Base editor comprising novel cas12a protein or variant of novel cas12a protein, and use of base editor for genome editing

Abstract

The present specification discloses a base editing system comprising a novel dead-Cas12a protein which has not been previously disclosed. The base editing system includes a base editor protein and a programmable guide RNA. This specification discloses a base editing method using the base editing system. The inventors of this specification have demonstrated that the base editing system can edit nucleobase pairs as intended at actually intended locations. Using the base editing system and base editing method disclosed in this specification: 1) A-T base pairs included in a target site of an intracellular target nucleic acid (double-stranded nucleic acid) can be changed to G-T base pairs; or 2) C-G base pairs included in a target site of an intracellular target nucleic acid (double-stranded nucleic acid) can be changed to T-A base pairs. Therefore, the base editing system can be used for the treatment of diseases caused by mutations in some base pairs in the genome.

Description

A base editor comprising a novel Cas12a protein or a variant of the novel Cas12a protein, and a use of the base editor for genome editing

[0001] This specification discloses technologies related to the CRISPR / Cas system and a base editing system using the same.

[0002]

[0003] Background Technology - CRISPR / Cas12a System

[0004] Overview of the CRISPR / Cas12a System

[0005] The CRISPR / Cas12a system, also known as the CRISPR / Cpf1 system, is a CRISPR / Cas system belonging to the A subtype of Type V of Class 2. The CRISPR / Cas12a system was first reported by Feng Zhang's research team at the Broad Institute. The CRISPR / Cas12a system, along with the CRISPR / Cas9 system, is a CRISPR / Cas system that has been extensively studied.

[0006] The CRISPR / Cas12a system, like the CRISPR / Cas9 system, is known to have target-specific cleavage activity for double-stranded nucleic acids, and therefore has high utility for gene editing in eukaryotic cells. Compared to the CRISPR / Cas9 system, the CRISPR / Cas12a system has the following characteristics: 1) the Cas12a protein has only one type of nucleic acid cleavage domain (the RuvC domain), 2) the guide RNA consists solely of crRNA, and 3) it cleaves nucleic acids in a sticky end form that generates a 4-5 nucleotide overhang during gene cleavage.

[0007] The above CRISPR / Cas12a system has advantages such as a simpler guide RNA structure than the CRISPR / Cas9 system, a lower off-target cleavage rate, and the ability to perform more precise gene editing, and many researchers are actively researching ways to utilize it.

[0008] Below, the CRISPR / Cas12a system and its components are described in more detail.

[0009] CRISPR / Cas12a classification

[0010] The CRISPR / Cas12a system belongs to the A subtype of the Class 2 type V CRISPR / Cas system, and is also called the CRISPR / Cpf1 system.

[0011] CRISPR / Cas12a system configuration and function

[0012] The CRISPR / Cas12a system comprises the Cas12a protein and a guide RNA. The Cas12a protein and the guide RNA function as a complex, exhibiting target-specific nucleic acid cleavage activity and secondary cleavage activity. The Cas12a protein recognizes a PAM sequence and functions to cleave nucleic acids of the target sequence targeted by the guide RNA. The guide RNA functions to target nucleic acids of the target sequence.

[0013] Structure of the Cas12a protein

[0014] The Cas12a protein can be broadly divided into the REC (RECognition) lobe and the NUC (NUClease) lobe, and the NUC lobe is further divided into the RuvC domain, NUC domain, WED domain, PI (PAM Interacting) domain, and BH (Bridge Helix) domain. The specific structure of the Cas12a protein has already been reported by several researchers and is described in detail in Paul et.al (CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, Vol.43, Issue 1, pp.8-17, 2020). Among these, the WED II, WED III, REC1, and PI domains play an important role in allowing the Cas12a protein to recognize the PAM sequence. Meanwhile, the domains that play an important role in the Cas12a protein cleaving the nucleic acid of the target sequence are the RuvC domain and the NUC domain.

[0015] Structure of guide RNA

[0016] Unlike the guide RNA of the CRISPR / Cas9 system, the guide RNA of the CRISPR / Cas12a system is composed of a single RNA molecule, called crRNA. The guide RNA includes a direct repeat (DR) and a guide domain. Specifically, the guide RNA is sequentially linked to the DR and the guide domain in the 3' to 5' end direction. The DR is a portion involved in the guide RNA interacting with the Cas12a protein to form a complex, and the guide domain is a portion that binds to the nucleic acid of the target sequence to enable the CRISPR / Cas12a system to exhibit target-specific cleavage activity and secondary cleavage activity.

[0017] Target-specific nucleic acid cleavage activity and PAM (Protospacer Adjacent Motif) of the CRISPR / Cas12a system

[0018] The CRISPR / Cas12a system has target-specific nucleic acid cleavage activity, but two conditions are required for this target-specific nucleic acid cleavage activity to occur. First, there must be a base sequence of a certain length that the Cas12a protein can recognize within the nucleic acid. Second, there must be a sequence around the base sequence of a certain length that can complementarily bind to the guide domain contained in the guide RNA. When the above two conditions are satisfied, 1) the Cas12a protein recognizes the base sequence of a certain length, and 2) the guide domain complementarily binds to a sequence portion around the base sequence of a certain length, nucleic acid cleavage activity occurs. At this time, the base sequence of a certain length recognized by the Cas12a protein is called a Protospacer Adjacent Motif (PAM) sequence.

[0019] The above PAM sequence is a unique sequence determined by the Cas12a protein. If the PAM sequence of the Cas12a protein is known, it can be used to design a CRISPR / Cas12a system that targets nucleic acids of a predetermined target sequence surrounding the PAM sequence.

[0020] Background Technology - Base Editing System

[0021] The base editing system is a type of gene editing technology that combines a deaminase with the CRISPR / Cas system to change a specific base pair to another base pair. The base editing system uses the CRISPR / Cas system, but with the double-strand cleavage activity removed or limited. As a result, it changes base pairs without double-stranded nucleic acid cleavage, so it has the advantage of being safer and having fewer side effects than the existing CRISPR / Cas system. The base editing system was first studied and reported by Professor David Liu and his research team (Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016). https: / doi.org / 10.1038 / nature17946). Since then, many research teams have studied the base editing system. The above base editing system is broadly divided into the Adenine Base Editor (ABE) and the Cytosine Base Editor (CBE). ABE converts adenine-thymine base pairs into guanine-cytosine base pairs, and CBE converts cytosine-guanine base pairs into thymine-adenine base pairs. Specifically, ABE includes adenine deaminase to deaminate adenine to inosine, while CBE includes cytosine deaminase to deaminate cytosine to uracil. These deaminated bases undergo a DNA repair process, where inosine is converted to guanine, and uracil to thymine, and the bases on the opposite strand that pair with them are also corrected.

[0022]

[0023] The technical task of this specification is to discover a novel Cas12a protein that can be used for base editing among several candidate Cas12a proteins and to present a base editing system that can change base pairs contained in an actual target nucleic acid.

[0024]

[0025] To address the above technical challenges, the inventors of this specification:

[0026] 1) Among several candidate Cas12a proteins, we identified a CRISPR / Cas12a system that recognizes and effectively binds to double-stranded nucleic acids in the genome. In this process, we identified a new scaffold for the guide RNA that had previously been incorrectly initiated.

[0027] 2) By analyzing the structure of the Cas12a protein included in the system, we found amino acid positions important for nucleic acid cleavage activity.

[0028] 3) A dead-Cas12a protein that lost its nucleic acid cleavage activity was produced by introducing a mutation into the identified location.

[0029] 4) We identified several mutations (PAM binding site mutation, R-loop mutation, and Ultra Nuclease mutation, etc.) that are expected to improve base editing efficiency, and produced a dead-Cas12a protein with additional mutations at these sites.

[0030] 5) An adenine base editor and a cytosine base editor containing the dead-Cas12a protein above were designed.

[0031] 6) A base editor of the corresponding configuration was manufactured.

[0032] 7) It was demonstrated that the manufactured base editor can change target base pairs in the cellular genome as intended.

[0033] Accordingly, this specification discloses a novel base editing system whose effectiveness has been proven, and a base editing method using the same.

[0034] Furthermore, the inventors introduced mutations into the dead-Cas12a protein that enable it to bind more strongly to the protospacer adjacent motif, and demonstrated the effect.

[0035] Accordingly, this specification discloses a novel base editing system in which the above mutation is additionally introduced, and a base editing method using the same.

[0036]

[0037] Using the base editing system and base editing method disclosed in this specification:

[0038] 1) AT base pairs contained in the target site of the intracellular target nucleic acid (double-stranded nucleic acid) can be changed to GC base pairs.

[0039] 2) CG base pairs contained in the target site of the intracellular target nucleic acid (double-stranded nucleic acid) can be changed to TA base pairs.

[0040] Therefore, the above base editing system can be used to treat diseases caused by mutations in some base pairs in the genome.

[0041]

[0042] Figure 1 shows the results of evaluating the CRISPR enzyme expression results according to Experimental Example 2.1 according to Experimental Example 1.4. (a) shows the results visualized by ethidium bromide staining after actual restriction enzyme digestion, and (b) shows the number labels for each position in the drawing (a). The meaning of each number label is shown in Table 3 of Experimental Example 2.2.

[0043] Figure 2 shows the results of the T7E1 assay according to Experimental Example 2.1. Here, each number represents a CRISPR / Cas12a complex with a label shown in Table 1, Lba represents a positive control, indicating the results of an experiment in which a CRISPR / LbaCas12a complex was introduced, and WT represents a negative control in which no CRISPR / Cas12a complex was administered.

[0044] Figure 3 shows the results of analyzing the pattern of indels that occurred in the endogenous EMX1 gene of HEK cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 218 into the cells according to Experimental Example 2.3. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0045] Figure 4 shows the results of analyzing the pattern of indels that occurred in the endogenous EMX1 gene of HEK cells after introducing a CRISPR / Cas12a complex containing the positive control LbaCas12a protein into the cells according to Experimental Example 2.3. Several mutations with a high number of reads as a result of NGS analysis are shown. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0046] Figure 5 schematically illustrates the CRISPR enzyme configuration according to Experimental Example 3. The numbers (1 to 7) in front of each schematic diagram represent the configurations of the corresponding NLS1 to NLS7, and the specific sequences, etc. are described in Table 5 of Experimental Example 3.1.

[0047] Figure 6 shows the results of evaluating the CRISPR enzyme expression results according to Experimental Example 3.1 by IPTG induction. The labels (NLS1 to NLS7) described in the experimental results represent the CRISPR enzymes of each configuration described in Table 5.

[0048] Figure 7 shows the results of evaluating the CRISPR enzyme expression results according to Experimental Example 3.1 using SDS-PAGE. The labels (NLS1 to NLS7) described in the experimental results represent the CRISPR enzymes of each composition described in Table 5.

[0049] Figure 8 shows the results of an experiment on the endogenous EMX1 gene editing activity of the CRISPR / Cas12a complex using the T7E1 assay according to Experimental Example 3.3. Here, #1 to #7 correspond to NLS1 to NLS7 of Table 5, respectively.

[0050] Figure 9 shows the results of evaluating the CRISPR enzyme expression results according to Experimental Example 4 according to Experimental Example 1.4. Each number label represents a CRISPR / Cas12a complex of the corresponding label in Table 8 of Experimental Example 4.1.

[0051] Figure 10 shows the results of evaluating the CRISPR enzyme expression results according to Experimental Example 4 by SDS-PAGE analysis according to Experimental Example 1.3. Each number label represents a CRISPR / Cas12a complex of the corresponding label in Table 8 of Experimental Example 4.1.

[0052] Figure 11 shows the results of analyzing the pattern of indels that occurred in the endogenous EMX1 gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the NLS5 CRISPR enzyme into the cells according to Experimental Example 3.4. Several mutations with a high number of reads as a result of NGS analysis are shown. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0053] Figure 12 shows the results of analyzing the pattern of indels that occurred in the endogenous MTAP gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 218 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion of each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0054] Figure 13 shows the results of analyzing the pattern of indels that occurred in the endogenous MTAP gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 233 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion of each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0055] Figure 14 shows the results of analyzing the pattern of indels that occurred in the endogenous MTAP gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 236 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion of each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0056] Figure 15 shows the results of analyzing the pattern of indels that occurred in the endogenous DYRK1A gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 218 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0057] Figure 16 shows the results of analyzing the pattern of indels that occurred in the endogenous DYRK1A gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 233 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0058] Figure 17 shows the results of analyzing the pattern of indels that occurred in the endogenous DYRK1A gene of HEK293T cells after introducing a CRISPR / Cas12a complex containing the Cas12a protein of ID 236 into cells according to Experimental Example 4. Several mutations with a high number of reads are shown in the NGS analysis results. The underlined portion in each mutation sequence represents the protospacer sequence, and the portion marked with a '-' represents the nucleotide deleted as a result of gene editing.

[0059] Figure 18 shows the in vitro nucleic acid cleavage activity of each CRISPR / Cas12a complex according to Experimental Example 4.3. Here, (+++) indicates the highest cleavage activity, (++) indicates an intermediate cleavage activity, (+) indicates a low cleavage activity, and (-) indicates no cleavage activity.

[0060] Figure 19 is a diagram illustrating the structure of a complex formed by binding a CRISPR / Cas12a system comprising the amino acid sequence of SEQ ID NO: 1 to a target nucleic acid, simulated using AlphaFold. Positions D828, E921, and D1145, which contribute to nucleic acid cleavage, are indicated.

[0061] Figure 20 is a diagram illustrating the structure of a complex formed by binding a CRISPR / Cas12a system comprising the amino acid sequence of SEQ ID NO: 3 to a target nucleic acid, simulated using AlphaFold. Positions D936, E1029, and D1254, which contribute to nucleic acid cleavage, are indicated.

[0062] Figure 21 shows the alignment of the amino acid sequences of SEQ ID NOs. 1 and 3 with the FnCpf1 sequence of a prior art document. The nucleic acid cleavage activity site disclosed in the prior art document is indicated by a bold box. This matches the location identified in Figures 19 and 20.

[0063] Figure 22 is a diagram illustrating an exemplary structure of a cytosine base editor protein.

[0064] Figure 23 is a diagram illustrating an exemplary structure of an adenine base editor protein.

[0065] Figure 24 shows, according to Experimental Example 5, whether a cytosine base editing system comprising a wild-type Cas12a protein or the dead-Cas12a protein identified in Experimental Example 5.1 introduces indels into each target nucleic acid, for each target nucleic acid. The vertical axis represents the indel introduction frequency in %.

[0066] Figure 25 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of the cytosine base editing system editing activity experiment according to Experimental Example 6. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, the solid box represents the protospacer region, and the gray box represents the SNP contained in the target nucleic acid of the cells used in the experiment.

[0067] Figure 26 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of the cytosine base editing system editing activity experiment according to Experimental Example 6. UNT represents a negative control group.

[0068] Figure 27 is a diagram illustrating the structure of a complex formed by binding a CRISPR / Cas12a system including a Cas12a protein of sequence number 1 to a target nucleic acid, simulated using AlphaFold.

[0069] Figure 28 is an enlarged view of the portion interacting with the PAM in the structure of a CRISPR / Cas12a system comprising the Cas12a protein of sequence number 1, which binds to a target nucleic acid to form a complex. The E155, K537, and G531 portions, which are likely to interact with the PAM sequence (DT, DT, DT, and DA) portions, are indicated.

[0070] Figure 29 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of the cytosine base editing system editing activity experiment according to Experimental Example 7. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0071] Figure 30 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 8. Each target nucleic acid sequence is described below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0072] Figure 31 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 8. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0073] Figure 32 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 8. UNT represents a negative control.

[0074] Figure 33 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 8. UNT represents a negative control.

[0075] Figure 34 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 9. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0076] Figure 35 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 9. Each target nucleic acid sequence is described below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0077] Figure 36 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 9. UNT represents a negative control.

[0078] Figure 37 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 9. UNT represents a negative control.

[0079] Figure 38 is a drawing illustrating the structure of a complex formed by a CRISPR / Cas12a system including a Cas12a protein of sequence number 3 binding to a target nucleic acid, simulated using AlphaFold.

[0080] Figure 39 is an enlarged view of the portion interacting with the PAM in the structure of a CRISPR / Cas12a system comprising the Cas12a protein of sequence number 3, which binds to a target nucleic acid to form a complex. The Q190, S579, and Y585 portions, which are likely to interact with the PAM sequence (DT, DT, DT, and DA) portions, are indicated.

[0081] Figure 40 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 10. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0082] Figure 41 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 10. UNT represents a negative control.

[0083] Figure 42 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 10. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0084] Figure 43 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 10. UNT represents a negative control.

[0085] Figure 44 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 11. Each target nucleic acid sequence is described below for reference. The dotted box represents the PAM region, the solid box represents the protospacer region, and the gray box represents the SNP contained in the target nucleic acid of the cells used in the experiment.

[0086] Figure 45 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 11. UNT represents a negative control.

[0087] Figure 46 is a portion of a diagram illustrating the structure of a CRISPR / Cas12a system comprising the amino acid sequence of SEQ ID NO: 1, bound to a target nucleic acid and forming a complex, simulated using AlphaFold. Position E791, which is expected to contribute to increasing the stability of the CRISPR / Cas12a complex, is indicated.

[0088] Figure 47 is a portion of a diagram illustrating the structure of a CRISPR / Cas12a system comprising the amino acid sequence of SEQ ID NO: 1, bound to a target nucleic acid and forming a complex, simulated using AlphaFold. Position N526, which is expected to interact significantly with the target nucleic acid, is indicated.

[0089] Figure 48 shows the results of aligning the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of AsCas12a (AsCpf1) in the prior literature. The results show that N526 and E791 correspond to positions M537 and F870, respectively, which were identified in the prior literature as contributing to enhanced nucleic acid cleavage activity. This supports the reliability of the results derived from structural analysis.

[0090] Figure 49 is a portion of a diagram illustrating the structure of a CRISPR / Cas12a system comprising the amino acid sequence of SEQ ID NO: 1, bound to a target nucleic acid and forming a complex, simulated using AlphaFold. Position W354, which is expected to contribute to an increase in R-loop formation time during the nucleic acid cleavage process of the CRISPR / Cas12a complex, is indicated.

[0091] Figure 50 shows the alignment of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of AsCas12a (AsCpf1) from a prior literature review. The W354 region corresponds to W355, a position previously identified as contributing to increased R-loop formation time. This supports the reliability of the results derived from structural analysis.

[0092] Figure 51 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 12. Each target nucleic acid sequence is described below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0093] Figure 52 shows a heatmap showing the degree of editing of each nucleobase, indicated by the intensity of each box, as a result of an adenine base editing system editing activity experiment according to Experimental Example 12. Each target nucleic acid sequence is provided below for reference. The dotted box represents the PAM region, and the solid box represents the protospacer region.

[0094] Figure 53 is a graph showing the frequency of indel introduction into each target nucleic acid as a result of an adenine base editing system editing activity experiment according to Experimental Example 12. UNT represents a negative control.

[0095] Figure 54 is a schematic diagram illustrating the process of self-maturation after multiple guide RNA sequences linked to one promoter are transcribed in the CRISPR / Cas12a system.

[0096] Figure 55 shows the results of confirming the base editing activity for multiple target nucleic acids according to Experimental Example 13 using a cytosine base editing system. A heat map is shown, with the degree of editing of each nucleobase indicated by the intensity of each box. The sequences of each target nucleic acid are provided for reference below. The dotted boxes represent the PAM region, the solid boxes represent the protospacer region, and the gray boxes represent the SNPs contained in the target nucleic acids of the cells used in the experiment.

[0097] Figure 56 shows the results of confirming the base editing activity for multiple target nucleic acids according to Experimental Example 13 using an adenine base editing system. A heat map is shown, with the degree of editing of each nucleobase indicated by the intensity of each box. The sequences of each target nucleic acid are provided for reference below. The dotted box indicates the PAM region, the solid box indicates the protospacer region, and the gray box indicates the SNPs contained in the target nucleic acids of the cells used in the experiment.

[0098]

[0099] Hereinafter, the best mode for carrying out the invention is exemplified. This includes some, but not all, implementations of the invention disclosed herein. The embodiments described in this paragraph are merely exemplary, and the implementations described in this paragraph should not be construed as the "best mode for carrying out the invention." Those skilled in the art will likely envision numerous variations and more desirable implementations of the examples described in this paragraph, and such variations should also be considered to be included within the best mode for carrying out the invention.

[0100] This specification discloses an adenine base editor composition comprising:

[0101] an adenine base editor protein, or a nucleic acid encoding said adenine base editor protein;

[0102] wherein the adenine base editor protein comprises a dead-Cas12a protein, one or more adenine deaminases, and one or more nuclear localization signals;

[0103] a guide RNA or a nucleic acid encoding said guide RNA,

[0104] Here, the guide RNA comprises a scaffold and a guide domain;

[0105] Here, the scaffold comprises a direct repeat portion,

[0106] The dead-Cas12a protein of the above direct repeat portion and the adenine base editor protein is a combination selected from the following:

[0107] A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 33; or

[0108] A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 35.

[0109] In one embodiment, the adenine base editor protein may have a structure that satisfies the following conditions:

[0110] wherein the dead-Cas12a protein and at least one adenine deaminase are linked directly or via an amino acid linker;

[0111] One or more nuclear localization signals are located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the above adenine base editor.

[0112] In one embodiment, the adenine base editor protein has the following structure:

[0113] [NLS1] - [Linker1] - [AD] - [Linker2] - [dead-Cas12a] - [Linker3] - [NLS2]

[0114] Here, the above NLS1 and NLS2 are each independently a nuclear localization signal or are absent,

[0115] The above Linker1 and Linker2 are each independently an amino acid linker or are absent,

[0116] The above AD is adenine deaminase,

[0117] The above dead-Cas12a is dead-Cas12a protein.

[0118] In one embodiment, the adenine base editor protein comprises an amino acid sequence selected from the following:

[0119] SEQ ID NO: 283 to SEQ ID NO: 294; SEQ ID NO: 412 to SEQ ID NO: 414; SEQ ID NO: 416; and SEQ ID NO: 418 to SEQ ID NO: 424.

[0120] This specification describes a method for changing A and T base pairs at a target site contained in a target nucleic acid within a cell to G and C base pairs.

[0121] A method is disclosed comprising:

[0122] Delivering the above adenine base editor composition to cells;

[0123] Here, the adenine base editor protein and the guide RNA combine to form a base editor complex,

[0124] The base editor complex contacts the target nucleic acid to change the A and T base pairs of the target site contained in the target nucleic acid into G and C base pairs.

[0125] As an example,

[0126] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0127] The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto,

[0128] When the above method is performed, A included in the second target site changes to G, and T in the corresponding first target site changes to C.

[0129] The above base editor complex recognizes a sequence consisting of the i-th nucleic acid to the (i+3)-th nucleic acid upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM),

[0130] The guide domain of the above guide RNA hybridizes with a second target site within the target strand and a sequence consisting of the first nucleic acid to the (i-1)th nucleic acid downstream from the 3' end of the second target site,

[0131] The above i can be an integer.

[0132] In one embodiment, i may be 7 or 8.

[0133] In one embodiment, the target region may be a double-stranded nucleic acid of 4 to 14 bp in length.

[0134] This specification discloses a cytosine base editor composition comprising:

[0135] a cytosine base editor protein, or a nucleic acid encoding said cytosine base editor protein;

[0136] wherein the cytosine base editor protein comprises a dead-Cas12a protein, one or more cytosine deaminases, one or more uracil glycosylase inhibitors, and one or more nuclear localization signals;

[0137] a guide RNA or a nucleic acid encoding said guide RNA,

[0138] Here, the guide RNA comprises a scaffold and a guide domain;

[0139] Here, the scaffold comprises a direct repeat portion,

[0140] The dead-Cas12a protein of the above direct repeat portion and the cytosine base editor protein is a combination selected from the following:

[0141] A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 33; or

[0142] A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 35.

