Novel cas12a protein, variant of novel cas12a protein, and use thereof for eukaryotic genome editing

Abstract

The present specification discloses a novel CRISPR / Cas12a system capable of eukaryotic genome editing. The novel CRISPR / Cas12a system comprises: a novel Cas12a protein or a variant thereof (or a nucleic acid encoding same); and a programmable guide RNA (or a nucleic acid encoding same). Here, the Cas12a protein or a variant thereof may be linked to one or more nuclear localization signals. A target nucleic acid in a eukaryotic genome can be edited by using the novel CRISPR / Cas12a system disclosed in the present specification and a method for editing a eukaryotic genome. Therefore, the novel CRISPR / Cas12a system can be utilized in techniques in various contexts requiring eukaryotic genome editing.

Description

Novel Cas12a proteins, variants of the novel Cas12a proteins, and their applications in eukaryotic genome editing

[0001] This specification covers technologies related to the CRISPR / Cas system, particularly the CRISPR / Cas12a system, and methods for editing eukaryotic genomes using the same.

[0002]

[0003] 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]

[0021] The technical task of this specification is to identify a novel Cas12a protein that can be used for eukaryotic genome editing among several candidate Cas12a proteins, and further to disclose a novel Cas12a variant that introduces additional mutations into the novel Cas12a protein. Furthermore, the technical task is to propose a CRISPR / Cas12a system that can be used for actual eukaryotic genome editing, comprising the novel Cas12a protein or a variant thereof.

[0022]

[0023] To address the above technical challenges, the inventors of this specification:

[0024] 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.

[0025] 2) Based on the novel Cas12a protein above, a variant of the above Cas12a protein was manufactured by introducing a new mutation that improves PAM recognition ability.

[0026] 3) A CRISPR / Cas12a system containing the above Cas12a protein and a variant of the Cas12a protein was manufactured.

[0027] 4) It was demonstrated that the above CRISPR / Cas12a system can edit the eukaryotic genome.

[0028] Accordingly, this specification discloses a novel CRISPR / Cas12a system that has been proven to be effective and a method for eukaryotic genome editing using the same.

[0029] Furthermore, by applying the eukaryotic genome editing method, a method for editing nucleic acids within the cytoplasm and a method for editing multiple target nucleic acids are also disclosed.

[0030]

[0031] The novel CRISPR / Cas12a system and eukaryotic genome editing method disclosed in this specification enable editing of target nucleic acids within the eukaryotic genome. Therefore, the novel CRISPR / Cas12a system can be utilized in a variety of contexts requiring eukaryotic genome editing.

[0032]

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 of each mutation sequence represents the protospacer sequence, and the portion marked with a "-" represents the nucleotide deleted as a result of gene editing.

[0048] 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 of each mutation sequence represents the protospacer sequence, and the portion marked with a "-" represents the nucleotide deleted as a result of gene editing.

[0049] 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 of each mutation sequence represents the protospacer sequence, and the portion marked with a "-" represents the nucleotide deleted as a result of gene editing.

[0050] 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.

[0051] Figure 19 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.

[0052] Figure 20 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.

[0053] Figure 21 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 5. Targeting the DYRK1A gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 5.2.

[0054] Figure 22 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 5. Targeting the MTAP gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 5.2.

[0055] Figure 23 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 5. Targeting the VEGFA gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 5.2.

[0056] Figure 24 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 5. Targeting the FANCF gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 5.2.

[0057] Figure 25 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 5. Targeting the CFRT gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 5.2.

[0058] Figure 26 shows a violin plot of the entire eukaryotic genome editing activity data according to Experimental Example 5. Results are shown for a Cas12a protein comprising the amino acid sequence of SEQ ID NO: 1 and a Cas12a protein with the E155R mutation introduced into the amino acid sequence.

[0059] 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 3 to a target nucleic acid, simulated using AlphaFold.

[0060] 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 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.

[0061] Figure 29 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 6. Targeting the DYRK1A gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 6.2.

[0062] Figure 30 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 6. Targeting the CFTR gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 6.2.

[0063] Figure 31 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 6. Targeting the VEGFA gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 6.2.

[0064] Figure 32 shows a violin plot of the entire eukaryotic genome editing activity data according to Experimental Example 6. Results are shown for a Cas12a protein comprising the amino acid sequence of SEQ ID NO: 3, and a Cas12a protein with the Q190R and S579R mutations introduced into the amino acid sequence.

[0065] Figure 33 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.

[0066] Figure 34 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.

[0067] Figure 35 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.

[0068] Figure 36 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.

[0069] Figure 37 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 results show that 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.

[0070] Figure 38 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the FANCF gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0071] Figure 39 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the VEGFA gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0072] Figure 40 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the Site3 region of HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0073] Figure 41 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the MTAP gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0074] Figure 42 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the CFTR gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0075] Figure 43 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 7. Targeting the DYRK1A gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 7.2.

