efficiency of the CRISPR-Cas system including.
  • the base conversion caused by the CRISPR-Cas system including the Cas protein, SsdA, and guide polynucleotide is a conversion from cytosine to uracil
  • uracil-specific removal of deaminated DNA The enzyme was treated with Uracil-Specific Excision Reagent (USER) enzyme, and the target site was amplified through PCR. As a result, it was confirmed that the number of reads in which cytosine was converted to uracil disappeared.
  • Uracil-Specific Excision Reagent USR
  • a plasmid expressing a protein bound to Cas9 was created. Then, HEK293 cells were distributed in a 48 well plate at a concentration of 6 x 10 4 cells/well, and the plasmid was transfected into the HEK293 cells. 48 hours after transfection, live cells were trypsinized and the number of HEK293 cells was counted using a hemocytometer.
  • Figure 4 is a graph showing the cytotoxicity of Cas9(D10A)-SsdA fusion protein, Cas9(D10A)-SsdA-UGI fusion protein, dCas9-SsdA fusion protein, and dCas9-SsdA-UGI fusion protein.
  • a plasmid expressing a protein bound to Cas9 was created and transfected into HEK293 cells. Then, the base sequence of the target site (HEK2, HEK3, HEK4, RNF2, EPAS1_e2, EPAS1_e5, HIF_e8, HIF_e9, or TFPi site) from the transfected cells was amplified by PCR and the mutation was identified using next-generation sequencing (NGS). The introduction and indel formation were analyzed, and the results are shown in Figure 5.
  • NGS next-generation sequencing
  • Cas9 used was spCas9 (Cas9(D10A)) or cjCas9(D8A).
  • Uracil-DNA glycosylase (UDG) expression suppressing cell line (UNG KD) contains an shRNA (5'-GTCTACAGACATAGAGGATTT -3: SEQ ID NO: 7) expression plasmid that knockdown UDG and an HIV-based packaging plasmid (genes: Gag/Pol, Rev , including VSV-G) were mixed with Lipofectamine 3000 (Invitrogen) reagent in Opti MEM (Invitrogen) to transfect HEK293 cells.
  • Figure 5 is a graph showing the base correction efficiency and indel formation efficiency of the CRISPR-Cas system containing Cas9 and SsdA fusion proteins:
  • Figure 5A shows the CRISPR-Cas system comprising Cas9(D10A)-SsdA fusion protein, Cas9(D10A)-SsdA-UGI fusion protein, dCas9-SsdA fusion protein, or dCas9-SsdA-UGI fusion protein for HEK2, HEK3, and It is a graph showing the base correction efficiency and indel formation efficiency in HEK4 and RNF2, and Figure 5b is a graph showing the cjCas9(D8A)-SsdA fusion protein, cjCas9(D8A)-SsdA-UGI fusion protein, or cjCas9(L58Y/D900K)(D8A).
  • both cjCas9(D8A)-SsdA-UGI and cjCas9(L58Y/D900K)(D8A)-SsdA-UGI showed a base correction efficiency of about 15%, making UGI It was confirmed that the base correction efficiency was significantly improved compared to the unbound cjCas9(D8A)-SsdA fusion protein.
  • Table 4 shows the results of analyzing the base-corrected sequences generated by Cas9(D10A)-Ssda-UGI and HEK2 targeting gRNA.
  • Uracil-DNA glycosylase is well known as a protein that repairs cytosine deamination in intracellular DNA.
  • SsdA actually causes cytosine deamination in intracellular DNA.
  • HEK293 UDG-KO HEK293 cell line
  • a fusion protein was prepared by varying the position of SsdA, and through this, the base Proofreading efficiency and indel formation efficiency were confirmed.
  • the pCMV plasmid shown in Figure 6 was constructed and introduced into HEK293 UDG-KO cells along with gRNA for each target site according to Example 3. Then, the base sequence of the target site (HEK2, HEK3, HEK4, RNF2, FANCF, TYRO3, CCR5, or EMX1 site) from the transfected cells was amplified by PCR and the introduction of mutations was determined using next-generation sequencing (NGS). and indel formation was analyzed, and the results are shown in Figure 7.