[0143] In one embodiment, the cytosine base editor protein may have a structure that satisfies the following conditions:

[0144] wherein the dead-Cas12a protein and at least one cytosine deaminase are linked directly or via an amino acid linker;

[0145] wherein the dead-Cas12a protein and at least one uracil glycosyltransferase inhibitor are linked directly, via a nuclear localization signal, or via an amino acid linker;

[0146] One or more nuclear localization signals are located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the cytosine base editor.

[0147] In one embodiment, the cytosine base editor protein may have the following structure:

[0148] [NLS1] - [Linker1] - [CD] - [Linker2] - [dead-Cas12a] - [Linker3] - [UGI] - [Linker4] - [NLS2]

[0149] Here, the NLS1 and NLS2 are each independently a nuclear localization signal or are absent,

[0150] The above Linker1 and Linker2 are each independently an amino acid linker or are absent,

[0151] The above Linker3 is an amino acid linker, a nuclear localization signal, or is absent,

[0152] The above CD is cytosine deaminase,

[0153] The above dead-Cas12a is a dead-Cas12a protein,

[0154] The above UGI is a uracil glycosylase inhibitor.

[0155] In one embodiment, the cytosine base editor protein may comprise an amino acid sequence selected from the following:

[0156] SEQ ID NO: 295 to SEQ ID NO: 296; and SEQ ID NO: 405.

[0157] This specification describes a method for changing C and G base pairs in a target site contained in a target nucleic acid within a cell into T and A base pairs.

[0158] A method is disclosed comprising:

[0159] Delivering the cytosine base editor composition to a cell;

[0160] Here, the cytosine base editor protein and the guide RNA combine to form a base editor complex,

[0161] The above base editor complex contacts the target nucleic acid and changes the C and G base pairs of the target site contained in the target nucleic acid into T and A base pairs.

[0162] As an example,

[0163] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0164] The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto,

[0165] When the above method is performed, C included in the second target site changes to T, and G in the corresponding first target site changes to A.

[0166] The above base editor complex recognizes a sequence consisting of the i-th nucleic acid to the (i+3)-th nucleic acid upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM),

[0167] The guide domain of the above guide RNA hybridizes with a second target site within the target strand and a sequence consisting of the first nucleic acid to the (i-1)th nucleic acid downstream from the 3' end of the second target site,

[0168] Here, i can be an integer.

[0169] In one embodiment, i may be an integer between 5 and 7.

[0170] In one embodiment, the target region may be a double-stranded nucleic acid of 7 bp to 14 bp in length.

[0171]

[0172] Hereinafter, the present invention will be described in more detail through specific implementations and examples with reference to the attached drawings. It should be noted that the attached drawings include some, but not all, implementations of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementations described herein. These implementations should be considered as provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementations of the invention disclosed herein. Therefore, the invention disclosed herein is not limited to the specific implementations described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0173] Definition of terms

[0174] approximately

[0175] The term "about" as used herein means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0176] Amino acid sequence notation

[0177] 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 in the N-terminal to C-terminal direction. For example, when written as PAAK, it means a peptide in which proline, alanine, alanine, and lysine are sequentially connected from the N-terminal to the C-terminal. Another example, when written as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the single-letter notation, other letters are used and additional explanations are provided.

[0178] 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).

[0179] Nucleic acid sequence notation

[0180] The symbols A, T, C, G, and U used herein are to be interpreted as meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively; when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide containing each of the above nucleosides.

[0181] CRISPR / Cas12a system comprising the Cas12a protein disclosed in KR 2023-0111399 A

[0182] Korean Publication No. KR 2023-0111399 A (hereinafter, "Prior Document 1") discloses dozens of novel CRISPR / Cas12a systems. Prior Document 1 discloses each Cas12a protein, guide RNA, PAM sequence recognized by the Cas12a protein, and in vitro nucleic acid cleavage activity constituting the novel CRISPR / Cas12a system. However, there is little evidence that a CRISPR / Cas12a system with the above configuration can actually operate in eukaryotic cells and exhibit gene editing activity. In other words, based solely on the disclosure of Prior Document 1, it is unclear whether each CRISPR / Cas12a system can target-specifically recognize and stably bind to double-stranded nucleic acids. Furthermore, the inventors of this specification have closely examined the contents of Prior Document 1 and discovered that the specification of the guide RNA of the CRISPR / Cas12a system, particularly the direct repeat, is incorrect.

[0183] Base editing system containing a novel variant of the Cas12a protein

[0184] Overview of a base editing system comprising a novel variant of the Cas12a protein.

[0185] This specification discloses a base editing system comprising a novel dead-Cas12a protein that has not been previously disclosed. The base editing system comprises a base editor protein and a programmable guide RNA. The base editor protein comprises a dead-Cas12a protein with a deleted nucleic acid cleavage activity and additional domains including a deaminase. The dead-Cas12a protein is a variant protein based on the Cas12a protein disclosed in the prior literature, which introduces mutations to inactivate the nucleic acid cleavage function and optionally introduces mutations to allow stronger interaction with a protospacer adjacent motif (PAM). The inventors of this specification 1) discovered a Cas12a protein that can recognize double-stranded nucleic acids and bind to targets specifically, 2) specified a guide RNA composition that works together with the Cas12a protein, 3) identified a site that exhibits nucleic acid cleavage activity, 4) identified a portion that interacts with PAM and designed additional mutations at this site, 5) manufactured a dead-Cas12a protein with inactivated nucleic acid cleavage activity, and 6) further developed a base editing system that can cause an intended base pair substitution at a target site. The components of the base editing system operate by forming a 'base editor complex', which is an RNA-protein complex. The base editing system is used in slightly different forms as a base editor complex, a base editor component expression vector, and a base editor composition, as needed. Hereinafter, the novel dead-Cas12a protein and the base editing system including the same will be described in more detail based on the 'base editor complex'.

[0186] Base Editor Complex Composition #1 - Base Editor Protein

[0187] This specification discloses a base editor protein comprising a novel dead-Cas12a protein. The base editor protein comprises a dead-Cas12a protein with abolished nucleic acid cleavage activity, and additional domains including a deaminase. Representative deaminase types include adenine deaminase and cytosine deaminase, and the protein is divided into an adenine base editor protein and a cytosine base editor protein depending on the type of deaminase included. A representative additional domain included in the base editor protein is a nuclear localization signal (NLS). The NLS is an element that significantly contributes to the transport of the base editor complex into the cell nucleus. The cytosine base editor protein may further comprise a uracil glycosylase inhibitor (UGI) to enhance cytosine base editing efficiency. The base editor protein disclosed in this specification is not particularly limited in its structure as long as it includes the above composition.

[0188] Base Editor Complex Composition #2 - Dead-Cas12a Protein Included in Base Editor Protein

[0189] The dead-Cas12a protein is a key component that enables the base editor complex to target-specifically recognize double-stranded nucleic acids. The novel dead-Cas12a protein disclosed in this specification was developed based on a novel Cas12a protein that has been proven to target-specifically recognize and cleave double-stranded nucleic acids. The inventors of this specification introduced mutations into the Cas12a protein to eliminate its nucleic acid cleavage function and confirmed that this function was inactivated. Furthermore, they further studied mutations that enhance the Cas12a protein's interaction with the PAM, and discovered that introducing some of these mutations improved base editing efficiency. The "novel dead-Cas12a protein," which encompasses the above, is also one of the key inventions covered in this specification. More specific details related to this are described in detail in the "Dead-Cas12a Protein" section.

[0190] Base Editor Complex Configuration #3 - Programmable Guide RNA

[0191] This specification discloses a programmable guide RNA that binds to a base editor protein to form a base editor complex. The programmable guide RNA comprises 1) a scaffold, which is a portion that significantly interacts with the base editor protein, and 2) a guide domain designed to be aligned with a target site. The CRISPR / Cas12a system is known to operate solely with crRNA, without a tracrRNA. The scaffold of the programmable guide RNA essentially includes the direct repeat (DR) portion of the crRNA. The inventors of this specification have discovered that the direct repeat portion of the guide RNA of the novel CRISPR / Cas12a system disclosed in the prior art was incorrectly specified, and have newly specified this portion to invent a CRISPR / Cas12a system that actually works. The scaffold may further include additional components that contribute to improving base editing efficiency in addition to the direct repeat portion. The guide domain can target a target sequence and is appropriately designed to be aligned with a target site containing a nucleotide to be edited. The specific definitions of the sequence of the above guide domain, target nucleic acid, target site, target sequence, etc. and their relationships are explained in more detail in the relevant section.

[0192] How to edit bass

[0193] This specification discloses a base editing method using the above base editing system. The above base editing method is performed by delivering the base editing system designed to cause a desired nucleobase pair edit at a desired location to a target cell. The target site and the nucleobase editing pattern differ depending on whether the above base editing system comprises an adenine base editor protein or a cytosine base editor protein. For example, an adenine base editor protein changes an AT base pair in the target site to a GC base pair. In another example, a cytosine base editor protein changes a CG base pair in the target site to a TA base pair. The scope of the specific target site and the relationship between the target site and the configuration of each base editing system are described in more detail in the relevant paragraphs.

[0194] Base Editing System Feature #1 - Development of a Novel Dead-Cas12a Protein

[0195] This specification discloses a novel dead-Cas12a protein having an unprecedented structure. The dead-Cas12a protein refers to a Cas12a protein with inactivated nucleic acid cleavage activity. The dead-Cas12a protein is basically manufactured by substituting the amino acid corresponding to the catalytic residue of the novel Cas12a protein with an inactive amino acid (e.g., alanine). Furthermore, some dead-Cas12a proteins are manufactured by introducing additional mutations in the site that interacts with the PAM sequence in addition to the mutation that 'eliminates nucleic acid cleavage activity'. The inventors of this specification developed a novel dead-Cas12a protein through a series of research processes, including identifying a Cas12a protein that recognizes a double-stranded nucleic acid and binds target-specifically, identifying the amino acid corresponding to the catalytic residue, introducing additional mutations related to the PAM sequence, and confirming through experiments that it actually functions as intended.

[0196] Base Editing System Feature #2 - New Guide RNA Specification

[0197] This specification discloses a guide RNA that operates in conjunction with the Cas12a protein selected above to enable the CRISPR / Cas12a complex to recognize a target nucleic acid. KR 2023-0111399 A also discloses guide RNAs for the Cas12a proteins, particularly direct repeat sequences. However, the inventors of this specification discovered that the direct repeat sequence disclosed in the above document was incorrectly specified, and through further research, they determined the correct sequence. In this specification, a guide RNA comprising a specific nucleic acid sequence (i.e., a direct repeat sequence) operates in conjunction with a corresponding Cas12a protein to enable the RNA-protein complex to recognize a double-stranded nucleic acid and bind to the target specifically.

[0198] Base Editing System Feature #3 - Nucleobase Editing Site (Target Site) Specificity

[0199] The inventors of this disclosure have developed an adenine base editing system and a cytosine base editing system using a novel dead-Cas12a protein. Furthermore, they designed various programmable guide RNAs for the base editing system and applied them to various target nucleic acids to analyze nucleobase editing patterns. The analysis results revealed that the target site where nucleobase editing occurs could be identified. Therefore, the base editing system disclosed in this disclosure has a reliable target site (the location where nucleobase editing occurs), and based on this, the structure of the base editing system can be designed according to the intended use.

[0200] Dead-Cas12a protein

[0201] Dead-Cas12a protein overview

[0202] This specification discloses a dead-Cas12a protein that is mutated to be unable to cleave nucleic acids. Specifically, based on a Cas12a protein comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3, the following mutations are introduced: 1) amino acids that function as catalytic residues in nucleic acid cleavage are replaced with inactive amino acids, for example, alanine (hereinafter, cleavage inactivation mutations); and 2) optionally, amino acids that interact with PAM upon nucleic acid-complex binding are replaced with amino acids having a stronger (+) charge, for example, arginine (hereinafter, PAM binding site mutations); For example, the Cas12a protein comprises the amino acid sequence of any one of SEQ ID NOs: 264 to 265, or any one of SEQ ID NOs: 266 to 274. The dead-Cas12a proteins recognize a specific PAM in a target double-stranded nucleic acid, and the guide domain of the guide RNA complexed with it binds to the target nucleic acid. The dead-Cas12a proteins bind target-specifically to nucleic acids but rarely cleave them. This is because their nucleic acid cleavage activity is either completely eliminated or severely inhibited. The inventors of this disclosure designed the dead-Cas12a proteins through a series of studies and demonstrated that they function as intended. The identification and validation of the cleavage-inactivating mutations and the PAM-binding domain mutations are described in more detail below.

[0203] Truncation Inactivation Mutation #1 - Overview

[0204] The inventors of this specification first selected a novel Cas12a protein capable of "recognizing and target-specifically binding double-stranded nucleic acids." Experimentally, they demonstrated that a Cas12a protein comprising the amino acid sequences of SEQ ID NOs: 1 and 3 can edit the eukaryotic genome, and used this as the basis for the dead-Cas12a protein. The ability to introduce indels into the eukaryotic genome implies the ability to recognize and target-specifically bind double-stranded nucleic acids. However, it is difficult to directly introduce a Cas12a protein with nucleic acid cleavage activity into a base editing system because the nucleic acid cleavage activity can introduce unintended mutations, i.e., indels. Therefore, the Cas12a protein must be designed to recognize and target-specifically bind double-stranded nucleic acids without cleaving them. Accordingly, the inventors of this specification analyzed the structure of the novel Cas12a protein discovered above, where it binds to a target nucleic acid, identified candidate sites that contribute critically to nucleic acid cleavage activity, confirmed these sites through comparison with prior studies, and confirmed that a dead-Cas12a protein can be produced by introducing cleavage-inactivating mutations at these sites. The structural prediction method and the process of identifying the catalytic residue sites are described in more detail below.

[0205] Truncation Inactivation Mutation #2 - Structure Prediction Using AlphaFold

[0206] The inventors of this specification analyzed the structures of Cas12a proteins having the amino acid sequence of SEQ ID NO: 1 and Cas12a proteins having the amino acid sequence of SEQ ID NO: 3, bound to target nucleic acids, respectively, using the AlphaFold model. The results of the structural analysis are shown in Figures X to X. As a result of the analysis, candidates for catalytic residues that significantly contribute to the cleavage of the target nucleic acid could be selected. Specifically, positions D828, E921, and D1145 in the amino acid sequence of SEQ ID NO: 1 are candidates for the catalytic residues. In addition, positions D936, E1029, and D1254 in the amino acid sequence of SEQ ID NO: 3 are candidates for the catalytic residues. It was predicted that if the above catalytic residue candidates were replaced with appropriate amino acids, a dead-Cas12a protein could be produced.

[0207] Cleavage inactivation mutation #3 - confirmed by alignment with known Cas12a sequences.

[0208] To more definitively identify the candidate catalytic residue positions, the inventors of this specification compared them with the FnCas12a protein (Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016 / j.cell.2015.09.038. Epub 2015 Sep 25. PMID: 26422227; PMCID: PMC4638220.), for which the catalytic residue positions are already known. Specifically, the sequences of the novel Cas12a protein and the FnCas12a protein were aligned, and the selected active site was compared with the known active site. The sequence alignment results are shown in Figures X to X. As a result of the comparison, we obtained confidence that the catalytic residue positions selected above are actually involved in the cleavage of the target nucleic acid. Accordingly, we judged that replacing the catalytic residue positions with alanine, a representative inactivating amino acid, would inactivate the nucleic acid cleavage activity. As confirmed through experimental examples, when all of the catalytic residue positions above are replaced with alanine (SEQ ID NO: 264 to SEQ ID NO: 265), the corresponding Cas12a protein does not exhibit nucleic acid cleavage activity. In other words, for each novel Cas12a protein, replacing the specific catalytic residues above with amino acids with low or no activity can be viewed as 'introducing a cleavage inactivating mutation into the Cas12a protein.'

[0209] PAM Binding Site Mutations #1 - Overview

[0210] This specification discloses a novel dead-Cas12a protein, in which a PAM binding site mutation is additionally introduced into the cleavage-inactivating mutation. It is well known that Cas proteins interact with and recognize the PAM sequence region, and that this is crucial for exhibiting "target-specific activity" in conjunction with the guide domain of a guide RNA. Previous research (Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, Aryee MJ, Joung JK. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol. 2019 Mar;37(3):276-282. doi: 10.1038 / s41587-018-0011-0. Epub 2019 Feb 11. Nat Biotechnol. 2020 Jul;38(7):901. PMID: According to (30742127; PMCID: PMC6401248.), when specific residues interacting with the PAM sequence portion of the Cas12a protein are replaced with residues with stronger activity, the function of recognizing target nucleic acids is improved. Specifically, a previous study identified three positions in the Cas12a protein that interact with PAM, and revealed that replacing all of them with arginine, which has a strong (+) charge, increased both gene editing activity and base editing activity. Inspired by the above study, the researchers in this specification attempted to improve nucleobase editing activity by additionally introducing mutations in the PAM binding site into the Cas12a protein that had introduced cleavage inactivating mutations.Specifically, we identified key residues interacting with the PAM sequence through structural analysis, introduced 'PAM binding site mutations' that replace these with amino acids with a strong (+) charge, and confirmed whether they affect base editing efficiency. In actual experiments, not all PAM binding site mutations led to improved nucleobase editing activity. The inventors of this specification, through rigorous experiments, identified mutations that enhance nucleobase editing activity, mutations that enhance activity only for specific targets, and 'usable' mutations that exhibit nucleobase editing activity on various targets. The following describes the structure prediction method and the process of specifying the PAM binding site introduction site in more detail.

[0211] PAM Binding Mutation #2 - Structure Prediction Using AlphaFold

[0212] The inventors of this specification analyzed the structures of Cas12a proteins having the amino acid sequence of SEQ ID NO: 1 and Cas12a proteins having the amino acid sequence of SEQ ID NO: 3, bound to target nucleic acids, respectively, using the AlphaFold model. The results of the structural analysis are shown in Figures X to X. As a result of the analysis, key residue positions interacting with the PAM sequence could be selected. Specifically, positions E155, G531, and K537 in the amino acid sequence of SEQ ID NO: 1 are positions that significantly interact with the PAM sequence. In addition, positions Q190, S579, and Y585 in the amino acid sequence of SEQ ID NO: 3 are positions that significantly interact with the PAM sequence. It was expected that substituting the above key residues with appropriate amino acids would allow stronger binding to the PAM sequence, which would ultimately contribute to improving base editing efficiency.

[0213] PAM binding site mutation #3 - specific mutations that enhance actual nucleobase editing activity

[0214] The inventors of this specification attempted to improve base editing efficiency by substituting each of the above key residues with strongly (+) charged arginine. Specifically, using a dead-Cas12a protein with cleavage-inactivating mutations, we constructed dead-Cas12a proteins with all possible combinations of PAM-binding site mutations (mutations at only one site, two sites, or all three sites) for each position derived above. Furthermore, we constructed a base editing system containing the dead-Cas12a protein and confirmed the base pair editing efficiency. As a result of the experiment, not all PAM-binding site mutations improved base editing efficiency (see Experimental Example). Through rigorous experiments, the inventors identified and specified 1) variants that exhibited higher nucleobase editing efficiency than the Cas12a protein with only cleavage-inactivating mutations introduced, 2) variants that showed good effects only for specific sequences, and 3) variants that showed a certain level of nucleobase editing effect and were thus usable. For example, dead-Cas12a proteins comprising amino acid sequences selected from SEQ ID NO: 266 and SEQ ID NO: 274 exhibited higher editing efficiency when incorporated into a base editing system than dead-Cas12a proteins comprising only cleavage-activating mutations, such as amino acid sequences selected from SEQ ID NO: 264 to SEQ ID NO: 265. As another example, dead-Cas12a proteins comprising amino acid sequences selected from SEQ ID NO: 273 exhibited sufficiently usable efficiency when incorporated into a base editing system.

[0215] R-loop mutation

[0216] This specification discloses a novel dead-Cas12a protein in which an R-loop mutation is additionally introduced into the cleavage-inactivating mutation. The inventors of this specification identified additional mutations by referring to a previous study (Naqvi, MM, Lee, L., Montaguth, OET et al. CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage. Nat Chem Biol 18, 1014-1022 (2022). https: / doi.org / 10.1038 / s41589-022-01082-8). The previous study described mutations that increase the duration of the R-loop formed during nucleic acid cleavage. The previous study only noted that a longer R-loop duration increases the nucleic acid cleavage efficiency, and only data related to this were disclosed. The inventors of the present invention hypothesized that as the R-loop formation time increases, one strand of the target double-stranded nucleic acid is exposed for a longer time, which increases the opportunity for the deaminase to function, and consequently, the nucleobase editing efficiency may be improved. Through sequence alignment and structural analysis, the inventors identified a novel amino acid position in Cas12a corresponding to the mutation position of lbCas12a introduced in a previous study, and designed an R-loop mutation that substituted this amino acid position with alanine. Subsequently, the R-loop mutation was introduced into a Cas12a protein to which a cleavage-inactivating mutation had been introduced, thereby producing a dead-Cas12a protein, and a base-editing protein containing this was produced and its nucleobase editing activity was confirmed.

[0217] Ultra Nuclease mutation

[0218] This specification discloses a novel dead-Cas12a protein in which an Ultra Nuclease mutation is additionally introduced into the cleavage inactivating mutation.

[0219] The inventors of this specification have discovered additional mutations by referring to previous studies (Zhang, L., Zuris, JA, Viswanathan, R. et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat Commun 12, 3908 (2021). https: / doi.org / 10.1038 / s41467-021-24017-8; Hsiung, C.CS., Wilson, CM, Sambold, NA et al. Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations. Nat Biotechnol (2024). https: / doi.org / 10.1038 / s41587-024-02224-0). The above previous studies are studies on mutations that increase the nucleic acid cleavage efficiency of the CRISPR / Cas12a system. The inventors of the present invention interpreted the M537R mutation in the AsCas12a protein as a mutation that enhances the DNA binding activity of the Cas12a protein and thus further enhances the PAM interaction, and the F870L mutation was viewed as a mutation that further stabilizes the CRISPR / Cas12a complex. Accordingly, the inventors speculated that applying a mutation corresponding to the above mutation (referred to as the Ultra Nuclease mutation) to the base editor complex would increase the nucleobase editing efficiency. Through sequence alignment and structural analysis, the inventors identified a novel amino acid position in Cas12a corresponding to the mutation position of AsCas12a introduced in the previous study, and designed an Ultra Nuclease mutation that substituted this amino acid position with arginine or leucine. Subsequently, the Ultra Nuclease mutation was introduced into the Cas12a protein to which the cleavage-inactivating mutation had been introduced, thereby producing a dead-Cas12a protein, and a base editing protein containing the same was produced and confirmed to have nucleobase editing activity.

[0220] Combination of the above mutations

[0221] This specification discloses a novel dead-Cas12a protein that additionally introduces a combination of the mutations mentioned above in addition to the cleavage-inactivating mutation. The PAM binding site mutation, R-loop mutation, and Ultra Nulcease mutation mentioned above all affect specific functions of the Cas12a protein. The inventors of this specification reported that when these additional mutations are combined, there may be a synergistic effect that further enhances base editing efficiency, and they tested the base editing efficiency by combining these mutations. As a result, it was confirmed that not all combinations increased base editing efficiency, and that certain combinations showed high base editing efficiency. For example, dead-Cas12a proteins comprising an amino acid sequence selected from SEQ ID NO: 271 showed higher editing efficiency when incorporated into a base editing system than dead-Cas12a proteins comprising only cleavage-activating mutations, including an amino acid sequence selected from SEQ ID NO: 264 to SEQ ID NO: 265. As another example, dead-Cas12a proteins comprising amino acid sequences selected from SEQ ID NO: 267 to SEQ ID NO: 270 and SEQ ID NO: 272 exhibited sufficiently usable levels of effectiveness when incorporated into a base editing system.