[0076] Figure 44 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the FANCF gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0077] Figure 45 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the VEGFA gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0078] Figure 46 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the CFTR gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0079] Figure 47 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the MTAP gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0080] Figure 48 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the DYRK1A gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0081] Figure 49 shows the results of a eukaryotic genome editing activity experiment according to Experimental Example 8. Targeting the Site3 gene in HEK cells, the frequency of indel introduction for each CRISPR / Cas12a system is depicted. The composition of each label in the chart is disclosed in the table in Experimental Example 8.1.

[0082] Figure 50 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.

[0083] Figure 51 shows the results of multiple eukaryotic genome editing according to Experimental Example 9. The experiment targeted VEGFA, MTAP, and DYRK1A simultaneously. The graph on the left for each target shows the results of editing using the actual CRISPR / Cas12a system, while the dots on the right represent the negative control group without any target editing.

[0084]

[0085] Hereinafter, the best mode for carrying out the invention is disclosed by way of example. This includes some, but not all, implementations of the invention disclosed in this specification. 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.

[0086] This specification discloses a method for editing a target nucleic acid within a eukaryotic genome, comprising:

[0087] A process for delivering a composition for eukaryotic genome editing to a eukaryotic cell,

[0088] Herein, the composition for editing the eukaryotic genome comprises:

[0089] a Cas12a protein having one or more nuclear localization signals (NLS) linked thereto, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked thereto; and

[0090] a guide RNA, or a nucleic acid encoding said guide RNA;

[0091] Here, the guide RNA comprises a guide domain and a scaffold;

[0092] Here, the guide domain targets the target nucleic acid,

[0093] The above Cas12a protein and the above scaffold are a combination selected from the following:

[0094] A scaffold comprising a Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, and a nucleic acid sequence of SEQ ID NO: 33; or

[0095] A scaffold comprising a Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 275 to SEQ ID NO: 279, and a nucleic acid sequence of SEQ ID NO: 35.

[0096] As an example,

[0097] A Cas12a protein linked to one or more of the above nuclear localization signals may have the following structure:

[0098] [NLS1] - [Cas12a] - [NLS2]

[0099] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94,

[0100] The above Cas12a is the Cas12a protein described above,

[0101] The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94,

[0102] At least one of the above NLS1 or NLS2 is present.

[0103] As an example,

[0104] In the structure of the Cas12a protein linked to one or more nuclear localization signals,

[0105] The above NLS1 is SEQ ID NO: 67, and the above NLS2 is SEQ ID NO: 67; or

[0106] The above NLS~1 may be absent, and the above NLS2 may be sequence number 70.

[0107] As an example,

[0108] The eukaryotic genome editing composition may include a CRISPR / Cas12a complex in which the guide RNA is bound to a Cas12a protein linked to one or more nuclear localization signals.

[0109] As an example,

[0110] The eukaryotic genome editing composition may comprise a nucleic acid encoding a Cas12a protein linked to one or more nuclear localization signals and a nucleic acid encoding the guide RNA.

[0111] This specification discloses a composition for eukaryotic genome editing comprising:

[0112] a Cas12a protein having one or more nuclear localization signals linked thereto, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked thereto; and

[0113] a guide RNA or a nucleic acid encoding said guide RNA,

[0114] Here, the guide RNA comprises a scaffold and a guide domain,

[0115] The 3' end of the above scaffold and the 5' end of the above guide domain are connected;

[0116] Here, the guide RNA binds to the Cas12a protein to form a complex,

[0117] The guide domain of the above guide RNA can target a predetermined target nucleic acid,

[0118] The above Cas12a protein and the above scaffold are a combination selected from the following:

[0119] A Cas12a protein comprising an amino acid sequence of SEQ ID NO: 1, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 33;

[0120] A Cas12a protein comprising the amino acid sequence of SEQ ID NO: 3, and a scaffold comprising the nucleic acid sequence of SEQ ID NO: 35; or

[0121] A Cas12a protein comprising an amino acid sequence of SEQ ID NO: 2, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 34.

[0122] As an example,

[0123] The composition may include a complex in which the Cas12a protein and the guide RNA are bound.

[0124] As an example,

[0125] The composition may include a nucleic acid encoding the Cas12a protein and a nucleic acid encoding the guide RNA.

[0126] As an example,

[0127] A Cas12a protein linked to one or more of the above nuclear localization signals may have the following structure:

[0128] [NLS1] - [Cas12a] - [NLS2]

[0129] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94,

[0130] The above Cas12a is the Cas12a protein described above,

[0131] The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94,

[0132] At least one of the above NLS1 or NLS2 is present.