  • NGS next-generation sequencing
  • Figure 6 is a schematic diagram showing a plasmid for producing a fusion protein in which SsdA and uracil glycosylase inhibitor (UGI) are bound to the C terminus, N terminus, and both N and C termini of the Cas protein.
  • SsdA and uracil glycosylase inhibitor URI
  • Figure 7 is a graph confirming the base correction efficiency according to the binding position of SsdA and Cas proteins:
  • Figure 7A shows Cas9(D10A)-SsdA fusion protein (SsdA-C), SsdA-Cas9(D10A) fusion protein (SsdA-N, SsCBE), or SsdA-Cas9(D10A)-SsdA(SsdA-NC) fusion protein.
  • FIG. 7b is a graph showing the efficiency of cytosine base correction in each of the CRISPR-Cas systems including HEK2, HEK3, HEK4, RNF2, FANCF, TYRO3, CCR5, or EMX1
  • Figure 7b is a graph showing the efficiency of cytosine base correction in each of the CRISPR-Cas systems including SsdA-Cas9(D10A) fusion protein (SsdA-N, CRISPR-Cas system containing a fusion protein combining one or two UGIs at the C terminus, N terminus, and both C and N termini of (SsCBE) HEK2, HEK3, HEK4, RNF2, FANCF, TYRO3, CCR5, or It is a graph showing the cytosine base proofreading efficiency in EMX1, and Figure 7c shows a UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2)
  • the base sequence of the target site of the target gene is shown in Table 5.
  • UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2) in which two UGIs with the highest base correction efficiency are linked to the N terminus;
  • UGI-UGI-SsdA-Cas9(D10A)-UGI-UGI fusion protein (SsCBE-UGI-N2C2), which combines two UGIs at both the N and C termini, was compared at 20 target positions. It was confirmed that the UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2) with two bonds to the N terminus showed slightly higher base correction efficiency.
  • UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2) in which two UGIs with the highest base correction efficiency are linked to the N terminus;
  • UGI-UGI-SsdA-Cas9(D10A)-UGI-UGI fusion protein (SsCBE-UGI-N2C2), which combines two UGIs at both the N and C termini; base correction range at 20 target positions (Editing window) ), it was confirmed that high base correction efficiency was observed at a position between 4 bp and 8 bp from the 5' end of the gRNA target base sequence.
  • the UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2), which was confirmed to have the highest base proofreading ability, was also confirmed to have base proofreading efficiency in wild-type HEK293 cells.
  • UGI-UGI-SsdA-Cas9(D10A) fusion protein SsCBE-UGI-N2
  • existing BE3 or BE4max were introduced into HEK293 cells along with gRNA for each target site according to Example 3.
  • the base sequence of the target site HEK2, HEK3, HEK2-2, HEK3-8, HEK4-2, or HEK4-7 site
  • NGS next-generation sequencing
  • Figure 8 shows UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2), BE3 at the target site (HEK2, HEK3, HEK2-2, HEK3-8, HEK4-2, or HEK4-7 site). This is a graph showing the substitution frequency (%) of BE4max.
  • UGI-UGI-SsdA-Cas9(D10A) fusion protein (SsCBE-UGI-N2) has six target sites (HEK2, HEK3, HEK2-2, HEK3-8, HEK4-2, or HEK4-7 site) successfully demonstrated base proofreading efficacy.
  • the G54D mutation in the catalytic active site of SsdA is the G302D mutation in the amino acid sequence (PAAR domaomn-containing protein) of SEQ ID NO: 1.
  • the G54D mutation in the catalytic active site of SsdA may have the sequence of SEQ ID NO: 17.
  • the amino acid sequence of the G54D mutation in the catalytic active site of SsdA is shown in Table 6.
  • Figure 9 includes Cas9(D10A)-SsdA(G54D)-UGI fusion protein, Cas9(D10A)-SsdA(G54D) fusion protein, dCas9-SsdA(G54D)-UGI fusion protein, or dCas9-SsdA(G54D) fusion protein.
  • This is a graph showing the base correction efficiency and indel formation efficiency in HEK2, HEK3, and HEK4 of each CRISPR-Cas system.
  • Table 7 shows the results of analyzing the base-corrected sequence generated by SsdA(G54D)-Cas9(D10A).

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