[0222] Base Editor Protein

[0223] Base Editor Protein Overview

[0224] This specification discloses a base editor protein comprising the dead-Cas12a protein disclosed above. The base editor protein comprises the dead-Cas12a protein; one or more deaminase(s); one or more nuclear localization signals (NLS), and optionally, additional domains. Representative deaminase(s) include adenine deaminase and cytosine deaminase. The nucleobase editing behavior of the base editor protein varies depending on the deaminase(s) included. Therefore, the deaminase(s) may be appropriately selected depending on the purpose. The base editor protein comprises one or more nuclear localization signals, which are configured to deliver the base editor protein into the nucleus of a eukaryotic cell. If base editing a prokaryotic genome is performed, the base editor protein does not necessarily need to include a nuclear localization signal. In some cases, the base editor protein may comprise additional domains. The additional domain described above is not limited to any specific domain, as long as it does not interfere with the function of the dead-Cas12a protein and the deaminase. For example, the additional domain may be any amino acid linker. For another example, if the deaminase is a cytosine deaminase, the additional domain may include a uracil glycosylase inhibitor. The structure of the base editor protein described above is well known to researchers and may include any known structure.

[0225] Deaminase #1 - Adenine Deaminase

[0226] The above deaminase may be an adenine deaminase. If the above base editor protein includes an adenine deaminase, it can be referred to as an adenine base editor. The above adenine deaminase can act on adenine in a nucleobase, deaminating it to inosine. This inosine can be converted to guanine during nucleic acid base repair, resulting in the change of A to G. The above adenine deaminase is not limited to a specific type as long as it performs the above function, and any known structure can be utilized. For example, the above adenine deaminase may be tRNA adenosine deaminase (TadA) or a variant of the above TadA. Specifically, the above TadA may be an enzyme derived from Escherichia coli. More diverse examples are described in the 'Possible Embodiments of the Invention' section.

[0227] Deaminase #2 - Cytosine Deaminase

[0228] The above deaminase may be a cytosine deaminase. If the above base editor protein includes a cytosine deaminase, it can be referred to as a cytosine base editor. The above cytosine deaminase can act on cytosine in the nucleobase, deaminating it to uracil. The above uracil can be converted to thymine during nucleic acid base repair, resulting in the conversion of C to T. The above cytosine deaminase is not limited to any type as long as it performs the above function, and any known structure can be utilized. For example, the above cytosine deaminase can be APOBEC1 or recombinant APOBEC1 (rAPOBEC1). More diverse examples are described in the 'Possible Embodiments of the Invention' section.

[0229] nuclear location signal

[0230] The base editor protein may include one or more nuclear localization signals (NLS). These nuclear localization signals play a crucial role in transporting the base editor protein into the nucleus of a eukaryotic cell. If a prokaryotic genome is base-edited, the base editor protein need not necessarily include a nuclear localization signal. The nuclear localization signal is not limited to any type as long as it performs the above function, and any known structure can be utilized. For example, the nuclear localization signal may be SV40. More diverse examples are described in the 'Possible Embodiments of the Invention' section.

[0231] Additional domain #1 - uracil glycosylase inhibitors

[0232] The above base editor protein may include one or more uracil glycosylase inhibitors (UGIs). The above uracil glycosylase inhibitors are domains that inhibit uracil glycosylase. Uracil glycosylase is an enzyme involved in the DNA repair mechanism that recognizes and removes the uracil base in DNA. The above uracil glycosylase inhibitors prevent uracil from being removed by uracil glycosylase when cytosine deaminase converts cytosine in a nucleobase to uracil. In other words, the above uracil glycosylase inhibitors are additional domains that help the cytosine base editor protein operate more efficiently. The type of the above uracil glycosylase inhibitors is not limited as long as they have the aforementioned functions. For example, the above uracil glycosylase inhibitor may be a protein comprising an amino acid sequence of SEQ ID NO: 281 to SEQ ID NO: 282.

[0233] Additional domain #2 - amino acid linker

[0234] The base editor protein may include one or more amino acid linkers. As discussed above, the base editor protein is a type of fusion protein in which domains with multiple functions are linked. When linking these domains, such as the dead-Cas12a protein, a deaminase, and a nuclear localization signal, the N- and C-termini of each domain can be directly linked, or they can be linked via an amino acid linker. The amino acid linker is not limited in type as long as it does not interfere with the activity and function of each domain. For example, the amino acid linker may be a linker having an amino acid sequence selected from SEQ ID NO: 95 to SEQ ID NO: 115.

[0235] programmable guide RNA

[0236] Overview of Programmable Guide RNA

[0237] The base editing system disclosed in this specification includes a programmable guide RNA (or a nucleic acid encoding the same). The programmable guide RNA can interact with a base editor protein to form an RNA-protein complex. The programmable guide RNA 1) interacts with the dead-Cas12a protein among the base editor proteins to form a complex, and 2) guides the RNA-protein complex to a target sequence near a PAM sequence, thereby enabling base pair editing to occur at the intended location. Among these, the portion that interacts with the dead-Cas12a protein to form a complex can be classified as a scaffold, and the portion designed to guide the RNA-protein complex to the target sequence can be classified as a guide domain. Each portion is described in detail below.

[0238] scaffold

[0239] The programmable guide RNA comprises a scaffold. The scaffold can interact with and bind to the Cas12a protein, thereby allowing the programmable guide RNA to form a complex with the Cas12a protein. In the base editing system, the scaffold interacts with the dead-Cas12a protein to form a complex, thereby forming a base editor complex. The scaffold comprises a direct repeat portion of the crRNA. The inventors of this specification have identified and disclose the direct repeat portion that actually binds to and forms a complex with the dead-Cas12a protein disclosed herein. For example, when the dead-Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 264 and SEQ ID NOs: 266 to 272, the direct repeat portion comprises a nucleic acid sequence of SEQ ID NO: 33. For another example, if the dead-Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 265 and SEQ ID NO: 273 to SEQ ID NO: 274, the direct repeat portion comprises the nucleic acid sequence of SEQ ID NO: 35. In addition to the direct repeat portion, the scaffold may further comprise an additional RNA domain that provides structural stability to the programmable guide RNA. For example, the scaffold may further comprise a stem-loop. The specific configuration of the scaffold, etc., is described in the 'Possible Embodiments of the Invention' section.

[0240] Guide domain

[0241] The above programmable guide RNA comprises a guide domain. The guide domain targets a target nucleic acid and guides an RNA-protein complex to bind to the target nucleic acid. In a base editing system, the guide domain targets the target nucleic acid and guides the base editor complex to the target nucleic acid. The guide domain is designed with a sequence related to the target nucleic acid and enables the base editor complex to edit a nucleotide at the intended location. For example, the guide domain is designed with a sequence complementary to all or part of the target strand of the target nucleic acid. In other words, the guide domain is designed with a sequence homologous to all or part of the non-target strand of the target nucleic acid. When the dead-Cas12a protein included in the base editor complex recognizes the PAM sequence of the non-target strand of the target nucleic acid, and the guide domain complementarily binds to the target strand of the target nucleic acid near the PAM sequence, the base editor complex stably binds to the location, and the deaminase included in the base editor complex edits the base pair at the intended location. The specific configuration of the above guide domain is described in the ‘Possible Embodiments of the Invention’ section.

[0242] Structure of programmable guide RNA and additional RNA sequences

[0243] The CRISPR / Cas system described in this specification belongs to the CRISPR / Cas12a system. Therefore, the programmable guide RNA structure is also identical to the known CRISPR / Cas12a system. Specifically, the programmable guide RNA has a structure in which a scaffold and a guide domain are sequentially connected from the 5' end to the 3' end. The 3' end of the scaffold and the 5' end of the guide domain can be directly connected, or can be connected via an arbitrary RNA linker. Furthermore, an additional RNA sequence can be further connected to the 5' end, the 3' end, or both ends of the RNA to which the scaffold and the guide domain are connected. The additional RNA sequence can have a function of imparting stability to the programmable guide RNA or increasing the efficiency with which the guide domain binds to the target sequence.

[0244] Base Editing System #1 - Common

[0245] Overview of the Base Editing System

[0246] This specification discloses a base editing system. The entity that actually edits nucleotides is a base editor complex comprising the base editor protein described above and a programmable guide RNA. However, with the advancement of biotechnology, it is now possible to deliver nucleic acids encoding proteins and RNAs to cells, and express each protein and RNA within the cells to form an RNA-protein complex. Therefore, the term "base editing system" used in this specification encompasses 1) the base editor complex, 2) a vector capable of expressing each component of the base editor complex (a base editor vector), and 3) the base editor composition.

[0247] Base editor complex

[0248] The above base editing system may refer to a base editor complex. The base editor complex refers to an RNA-protein complex formed by combining the base editing protein described above with a programmable guide RNA. The base editor complex actually recognizes the target nucleic acid and edits the nucleotide at the intended target site.

[0249] Base Editor Vector

[0250] The base editing system described above may refer to a base editor vector. The base editor vector refers to a vector capable of expressing each component of the base editor complex. The base editor vector comprises a nucleic acid encoding the base editor protein described above and a nucleic acid encoding the programmable guide RNA. The base editor vector is configured to express the base editor protein and the programmable guide RNA when delivered to a cell. Consequently, the base editor protein expressed within the cell and the programmable guide RNA combine to form the base editor complex, enabling it to function as intended. In addition to the nucleic acids encoding each component, the base editor vector also includes components for proper expression within the cell. The vector can be constructed using any known technology. Specific examples of vector construction are described in detail in the "Possible Embodiments of the Invention" section.

[0251] Base editor composition

[0252] The base editing system may refer to a base editor composition. The base editor composition includes a base editor protein or a nucleic acid encoding the base editor protein; and a programmable guide RNA or a nucleic acid encoding the programmable guide RNA. The base editor composition encompasses the base editor complex, base editor vector, and various other implementation technologies described above. For example, the base editor composition may include mRNA encoding the base editor protein and a programmable guide RNA.

[0253] Base Editing System #2 - Adenine Base Editing System

[0254] Contains adenine deaminase

[0255] In a base editing system, the function of editing base pairs in a target nucleic acid is performed by a deaminase contained in a base editor protein. When the deaminase contained in a base editing system is adenine deaminase, it is called an adenine base editing system.

[0256] Nucleobase editing patterns

[0257] The adenine base editing system edits the adenine base within the target site (also known as the editing window). Specifically, when the adenine base editing system binds to the target nucleic acid via the dead-Cas12a protein and programmable guide RNA, the adenine deaminase deaminizes the adenine base within the target site to inosine. The cellular DNA repair mechanism then corrects the inosine to guanine, and the thymine complementary to the adenine base to cytosine. Consequently, the AT base pair within the target site is edited to a GC base pair.

[0258] Location of the target area

[0259] The target site of the adenine base editing system is described as follows based on the target nucleic acid, the PAM sequence, and the programmable guide RNA contained therein:

[0260] 1) The target nucleic acid is a double-stranded nucleic acid, including a target strand and a non-target strand;

[0261] 2) The above non-target strand contains a PAM that can be recognized by the dead-Cas12a protein among the base editor proteins of the above base editing system;

[0262] 3) A sequence of a certain length downstream of the PAM in the non-target strand, i.e., in the 3' end direction from the PAM, is referred to as a protospacer;

[0263] 4) The portion of the target strand that complementarily binds to the above protospacer is referred to as the target sequence;

[0264] 5) The guide domain of the programmable guide RNA binds complementarily to the target sequence;

[0265] 6) The target site includes nucleobases 3 to 21, where the nucleobase at the 5' end of the protospacer is numbered 1 and numbered in the 3' end direction, and the target sequence of the corresponding target strand.

[0266] 7) Adenine (A) in the non-target strand is changed to guanine (G), and thymine (T) in the corresponding target strand is changed to cytosine (C).

[0267] Base Editing System #3 - Cytosine Base Editing System

[0268] Contains cytosine deaminase

[0269] In a base editing system, the function of editing base pairs in a target nucleic acid is performed by a deaminase contained in a base editor protein. When the deaminase contained in a base editing system is cytosine deaminase, it is called a cytosine base editing system.

[0270] Nucleobase editing patterns

[0271] The cytosine base editing system edits the cytosine base within the target site (also known as the proofreading window). Specifically, when the cytosine base editing system binds to the target nucleic acid via the dead-Cas12a protein and a programmable guide RNA, the cytosine deaminase deamidates the cytosine base within the target site, converting it to uracil. Subsequently, the cellular DNA repair mechanism corrects the uracil to thymine, and the guanine complementary to the cytosine base to adenine. While the uracil base can be removed from the target nucleic acid by intracellular uracil glycosylase, the inclusion of a uracil glycosylase inhibitor within the base editing system can inhibit this process, thereby increasing proofreading efficiency. Consequently, the CG base pair within the target site is edited to a TA base pair.

[0272] Location of the target area

[0273] The target site of the cytosine base editing system is described as follows based on the target nucleic acid, the PAM sequence, and the programmable guide RNA contained therein:

[0274] 1) The target nucleic acid is a double-stranded nucleic acid, including a target strand and a non-target strand;

[0275] 2) The above non-target strand contains a PAM that can be recognized by the dead-Cas12a protein among the base editor proteins of the above base editing system;

[0276] 3) A sequence of a certain length downstream of the PAM in the non-target strand, i.e., in the 3' end direction from the PAM, is referred to as a protospacer;

[0277] 4) The portion of the target strand that complementarily binds to the above protospacer is referred to as the target sequence;

[0278] 5) The guide domain of the programmable guide RNA binds complementarily to the target sequence;

[0279] 6) The target site includes nucleobases 3 to 21, where the nucleobase at the 5' end of the protospacer is numbered 1 and numbered in the 3' end direction, and the target sequence of the corresponding target strand.

[0280] 7) Cytosine (C) in the non-target strand is changed to thymine (T), and the corresponding guanine (G) in the target strand is changed to adenine (A).

[0281] How to Edit Base - Common

[0282] Overview of bass editing methods

[0283] This specification discloses a method for base editing a target nucleic acid using the base editing system described above. The base editing method is performed by delivering the base editing system to a target cell. The base editing method is a method for editing base pairs at a target site, and therefore, the base editing system used therein is designed to be capable of editing base pairs at the target site. The specific relationship between the target site and the configuration of the base editing system is described in more detail below.

[0284] target cells

[0285] In the above base editing method, the type of target cell is not specifically limited. Examples of specific target cells are described in the "Possible Embodiments of the Invention" section.

[0286] Target nucleic acid and target site

[0287] In the above base editing method, the target nucleic acid refers to double-stranded DNA at a specific location. For example, the target nucleic acid may refer to double-stranded DNA contained at a specific location within a specific genome of a target cell. The target nucleic acid includes a target strand and a non-target strand. The target site may be a portion of the target nucleic acid and is a term conceptualizing the range of base pairs edited by the base editing system. The above base editing method is performed to edit specific base pairs contained in the target site.

[0288] Delivering a base editing system

[0289] The above base editing method is performed by delivering the above base editing system to the target cell. The above base editing system can be implemented as a base editor complex, a base editor vector, and a base editor composition, and the specific configuration may vary depending on each type. Therefore, the above base editing method can be implemented using various 'delivery methods' depending on the specific configuration of the base editing system to be delivered. The above delivery method is not particularly limited as long as it can deliver the above base editing system to the cell, and any known technology can be appropriately used. For example, when delivering the base editor complex in the above base editing method, it can be delivered via lipid nanoparticles.

[0290] How to Base Edit - Adenine Base Editing

[0291] Overview of base editing methods using adenine deaminase

[0292] As mentioned above, when the base editing system includes adenine deaminase, it is referred to as an adenine base editing system. Similarly, a base editing method using an adenine base editing system can be referred to as an adenine base editing method. The adenine base editing method is a method of changing an AT base pair contained in a target site to a GC base pair. Specifically, the adenine base editing system deamines the adenine base contained in the target site to change it to inosine, and the inosine is changed to guanine by the cell's DNA repair mechanism, and the thymine complementarily bonded to the adenine base is changed to cytosine. As a result, the AT base pair contained in the target site is edited to a GC base pair.

[0293] Relationship between target site, target nucleic acid, and programmable guide RNA

[0294] 1) The base editing system is designed so that, when delivered to a cell, the base editor complex can come into contact with the target nucleic acid.

[0295] 2) The target nucleic acid is a double-stranded nucleic acid and includes a target strand and a non-target strand.

[0296] 3) The target site is a double-stranded nucleic acid with a length of l bp at a 'specific location' within the target nucleic acid.

[0297] 4) For convenience, the portion of the target site included in the target strand is referred to as the first target site, and the portion included in the non-target strand is referred to as the second target site.

[0298] 5) As a result of performing the base editing method, A in the first target site is changed to G, and T in the corresponding second target site is changed to C.

[0299] 6) The above base editor complex is designed to satisfy the following conditions:

[0300] 7) The base editor protein recognizes a nucleic acid sequence including the (i+3)th base from the i-th base upstream from the 5' end of the first target site as a PAM.

[0301] 8) The guide domain of the programmable guide RNA hybridizes with a second target site and a sequence consisting of the 1st to i-1th nucleic acid downstream from the 3' end of the second target site.

[0302] The specific location and specific length of the target site, the range recognized by the PAM sequence, and the range over which the guide domain hybridizes may vary somewhat depending on the implementation of the base editor. For example, l may be 4 and i may be 8. For another example, l may be 6 and i may be 17. Furthermore, there may be more than one target site. For example, l may be 4 and i may be 8 for the first target site, and l may be 6 and i may be 17 for the second target site. Here, for both target sites, the site recognized by the base editor protein as the PAM and the range over which the guide domain hybridizes are the same.

[0303] Base Editing Methods - Cytosine Base Editing

[0304] Overview of Base Editing Methods Using Cytosine Deaminase and Uracil Glycosylase Inhibitors

[0305] As mentioned above, when a base editing system includes cytosine deaminase, it is referred to as a cytosine base editing system. Similarly, a base editing method using a cytosine base editing system can be referred to as a cytosine base editing method. A cytosine base editing method is a method of changing a CG base pair contained in a target site into a TA base pair. Specifically, the cytosine base editing system deamines the cytosine base contained in the target site to change it into uracil, and the uracil is corrected to thymine and the guanine complementarily bonded to the cytosine base is corrected to adenine by the cell's DNA repair mechanism. Here, there is a possibility that the uracil is removed from the target nucleic acid by intracellular uracil glycosylase. If the base editing system includes a uracil glycosylase inhibitor, this is inhibited, thereby increasing the correction efficiency. As a result, the CG base pair contained in the target site is edited to a TA base pair.

[0306] Relationship between target site, target nucleic acid, and guide RNA

[0307] 1) The base editing system is designed so that, when delivered to a cell, the base editor complex can come into contact with the target nucleic acid.

[0308] 2) The target nucleic acid is a double-stranded nucleic acid and includes a target strand and a non-target strand.

[0309] 3) The target site is a double-stranded nucleic acid with a length of l bp at a 'specific location' within the target nucleic acid.

[0310] 4) For convenience, the portion of the target site included in the target strand is referred to as the first target site, and the portion included in the non-target strand is referred to as the second target site.

[0311] 5) The above base editor complex is designed to satisfy the following conditions:

[0312] 5) As a result of performing the base editing method, C in the first target site is changed to T, and G in the corresponding second target site is changed to A.

[0313] 7) The base editor protein recognizes a nucleic acid sequence including the (i+3)th base from the i-th base upstream from the 5' end of the first target site as a PAM.

[0314] 8) The guide domain of the programmable guide RNA hybridizes with a second target site and a sequence consisting of the 1st to i-1th nucleic acid downstream from the 3' end of the second target site.

[0315] The specific location and specific length of the target site, the range recognized by the PAM sequence, and the range over which the guide domain hybridizes may vary somewhat depending on the implementation of the base editor. For example, l may be 8 and i may be 7. For another example, l may be 6 and i may be 8. Furthermore, there may be more than one target site. For example, l may be 4 and i may be 8 for the first target site, and l may be 6 and i may be 17 for the second target site. Here, for both target sites, the site recognized by the base editor protein as the PAM and the range over which the guide domain hybridizes are the same.

[0316] Possible embodiments of the invention

[0317] Novel Cas12a protein

[0318] Example 1: Novel Cas12a protein

[0319] A Cas12a protein comprising a sequence selected from the following:

[0320] and

[0321] .

[0322] A novel variant of the Cas12a protein (Dead-Form)

[0323] Example 2: Dead Form of Novel Cas12a Protein

[0324] A variant of the Cas12a protein of Example 1, wherein the nucleic acid cleavage activity is inhibited, reduced, inactivated, and / or removed.

[0325] Hereinafter, the variant for the above Cas12a protein is referred to as dead-Cas12a protein.

[0326] Example 3, dead-form mutation location

[0327] In Example 2, the dead-Cas12a protein comprises a mutation selected from the following:

[0328] Based on the amino acid sequence of SEQ ID NO: 1, D828A, E921A, D1145A, or any combination thereof;

[0329] Based on the amino acid sequence of SEQ ID NO: 3, D936A, E1029A, D1254A, or any combination thereof.

[0330] Example 4, dead-form mutant sequence

[0331] In Example 3, the dead-Cas12a protein comprises an amino acid sequence selected from the following:

[0332] Sequence number 264 to sequence number 265.

[0333] Example 5, PAM

[0334] In any one of Examples 2 to 4, the dead-Cas12a protein recognizes a PAM sequence of 5'-TTTV-3' or 5'-TTTN-3';

[0335] Here, V is any one of A, G, or C, and N is any one of A, T, C, or G.

[0336] Novel Cas12a protein variants

[0337] Example 6, variants of novel Cas12a proteins

[0338] A variant of a Cas12a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, or deleted, based on the dead-Cas12a protein of any one of Examples 2 to 5.

[0339] Example 7, PAM binding site mutation

[0340] In Example 6, the variant of the Cas12a protein is characterized by an improved ability to bind to a protospacer adjacent motif (PAM) compared to the reference dead-Cas12a protein.

[0341] Example 8, R-loop mutation

[0342] In any one of Examples 6 to 7, the variant of the Cas12a protein is characterized in that the R-loop duration upon protein-target nucleic acid binding is longer compared to the reference dead-Cas12a protein.

[0343] Example 9, Ultra Nuclease variant #1

[0344] In any one of Examples 6 to 8, the variant of the Cas12a protein is characterized by having increased DNA binding ability compared to the reference dead-Cas12a protein.

[0345] Example 10, Ultra Nuclease Variant #2

[0346] In any one of Examples 6 to 9, the variant of the Cas12a protein is characterized by increased stability of the RNA-protein complex compared to the reference dead-Cas12a protein.

[0347] Example 11, variant location

[0348] In Example 7, the variant of the Cas12a protein comprises a mutation selected from the following:

[0349] Based on the amino acid sequence of SEQ ID NO: 1, D828A, E921A, D1145A, or any combination thereof, and E155R, G531R, K537R, S1137A, or any combination thereof;

[0350] Based on the amino acid sequence of SEQ ID NO: 1, D828A, E921A, D1145A, or any combination thereof, and W354A;

[0351] Based on the amino acid sequence of SEQ ID NO: 1, D828A, E921A, D1145A, or any combination thereof, and N526R, E791L, or any combination thereof;

[0352] Based on the amino acid sequence of SEQ ID NO: 1, D828A, E921A, D1145A, or any combination thereof, and E155R, G531R, K537R, S1137A, N526R, E791L, N526R, E791L, or any combination thereof;

[0353] Based on the amino acid sequence of SEQ ID NO: 3, D936A, E1029A, D1254A, or any combination thereof, and Q190R, S579R, Y585R, or any combination thereof.

[0354] Example 12, variant sequence

[0355] In any one of Examples 6 to 11, the variant of the Cas12a protein comprises an amino acid sequence selected from the following:

[0356] ; ; ; ; ; ; ; ; and .

[0357] Example 13, PAM

[0358] In any one of Examples 6 to 12, the dead-Cas12a protein recognizes a PAM sequence of 5'-TTTV-3' or 5'-TTTN-3';

[0359] Here, V is any one of A, G, or C, and N is any one of A, T, C, or G.

[0360] nuclear localization signal

[0361] Example 14, nuclear localization signal

[0362] Nuclear Localization Signal (NLS).

[0363] Example 15, nuclear localization signal, sequence

[0364] In Example 14, the nuclear localization signal comprises an amino acid sequence selected from the following, or any combination of one or more amino acid sequences selected from the following:

[0365] PKKKRKV (SEQ ID NO: 67); KRPAATKKAGQAKKKK (SEQ ID NO: 68); PKKKRKVKRPAATKKAGQAKKKK (SEQ ID NO: 69); KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70); PKKKRKVPKKKRKVPKKKRKV (SEQ ID NO: 71); PAAKRVKLD (SEQ ID NO: 72); RQRRNELKRSP (SEQ ID NO: 73); NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 74); RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 75); VSRKRPRP (SEQ ID NO: 76); PPKKARED (SEQ ID NO: 77); PQPKKKPL (SEQ ID NO: 78); SALIKKKKKMAP (SEQ ID NO: 79); DRLRR (SEQ ID NO: 80); PKQKKRK (SEQ ID NO: 81); RKLKKKIKKL (SEQ ID NO: 82); REKKKFLKRR (SEQ ID NO: 83); KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 84); RKCLQAGMNLEARKTKK (SEQ ID NO: 85); PKTKRKV (SEQ ID NO: 86); PAAKTKRKVLD (SEQ ID NO: 87); PAAKTKRLD (SEQ ID NO: 88); KRXXXXXXXXXPKTKRKV (SEQ ID NO: 89); KRXXXXXXXXXXKKKKLD (SEQ ID NO: 90); AAAKKKLD (SEQ ID NO: 91); PAAKKKKLD (SEQ ID NO: 92); PAAKKKK (SEQ ID NO: 93); and PAAKRVKLD (SEQ ID NO: 94).

[0366] Example 16, Nuclear localization signal, meaning of combinations

[0367] In Example 15, 'any combination of one or more amino acid sequences' means that one or more amino acid sequences are directly connected or connected via a linker selected from the following:

[0368] GGGGSGGGGSGGGGS (SEQ ID NO: 95); GGGGSGGGGS (SEQ ID NO: 96); GGGGS (SEQ ID NO: 97); GGGGGG (SEQ ID NO: 98); GGGGGGGG (SEQ ID NO: 99); EAAAK (SEQ ID NO: 100); EAAAKEAAAK (SEQ ID NO: 101); EAAAKEAAAKEAAAK (SEQ ID NO: 102); AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 103); PAPAP (SEQ ID NO: 104); AEAAAKEAAAKA (SEQ ID NO: 105); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 106); VSQTSKLTRAETVFPDV (SEQ ID NO: 107); PLGLWA (SEQ ID NO: 108); RVLAEA (SEQ ID NO: 109); EDVVCCSMSY (SEQ ID NO: 110); GGIEGRGS (SEQ ID NO: 111); TRHRQPRQWE (SEQ ID NO: 112); AGNRVRRSVG (SEQ ID NO: 113); RRRRRRRRR (SEQ ID NO: 114); and GFLG (SEQ ID NO: 115).

[0369] amino acid linker

[0370] Example 17, amino acid linker

[0371] Amino acid linker.

[0372] Example 18, amino acid linker sequence

[0373] In Example 17, the amino acid linker comprises a sequence selected from the following:

[0374] Sequence number 95 to sequence number 115; and

[0375] Sequence number 67 to sequence number 94.

[0376] Base Editing Domain #1 - Adenine Deaminase

[0377] Example 19, adenine deaminase

[0378] Adenine deaminase.

[0379] Example 20, TadA and its variants

[0380] In Example 19, the adenine deaminase is tRNA adenosine deaminase (TadA) derived from Escherichia coli or a variant of the TadA.

[0381] Example 21, specific sequence

[0382] In any one of Examples 19 to 20, the adenine deaminase comprises an amino acid sequence selected from the following:

[0383] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 275); MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 276); and SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 277).

[0384] Base Editing Domain #2 - Cytosine Deaminase

[0385] Example 22, cytosine deaminase

[0386] Cytosine deaminase.

[0387] Example 23, cytosine deaminase type

[0388] In Example 22, the cytosine deaminase is selected from the following:

[0389] activation-induced cytidine deaminase (AID); APOBEC3G; APOBEC1; rAPOBEC1; APOBEC3A; APOBEC3B; CDA; Anc689 APOBEC; and PmCDA1.

[0390] Example 24, specific sequence

[0391] In any one of Examples 22 to 23, the cytosine deaminase comprises an amino acid sequence selected from the following:

[0392] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIA RLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 278); SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLYEIKWGTSHKIWRHSSKNTTKHVEVNFIEKFTSERHFCPSTSCSITWFLSWSPCGECSKAITEFLSQHPNVTLVIYVA RLYHHMDQQNRQGLRDLVNSGVTIQIMTAPEYDYCWRNFVNYPPGKEAHWPRYPPLWMKLYALELHAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 279); and SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIA RLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 280).

[0393] uracil glycosylase inhibitors

[0394] Example 25, uracil glycosylase inhibitor

[0395] Uracil Glycosylase Inhibitor (UGI).

[0396] Example 26, UGI sequence

[0397] In Example 25, the uracil glycosylase inhibitor comprises an amino acid sequence selected from the following:

[0398] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 281); and TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 282).

[0399] Base Editor Protein #1 - Adenine Base Editor

[0400] Example 27, ABE protein

[0401] Adenine Base Editor (ABE) proteins containing:

[0402] A dead-Cas12a protein of any one of Examples 2 to 5, or a variant of the dead-Cas12a protein of any one of Examples 6 to 13;

[0403] A nuclear location signal of any one of Examples 14 to 16; and

[0404] An adenine deaminase according to any one of Examples 19 to 21.

[0405] Example 28, ABE protein structure

[0406] In Example 27,

[0407] The adenine base editor protein is a fusion protein comprising a dead-Cas12a protein or a variant of the dead-Cas12a protein; one or more nuclear localization signals; and one or more adenine deaminase enzymes, wherein each of the components is directly linked or linked via an amino acid linker.

[0408] Example 29, ABE protein structure #1

[0409] In Examples 27 to 28, the adenine base editor has the following structure:

[0410] [NLS1] - [Linker1] - [AD] - [Linker2] - [dead-Cas12a] - [Linker3] - [NLS2]

[0411] Here, the NLS1 is a nuclear localization signal of any one of Examples 14 to 16, or is absent,

[0412] The above Linker1 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0413] The above AD is an adenine deaminase of any one of Examples 19 to 21,

[0414] The above Linker2 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0415] The dead-Cas12a is a variant of the dead Cas12a protein of any one of Examples 2 to 5, or the dead-Cas12a protein of any one of Examples 6 to 13,

[0416] The above Linker3 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0417] The above NLS2 is a nuclear localization signal of any one of Examples 14 to 16, or is absent.

[0418] Example 30, ABE Protein Structure #2

[0419] In Examples 27 to 29, the adenine base editor has the following structure:

[0420] [NLS1] - [Linker1] - [dead-Cas12a] - [Linker2] - [AD] - [Linker3] - [NLS3]

[0421] Here, the NLS1 is a nuclear localization signal of any one of Examples 14 to 16, or is absent,

[0422] The above Linker1 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0423] The above AD is an adenine deaminase of any one of Examples 19 to 21,

[0424] The above Linker2 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0425] The dead-Cas12a is a variant of the dead Cas12a protein of any one of Examples 2 to 5, or the dead-Cas12a protein of any one of Examples 6 to 13,

[0426] The above Linker3 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0427] The above NLS2 is a nuclear localization signal of any one of Examples 14 to 16, or is absent.

[0428] Example 31, ABE protein sequence

[0429] In any one of Examples 27 to 30, the adenine base editor protein comprises a sequence selected from the following:

[0430] ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

[0431] Base Editor Protein #2 - Cytosine Base Editor

[0432] Example 32, CBE protein

[0433] Cytidine Base Editor (CBE) proteins, including:

[0434] A dead-Cas12a protein of any one of Examples 2 to 5, or a variant of the dead-Cas12a protein of any one of Examples 6 to 13;

[0435] A nuclear location signal of any one of Examples 14 to 16;

[0436] A cytosine deamination enzyme of any one of Examples 22 to 24; and

[0437] A uracil glycosylase inhibitor according to any one of Examples 25 to 26.

[0438] Example 33, CBE protein structure

[0439] In Example 32,

[0440] The adenine base editor protein is a fusion protein comprising a dead-Cas12a protein or a variant of the dead-Cas12a protein; one or more nuclear localization signals; one or more cytosine deaminase enzymes; and one or more uracil glycosylase inhibitors, wherein each of the components is directly linked or linked via an amino acid linker.

[0441] Example 34, CBE protein structure #1

[0442] In any one of Examples 32 to 33, the cytosine base editor protein has the following structure:

[0443] [NLS1] - [Linker1] - [CD] - [Linker2] - [dead-Cas12a] - [Linker3] - [UGI] - [Linker4] - [NLS2]

[0444] Here, the NLS1 is a nuclear localization signal of any one of Examples 14 to 16, or is absent,

[0445] The above Linker1 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0446] The above CD is an adenine deaminase of any one of Examples 22 to 24,

[0447] The above Linker2 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0448] The dead-Cas12a is a variant of the dead Cas12a protein of any one of Examples 2 to 5, or the dead-Cas12a protein of any one of Examples 6 to 13,

[0449] The above Linker3 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0450] The above UGI is a uracil glycosylase inhibitor of any one of Examples 25 to 26,

[0451] The above Linker4 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0452] The above NLS2 is a nuclear localization signal of any one of Examples 14 to 16, or is absent.

[0453] Example 35, CBE protein structure #2

[0454] In any one of Examples 32 to 34, the cytosine base editor protein has the following structure:

[0455] [NLS1] - [Linker1] - [UGI] - [Linker2] - [CD] - [Linker3] - [dead-Cas12a] - [Linker4] - [NLS2]

[0456] Here, the NLS1 is a nuclear localization signal of any one of Examples 14 to 16, or is absent,

[0457] The above Linker1 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0458] The above CD is an adenine deaminase of any one of Examples 22 to 24,

[0459] The above Linker2 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0460] The dead-Cas12a is a variant of the dead Cas12a protein of any one of Examples 2 to 5, or the dead-Cas12a protein of any one of Examples 6 to 13,

[0461] The above Linker3 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0462] The above UGI is a uracil glycosylase inhibitor of any one of Examples 25 to 26,

[0463] The above Linker4 is an amino acid linker of any one of Examples 17 to 18, or is absent,

[0464] The above NLS2 is a nuclear localization signal of any one of Examples 14 to 16, or is absent.

[0465] Example 36, CBE protein sequence

[0466] In any one of Examples 32 to 35, the cytosine base editor protein comprises a sequence selected from the following:

[0467] ; ; and .

[0468] programmable guide RNA

[0469] Example 37, Programmable Guide RNA

[0470] Programmable guide RNAs containing:

[0471] scaffold,

[0472] The scaffold can interact with any one of the Cas12a proteins of Example 1, or a variant of any one of the Cas12a proteins of Examples 6 to 13 to form a complex; and

[0473] Guide domain,

[0474] Here, the guide domain is designed to target a target nucleic acid.

[0475] Example 38, Scaffold

[0476] In Example 37, the scaffold comprises a direct repeat (DR),

[0477] The above direct repeat portion comprises a nucleic acid sequence selected from among TAATTTCTACTAAGTGTAGAT (SEQ ID NO: 33), AAAATTTCTACTCTTGTAGAT (SEQ ID NO: 34), and TAAATTTCTACTGTTGTAGAT (SEQ ID NO: 35).

[0478] Example 39, with stem loop

[0479] In Example 38, the scaffold further comprises a stem-loop,

[0480] The above stem-loop comprises the nucleic acid sequence GTTTCAAAGATTAAAT (SEQ ID NO: 49),

[0481] The above stem-loop is connected to the 5' end of the above direct repeat.

[0482] Example 40, Meaning of Targeting a Target Nucleic Acid

[0483] In any one of Examples 37 to 39, the description that 'the guide domain targets the target nucleic acid' has the following meaning:

[0484] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0485] The above guide domain and the above target nucleic acid are in a relationship selected from the following:

[0486] The nucleic acid sequence of the above guide domain is a sequence complementary to the nucleic acid sequence of all or part of the target strand;

[0487] The guide domain hybridizes or complementarily binds to all or part of the target strand;

[0488] The nucleic acid sequence of the above guide domain is a sequence equivalent to the nucleic acid sequence of all or part of the above non-target strand; or

[0489] Any combination of the above relationships.

[0490] Example 41, length of guide domain

[0491] In any one of Examples 37 to 40, the guide domain has a length selected from the following:

[0492] 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt; or

[0493] A length within the two numerical ranges described above, for example, 16nt to 23nt.

[0494] Example 42, including mismatches

[0495] In Example 40, the guide domain hybridizes or complementarily binds to all or part of the target strand,

[0496] The nucleic acid sequence of the above guide domain and the nucleic acid sequence of the portion of the target strand that hybridizes or complementarily binds to the guide domain are in a relationship selected from the following:

[0497] having nucleic acid sequences that are completely complementary to each other;

[0498] The remaining nucleic acid sequences are complementary, excluding one, two, three, four, or five mismatches;

[0499] There is at least one RNA buldge portion in the nucleic acid sequence of the guide domain that does not hybridize with the target strand;

[0500] There is at least one DNA bulge portion in the nucleic acid sequence of the target strand that is not hybridized with the guide domain; or

[0501] Any combination of the above relationships.

[0502] Example 43, Guide Domain Sequence Homology

[0503] In Example 40, the nucleic acid sequence of the guide domain comprises a nucleic acid sequence selected from the following:

[0504] A nucleic acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to all or part of the nucleic acid sequence of the target strand; or

[0505] A sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous, identical, or equivalent to all or part of the nucleic acid sequence of the non-target strand.

[0506] Example 44, Guide RNA Structure

[0507] In any one of Examples 37 to 43,

[0508] The above programmable guide RNA may have a structure in which a scaffold and a guide domain are sequentially connected in the direction from the 5' end to the 3' end.

[0509] Here, the 3' end of the scaffold and the 5' end of the guide domain are connected directly or via an arbitrary RNA sequence.

[0510] Example 45, structural formula limitation

[0511] In any one of Examples 37 to 44, the programmable guide RNA has the following structure:

[0512] 5' - [scaffold] - [RNA linker] - [guide domain] - 3'

[0513] Here, the RNA linker is an arbitrary RNA sequence or is absent,

[0514] In the absence of the above RNA linker, the above structure is as follows:

[0515] 5' - [Scaffold] - [Guide Domain] - 3'.

[0516] Example 46, including additional domains

[0517] In Example 45, the programmable guide RNA further comprises one or more additional domains,

[0518] The above additional domain is RNA and is linked to the 5' end, the 3' end, or both the 5' end and the 3' end of the programmable guide RNA.

[0519] CRISPR / Cas12a complex (Dead-Form)

[0520] Example 47, CRISPR / Cas12a complex

[0521] CRISPR / Cas12a complex containing:

[0522] A dead-Cas12a protein of any one of Examples 2 to 5, or a variant of the dead-Cas12a protein of any one of Examples 6 to 13 (hereinafter collectively referred to as dead-Cas12a protein); and

[0523] The programmable guide RNA of any one of Examples 37 to 46;

[0524] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas complex,

[0525] The above CRISPR / Cas12a complex has a function of recognizing and binding to a target nucleic acid targeted by the guide domain of the programmable guide RNA.

[0526] Hereinafter, the CRISPR / Cas complex is also referred to as an RNA-protein complex, or ribonucleoprotein (RNP).

[0527] Example 48, dead-Cas12a protein and scaffold combination

[0528] The scaffold of the dead-Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0529] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0530] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0531] CRISPR / Cas12a vector (Dead-Form)

[0532] Example 49, CRISPR / Cas12a vector

[0533] CRISPR / Cas12a component expression vectors containing:

[0534] A nucleic acid encoding a dead-Cas12a protein of any one of Examples 2 to 5, or a variant of a dead-Cas12a protein of any one of Examples 6 to 13 (hereinafter collectively referred to as dead-Cas12a protein); and

[0535] A nucleic acid encoding a programmable guide RNA of any one of Examples 37 to 46;

[0536] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas complex,

[0537] The above CRISPR / Cas12a complex has a function of recognizing and binding to a target nucleic acid targeted by the guide domain of the programmable guide RNA.

[0538] Example 50, including additional components

[0539] In Example 49, the component expression vector further comprises one or more selected from the following:

[0540] Promoter; enhancer; intron; polyadenylation signal; Kozak consensus sequence; internal ribosome entry site (IRES); splice acceptor; 2A sequence; and replication origin.

[0541] Example 51, Promoter Addition

[0542] In Example 50, the promoter is at least one selected from the following:

[0543] SV40 early promoter; mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE); rous sarcoma virus (RSV) promoter; human U6 small nuclear promoter (U6) (Miyagishi et al.; Nature Biotechnology 20; 497-500 (2002)); enhanced U6 promoter (e.g., Xia et al.; Nucleic Acids Res. 2003 Sep 1; 31(17)); human H1 promoter (H1); and 7SK.

[0544] Example 52, adding a replication origin

[0545] In any one of Examples 50 to 51, the replication origin is one or more selected from the following:

[0546] f1 origin of replication; SV40 origin of replication; pMB1 origin of replication; adeno origin of replication; AAV origin of replication; and BBV origin of replication.

[0547] Example 53, vector types

[0548] In any one of Examples 49 to 52, the vector is a viral vector, a non-viral vector, or any combination thereof.

[0549] Example 54, viral vector

[0550] In Example 53, the vector is a viral vector selected from the following:

[0551] Retrovirus; lentivirus; adenovirus; adeno-associated virus; vaccinia virus; poxvirus; herpes simplex virus; and any combination thereof.

[0552] Example 55, non-viral vector

[0553] In Example 53, the vector is a non-viral vector selected from the following:

[0554] Plasmid; phage; naked DNA; DNA complex; PCR amplicon; mRNA; and any combination thereof.

[0555] Example 56, plasmid

[0556] In Example 55, the plasmid is one selected from the following:

[0557] pcDNA series; pS456; p326; pACYC177; ColE1; pKT230; pME290; pBR322; pUC8 / 9; pUC6; pBD9; pHC79; pIJ61; pLAFR1; pHV14; pGEX series; pET series; pUC19 and any combination thereof.

[0558] Example 57, Phage

[0559] In Example 55, the phage is one selected from the following:

[0560] λgt4λB; λ-Charon; λΔz1; M13; and any combination thereof.

[0561] Example 58, single vector

[0562] In any one of Examples 49 to 57, the vector is a single vector composed of one molecule.

[0563] Example 59, multiple vectors

[0564] In any one of Examples 49 to 58, the vector is a plurality of vectors composed of two or more molecules.

[0565] Example 60, each component contained in a different vector

[0566] In Example 59, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are contained in vectors of different molecules.

[0567] Example 61, DNA and RNA embodiment

[0568] In any one of Examples 49 to 60, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are each independently DNA or RNA.

[0569] Example 62, Cas12a protein and scaffold combination

[0570] In any one of Examples 49 to 61, the scaffold of the dead-Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0571] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0572] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 35.

[0573] CRISPR / Cas12a composition (Dead-Form)

[0574] Example 63, CRISPR / Cas12a composition

[0575] A CRISPR / Cas12a composition comprising:

[0576] A dead-Cas12a protein of any one of Examples 2 to 5, a variant of a dead-Cas12a protein of any one of Examples 6 to 13 (hereinafter collectively referred to as dead-Cas12a protein), or a nucleic acid encoding the dead-Cas12a protein; and

[0577] A programmable guide RNA of any one of Examples 37 to 46, or a nucleic acid encoding the guide RNA;

[0578] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0579] The above CRISPR / Cas12a complex has a function of recognizing and binding to a target nucleic acid targeted by the guide domain of the programmable guide RNA.

[0580] Example 64, including a CRISPR / Cas12a complex

[0581] In Example 63, the CRISPR / Cas12a composition comprises a CRISPR / Cas12a complex of any one of Examples 47 to 48.

[0582] Example 65, including CRISPR / Cas12a vector

[0583] In Example 63, the CRISPR / Cas12a composition comprises an expression vector of a CRISPR / Cas12a system component of any one of Examples 49 to 62.

[0584] Example 66, including mRNA and guide RNA

[0585] In Example 63, the CRISPR / Cas12a composition comprises an mRNA encoding the Cas12a protein and the programmable guide RNA.

[0586] Example 67, Cas12a protein and scaffold combination

[0587] In any one of Examples 63 to 66, the scaffold of the dead-Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0588] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0589] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0590] Base editor complex

[0591] Example 68, Base Editor Complex

[0592] Base editor complex including:

[0593] An adenine base editor protein of any one of Examples 27 to 31, or a cytosine base editor protein of any one of Examples 32 to 36; and

[0594] The programmable guide RNA of any one of Examples 37 to 46;

[0595] Here, the base editor protein and the programmable guide RNA combine to form a complex,

[0596] The above base editor complex has a function of changing the base pairing of a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0597] Example 69, dead-Cas12a protein and scaffold assembly

[0598] In Example 68, the dead-Cas12a protein included in the base editor protein and the scaffold of the programmable guide RNA are a combination selected from the following:

[0599] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0600] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0601] Example 70, Adenine Base Editor Complex

[0602] In any one of Examples 68 to 69,

[0603] The above base editor complex comprises an adenine base editor protein of any one of Examples 27 to 31,

[0604] The above base editor complex has a function of changing the A and T base pairs of the target nucleic acid targeted by the guide domain of the programmable guide RNA, or an adjacent nucleic acid, to C and G base pairs.

[0605] Example 71, Adenine Base Editor Complex, Correction Window

[0606] In Example 70,

[0607] The target nucleic acid targeted by the guide domain of the programmable guide RNA of the above base editor complex is double-stranded DNA,

[0608] The target nucleic acid comprises a target strand and a non-target strand,

[0609] The non-target strand comprises a protospacer adjacent motif (PAM) and a protospacer,

[0610] The target strand comprises a target site that complementarily binds to the protospacer,

[0611] The base editor protein recognizes the PAM, and the guide domain of the programmable guide RNA hybridizes with the target site,

[0612] The above base editor complex can change adenine contained in the 7th to 14th nucleobase, when the 5'-end nucleobase of the protospacer is referred to as the 1st nucleobase, and thymine of the target strand complementarily bound thereto to cytosine and guanine, respectively.

[0613] Example 72, cytosine base editor complex

[0614] In any one of Examples 68 to 69,

[0615] The above base editor complex comprises a cytosine base editor protein of any one of Examples 32 to 36,

[0616] The above base editor complex has a function of changing the C and G base pairs of the target nucleic acid targeted by the guide domain of the above programmable guide RNA, or an adjacent nucleic acid, to T and A base pairs.

[0617] Example 73, cytosine base editor complex, proofreading window

[0618] In Example 72,

[0619] The target nucleic acid targeted by the guide domain of the programmable guide RNA of the above base editor complex is double-stranded DNA,

[0620] The target nucleic acid comprises a target strand and a non-target strand,

[0621] The non-target strand comprises a protospacer adjacent motif (PAM) and a protospacer,

[0622] The target strand comprises a target site that complementarily binds to the protospacer,

[0623] The base editor protein recognizes the PAM, and the guide domain of the programmable guide RNA hybridizes with the target site,

[0624] The above base editor complex can change the cytosine contained in the Nth to Mth nucleobase and the guanine of the target strand complementarily bound thereto, when the 5'-end nucleobase of the protospacer is referred to as the 1st, to thymine and adenine, respectively.

[0625] Base Editor Component Expression Vector

[0626] Example 74, Base Editor Component Expression Vector

[0627] Base editor component expression vector, including:

[0628] A nucleic acid encoding an adenine base editor protein of any one of Examples 27 to 31, or a nucleic acid encoding a cytosine base editor protein of any one of Examples 32 to 36; and

[0629] A nucleic acid encoding a programmable guide RNA of any one of Examples 37 to 46;

[0630] Here, the base editor protein and the programmable guide RNA can be combined to form a base editor complex of any one of Examples 68 to 73,

[0631] The above base editor complex has a function of changing the base pairing of a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0632] Example 75, including additional components

[0633] In Example 74, the component expression vector further comprises one or more selected from the following:

[0634] Promoter; enhancer; intron; polyadenylation signal; Kozak consensus sequence; internal ribosome entry site (IRES); splice acceptor; 2A sequence; and replication origin.

[0635] Example 76, Promoter Addition

[0636] In Example 75, the promoter is one or more selected from the following:

[0637] SV40 early promoter; mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE); rous sarcoma virus (RSV) promoter; human U6 small nuclear promoter (U6) (Miyagishi et al.; Nature Biotechnology 20; 497-500 (2002)); enhanced U6 promoter (e.g., Xia et al.; Nucleic Acids Res. 2003 Sep 1; 31(17)); human H1 promoter (H1); and 7SK.

[0638] Example 77, adding a replication origin

[0639] In any one of Examples 75 to 76, the replication origin is one or more selected from the following:

[0640] f1 origin of replication; SV40 origin of replication; pMB1 origin of replication; adeno origin of replication; AAV origin of replication; and BBV origin of replication.

[0641] Example 78, vector types

[0642] In any one of Examples 74 to 77, the vector is a viral vector, a non-viral vector, or any combination thereof.

[0643] Example 79, viral vector

[0644] In Example 78, the vector is a viral vector selected from the following:

[0645] Retrovirus; lentivirus; adenovirus; adeno-associated virus; vaccinia virus; poxvirus; herpes simplex virus; and any combination thereof.

[0646] Example 80, non-viral vector

[0647] In Example 78, the vector is a non-viral vector selected from the following:

[0648] Plasmid; phage; naked DNA; DNA complex; PCR amplicon; mRNA; and any combination thereof.

[0649] Example 81, plasmid

[0650] In Example 80, the plasmid is one selected from the following:

[0651] pcDNA series; pS456; p326; pACYC177; ColE1; pKT230; pME290; pBR322; pUC8 / 9; pUC6; pBD9; pHC79; pIJ61; pLAFR1; pHV14; pGEX series; pET series; pUC19 and any combination thereof.

[0652] Example 82, Phage

[0653] In Example 80, the phage is one selected from the following:

[0654] λgt4λB; λ-Charon; λΔz1; M13; and any combination thereof.

[0655] Example 83, single vector

[0656] In any one of Examples 74 to 82, the vector is a single vector composed of one molecule.

[0657] Example 84, multiple vectors

[0658] In any one of Examples 74 to 83, the vector is a plurality of vectors composed of two or more molecules.

[0659] Example 85, each component contained in a different vector

[0660] In Example 84, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are contained in vectors of different molecules.

[0661] Example 86, DNA and RNA embodiment

[0662] In any one of Examples 74 to 85, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are each independently DNA or RNA.

[0663] Example 87, Cas12a protein and scaffold combination

[0664] In any one of Examples 74 to 86, the scaffold of the dead-Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0665] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0666] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0667] Base editor composition

[0668] Example 88, base editor composition

[0669] Base editor composition comprising:

[0670] An adenine base editor protein of any one of Examples 27 to 31 or a nucleic acid encoding the adenine base editor protein, or

[0671] A cytosine base editor protein of any one of Examples 32 to 36 or a nucleic acid encoding the cytosine base editor protein; and

[0672] The programmable guide RNA of any one of Examples 37 to 46;

[0673] Here, the base editor protein and the programmable guide RNA can be combined to form a base editor complex,

[0674] The above base editor complex has a function of changing the base pairing of a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0675] Example 89, including a base editor complex

[0676] In Example 88, the base editor composition comprises a base editor complex of any one of Examples 68 to 73.

[0677] Example 90, including base editor vector

[0678] In Example 88, the base editor composition comprises a base editor complex component expression vector of any one of Examples 74 to 87.

[0679] Example 91, including mRNA and guide RNA

[0680] In Example 88, the base editor composition comprises an mRNA encoding the base editor protein and the programmable guide RNA.

[0681] Example 92, dead-Cas12a protein and scaffold combination

[0682] In any one of Examples 88 to 91, the scaffold of the dead-Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0683] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0684] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0685] How to Edit Base - Common

[0686] Example 93, Base Editing Method

[0687] A method of changing a specific base pair of a target nucleic acid within a cell to a different base pair.

[0688] The above method includes:

[0689] Delivering the base editor composition of any one of Examples 88 to 92 to the cell;

[0690] Here, a base editor protein included in or derived from the base editor composition and a programmable guide RNA included in or derived from the base editor composition are combined to form a base editor complex,

[0691] The base editor complex contacts the target nucleic acid and changes the specific base pair into another base pair.

[0692] Example 94, Common Base Editing Method #1

[0693] A method for changing the base pairing of a target site contained in a target nucleic acid within a cell,

[0694] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0695] The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto,

[0696] The above method includes:

[0697] Delivering the base editor composition of any one of Examples 88 to 92 to the cell;

[0698] Here, a base editor protein included in or derived from the base editor composition and a programmable guide RNA included in or derived from the base editor composition are combined to form a base editor complex,

[0699] The above base editor complex recognizes the i-th nucleobase to the (i+3)-th nucleobase upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM),

[0700] The guide domain of the above programmable guide RNA hybridizes with a second target site in the target strand and a sequence consisting of the first to (i-1)th nucleic acid downstream from the 3' end of the second target site,

[0701] Here, i is an integer,

[0702] Here, the scaffold of the dead-Cas12a protein and the programmable guide RNA included in the base editor protein is a combination selected from the following:

[0703] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0704] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0705] Example 95, Alternative Expression for Transmission

[0706] In Example 94, the phrase 'deliver to cells' is replaced with the following expression:

[0707] Introduce into a cell; administer into a cell; inject into a cell; transfect into a cell; transduct into a cell; or any combination of the above expressions.

[0708] Example 96, Base Editing Method Joint #2

[0709] A method for changing the base pairing of a target site contained in a target nucleic acid within a cell,

[0710] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0711] The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto,

[0712] The above method includes:

[0713] Contacting or inducing contact between the base editor complex of any one of Examples 68 to 73 and the target nucleic acid;

[0714] The above base editor complex recognizes the i-th nucleobase to the (i+3)-th nucleobase upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM),

[0715] The guide domain of the above programmable guide RNA hybridizes with a second target site in the target strand and a sequence consisting of the first to (i-1)th nucleic acid downstream from the 3' end of the second target site,

[0716] Here, i is an integer,

[0717] Here, the scaffold of the dead-Cas12a protein and the programmable guide RNA included in the base editor protein is a combination selected from the following:

[0718] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0719] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0720] Example 97, PAM

[0721] In any one of Examples 93 to 96, the protospacer adjacent motif is 5'-TTTV-3' or 5'-TTTN-3',

[0722] Here, V is A, C, or G, and N is A, C, T, or G.

[0723] Example 98, Base Editing Method Joint #3

[0724] A method for changing the base pairing of a target site contained in a target nucleic acid within a cell,

[0725] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0726] The above method includes:

[0727] Delivering the base editor composition of any one of Examples 88 to 92 to the cell;

[0728] Here, a base editor protein included in or derived from the base editor composition and a programmable guide RNA included in or derived from the base editor composition are combined to form a base editor complex,

[0729] The non-target strand of the target nucleic acid comprises a protospacer adjacent motif (PAM) and a protospacer,

[0730] The target strand of the target nucleic acid comprises a target sequence that complementarily binds to the protospacer,

[0731] When the 5'-end nucleobase of the above protospacer is considered as the 1st, the 1st nucleobase among the i-th to (i + j)-th nucleobases is changed to the 2nd base,

[0732] The third base complementarily binding to the first base in the above target sequence is changed to the fourth base,

[0733] Here, i and j are integers,

[0734] Here, the scaffold of the dead-Cas12a protein and the programmable guide RNA included in the base editor protein is a combination selected from the following:

[0735] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0736] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0737] Example 99, including additional parts

[0738] In Example 98, additionally, when the 5'-end nucleobase of the protospacer is considered as the 1st, the 1st base among the kth to (k + 1)th nucleobase is changed to the 2nd base,

[0739] The third base that complementarily binds to the first base in the above target sequence is changed to the fourth base,

[0740] Here, k and l are integers.

[0741] Example 100, Base Editing Method Joint #4

[0742] A method for changing the base pairing of a target site contained in a target nucleic acid within a cell,

[0743] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0744] The above method includes:

[0745] Contacting or inducing contact between the base editor complex of any one of Examples 68 to 73 and the target nucleic acid;

[0746] The non-target strand of the target nucleic acid comprises a protospacer adjacent motif (PAM) and a protospacer,

[0747] The target strand of the target nucleic acid comprises a target sequence that complementarily binds to the protospacer,

[0748] When the 5'-end nucleobase of the above protospacer is considered as the 1st, the 1st nucleobase among the i-th to (i + j)-th nucleobases is changed to the 2nd base,

[0749] The third base complementarily binding to the first base in the above target sequence is changed to the fourth base,

[0750] Here, i and j are integers,

[0751] Here, the scaffold of the dead-Cas12a protein and the programmable guide RNA included in the base editor protein is a combination selected from the following:

[0752] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat sequence of SEQ ID NO: 33; or

[0753] A scaffold comprising a dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat sequence of SEQ ID NO: 35.

[0754] Example 101, including additional parts

[0755] In Example 100, additionally, when the 5'-end nucleobase of the protospacer is considered as the 1st, the 1st base among the kth to (k + 1)th nucleobase is changed to the 2nd base,

[0756] The third base that complementarily binds to the first base in the above target sequence is changed to the fourth base,

[0757] Here, k and l are integers.

[0758] Example 102, PAM

[0759] In any one of Examples 93 to 101, the protospacer adjacent motif is 5'-TTTV-3' or 5'-TTTN-3',

[0760] Here, V is A, C, or G, and N is A, C, T, or G.

[0761] Example 103, Target Cell Type

[0762] In any one of Examples 93 to 102,

[0763] The target cell is a eukaryotic cell or a prokaryotic cell.

[0764] Example 104, Eukaryotic Cell Types

[0765] In Example 103, the target cell is a eukaryotic cell,

[0766] Selected from plant cells, human cells, and non-human animal cells.

[0767] Example 105, Isolated Cells

[0768] In any one of Examples 93 to 104, the target cell is a cell isolated from an individual.

[0769] Example 106, Method Execution Environment

[0770] In any one of Examples 93 to 105, the method is performed in an in vivo, in vitro or ex vivo environment.

[0771] Example 107, limited delivery method

[0772] In any one of Examples 93 to 106, delivering the base editor composition to the cell is performed by a method selected from the following:

[0773] Electroporation; gene gun; sonoporation; magnetofection; transient cell compression or squeezing; cationic liposome method; lithium acetate-DMSO; lipid-mediated transfection; calcium phosphate precipitation; lipofection; polyethyleneimine (PEI)-mediated transfection; DEAE-dextran-mediated transfection; nanoparticle-mediated nucleic acid delivery (see Panyam et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023); or any combination of the above methods.

[0774] Base Editing Method #1 - Adenine Base Editing Method

[0775] Example 108, Adenine Base Editing Method

[0776] In Example 93, or any one of Examples 103 to 107 citing the same,

[0777] The above base editor complex is an adenine base editor complex of any one of Examples 70 to 71,

[0778] The above method is a method of changing the A and T base pairs of the target nucleic acid in the cell to G and C base pairs.

[0779] Example 109, Adenine Base Editing Method #1

[0780] In any one of Examples 94 to 97, or Examples 103 to 107 citing the same,

[0781] The above base editor complex is an adenine base editor complex of any one of Examples 70 to 71,

[0782] The above method is a method of changing the A and T base pairs of the target site contained in the target nucleic acid within the cell to G and C base pairs,

[0783] When the above method is performed, A included in the second target site is changed to G, and T of the corresponding first target site is changed to C.

[0784] The above i is an integer selected from 7 to 22, preferably 8 or 17.

[0785] Example 110, target region length limitation

[0786] In Example 109, the target site is a double-stranded nucleic acid of 3 bp, 4 bp, 5 bp, 6 bp, or 7 bp.

[0787] Example 111, Adenine Base Editing Method #2

[0788] In any one of Examples 98 to 102, or Examples 103 to 107 citing the same,

[0789] The above base editor complex is an adenine base editor complex of any one of Examples 70 to 71,

[0790] The first base is adenine, the second base is guanine, the third base is thymine, and the fourth base is cytosine.

[0791] wherein i is an integer selected from 7 to 22, and j is 3, 4, 5, or 6, preferably i is 8 and j is 3, or i is 17 and j is 5,

[0792] When the kth to (k + l)th base pairs are additionally changed, k is an integer selected from 15 to 22, and l is an integer selected from 3 to 6, preferably k is 17 and l is 5.

[0793] Base Editing Method #2 - Cytosine Base Editing Method

[0794] Example 112, Adenine Base Editing Method

[0795] In Example 93, or any one of Examples 103 to 107 citing the same,

[0796] The above base editor complex is a cytosine base editor complex of any one of Examples 72 to 73,

[0797] The above method is a method of changing the C and G base pairs of the target nucleic acid in the cell to T and A base pairs.

[0798] Example 113, cytosine base editing method

[0799] In any one of Examples 98 to 102, or Examples 103 to 107 citing the same,

[0800] The above base editor complex is an adenine base editor complex of any one of Examples 72 to 73,

[0801] The above method is a method of changing the C and G base pairs of the target site contained in the target nucleic acid within the cell to T and A base pairs,

[0802] When the above method is performed, C included in the second target site is changed to T, and G in the corresponding first target site is changed to A.

[0803] The above i is an integer selected from 7 to 14, preferably 7 or 8.

[0804] Example 114, target region length limitation

[0805] In Example 113, the target site is a double-stranded nucleic acid of 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, or 10 bp.

[0806] Example 115, Cytosine Base Editing Method #2

[0807] In any one of Examples 98 to 102, or Examples 103 to 107 citing the same,

[0808] The above base editor complex is a cytosine base editor complex of any one of Examples 72 to 73,

[0809] The first base is cytosine, the second base is thymine, the third base is guanine, and the fourth base is adenine.

[0810] wherein i is an integer selected from 7 to 14, and j is an integer selected from 6 to 10, preferably, i is 7 and j is 7, or i is 8 and j is 6.

[0811] Bass Editing Method #3 - Multiplexing Bass Editing

[0812] Example 116, vector structure

[0813] A multiplex vector structure (construct) containing:

[0814] promoter;

[0815] DNA encoding the first guide RNA; and

[0816] DNA encoding a second guide RNA;

[0817] Here, the first guide RNA is any one selected from Examples 37 to 46,

[0818] The second guide RNA is any one selected from Examples 37 to 46,

[0819] The DNA encoding the first guide RNA is operably linked to a promoter,

[0820] The 5' end of the DNA encoding the second guide RNA is directly linked to the 3' end of the first guide RNA,

[0821] The first guide domain and the second guide domain have different nucleic acid sequences.

[0822] Example 117, including 3 or more guide RNAs

[0823] A multiplex vector structure containing the following structures:

[0824] [Promoter] - [1st guide RNA encoding DNA] - [2nd guide RNA encoding DNA] - [Nth guide RNA encoding DNA]

[0825] Here, n is an integer greater than or equal to 3,

[0826] The first guide RNA-encoding DNA, the second guide RNA-encoding DNA, and the n-th guide RNA-encoding DNA are each independently DNAs encoding any one programmable guide RNA selected from Examples 37 to 46,

[0827] The above first guide RNA encoding DNA is operably linked to the above promoter,

[0828] The first guide RNA encoding DNA, the second guide RNA encoding DNA, and the nth guide RNA encoding DNA are directly connected.

[0829] Example 118, Method for Editing Two or More Target Nucleic Acids

[0830] A method for replacing a base pair contained in a target region of each target nucleic acid with a different base pair, targeting two or more target nucleic acids within a target cell, including:

[0831] The above method includes:

[0832] Delivering to said target cell a composition comprising:

[0833] The base editor protein of any one of Examples 27 to 31, or a nucleic acid encoding the base editor protein, or

[0834] A base editor protein of any one of Examples 32 to 36, or a nucleic acid encoding said base editor protein; and

[0835] A multiplex vector structure of any one of Examples 116 to 117;

[0836] Here, depending on the combination of each guide RNA and base editor protein included in the multiplex vector structure,

[0837] Any one of the adenine base editing methods of Examples 108 to 111 is performed, or

[0838] Any one of the cytidine base editing methods of Examples 112 to 115 is performed.

[0839] Experimental example

[0840] 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.

[0841] Experimental Example 1. Experimental Method and Materials

[0842] Experimental Example 1.1. CRISPR Enzyme Expression Vector Production #1

[0843] The Cas12a protein coding sequence was codon-optimized for E. coli using the Twist Bioscience algorithm, and the gene was cloned into the pET21(+) or pET28 vector via the BamHI and XhoI sites. A nucleic acid capable of expressing a 6-His-tag and NLS was added to the PET21 vector, thereby producing a vector capable of expressing a CRISPR enzyme in which an NLS is linked to the N-terminus and / or C-terminus of the Cas12a protein, and a 6-His-tag is linked to the C-terminus.

[0844] Experimental Example 1.2. Guide RNA Preparation

[0845] In vitro transcription of sgRNA was performed using the MEGAscript(TM) T7 transcription kit (ThermoFisher Scientific). The synthesized fragment was PCR amplified (CloneAmp HiFi PCR Premix, Takara) to generate a template for sgRNA transcription. The DNA template was then removed with DNaseI (ThermoFisher Scientific), and the transcribed RNA product was washed with a PCR purification kit (MEGAclear(TM) Transcription Clean-Up Kit) and eluted in nuclease-free water. The concentration and purity of the RNA were determined by NanoDrop, and RNA integrity was visualized by ethidium bromide staining of the separated reaction products on a 2% agarose gel containing 1x TAE (Tris acetate EDTA) buffer.

[0846] Experimental Example 1.3. CRISPR Enzyme Production and Purification

[0847] The vector prepared according to Experimental Example 1.1 was expressed in E. coli strains (NiCo21(DE3), T7 Express lysY / Iq Competent E. coli; or BL21(DE3), Competent cells from ThermoFisher Scientific, #EC0114). Briefly, E. coli cells were transformed with the plasmid vector according to the manufacturer's instructions. Single colonies were grown overnight in Luria-Bertani medium containing 100 μg ml-1 ampicillin at 37_. The cells were diluted 1:200 in 250 ml of the same medium and grown at 37_ until OD600= 0.70-0.75. Cultures were incubated at room temperature for 1 h and then incubated at 23°C with shaking (220 rpm) for 16–18 h to induce protein expression with 0.5 mM isopropyl-bD-1-thiogalactopyranoside. Cell pellets were analyzed for protein expression using 10% SDS-PAGE. Protein purification was performed using the Ni-NTA Fast start kit (Qiagen; 30600) and then purified on a HiTrap HP SP (GE Healthcare, 17115101). The amount of protein produced by the cultures was measured by SDS-PAGE analysis. Purified Cas12a protein was stored at -80°C in 20 mM sodium acetate (pH 6.0), 500 mM NaCl, 0.1 mM EDTA, 0.1 mM TCEP, and 50% (v / v) glycerol.

[0848] Experimental Example 1.4. Verification of the Manufactured CRISPR Enzyme

[0849] The CRISPR enzyme prepared according to Experimental Example 1.3 was verified by Restriction Digest Analysis. Briefly, 1 μg of plasmid sample (NiCo21(DE3), T7 Express lysY / Iq Competent E. coli; or BL21(DE3) Competent cells) was mixed with 1 μL of restriction enzyme, 2 μL of digestion buffer, and nuclease-free water to a final volume of 20 μL, and incubated at 37°C for 30 min. The reaction products were separated on a 1% agarose gel using 1x TAE (Tris acetate EDTA) buffer and visualized by Ethidium Bromide staining.

[0850] Experimental Example 1.5. Preparation of CRISPR / Cas12a Complex

[0851] RNP complexes were individually pre-assembled by incubating 0.5 uM (or 20 ng) of CRISPR enzyme with 1 uM (or 5 ng) of guide RNA in NEB r2.1 buffer containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / mL BSA (pH=7.9) at room temperature for 15 min.

[0852] Experimental Example 1.6. Cell Culture

[0853] HEK293T (ATCC, CRL-11268) was used to evaluate the in vivo cleavage activity of the CRISPR / Cas12a system. The HEK cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Hyclone, SH30243.01) supplemented with 10% fetal bovine serum (Gibco, 16000-044) and 1% penicillin-streptomycin (ThermoFisher, 15070063) at 37 °C in a 5% CO2 atmosphere.

[0854] Experimental Example 1.7. Measurement of in vivo gene editing activity #1

[0855] The CRISPR / Cas12a complex prepared according to Experimental Example 1.5 was transfected into HEK293T cells cultured according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019). After culturing the transfected cells for 3 days, the cells were harvested and the genomic DNA of the cells was extracted using the TIANamp genomic DNA kit (Tiagen; 4992254) for T7E1 analysis (NEB; M0302S). The gene editing activity of the extracted genomic DNA of the cells was measured through the T7E1 assay (NEB; M0302S). The experimental results were confirmed through the NSG assay.

[0856] Experimental Example 1.8. Measurement of in vivo gene editing activity #2

[0857] The CRISPR / Cas12a complex prepared according to Experimental Example 1.5 was transfected into HEK293T cells cultured according to Experimental Example 1.6 using the Neon(R) Transfection System (ThermoFisher Scientific, MPK1096). After thawing the cells, the cells were passaged 5 times, and 1 Υ 106 cells were treated at a ratio of (CRISPR enzyme: guide RNA = 4: 1) and used for cell transfection according to the manufacturer's protocol under the following conditions: pulse voltage: 1500 V; pulse width: 30 ms; pulse number: 1. The cells were then cultured at 37°C under 5% CO2 for 3 days.

[0858] The cells cultured in 48-well plates were harvested with a mixture containing 5 μL Proteinase K and 100 μL of lysis buffer containing 1 M Tris-HCl (pH 7.5), 0.5 M EDTA (pH = 8.0), and 10% SDS. The cell lysis solution was then incubated at 50°C for 1 h, 80°C for 20 min, and stored at 4°C overnight. PCR reactions were performed in PrimeSTAR GXL DNA polymerase buffer (Takara, #R050A), and NSG analysis was performed on an iSeq100 (Illumnia). NSG results were verified using a web tool (http: / www.rgenome.net / ).

[0859] Experimental Example 1.9. Plasmid Construction for Base Editor Experiments

[0860] pCMV-dCpf1-BE (Addgene, #107685) and ABE8e (Addgene, #138489) were modified with the novel Cas12a using Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, NEB #E5520). The novel Cas12a sequence was optimized for human codons using the Codon Optimization Tool from IDT (Integrated DNA Technologies) and ordered as gBlocks from IDT. The novel Cas12a variant was constructed using site-directed mutagenesis (Q5-site directed mutagenesis kit, NEB #E0554S). The gRNA-encoding plasmid was constructed using PCR-amplified oligonucleotides into pLb-Cpf1-pGL3-U6-sgRNA (Addgene, #107682) using Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, NEB #E5520).

[0861] Experimental Example 1.10. Base Editing System Transfection

[0862] A plasmid encoding a base editor protein manufactured according to Experimental Example 1.9 and a plasmid encoding a guide RNA were transfected into cells cultured according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019).

[0863] Experimental Example 1.11. Measuring Base Editing Efficiency

[0864] The genomes of cells transfected with the base editing system and subjected to nucleotide editing according to Experimental Example 1.10 were extracted. To analyze the frequency of nucleotide editing in the extracted cell genomes, the target region was amplified through nested first-round PCR, second-round PCR, and third-round PCR using primers containing the Tru-Seq HT Dual index and PrimeSTAR® GXL DNA Polymerase (TAKARA) to generate a deep sequencing library. The library was sequenced using an Illumina iSeq paired-end sequencing system. The frequency of base editing and unwanted indels was expressed as the percentage of sequencing reads containing correct edits or indels among the total number of sequencing reads. The computer program used to analyze the frequency of editing is available at the prime_editor_analysis site on GitHub.

[0865] Experimental Example 2. Confirmation of the in vivo gene editing activity of the CRISPR / Cas12a system.

[0866] Experimental Example 2.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0867] To verify whether the CRISPR / Cas12a system disclosed herein can edit genes in eukaryotic cells, an in vivo gene editing activity experiment was performed targeting the EMX1 gene in immortalized HEK cells.

[0868] The target nucleic acid sequence in the EMX1 gene is as follows: CTCATCTGTGCCCCTCCCTCCCTG (SEQ ID NO: 136)

[0869] The composition of each CRISPR / Cas12a complex used in the above experiments is shown in the following table:

[0870] Composition of the CRISPR / Cas12a complex used in the experiment: LabelCas12a protein(SEQ ID NO)CRISPR enzyme(Cas12a + NLS)(SEQ ID NO)Direct repeat ofguide RNAGuide Domain ofguide RNAID2044125UAAUUUCUACUAUUCGUAGAU (SEQ ID NO: 36)CUCAUCUGUGCCCCUCCCUCCCUG (SEQ ID NO: 137)ID2065126UUAAUUUCUACUUUCGUAGAU (SEQ ID NO: 37)SEQ ID NO: 137ID2106127CAAUUUCUACUUUCAGUAGAU (SEQ ID NO: 38)SEQ ID NO: 137ID2117128UUAAUUUCUACUUGUGUAGAU (SEQ ID NO: 39)SEQ ID NO: 137ID2181120UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)SEQ ID NO: 137ID2199129UAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 41)SEQ ID NO: 137ID22310130AAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 42)SEQ ID NO: 137ID22411131AAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 43)SEQ ID NO: 137ID23012132UAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 44)SEQ ID NO: 137ID23813133AUAAUUUCUACUGUUGUAGAU (SEQ ID NO: 45)SEQ ID NO: 137ID24515134AUAAUUUCUACUGUUGUAGAU (SEQ ID NO: 47)SEQ ID NO: 137

[0871] Among the components of the above CRISPR / Cas12a complex, the sequence of the Cas12a protein was obtained from KR 2023-0111399 A, and the composition of the guide RNA was determined by the inventors of the present specification through further research.

[0872] The specific experimental method is as follows:

[0873] 1) A CRISPR enzyme expression vector was prepared according to Experimental Example 1.1, and the CRISPR enzyme was prepared and purified using the vector according to Experimental Example 1.3.

[0874] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0875] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0876] 4) The in vivo gene editing activity of the CRISPR / Cas12a complex prepared above was measured according to Experimental Example 1.7 for immortalized HEK cells prepared according to Experimental Example 1.6.

[0877] Experimental Example 2.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0878] The results of expressing the CRISPR enzyme according to Experimental Example 2.1 are shown in Fig. 1, and the label, restriction enzyme, and fragment bp for each defense in Fig. 1 are shown in the following table.

[0879] NoLabelRestriction EnzymeFragment 1 (bp)Fragment 2 (bp)Fragment 3 (bp)11 kb DNA Ladder----2ID204EcoRV1.54472.4083-3ID206EcoRV1.65872.26253.1024ID210SphI1.66 722.2438-5ID211EcoRV1.65872.2523-6ID218HindIII1.73422.1738-7ID219EcoRV1.54442 .3786-8ID223KpnI1.80082.1138-9ID224EcoRV1.74372.2087-10ID230SphI1.65192.2648- 11ID233HindIII1.76882.1695-12ID238MluI1.71812.1932-13ID245HindIII1.7992.1249-

[0880] Looking at the above results, it can be seen that each Cas12a protein was expressed well according to Experimental Example 2.1.

[0881] Experimental Example 2.3. Results of in vivo gene editing activity experiments

[0882] The results of the T7E1 assay performed according to Experimental Example 2.1 are shown in Fig. 2.

[0883] Experimental results show that only the CRISPR / Cas12a system containing the Cas12a protein of ID218 can edit the EMX1 target in the HEK cells, resulting in the appearance of the relevant band.

[0884] To quantitatively confirm the results, NGS sequence analysis was performed and the results are shown in the following table:

[0885] NGS results for endogenous EMX1 gene editing activity in HEK cells: ID Total Read Wild type Insertion Deletion In-del (%) 20450175017000.0 (0.0%) 20644784478000.0 (0.0%) 21041924192000.0 (0.0%) 21151445144022 (0.0%) 2183636324413379392 (10.8%) 21946524646066 (0.0%) 22352485248000.0 (0.0%) 22439123912000.0 (0.0%) 23041014101000.0 (0.0%) 23854855485000.0 (0.0%)24561726172000.0 (0.0%)LB5147514706464 (1.2%)

[0886] As confirmed in Fig. 2, the experimental results confirmed that only the CRISPR / Cas12a system including the Cas12a protein of ID218 induced the occurrence of indels in the endogenous EMX1 gene of the HEK cells. Therefore, it can be concluded that the CRISPR / Cas12a system including the Cas12a protein of ID218 has eukaryotic gene editing activity.

[0887] Furthermore, mutation analysis was performed on the results of the above indel introduction and is shown in Fig. 3.

[0888] It can be confirmed that the CRISPR / Cas12a system including the Cas12a protein of the above ID218 caused various types of mutations in the endogenous EMX1 gene of HEK cells.

[0889] For reference, the results of mutation analysis for LbCas12a used as a control group are shown in Figure 4.

[0890] Experimental Example 3. Confirmation of Gene Editing Activity According to Nuclear Localization Signal Composition

[0891] Experimental Example 3.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0892] As confirmed in Experimental Example 2, the CRISPR / Cas12a system containing the Cas12a protein of ID218 exhibits eukaryotic editing activity. Furthermore, to examine the influence of the Nuclear Localization Signal on eukaryotic editing activity, the in vivo gene editing activity of the CRISPR / Cas12a system containing the Cas12a protein of ID218 and various combinations of NLSs was measured.

[0893] As in Experimental Example 2, an in vivo gene editing activity experiment was performed targeting the EMX1 gene of immortalized HEK cells.

[0894] The target nucleic acid sequence in the EMX1 gene is as follows: CTCATCTGTGCCCCTCCCTCCCTG (SEQ ID NO: 136)

[0895] The configuration of each CRISPR / Cas12a system used in the experiment is schematically shown in Figure 5, and its specific sequence configuration is shown in the following table:

[0896] Configuration of each CRISPR / Cas12a system used in Experimental Example 3: LabelNLS at N-terminalCas12a protein (SEQ ID NO)NLS at C-terminalCRISPR enzyme (SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNANLS1 (#1)PKKKRKV (SEQ ID NO: 67)1PKKKRKVKRPAATKKAGQAKKKK (SEQ ID NO: 69)118UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)CUCAUCUGUGCCCCUCCCUCCCUG (SEQ ID NO: 137)NLS2 (#2)-1SEQ ID NO: 69119SEQ ID NO: 33SEQ ID NO: 137NLS3 (#3)SEQ ID NO: 671KRPAATKKAGQAKKKK (SEQ ID NO: 68)120SEQ ID NO: 33SEQ ID NO: 137NLS4 (#4)-1SEQ ID NO: 68121SEQ ID NO: 33SEQ ID NO: 137NLS5 (#5)SEQ ID NO: 671KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70)122SEQ ID NO: 33SEQ ID NO: 137NLS6 (#6)-1SEQ ID NO: 70123SEQ ID NO: 33SEQ ID NO: 137NLS7 (#7)SEQ ID NO: 671PKKKRKVPKKKRKVPKKKRKV (SEQ ID NO: 71)124SEQ ID NO: 33SEQ ID NO: 137

[0897] The specific experimental method is as follows:

[0898] 1) A CRISPR enzyme expression vector was prepared according to Experimental Example 1.1, and the CRISPR enzyme was prepared and purified using the vector according to Experimental Example 1.3. Here, BL21 competent cells (ThermoFisher) were used as competent cells, and grown until OD600 = 0.5 to 0.7. The culture was incubated at room temperature for 1 hour, and then incubated at 23°C with shaking (180 rpm) for 18 hours, thereby inducing protein expression with 0.5 mM isopropyl-bD-1-thiogalactopyranoside.

[0899] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0900] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0901] 4) The in vivo gene editing activity of the CRISPR / Cas12a complex prepared above was measured according to Experimental Example 1.7 for immortalized HEK cells prepared according to Experimental Example 1.6.

[0902] Experimental Example 3.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0903] The results of expressing the CRISPR enzyme according to Experimental Example 3.1 and evaluating the results according to Experimental Example 1.4 are shown in Figures 6 and 7.

[0904] Looking at the above results, it can be seen that each CRISPR enzyme was expressed well according to Experimental Example 3.1.

[0905] The molecular weight and concentration of each expression result are shown in the following table:

[0906] Molecular weight and concentration of CRISPR enzyme expressed according to Experimental Example 3.1: LabelMW(Kda)Conc(mg / ml)NLS11498.565NLS21488.757NLS314813.363NLS414711.594NLS515111.22NLS615010.15NLS71497.491

[0907] Experimental Example 3.3. Results of in vivo gene editing activity experiment The results of the gene editing activity experiment of the CRISPR / Cas12a system targeting the endogenous EMX1 gene of HEK cells according to Experimental Example 3.1 are shown in Fig. 8.

[0908] As a result of the experiment, it was confirmed that a band indicating that the EMX1 gene was edited was clearly visible in NLS1 to NLS6, and it was confirmed that the editing activity was different depending on the combination of NLSs used.

[0909] Experimental Example 3.4. In Vivo Gene Editing Activity Experiment Results #2

[0910] Among the CRISPR / Cas12a systems in Table 5 above, the results of confirming the in vivo gene editing activity of the CRISPR / Cas12a system corresponding to NLS5 through Mutation Analysis are shown in Figure 11.

[0911] Experimental results confirmed that the CRISPR / Cas12a system of ID218 containing the NLS configuration corresponding to NLS5 exhibited significantly high in vivo gene editing activity.

[0912] Experimental Example 4. Confirmation of the in vivo gene editing activity of the CRISPR / Cas12a system #2

[0913] Experimental Example 4.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0914] To determine whether the CRISPR / Cas12a system disclosed herein can edit genes in eukaryotic cells, in vivo gene editing activity experiments were performed targeting several genes (EMX1, MTAP, DYRK1A, and DNMT1A) in the HEK293T cell genome.

[0915] The gene sequences and protospacer sequences used in the above experiments are shown in the following table:

[0916] Gene editing target gene and protospacer sequence:Gene nameTarget sequenceProtospacer sequenceMTAPCCATCTGTACCCCCAAAAACTATTGAAATTAAAAAAAAATTGTGATTGATGTGGTTTCGCTGCATCCTCGCCCAAATCTCATTTGTATTTTAGCCCCAATAATCCCCACATGTCATGGAAGGGACCTGGTGGCAGGTAATTGAATCATGGAGGTGGGTCTTTCTCGTGCTGTTCTCGTGATAGTGAATAAATCTCACAAGATCTGATGGTTTTATAAAGGGGAGTTCCCCTGCACATGC (SEQ ID NO: 138)GCCCCAATAATCCCCACATGTCA (SEQ ID NO: 139)DYRK1ATCTTAAAACCTTGTCACACACAATGAAACTTTGCTGTTCACTGTCAGTTATAACTTACATGAGGTGACCCATTTCCATTCAAGGGTTTTAGAAGCACATCAAGGACATTCTAAGGATGATTGACTTACACAATGATCTCTGAACATGCCTCCTGCCTTCTCCTCACTCTTGAGTATTTGCTTAGGGGAGC (SEQ ID NO: 141)GAAGCACATCAAGGACATTCTAA (SEQ ID NO: 142)

[0917] The composition of each CRISPR / Cas12a complex used in the above experiments is shown in the following table:

[0918] Composition of the CRISPR / Cas12a complex used in the experiment: LabelCas12a protein (SEQ ID NO)CRISPR enzyme (SEQ ID NO)Direct repeat of guide RNA (SEQ ID NO)Guide domain of guide RNA (SEQ ID NO)204425636MTAP: 140DYRK1A: 143206525737MTAP: 140DYRK1A: 143210625838MTAP: 140DYRK1A: 143211725939MTAP: 140DYRK1A: 143217826040MTAP: 140DYRK1A: 143218125333MTAP: 140DYRK1A: 1432301226144MTAP: 140DYRK1A: 143233225434MTAP: 140DYRK1A: 143236325535MTAP: 140DYRK1A: 1432391426246MTAP: 140DYRK1A: 1432501626348MTAP: 140DYRK1A: 143

[0919] In the table above, the CRISPR enzyme sequence represents the sequence containing Cas12a and NLS.

[0920] Among the components of the above CRISPR / Cas12a complex, the sequence of the Cas12a protein was obtained from KR 2023-0111399 A, and the composition of the guide RNA was determined by the inventors of the present specification through further research.

[0921] The specific experimental method is as follows:

[0922] 1) A CRISPR enzyme expression vector was prepared according to Experimental Example 1.1, and the CRISPR enzyme was prepared and purified using the vector according to Experimental Example 1.3.

[0923] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0924] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0925] 4) The in vivo gene editing activity of the CRISPR / Cas12a complex prepared above was measured according to Experimental Example 1.8 for immortalized HEK cells prepared according to Experimental Example 1.6.

[0926] Information on each primer used in the above experiment is shown in Figures 19 to 22:

[0927] Experimental Example 4.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0928] The results of evaluating the CRISPR enzyme manufactured according to Experimental Example 4.1 according to Experimental Example 1.4 are shown in Fig. 9, and the results of SDS-PAGE analysis according to Experimental Example 1.3 are shown in Fig. 10.

[0929] Looking at the above results, it can be seen that each CRISPR enzyme was expressed well according to Experimental Example 4.1.

[0930] Experimental Example 4.3. In vitro gene editing activity experiment

[0931] To evaluate the in vitro cleavage activity of the CRISPR enzyme manufactured according to Experimental Example 4.1, the following experiment was conducted:

[0932] 1) According to Experimental Example 1.5, the CRISPR / Cas12a complex was assembled with the manufactured CRISPR enzyme and guide RNA. Here, to determine the activity according to pH conditions, the CRISPR / Cas12a complex was assembled in buffers of pH = 7.9 (NEB buffer 2.1), pH = 6.5 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 ug / mL BSA), and pH = 4.5 (50 mM NaCl, 10 mM sodium acetate, 10 mM MgCl2, 0.1 mM DTT), respectively.

[0933] 2) The CRISPR / Cas12a complex assembled in 1) was added to 1 μg of the DNA template of the SCN1A gene to initiate the cleavage reaction. Here, to determine the activity according to the reaction temperature, each reaction mixture was incubated at 25°C, 37°C, and 60°C for 1 hour.

[0934] 3) The cleavage activity of the CRISPR / Cas12a complex was visualized by staining the reaction products separated on a 1% agarose gel using 1x TAE (Tris acetate EDTA) buffer with ethidium bromide.

[0935] The results of the in vitro cleavage activity experiment of each CRISPR / Cas12a complex are shown in Figure 23.

[0936] Experimental results showed that most CRISPR / Cas12a complexes exhibited in vitro nucleic acid cleavage activity.

[0937] Experimental Example 4.4. In Vivo Gene Editing Activity Experiment #1 - Experimental Method

[0938] To confirm the in vivo gene editing activity of the CRISPR enzyme according to Experimental Example 4.1, the following experiment was conducted:

[0939] 1) According to Experimental Example 1.5, a CRISPR / Cas12a complex was assembled using the CRISPR enzyme and guide RNA manufactured in Experimental Example 4.1.

[0940] 2) The CRISPR / Cas12a complex assembled in 1) was transfected into HEK293T cells according to Experimental Example 1.8, and gene editing activity was measured.

[0941] Experimental Example 4.5. In vivo gene editing activity experiment #2 - MTAP gene

[0942] The results of measuring the in vivo gene editing activity of each CRISPR / Cas12a complex targeting the MTAP gene are shown in the following table:

[0943] MTAP gene editing activity: Label Total Reads Wild Type Insertion Deletion Indel (%) 2041427514270055 (0.0%) 2061500014998022 (0.0%) 21034023402000 (0.0%) 2111672216718044 (0.0%) 217139531392822325 (0.2%) 218389437213633863522 (90.4%) 23037693769000 (0.0%) 23314303121839520252120 (14.8%) 2364346360752686739 (17.0%)23933113311000 (0.0%)2501286012860000 (0.0%)

[0944] The results of sequencing the MTAP gene editing results of ID 218, ID 233, and ID 236 using NSG are shown in Figures 12 to 14.

[0945] Experimental results showed that the CRISPR / Cas12a complex containing Cas12a proteins of ID 218, ID 233, and ID 236 introduced a significant proportion of Indels into the MTAP gene.

[0946] Experimental Example 4.6. In vivo gene editing activity experiment #3 - DYRK1A gene

[0947] The results of measuring the in vivo gene editing activity of each CRISPR / Cas12a complex targeting the DYRK1A gene are shown in the following table:

[0948] DYRK1A gene editing activity: Label Total Reads Wild Type Insertion Deletion Indel (%) 2041558415582022 (0.0%) 2061659116588033 (0.0%) 21041614159022 (0.0%) 211184171838603131 (0.2%) 2171389413894000 (0.0%) 2184199121912128592980 (71.0%) 23035313531000 (0.0%) 23314386126285617021758 (12.2%) 236477945314244248 (5.2%)23936973695022 (0.1%)2501423414229055 (0.0%)

[0949] The results of sequencing the DYRK1A gene editing results of ID 218, ID 233, and ID 236 using NSG are shown in Figures 15 to 17.

[0950] Experimental results showed that the CRISPR / Cas12a complex containing Cas12a proteins of ID 218, ID 233, and ID 236 introduced a significant proportion of Indels into the DYRK1A gene.

[0951] Experimental Example 4.7. In Vivo Gene Editing Activity Experiment #4 - Interpreting Experimental Results

[0952] Experimental results showed that not all manufactured CRISPR enzymes had in vivo gene editing activity, and only the novel Cas12a proteins having amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 were confirmed to have gene editing activity. The novel Cas12a proteins of the above sequences can sufficiently edit the cellular genome in vivo when forming a CRISPR / Cas12a system together with an appropriate NLS and an appropriate guide RNA, although the editing efficiency may vary slightly depending on the gene sequence.

[0953] Experimental Example 5. Production of Dead-Form Cas12a

[0954] Experimental Example 5.1. Exploring Dead-Form Cas12a Mutation Sites

[0955] The inventors of this specification sought to utilize the Cas12a proteins comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3, which were confirmed to have eukaryotic genome editing activity in Experimental Examples 2 to 4, in a base editing system. First, they sought to create a dead form in which the nucleic acid cleavage activity of the above Cas12a proteins was eliminated. The inventors analyzed the structure of the CRISPR / Cas12a system comprising the above Cas12a proteins bound to the target nucleic acid through alphafold structural analysis. The analysis results are shown in Figures 19 to 21. As a result of the analysis, positions E921, D828, and D1145 in the amino acid sequence of SEQ ID NO: 1 were confirmed to be important positions for nucleic acid cleavage activity. In addition, positions D936, E1029, and D1254 in the amino acid sequence of SEQ ID NO: 3 were confirmed to be important positions for nucleic acid cleavage activity.

[0956] Additionally, the inventors compared the active site by aligning the amino acid sequence of FnCpf1, in which the nucleic acid cleavage active site was identified in the prior literature (Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016 / j.cell.2015.09.038. Epub 2015 Sep 25. PMID: 26422227; PMCID: PMC4638220.) with the above SEQ ID NO: 1 and SEQ ID NO: 3. The comparison results are shown in Figure 21. The comparison confirmed that the candidate sites identified through AlphaFold structural analysis correspond to the cleavage activity sites of FnCpf1 disclosed in prior literature. This suggests that the sites identified through structural analysis above are accurate.

[0957] Consequently, it was predicted that introducing mutations E921A, D828A, and D1145A into the amino acid sequence of SEQ ID NO: 1 would result in the production of Dead-Form Cas12a with eliminated nucleic acid cleavage activity. Furthermore, it was predicted that introducing mutations D936A, E1029A, and D1254A into the amino acid sequence of SEQ ID NO: 3 would result in the production of Dead-Form Cas12a with eliminated nucleic acid cleavage activity.

[0958] Experimental Example 5.2. Dead-Form Cas12a Activity Evaluation Experiment

[0959] To confirm whether the Dead-Form Cas12a protein derived from Experimental Example 5.1 actually cannot cleave the target nucleic acid, the Cas12a protein of the actual sequence number 264 was prepared, and the cytosine base editing system containing it was tested on various targets.

[0960] Specifically, plasmids encoding base editor proteins and plasmids encoding guide RNAs targeting various target nucleic acids were prepared according to Experimental Example 1.9. Each of the constructs prepared above was delivered into eukaryotic cells according to Experimental Example 1.10. The genomes of the eukaryotic cells were analyzed according to Experimental Example 1.8 to determine whether indels were introduced.

[0961] The configurations used in each experimental example are as follows:

[0962] LabelCBE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)WT-CBE406UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)RAPSN: 440IFTAP: 441EMX1: 137PD1: 442DNMT3B: 443FANCF: 434RUNX1: 437DNMT1: 146CFTR: 435Site3: 436DYRK1A: 143Site6: 444VEGFA: 433MTAP: 140dead-CBE295(Same as above)(Same as above)Control---

[0963] Experimental Example 5.3. Dead-Form Cas12a Activity Evaluation Experiment Results

[0964] The experimental results are shown in Fig. 24. As a result, in the case of the cytosine base editor protein including the amino acid sequence of SEQ ID NO: 1, it showed the activity of introducing an indel by targeting the target sequence, but in the case of the cytosine base editor protein including the amino acid sequence of SEQ ID NO: 264, it did not introduce an indel into the target sequence. This indicates that the nucleic acid cleavage activity of the Dead-Form Cas12a prepared above was successfully suppressed. Subsequently, various variants were prepared based on the Dead-Form Cas12a above, and base editing experiments were performed, and as a result, no base editor protein showed the activity of introducing an indel into the target nucleic acid (see Figs. 26, 32, 33, 36, 37, 41, 43, 45, and 53). Therefore, it can be seen that the Dead-Form Cas12a disclosed in this specification has the nucleic acid cleavage activity eliminated or extremely suppressed as intended.

[0965] Experimental Example 6. Confirming Editing Activity of the Cytosine Base Editing System #1

[0966] Experimental Example 6.1. Experimental Method and Configuration Used in the Experiment

[0967] A cytosine base editor containing a specific dead-Cas12a protein was constructed above to determine whether it has the activity of editing CG base pairs into TA base pairs. Specifically, plasmids encoding each cytidine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also constructed. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[0968] The configuration of each base editing system used in Experimental Example 6 is shown in the following table:

[0969] LabelChart LabelCBE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 6.1dCas12a (236)296UAAAUUUCUACUGUUGUAGAU (SEQ ID NO: 35)MTAP: 140DYRK1A: 143RUNX1: 437ControlUNT---

[0970] Experimental Example 6.2 Experimental Results

[0971] The experimental results are shown in Figures 25 and 26. Among these, quantitative data on the results of nucleotide editing targeting the MTAP gene are shown in the following table:

[0972] PositionNucleobasedCas12a(236)-CBEPAMT0.00PAMT0.00PAMT0.00PAMA0.00#1G0.07#2C0.19#3C0.15#4C0.91#5C0.14#6A0.00#7A0.00#8T0. 00#9A0.00#10A0.00#11T0.00#12C6.54#13C3.14#14C1.45#15C1.04#16 A0.00#17C0.20#18A0.00#19T0.00#20G0.11#21T0.00#22C0.14#23A0.00

[0973] Experimental results showed that the cytosine-based editor of the above configuration effectively changed the 7th to 14th CG base pairs in the protospacer sequence to AT base pairs. Furthermore, when the frequency of indel introduction was measured, it was nearly identical to that of the negative control, UNT, indicating that the nucleic acid cleavage function was eliminated.

[0974] Experimental Example 7. Confirming Editing Activity of the Cytosine Base Editing System #2

[0975] Experimental Example 7.1. Experimental Method and Configuration Used in the Experiment

[0976] A cytosine base editor containing a specific dead-Cas12a protein was constructed above to determine whether it has the activity of editing CG base pairs into TA base pairs. Specifically, plasmids encoding each cytidine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also constructed. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[0977] The configuration of each base editing system used in Experimental Example 7 is shown in the following table:

[0978] LabelChart LabelCBE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 7.1NLS6-dCas12a (218)-CBE405UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)GCCCCAAUAAUCCCCACAUGUCAUGG (SEQ ID NO: 420)Example 7.2dCas12a (218)-CBE295(Same as above)(Same as above)Example 7.3WT-Cas12a (218)-CBE406(Same as above)(Same as above)ControlUNT---

[0979] Experimental Example 7.2 Experimental Results

[0980] The experimental results are shown in Fig. 29.

[0981] Experimental results showed that the cytosine base editors of Example 7.1 and Example 7.2 were effective in changing CG base pairs to AT base pairs within the protospacer sequence range.

[0982] Experimental Example 8. Confirming Adenine Base Editing System Editing Activity #1

[0983] Experimental Example 8.1. Exploring the Location of Mutations in the PAM Recognition Site

[0984] Prior literature (Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, Aryee MJ, Joung JK. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol. 2019 Mar;37(3):276-282. doi: 10.1038 / s41587-018-0011-0. Epub 2019 Feb 11. Nat Biotechnol. 2020 Jul;38(7):901. PMID: 30742127; PMCID: PMC6401248.) was used to find the PAM recognition site mutation position of the Cas12a protein discovered above. Specifically, the structure of the CRISPR / Cas12a system including the Cas12a protein of SEQ ID NO: 1 bound to the target nucleic acid was analyzed using AlphaFold. The analysis results are shown in Figs. 27 and 28. The analysis results revealed that positions E155, G531, and K537 in the amino acid sequence of SEQ ID NO: 1 are positions that significantly interact with the PAM sequence. The inventors introduced the PAM recognition site mutation discovered above into the dead-Cas12a protein including the amino acid sequence of SEQ ID NO: 264 to produce a mutant of the dead-Cas12a protein. In addition, an adenine base editing system including the mutant of the dead-Cas12a protein was produced and the nucleobase editing activity was measured.

[0985] Experimental Example 8.2. Experimental Method and Configuration Used in the Experiment

[0986] A mutant of the dead-Cas12a protein of SEQ ID NO: 264, in which an amino acid at a specific position is substituted with arginine in Experimental Example 8.1, was prepared and used in the experiment. An adenine base editor containing the dead-Cas12a protein prepared above was prepared and its activity in editing AT base pairs into GC base pairs was confirmed. Specifically, plasmids encoding each adenine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also prepared. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[0987] The configuration of each base editing system used in Experimental Example 8 is shown in the following table:

[0988] LabelChart LabelABE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 8.1dCas12a(218)_ABE283UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)Site3: 436VEGFA: 433VEGFAT: 438FANCF: 434CFTR: 435DYRK1A: 143Example 8.2dCas12a(218)_ABE_NLS6284(Same as above)(Same as above)Example 8.3dCas12a(218)_ABE_RRR421(Same as above)(Same as above)Example 8.4dCas12a(218)_ABE_RRR_NLS6422(Same as above)(Same as above)ControlUNT---

[0989] Experimental Example 8.3. Experimental Results

[0990] The experimental results are shown in Figs. 30 to 33.

[0991] As a result of the experiment, Example 8.1 and Example 8.2 showed activity of changing adenine to guanine within the range of about the 8th to 11th base based on the protospacer sequence.

[0992] Meanwhile, the base editing system of Example 8.4 introduced indels into the target nucleic acid. The other base editing systems did not introduce indels into the target nucleic acid.

[0993] Experimental Example 9. Confirming Adenine Base Editing System Editing Activity #2

[0994] Experimental Example 9.1. Experimental Method and Configuration Used in the Experiment

[0995] A mutant of the dead-Cas12a protein of SEQ ID NO: 264, in which an amino acid at a specific position is substituted with arginine in Experimental Example 7.1, was prepared and used in the experiment. An adenine base editor containing the dead-Cas12a protein prepared above was prepared to confirm whether it had the activity of editing AT base pairs into GC base pairs. Specifically, plasmids encoding each adenine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also prepared. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[0996] The configuration of each base editing system used in Experimental Example 9 is shown in the following table:

[0997] LabelChart LabelABE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 9.1218_ABE283UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)DYRK1A: 143Site3: 436CFTR: 435VEGFA: 433FANCF: 434Example 9.2218_ABE_E155R285(Same as above)(Same as above)ControlUNT---

[0998] Experimental Example 9.2. Experimental Results

[0999] The experimental results are shown in Figs. 34 to 37.

[1000] As a result of the experiment, Example 9.1 and Example 9.2 showed activity of changing adenine to guanine within the range of about the 8th to 14th base based on the protospacer sequence.

[1001] Additionally, not all base editing systems introduced indels into the target nucleic acid.

[1002] Experimental Example 10. Confirming Adenine Base Editing System Editing Activity #3

[1003] Experimental Example 10.1. Exploring the Location of PAM Recognition Site Mutations

[1004] Using the same method as Experimental Example 8.1, we found a position that significantly interacts with PAM in the amino acid sequence of SEQ ID NO: 3. The analysis results are shown in Figures 38 and 39. As a result of the analysis, it was confirmed that positions Q190, S579, and Y585 in the amino acid sequence of SEQ ID NO: 3 are positions that significantly interact with the PAM sequence. The inventors introduced the PAM recognition site mutation discovered above into the dead-Cas12a protein including the amino acid sequence of SEQ ID NO: 265 to prepare a mutant of the dead-Cas12a protein. In addition, an adenine base editing system including the mutant of the dead-Cas12a protein was prepared and the nucleobase editing activity was measured.

[1005] Experimental Example 10.2. Experimental Method and Configuration Used in the Experiment

[1006] A mutant of the dead-Cas12a protein of SEQ ID NO: 265 was prepared by substituting an amino acid at a specific position with arginine in Experimental Example 10.1 and used in the experiment. An adenine base editor containing the dead-Cas12a protein prepared above was prepared and its activity in editing AT base pairs into GC base pairs was confirmed. Specifically, plasmids encoding each adenine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also prepared. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[1007] The configuration of each base editing system used in Experimental Example 10 is shown in the following table:

[1008] LabelChart LabelABE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 10.1dCas12a (236-RRR)-ABE-NLS6410UAAAUUUCUACUGUUGUAGAU (SEQ ID NO: 35)MTAP: 140DYRK1A: 143FANCF: 434VEGFA: 433Site3: 436Example 10.2dCas12a (236)-ABE-NLS6409(Same as above)(Same as above)Example 10.3dCas12a (236-RRR)-ABE411(Same as above)(Same as above)Example 10.4dCas12a (236-RR)-ABE274(Same as above)(Same as above)Example 10.5dCas12a (236-R)-ABE412(Same as above)(Same as above)Example 10.6dCas12a (236)-ABE292(Same as above)(Same as above)ControlUNT---

[1009] Experimental Example 10.3. Experimental Results

[1010] The experimental results are shown in Figs. 40 to 43.

[1011] Quantitative data for Experimental Example 10 are shown in the following table:

[1012] Target: MTAPPositionNucleobasedCas12a (236-RRR)-ABE-NLS6dCas12a (236)-ABE-NLS6dCas12a (236-RRR)-ABEdCas12a (236-RR)-ABEdCas12a (236-R)-ABEdCas12a (236)-ABEUNTPAMT0.010.010.000.000.000.010.01PAMT0.000.010.020.010.010.000.01PAMT0.000.010.020.000.010.010.03PAMA0.030.010.020.020.020.060.02#1G0.050.020.020.010.020.040.02#2C0.060.080.050.050.080.080.05#3C0.050.030.040.070.030.010.02#4C0.010.070.040.050.060.040.04#5C0.100.100.100.160.090.100.11#6A0.020.010.020.030.030.020.02#7A0.020.010.020.070.080.030.03#8T0.020.010.000.030.020.010.01#9A3.265.962.0111.628.564.910.02#10A0.070.090.030.210.130.050.01#11T0.000.000.010.000.000.020.00#12C0.050.090.050.060.090.030.08#13C0.070.040.060.050.030.040.04#14C0.090.080.020.050.070.070.04#15C0.090.020.040.040.040.100.07#16A0.070.110.050.120.060.020.01#17C0.020.030.020.040.030.040.03#18A0.090.150.080.300.150.130.03#19T0.020.010.020.010.020.020.02#20G0.020.020.020.030.040.040.03#21T0.010.020.020.030.020.020.02#22C0.040.020.040.020.040.010.02#23A0.190.330.100.510.350.320.01.

[1013] Target: DYRK1APositionNucleobasedCas12a (236-RRR)-ABE-NLS6dCas12a (236)-ABE-NLS6dCas12a (236-RRR)-ABEdCas12a (236-RR)-ABEdCas12a (236-R)-ABEdCas12a (236)-ABEUNTPAMT0.010.000.010.020.000.010.00PAMT0.010.010.000.010.000.040.01PAMT0.000.000.010.010.000.020.01PAMA0.030.020.030.040.000.010.02#1G0.080.070.060.100.050.070.09#2A0.010.020.000.010.030.010.01#3A0.020.020.030.010.060.030.01#4G0.050.040.030.040.000.020.02#5C0.020.020.030.020.030.020.03#6A0.030.020.010.140.050.030.01#7C0.030.010.040.010.000.040.01#8A1.023.830.6213.566.403.020.00#9T0.010.000.010.010.000.030.02#10C0.010.010.010.000.000.010.02#11A0.150.420.081.330.650.310.02#12A0.030.040.030.110.020.010.02#13G0.040.070.040.040.050.030.05#14G0.030.040.030.020.000.040.04#15A0.060.040.010.240.080.000.01#16C0.040.010.010.020.050.020.01#17A0.080.200.030.930.120.050.01#18T0.010.010.020.010.050.010.04#19T0.010.010.000.020.000.010.01#20C0.000.010.020.020.000.010.01#21T0.020.010.000.000.020.010.00#22A0.040.160.020.540.140.070.01#23A0.030.040.020.020.030.010.01.

[1014] Target: FANCFPositionNucleobasedCas12a (236-RRR)-ABE-NLS6dCas12a (236)-ABE-NLS6dCas12a (236-RRR)-ABEdCas12a (236-RR)-ABEdCas12a (236-R)-ABEdCas12a (236)-ABEUNTPAMT0.000.010.010.000.000.000.02PAMT0.010.030.010.020.010.020.00PAMT0.040.020.010.010.000.010.00PAMG0.030.050.100.060.050.040.05#1G0.110.060.050.090.080.060.06#2G0.070.060.040.050.060.050.09#3A0.030.020.020.020.030.010.03#4T0.000.020.010.040.000.010.02#5A0.000.010.010.050.010.000.00#6A0.000.030.020.010.020.010.02#7G0.050.050.030.040.070.040.02#8C0.010.010.010.010.030.030.03#9A0.020.160.032.441.170.170.03#10C0.000.010.010.010.020.020.02#11A0.020.030.030.100.060.010.02#12G0.040.030.040.040.030.030.03#13T0.010.040.030.010.010.010.00#14T0.020.020.010.010.010.000.02#15T0.000.010.010.010.020.010.00#16T0.000.030.040.000.010.010.03#17A0.000.010.020.020.030.010.00#18A0.030.000.000.000.010.010.00#19A0.010.030.020.010.020.020.00#20T0.000.020.010.000.000.030.02#21A0.000.010.010.020.030.010.02#22G0.020.030.020.020.000.020.00#23T0.020.020.020.020.010.030.02.

[1015] Target: Site3PositionNucleobasedCas12a (236-RRR)-ABE-NLS6dCas12a (236)-ABE-NLS6dCas12a (236-RRR)-ABEdCas12a (236-RR)-ABEdCas12a (236-R)-ABEdCas12a (236)-ABEUNTPAMT0.030.020.020.000.000.020.05PAMT0.000.020.000.020.020.000.02PAMT0.010.010.010.010.010.010.01PAMG0.010.060.030.070.040.070.08#1G0.040.000.010.020.030.030.06#2T0.000.010.020.010.020.010.02#3T0.030.020.020.020.040.000.00#4T0.010.010.020.030.010.040.02#5A0.110.150.140.040.120.080.05#6A0.040.050.050.020.030.010.02#7A0.010.010.020.080.060.020.00#8C0.050.050.030.060.010.060.05#9A0.100.580.045.662.630.300.00#10C0.030.010.000.020.020.020.02#11A0.050.380.022.761.320.240.02#12C0.050.010.030.040.010.080.01#13C0.070.060.080.090.070.110.09#14G0.090.150.080.140.120.140.17#15G0.090.080.080.120.140.160.08#16G0.060.080.010.050.050.040.05#17T0.020.010.000.010.000.010.00#18T0.020.030.020.020.010.010.00#19A0.010.060.000.120.040.010.00#20A0.000.010.030.010.040.010.00#21T0.000.010.000.020.010.010.03#22A0.040.070.030.770.500.080.02#23A0.040.050.020.040.040.010.04.

[1016] Target: VEGFAPositionNucleobasedCas12a (236-RRR)-ABE-NLS6dCas12a (236)-ABE-NLS6dCas12a (236-RRR)-ABEdCas12a (236-RR)-ABEdCas12a (236-R)-ABEdCas12a (236)-ABEUNTPAMT0.010.010.000.020.010.030.03PAMT0.010.020.000.000.020.010.03PAMT0.020.020.020.020.020.000.01PAMG0.050.010.050.050.040.010.02#1C0.030.020.010.060.030.050.04#2T0.030.010.010.030.050.020.01#3C0.010.020.020.040.030.020.01#4T0.070.040.040.040.060.020.03#5C0.020.060.030.040.040.010.02#6A0.000.010.020.010.030.000.02#7A0.040.060.000.160.100.010.03#8G0.030.020.010.020.020.020.04#9A0.110.380.093.302.040.290.02#10C0.030.050.050.020.040.050.02#11C0.030.040.060.030.070.020.06#12C0.050.040.040.050.010.040.04#13A0.020.010.060.030.050.030.01#14C0.020.020.040.010.000.020.01#15A0.010.010.030.000.070.030.01#16A0.020.000.040.030.020.020.01#17T0.040.010.030.040.010.020.01#18C0.030.050.030.050.030.060.02#19C0.040.020.060.040.050.030.05#20A0.030.050.030.020.080.060.03#21G0.070.040.060.070.050.060.03#22G0.040.050.060.030.030.020.07#23C0.020.050.050.060.070.040.03.

[1017] Experimental results showed that the base editing system of the above configuration was active in converting adenine to guanine within the range of approximately the 8th to 11th bases based on the protospacer sequence. Among these, the activity of Example 10.4 and Example 10.5 appeared to be particularly strong. In addition, depending on the target, the system also showed activity in converting adenine to guanine within the range of the 17th to 22nd bases. Furthermore, all base editing systems did not introduce indels into the target nucleic acid.

[1018] Experimental Example 11. Confirming Adenine Base Editing System Editing Activity #4

[1019] Experimental Example 11.1. Experimental Method and Configuration Used in the Experiment

[1020] A mutant of the dead-Cas12a protein of SEQ ID NO: 265 was prepared by substituting an amino acid at a specific position with arginine in Experimental Example 10.1 and used in the experiment. An adenine base editor containing the dead-Cas12a protein prepared above was prepared and its activity in editing AT base pairs into GC base pairs was confirmed. Specifically, plasmids encoding each adenine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also prepared. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[1021] The configuration of each base editing system used in Experimental Example 11 is shown in the following table:

[1022] LabelChart LabelABE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 11.1d236-ABE-NLS6409UAAAUUUCUACUGUUGUAGAU (SEQ ID NO: 35)MTAP: 140DYRK1A: 143FANCF: 434Example 11.2d236-ABE (Q190R)-NLS6413(Same as above)(Same as above)Example 11.3d236-ABE (S579R)-NLS6414(Same as above)(Same as above)Example 11.4d236-ABE (Y585R)-NLS6415(Same as above)(Same as above)Example 11.5d236-ABE (Q190R+S579R)-NLS6416(Same as above)(Same as above)Example 11.6d236-ABE (S579R+Y585R)-NLS6417(Same as above)(Same as above)Example 11.7d236-ABE (Q190R+S579R+Y585R)-NLS6410(Same as above)(Same as above)ControlUNT--

[1023] Experimental Example 11.2. Experimental Results

[1024] The experimental results are shown in Figs. 44 and 45.

[1025] Quantitative data for Experimental Example 11 are shown in the following table:

[1026] Target: MTAPPositionNucleobased236-ABE-NLS6d236-ABE (Q190R)-NLS6d236-ABE (S579R)-NLS6d236-ABE (Y585R)-NLS6PAMT0.010.010.020.00PAMT0.080.100.060.11PAMT0.010.020.000.03PAMA0.020.010.030.02#1G0.020.020.020.01#2C0.070.060.070.05#3C0.070.080.050.06#4C0.110.100.120.12#5C0.160.110.110.10#6A0.020.010.010.00#7A0.030.070.040.00#8T0.020.020.010.01#9A2.316.595.390.02#10A0.020.060.050.01#11T0.010.010.010.01#12C0.040.030.050.04#13C0.020.050.040.05#14C0.040.040.030.05#15C0.080.120.060.08#16A0.030.110.080.01#17C0.040.020.020.02#18A0.050.210.140.02#19T0.010.010.010.01#20G0.030.030.040.01#21T0.020.020.020.02#22C0.030.020.020.02#23A0.110.240.260.03

[1027] Target: MTAPPositionNucleobased236-ABE (Q190R+S579R)-NLS6d236-ABE (S579R+Y585R)-NLS6d236-ABE (Q190R+S579R+Y585R)-NLS6UNTPAMT0.000.010.010.00PAMT0.080.050.080.06PAMT0.010.010.020.01PAMA0.000.030.010.03#1G0.010.020.010.02#2C0.050.060.050.06#3C0.100.090.100.08#4C0.100.090.100.13#5C0.100.120.140.15#6A0.010.020.000.02#7A0.050.020.030.01#8T0.010.010.010.00#9A8.640.100.330.01#10A0.080.000.010.02#11T0.010.000.010.01#12C0.030.040.050.06#13C0.070.050.070.05#14C0.040.050.050.04#15C0.090.090.100.06#16A0.120.020.000.01#17C0.020.010.030.03#18A0.290.020.030.02#19T0.010.000.020.00#20G0.050.010.030.02#21T0.010.000.010.02#22C0.030.020.030.04#23A0.290.020.050.03

[1028] Target: DYRK1APositionNucleobased236-ABE-NLS6d236-ABE (Q190R)-NLS6d236-ABE (S579R)-NLS6d236-ABE (Y585R)-NLS6PAMT0.000.010.010.00PAMT0.010.020.020.03PAMT0.010.010.020.01PAMA0.020.030.010.01#1G0.020.010.020.01#2A0.020.020.040.01#3A2.452.602.342.21#4G0.060.040.020.03#5C0.030.030.030.02#6A0.030.060.010.01#7C0.010.020.020.01#8A3.2610.656.350.03#9T0.010.000.010.02#10C0.030.020.020.03#11A0.230.740.420.01#12A0.030.080.070.02#13G0.030.070.040.05#14G0.030.030.020.02#15A0.100.480.150.02#16C0.020.030.010.01#17A0.292.040.800.00#18T0.020.020.010.02#19T0.010.010.010.01#20C0.020.010.020.00#21T0.020.020.010.00#22A0.060.240.100.02#23A0.020.020.020.01

[1029] Target: DYRK1APositionNucleobased236-ABE (Q190R+S579R)-NLS6d236-ABE (S579R+Y585R)-NLS6d236-ABE (Q190R+S579R+Y585R)-NLS6UNTPAMT0.010.010.000.01PAMT0.010.000.010.02PAMT0.020.010.020.01PAMA0.030.010.030.01#1G0.020.020.010.01#2A0.020.020.040.01#3A2.372.362.342.28#4G0.050.020.060.03#5C0.010.030.030.02#6A0.090.000.020.02#7C0.010.010.030.02#8A8.850.090.680.04#9T0.010.010.020.01#10C0.040.020.020.02#11A0.490.020.050.02#12A0.070.030.020.03#13G0.050.050.030.05#14G0.030.020.050.02#15A0.230.010.010.00#16C0.020.010.010.03#17A0.810.020.060.02#18T0.010.010.000.01#19T0.010.010.010.00#20C0.000.010.020.00#21T0.010.010.000.01#22A0.180.010.020.02#23A0.010.020.020.02

[1030] Target: FANCFPositionNucleobased236-ABE-NLS6d236-ABE (Q190R)-NLS6d236-ABE (S579R)-NLS6d236-ABE (Y585R)-NLS6PAMT0.000.010.010.01PAMT0.010.020.030.03PAMT0.010.000.020.00PAMG0.020.040.040.04#1G0.070.060.040.03#2G0.080.080.030.05#3A0.020.010.020.01#4T0.000.000.000.01#5A0.010.030.040.01#6A2.222.152.282.14#7G0.060.020.020.05#8C0.010.010.020.02#9A0.220.910.830.02#10C0.000.020.030.00#11A0.030.060.040.02#12G0.010.040.050.03#13T0.020.010.000.02#14T0.020.010.020.02#15T0.010.040.010.01#16T0.000.000.000.02#17A0.020.030.010.00#18A0.000.010.000.01#19A0.010.010.020.02#20T0.010.010.010.00#21A0.010.010.020.01#22G0.010.010.020.02#23T0.030.010.030.02

[1031] Target: FANCFPositionNucleobased236-ABE (Q190R+S579R)-NLS6d236-ABE (S579R+Y585R)-NLS6d236-ABE (Q190R+S579R+Y585R)-NLS6UNTPAMT0.030.030.020.01PAMT0.030.010.010.02PA MT0.000.030.020.00PAMG0.050.030.040.03#1G0.060.090.030.06#2G0.030.050. 060.05#3A0.000.010.010.02#4T0.000.000.010.03#5A0.040.010.020.01#6A2.28 2.282.202.03#7G0.070.050.040.05#8C0.010.000.020.06#9A1.510.030.070.01# 10C0.000.010.000.01#11A0.070.010.020.02#12G0.030.000.030.02#13T0.040. 000.000.00#14T0.000.020.030.01#15T0.000.010.000.00#16T0.010.000.020.00 #17A0.000.010.010.01#18A0.010.010.020.01#19A0.010.030.020.03#20T0.010. 010.000.02#21A0.030.000.000.01#22G0.020.010.020.04#23T0.010.010.040.02

[1032] Experimental results showed that the base editing systems of the above configuration were active in converting adenine to guanine within the range of approximately the 8th to 11th bases relative to the protospacer sequence. Among these, Example 11.1, Example 11.2, and Example 11.5 appeared to be particularly active. Furthermore, all base editing systems did not introduce indels into the target nucleic acid.

[1033] Experimental Example 12. Confirming Adenine Base Editing System Editing Activity #5

[1034] Experimental Example 12.1. Exploration of R-loop and Ultra Nuclease Mutation Locations

[1035] Referring to previous literature (Zhang, L., Zuris, JA, Viswanathan, R. et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat Commun 12, 3908 (2021). https: / doi.org / 10.1038 / s41467-021-24017-8; Hsiung, C.CS., Wilson, CM, Sambold, NA et al. Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations. Nat Biotechnol (2024). https: / doi.org / 10.1038 / s41587-024-02224-0), we found the Ultra Nuclease mutation site in the Cas12a protein containing the amino acid sequence of SEQ ID NO: 1. In particular, we sought to find the positions corresponding to M537 and F870 of the AsCas12a protein disclosed in the prior literature. In addition, we sought to find the position of the R-loop mutation by referring to the prior literature (Naqvi, MM, Lee, L., Montaguth, OET et al. CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage. Nat Chem Biol 18, 1014-1022 (2022). https: / doi.org / 10.1038 / s41589-022-01082-8). The analysis results are shown in Figures 46 to 50. As a result of the analysis, the positions corresponding to the above Ultra Nuclease mutation were found to be N526 and E791 of the amino acid sequence of SEQ ID NO: 1. In addition, the position corresponding to the above R-loop mutation was found to be W354 of the amino acid sequence of SEQ ID NO: 1.

[1036] The inventors introduced the additional mutations identified above into the dead-Cas12a protein containing the amino acid sequence of SEQ ID NO: 264 to produce a mutant of the dead-Cas12a protein. Furthermore, they produced an adenine base editing system containing the mutant of the dead-Cas12a protein and measured its nucleotide editing activity.

[1037] Experimental Example 12.2. Experimental Method and Configuration Used in the Experiment

[1038] In Experimental Example 7.1, variants were prepared by substituting amino acids at specific positions with arginine, leucine, and alanine, respectively, and used in the experiment. All possible combinations of proteins were prepared, with each mutant position replaced with a suitable amino acid.

[1039] An adenine base editor containing the dead-Cas12a protein prepared above was prepared to determine whether it had the activity of editing AT base pairs into GC base pairs. Specifically, plasmids encoding each adenine base editor protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of operating with the corresponding base editor proteins were also prepared. Each plasmid was delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing occurred.

[1040] The configuration of each base editing system used in Experimental Example 12 is shown in the following table:

[1041]

[1042] LabelChart LabelABE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 12.1d218-ABE-NLS6284UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)DYR1A: 143CFTR: 435FANCF: 434Example 12.2d218-ABE (E155R)-NLS6418(Same as above)(Same as above)Example 12.3d218-ABE (E791L)-NLS6419(Same as above)(Same as above)Example 12.4d218-ABE (N526R+E791L)-NLS6420(Same as above)(Same as above)Example 12.5d218-ABE (E155R+W354A)-NLS6421(Same as above)(Same as above)Example 12.6d218-ABE (E155R+N526R)-NLS6422(Same as above)(Same as above)Example 12.7d218-ABE (E155R+E791L)-NLS6423(Same as above)(Same as above)Example 12.8d218-ABE (E155R+N526R+E791L)-NLS6424(Same as above)(Same as above)Example 12.9d218-ABE (W354A)-NLS6425(Same as above)(Same as above)Example 12.10d218-ABE (N526R)-NLS6426(Same as above)(Same as above)ControlUNT---

[1043] Experimental Example 12.3. Experimental Results

[1044] The experimental results are shown in Figs. 51 to 53.

[1045] Quantitative data for Experimental Example 12 are shown in the following table:

[1046] Target: DYRK1APositionNucleobased218-ABE-NLS6d218-ABE (E155R)-NLS6d218-ABE (E791L)-NLS6d218-ABE (N526R+E791L)-NLS6d218-ABE (E155R+W354A)-NLS6PAMT0.020.010.010.010.00PAMT0.020.000.010.020.03PAMT0.010.000.010.000.01PAMA0.030.020.040.020.00#1G0.040.020.010.000.01#2A0.010.010.020.000.00#3A0.050.000.030.010.05#4G0.040.040.040.020.05#5C0.040.020.010.020.03#6A0.040.060.070.020.01#7C0.010.000.010.030.01#8A24.5312.8821.6611.1921.10#9T0.010.020.000.020.03#10C0.010.000.010.010.03#11A9.403.958.964.225.19#12A0.540.130.500.240.34#13G0.010.050.110.030.04#14G0.030.020.030.010.03#15A0.340.090.310.100.58#16C0.020.000.040.020.01#17A1.130.331.160.461.37#18T0.040.000.050.020.04#19T0.020.020.000.020.01#20C0.010.020.020.020.00#21T0.050.000.020.010.03#22A0.040.020.070.020.00#23A0.010.020.050.030.05

[1047]

[1048] Target: DYRK1APositionNucleobased218-ABE (E155R+N526R)-NLS6d218-ABE (E155R+E791L)-NLS6d218-ABE (E155R+N526R+E791L)-NLS6d218-ABE (W354A)-NLS6d218-ABE (N526R)-NLS6UNTPAMT0.000.010.020.000.000.00PAMT0.010.020.000.010.000.00PAMT0.010.010.000.010.020.01PAMA0.020.030.030.010.000.03#1G0.030.030.030.010.020.01#2A0.030.020.000.010.000.02#3A0.030.010.020.020.010.02#4G0.050.050.060.030.030.03#5C0.020.060.030.030.060.02#6A0.020.090.010.030.020.01#7C0.030.020.010.030.040.01#8A14.6128.4521.270.927.650.03#9T0.010.010.030.030.010.02#10C0.020.040.030.000.050.02#11A3.959.166.060.202.820.00#12A0.190.510.250.030.110.01#13G0.070.060.050.080.030.01#14G0.030.040.020.020.020.01#15A0.130.670.200.000.080.00#16C0.020.020.040.020.010.03#17A0.371.960.630.030.340.01#18T0.030.000.020.050.000.05#19T0.010.020.000.010.000.01#20C0.010.010.010.000.000.01#21T0.000.070.030.020.010.02#22A0.050.040.070.000.030.01#23A0.020.020.010.040.060.03

[1049]

[1050] Target: CFTRPositionNucleobased218-ABE-NLS6d218-ABE (E155R)-NLS6d218-ABE (E791L)-NLS6d218-ABE (N526R+E791L)-NLS6d218-ABE (E155R+W354A)-NLS6PAMT0.020.000.040.000.01PAMT0.010.000.000.010.00PAMT0.010.000.010.000.00PAMC0.490.640.620.640.72#1C0.070.070.060.040.14#2T0.080.040.030.010.03#3G0.120.110.090.130.08#4A0.000.050.010.010.04#5C0.020.040.010.000.03#6T0.000.000.000.020.00#7G0.050.040.100.100.06#8C0.010.020.000.040.05#9T0.010.000.030.030.04#10C0.060.080.050.050.07#11A2.785.856.720.467.18#12C0.080.020.040.070.06#13T0.020.030.030.030.02#14A0.391.251.040.053.08#15G0.090.050.070.030.02#16A0.010.050.090.010.09#17T0.020.000.010.000.02#18G0.000.000.010.010.01#19T0.010.020.010.010.00#20T0.020.000.010.010.00#21G0.060.040.010.020.04#22A0.030.010.010.030.03#23A0.030.080.020.040.01

[1051] Target: CFTRPositionNucleobased218-ABE (E155R+N526R)-NLS6d218-ABE (E155R+E791L)-NLS6d218-ABE (E155R+N526R+E791L)-NLS6d218-ABE (W354A)-NLS6d218-ABE (N526R)-NLS6UNTPAMT0.030.000.010.020.050.01PAMT0.030.040.000.000.010.03PAMT0.010.000.010.000.010.00PAMC0.790.540.500.530.470.47#1C0.040.120.030.030.050.03#2T0.050.040.040.000.060.00#3G0.100.120.110.110.060.06#4A0.020.000.040.000.010.03#5C0.040.020.010.000.060.01#6T0.010.020.000.000.030.00#7G0.050.050.050.100.030.10#8C0.040.040.040.030.030.01#9T0.030.010.070.010.010.02#10C0.050.060.050.130.040.10#11A1.6916.833.350.030.150.02#12C0.000.090.050.090.060.07#13T0.030.020.050.040.000.01#14A0.393.580.600.010.000.00#15G0.020.010.010.040.010.08#16A0.000.090.050.010.010.03#17T0.000.000.000.000.010.00#18G0.010.050.040.020.010.04#19T0.010.010.000.060.000.00#20T0.000.010.000.010.000.01#21G0.050.010.030.060.040.04#22A0.010.080.010.040.010.01#23A0.000.010.000.000.020.04

[1052]

[1053] <h2 style=";text-align:left;direction:ltr">Target: FANFPositionNucleobased218-ABE-NLS6d218-ABE (E155R)-NLS6d218-ABE (E791L)-NLS6d218-ABE (N526R+E791L)-NLS6d218-ABE (E155R+W354A) - NLS6PAMT0.010.000.010.000.01PAMT0.040.020.040.030.01PAMT0.000.020.01 0.000.01PAMG0.030.070.040.050.01#1G0.040.060.040.040.03#2G0.070.020.040.050.01#3A0 .000.030.030.010.00#4T0.000.010.000.000.01#5A0.010.010.000.010.02#6A0.040.020.000.020.01#7G0.020.030.040.040.02#8C0.010.020.010.000.02#9A3.631.612.881.276.86#10C0.0 50.010.000.020.03#11A1.430.630.990.621.11#12G0.020.020.030.030.00#13T0.020.010.010 .000.00#14T0.000.000.000.020.02#15T0.010.010.010.000.00#16T0.020.000.020.020.00#17 A0.030.010.030.020.12#18A0.000.010.000.000.01#19A0.020.050.020.010.03#20T0.010.010 .000.000.01#21A0.030.040.000.020.01#22G0.000.020.020.030.00#23T0.020.010.020.020.01<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[1054] Target: FANCFPositionNucleobased218-ABE (E155R+N526R)-NLS6d218-ABE (E155R+E791L)-NLS6d218-ABE (E155R+N526R+E791L)-NLS6d218-ABE (W354A)-NLS6d218-ABE (N526R)-NLS6UNTPAMT0.010.010.010.010.010.01PAMT0.030.020.010.000.050.02PAMT0.030.010.040.000.010.01PAMG0.030.060.020.020.040.05#1G0.040.040.020.080.070.04#2G0.030.060.000.060.020.03#3A0.030.030.010.010.020.04#4T0.000.010.000.010.000.02#5A0.010.000.010.020.010.01#6A0.030.040.030.010.000.02#7G0.020.010.010.050.060.07#8C0.010.010.020.020.000.00#9A2.067.953.390.100.600.04#10C0.020.010.010.000.020.06#11A1.193.691.970.010.270.02#12G0.060.000.000.010.020.01#13T0.010.000.010.030.000.01#14T0.000.000.010.020.010.00#15T0.010.010.010.000.010.03#16T0.010.040.020.010.010.02#17A0.000.050.020.000.020.00#18A0.020.010.000.000.010.02#19A0.010.010.010.020.030.02#20T0.000.030.030.010.010.02#21A0.000.040.030.010.030.01#22G0.010.030.000.000.010.03#23T0.000.000.010.000.020.02

[1055] Experimental results showed that the base-editing systems of the above configurations were active in converting adenines to guanines within the range of approximately the 8th to 11th bases relative to the protospacer sequence. Among these, Example 12.2 and Example 12.7 appeared to be particularly active. Furthermore, none of the base-editing systems introduced indels into the target nucleic acid.

[1056] Experimental Example 13. Confirmation of Base Editing Activity for Multiple Target Nucleic Acids

[1057] Experimental Example 13.1. Experimental Method and Configuration Used in the Experiment

[1058] If the novel CRISPR / Cas12a system disclosed in this specification possesses the characteristics of known CRISPR / Cas12a, the nucleic acid encoding the guide RNA will be capable of self-maturation without the need for additional components such as terminators. Furthermore, even if DNAs encoding multiple guide RNAs are linked and transcription is induced without any additional spacers or terminators, each guide RNA will mature without any problems. The process of self-maturation of multiple guide RNA sequences linked to a single promoter is schematically illustrated in Figure 54. Applying these characteristics, it is very easy to design a vector construct that induces simultaneous editing of multiple target nucleic acids. Referring to Experimental Example 1, a vector construct containing the encoding nucleic acids in the following table was prepared:

[1059] PromoterDirect Repeat #1Guide Domain #1Direct Repeat #2Guide Domain #2Direct Repeat #3Guide Domain #3GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC (SEQ ID NO: 439)TAATTTCTACTAAGTGTAGAT (SEQ ID NO: 50)CTCTCAAGACCCACAATCCAGGC (SEQ ID NO: 427)SEQ ID NO: 50GCCCCAATAATCCCCACATGTCA (SEQ ID NO: 139)SEQ ID NO: 50GAAGCACATCAAGGACATTCTAA (SEQ ID NO: 142)

[1060] A vector construct containing DNA sequences sequentially linked from the 5' end to the 3' end was prepared according to the configuration of the table above. The vector construct prepared above and a plasmid encoding a base editor protein prepared according to Experimental Example 1.9 were delivered to cells according to Experimental Example 1.10. According to Experimental Example 1.11, the cell genome sequence was analyzed to determine whether base editing had occurred.

[1061] The configuration of each base editing system used in Experimental Example 13 is shown in the following table:

[1062] LabelChart LabelBE Protein(SEQ ID NO)Direct repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 13.1Cas12a (218)-ABE283UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)MTAP: 140DYRK1A: 143VEGFA: 433Example 13.2Cas12a (218)-CBE295(Same as above)(Same as above)ControlUNT---

[1063] Experimental Example 13.2. Experimental Results

[1064] The experimental results are shown in Figs. 55 and 56.

[1065] Quantitative data for Experimental Example 13 are shown in the following table:

[1066] Data for CBEPositionVAGFANucleobaseVAGFAEfficiencyVEGFAControlDYRK1ANucleobaseDYRK1AEfficiencyDYRK1AControlMTAPNucleobaseMTAPEfficiencyMTAPControlPAMT0.020.01T0.020.02T0.010.01PAMT0.020.00T0.020.02T0.040.02PAMT0.000.01T0.010.00T0.010.01PAMG0.020.04A0.020.01A0.010.04#1C0.050.03G0.030.04G0.010.02#2T0.010.01A0.010.01C0.070.08#3C0.040.04A0.020.03C0.050.05#4T0.010.02G0.060.07C0.070.04#5C0.350.05C0.020.02C0.110.16#6A0.010.02A0.010.04A0.010.01#7A0.020.01C0.510.01A0.010.02#8G0.050.04A0.020.01T0.030.02#9A0.010.04T0.010.02A0.040.03#10C0.560.02C1.360.03A0.000.02#11C0.520.04A0.010.03T0.000.02#12C0.270.06A0.010.04C2.650.05#13A0.000.00G0.060.07C0.650.05#14C0.020.01G0.120.03C0.040.02#15A0.000.02A0.020.02C0.070.06#16A0.010.04C0.150.01A0.010.02#17T0.030.04A0.020.01C0.020.03#18C0.150.03T0.020.00A0.020.00#19C0.080.01T0.010.02T0.010.02#20A0.050.05C0.340.00G0.020.02#21G0.030.02T0.010.01T0.020.00#22G0.040.06A0.000.02C0.050.04#23C0.040.06A0.020.02A0.010.03

[1067] Data for ABEPositionVAGFANucleobaseVAGFAEfficiencyVEGFAControlDYRK1ANucleobaseDYRK1AEfficiencyDYRK1AControlMTAPNucleobaseMTAPEfficiencyMTAPControlPAMT0.000.01T0.000.02T0.000.01PAMT0.010.00T0.020.02T0.000.02PAMT0.000.01T0.020.00T0.000.01PAMG0.020.04A0.040.01A0.010.04#1C0.030.03G0.040.04G0.020.02#2T0.020.01A0.010.01C0.060.08#3C0.010.04A0.020.03C0.060.05#4T0.050.02G0.010.07C0.040.04#5C0.060.05C0.050.02C0.150.16#6A0.170.02A0.050.04A0.020.01#7A1.800.01C0.010.01A0.490.02#8G0.030.04A13.920.01T0.010.02#9A23.510.04T0.010.02A19.210.03#10C0.070.02C0.020.03A5.010.02#11C0.070.04A4.600.03T0.010.02#12C0.050.06A0.180.04C0.030.05#13A0.530.00G0.060.07C0.080.05#14C0.010.01G0.020.03C0.050.02#15A0.060.02A0.180.02C0.100.06#16A0.010.04C0.020.01A0.170.02#17T0.050.04A0.630.01C0.010.03#18C0.030.03T0.010.00A0.490.00#19C0.060.01T0.000.02T0.000.02#20A0.040.05C0.030.00G0.030.02#21G0.040.02T0.010.01T0.010.00#22G0.010.06A0.000.02C0.020.04#23C0.040.06A0.030.02A0.040.03

[1068] Experimental results confirmed that base editing can be induced simultaneously in multiple target nucleic acids using a vector construct that induces transcription by linking DNA encoding multiple guide RNAs without any additional spacers or terminators.

[1069] Experimental Example 14. Primer sequences used in the experiment

[1070] The primer sequences used in the experimental examples are shown in the following table:

[1071] PRIMER NAMEPRIMER SEQUENCESEQ ID NOID_204_FWDGAAATTAATACGACTCACTATAGGTAATTTCTACTATTCGTAGAT147ID_204_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACGAATAGTAGAAATTA148ID_206_FWDGAAATTAATACGACTCACTATAGGTTAATTTCTACTTTCGTAGAT149ID_206_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACGAAAGTAGAAATTAA150 ID_210_FWDGAAATTAATACGACTCACTATAGGCAATTTCTACTTTCAGTAGAT151ID_210_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACTGAAAGTAGAAATTG152ID_211_FWDGAAATTAATACGACTCACTATAGGTTAATTTCTACTTGTGTAGAT153ID_211_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACACAAGTAGAAATTAA154ID_ 218_FWDGAAATTAATACGACTCACTATAGGTAATTTCTACTAAGTGTAGAT155ID_218_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACACTTAGTAGAAATTA156ID_219_230_FWDGAAATTAATACGACTCACTATAGGTAAATTTCTACTATTGTAGAT157ID_219_230_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACAATAGTAGAAATTTA 158ID_223_FWDGAAATTAATACGACTCACTATAGGAAAATTTCTACTATTGTAGAT159ID_223_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACAATAGTAGAAATTTT160ID_238_FWDGAAATTAATACGACTCACTATAGGATAATTTCTACTGTTGTAGAT161ID_238_RG1_REVCAGGGAGGGGAGGGGCACAGATGAATCTACAACAGTAGAAAATTAT162

[1072]

[1073]

[1074]

[1075]

[1076]

[1077]

[1078] Using the base editing system and base editing method disclosed in this specification:

[1079] 1) AT base pairs contained in the target site of the intracellular target nucleic acid (double-stranded nucleic acid) can be changed to GC base pairs.

[1080] 2) CG base pairs contained in the target site of the intracellular target nucleic acid (double-stranded nucleic acid) can be changed to TA base pairs.

[1081] Therefore, the above base editing system can be used to treat diseases caused by mutations in some base pairs in the genome.

Claims

1. Adenine-based editor composition comprising: an adenine base editor protein, or a nucleic acid encoding said adenine base editor protein; wherein the adenine base editor protein comprises a dead-Cas12a protein, one or more adenine deaminases, and one or more nuclear localization signals; a guide RNA or a nucleic acid encoding said guide RNA, Here, the guide RNA comprises a scaffold and a guide domain; Here, the scaffold comprises a direct repeat portion, The dead-Cas12a protein of the above direct repeat portion and the adenine base editor protein is a combination selected from the following: A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 33; or A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO:

35.

2. In the first paragraph, the adenine base editor protein has a structure that satisfies the following conditions: wherein the dead-Cas12a protein and at least one adenine deaminase are linked directly or via an amino acid linker; One or more nuclear localization signals are located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the above adenine base editor.

3. In any one of Examples 27 to 31, the adenine base editor protein has the following structure: [NLS1] - [Linker1] - [AD] - [Linker2] - [dead-Cas12a] - [Linker3] - [NLS2] Here, the above NLS1 and NLS2 are each independently a nuclear localization signal or are absent, The above Linker1 and Linker2 are each independently an amino acid linker or are absent, The above AD is adenine deaminase, The above dead-Cas12a is dead-Cas12a protein.

4. In any one of claims 1 to 3, the adenine base editor protein comprises an amino acid sequence selected from the following: SEQ ID NO: 283 to SEQ ID NO: 294; SEQ ID NO: 412 to SEQ ID NO: 414; SEQ ID NO: 416; and SEQ ID NO: 418 to SEQ ID NO:

424.

5. A method of changing the A and T base pairs of the target site contained in the target nucleic acid within the cell to G and C base pairs. The above method includes: Delivering to a cell an adenine base editor composition of any one of claims 1 to 4; Here, the adenine base editor protein and the guide RNA combine to form a base editor complex, The base editor complex contacts the target nucleic acid to change the A and T base pairs of the target site contained in the target nucleic acid into G and C base pairs.

6. In paragraph 5, The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand, The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto, When the above method is performed, A included in the second target site changes to G, and T in the corresponding first target site changes to C. The above base editor complex recognizes a sequence consisting of the i-th nucleic acid to the (i+3)-th nucleic acid upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM), The guide domain of the above guide RNA hybridizes with a second target site within the target strand and a sequence consisting of the first nucleic acid to the (i-1)th nucleic acid downstream from the 3' end of the second target site, The above i is an integer.

7. In paragraph 6, i is 7 or 8.

8. In any one of paragraphs 5 to 7, the target portion is a double-stranded nucleic acid having a length of 4 bp to 14 bp.

9. A cytosine base editor composition comprising: a cytosine base editor protein, or a nucleic acid encoding said cytosine base editor protein; wherein the cytosine base editor protein comprises a dead-Cas12a protein, one or more cytosine deaminases, one or more uracil glycosylase inhibitors, and one or more nuclear localization signals; a guide RNA or a nucleic acid encoding said guide RNA, Here, the guide RNA comprises a scaffold and a guide domain; Here, the scaffold comprises a direct repeat portion, The dead-Cas12a protein of the above direct repeat portion and the cytosine base editor protein is a combination selected from the following: A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264, and SEQ ID NO: 264, and SEQ ID NO: 266 to SEQ ID NO: 272, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO: 33; or A dead-Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 265, and SEQ ID NO: 265, and SEQ ID NO: 273 to SEQ ID NO: 274, and a direct repeat comprising a nucleic acid sequence of SEQ ID NO:

35.

10. In the 9th paragraph, the cytosine base editor protein has a structure that satisfies the following conditions: wherein the dead-Cas12a protein and at least one cytosine deaminase are linked directly or via an amino acid linker; wherein the dead-Cas12a protein and at least one uracil glycosyltransferase inhibitor are linked directly, via a nuclear localization signal, or via an amino acid linker; One or more nuclear localization signals are located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the cytosine base editor.

11. In any one of Examples 32 to 36, the cytosine base editor protein has the following structure: [NLS1] - [Linker1] - [CD] - [Linker2] - [dead-Cas12a] - [Linker3] - [UGI] - [Linker4] - [NLS2] Here, the NLS1 and NLS2 are each independently a nuclear localization signal or are absent, The above Linker1 and Linker2 are each independently an amino acid linker or are absent, The above Linker3 is an amino acid linker, a nuclear localization signal, or is absent, The above CD is cytosine deaminase, The above dead-Cas12a is a dead-Cas12a protein, The above UGI is a uracil glycosylase inhibitor.

12. In any one of claims 9 to 11, the cytosine base editor protein comprises an amino acid sequence selected from the following: SEQ ID NO: 295 to SEQ ID NO: 296; and SEQ ID NO:

405.

13. A method of changing the C and G base pairs of the target site contained in the target nucleic acid within the cell to T and A base pairs. The above method includes: Delivering to a cell a cytosine base editor composition of any one of claims 9 to 12; Here, the cytosine base editor protein and the guide RNA combine to form a base editor complex, The above base editor complex contacts the target nucleic acid and changes the C and G base pairs of the target site contained in the target nucleic acid into T and A base pairs.

14. In paragraph 13, The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand, The target site comprises a first target site that is part of the target strand, and a second target site that is part of the non-target strand, complementary thereto, When the above method is performed, C included in the second target site changes to T, and G in the corresponding first target site changes to A. The above base editor complex recognizes a sequence consisting of the i-th nucleic acid to the (i+3)-th nucleic acid upstream from the 5' end of the first target site as a protospacer adjacent motif (PAM), The guide domain of the above guide RNA hybridizes with a second target site within the target strand and a sequence consisting of the first nucleic acid to the (i-1)th nucleic acid downstream from the 3' end of the second target site, Here, i is an integer.

15. In paragraph 14, i is an integer between 5 and 7.

16. In any one of claims 13 to 15, the target region is a double-stranded nucleic acid having a length of 7 bp to 14 bp.

Images