[0133] This specification discloses a CRISPR / Cas12a composition comprising:

[0134] Cas12a protein, or a nucleic acid encoding said Cas12a protein; and

[0135] a guide RNA or a nucleic acid encoding said guide RNA,

[0136] Here, the guide RNA comprises a scaffold and a guide domain,

[0137] The 3' end of the above scaffold and the 5' end of the above guide domain are connected;

[0138] Here, the guide RNA binds to the Cas12a protein to form a complex,

[0139] The guide domain of the above guide RNA can target a predetermined target nucleic acid,

[0140] The above Cas12a protein and the above scaffold are a combination selected from the following:

[0141] A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264 to SEQ ID NO: 274, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 33;

[0142] A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 275 to SEQ ID NO: 279, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 35.

[0143] As an example,

[0144] The composition may include a complex in which the Cas12a protein and the guide RNA are bound.

[0145] As an example,

[0146] The composition may include a nucleic acid encoding the Cas12a protein and a nucleic acid encoding the guide RNA.

[0147] As an example,

[0148] The above Cas12a protein further comprises one or more nuclear localization signals,

[0149] The above Cas12a protein may have the following structure:

[0150] [NLS1] - [Cas12a] - [NLS2]

[0151] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94,

[0152] The above Cas12a is the Cas12a protein described above,

[0153] The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94,

[0154] At least one of the above NLS1 or NLS2 is present.

[0155] This specification discloses a method of editing two or more target nucleic acids within a eukaryotic genome, the method comprising:

[0156] (a) delivering a Cas12a protein having one or more nuclear localization signals linked to said eukaryotic cell, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked to said eukaryotic cell;

[0157] (b) delivering the vector construct to the eukaryotic cell;

[0158] Here, the vector structure comprises DNA encoding a first guide RNA and DNA encoding a second guide RNA,

[0159] The first guide RNA is composed of a first guide domain and a first scaffold,

[0160] The first guide domain targets a first target nucleic acid in the eukaryotic genome,

[0161] The second guide RNA is composed of a second guide domain and a second scaffold,

[0162] The second guide domain targets a second target nucleic acid within the eukaryotic genome,

[0163] The DNA encoding the first guide RNA is operably linked to a promoter,

[0164] The DNA encoding the second guide RNA is directly linked to the 3' end of the first guide RNA;

[0165] Here, the above processes (a) and (b) may be performed simultaneously or in any order,

[0166] The Cas12a protein, the first scaffold, and the second scaffold are a combination selected from the following:

[0167] A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, a first scaffold comprising a nucleic acid sequence of SEQ ID NO: 33, and a second scaffold comprising a nucleic acid sequence of SEQ ID NO: 33; or

[0168] A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 275 to SEQ ID NO: 279, a first scaffold comprising a nucleic acid sequence of SEQ ID NO: 35, and a second scaffold comprising a nucleic acid sequence of SEQ ID NO: 35.

[0169]

[0170] Hereinafter, the present invention will be described in more detail with reference to the attached drawings, through specific implementation examples and examples. It should be noted that the attached drawings include some, but not all, implementation examples of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementation examples described herein. These implementation examples 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 in this specification. Therefore, the invention disclosed in this specification is not limited to the specific implementation examples described herein, and it should be understood that modifications and other implementations thereof are included within the scope of the claims.

[0171]

[0172] Definition of terms

[0173] approximately

[0174] The term "about" as used in this specification means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0175] Amino acid sequence notation

[0176] 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.

[0177] 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).

[0178] Nucleic acid sequence notation

[0179] The symbols A, T, C, G, and U used in this specification are to be interpreted as meanings understood by a person of ordinary skill 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.

[0180] target gene or target nucleic acid

[0181] As used herein, "target gene" or "target nucleic acid" basically refers to a gene or nucleic acid within a cell that is the target of gene editing. The terms "target gene" and "target nucleic acid" may be used interchangeably and may refer to the same subject. Unless otherwise specified, the target gene or target nucleic acid may refer to either a gene or nucleic acid inherent to the target cell or an exogenous gene or nucleic acid, and is not particularly limited as long as it can be the target of gene editing. The target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and / or RNA. Furthermore, the terms encompass all meanings that can be recognized by those skilled in the art and may be appropriately interpreted depending on the context.

[0182] Target sequence

[0183] As used herein, "target sequence" refers to a specific sequence that a CRISPR / Cas complex recognizes to cleave a target gene or target nucleic acid. The target sequence may be appropriately selected depending on the purpose. Specifically, the "target sequence" refers to a sequence contained within a target gene or target nucleic acid sequence, and a sequence that is complementary to a spacer sequence contained in a guide RNA or engineered guide RNA provided herein. Generally, the spacer sequence is determined in consideration of the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR / Cas system. The target sequence may refer only to a specific strand that complementarily binds to the guide RNA of the CRISPR / Cas complex, or may refer to the entire target double strand including a portion of the specific strand, which shall be appropriately interpreted depending on the context. In addition, the term includes all meanings that can be recognized by a person skilled in the art, and may be appropriately interpreted depending on the context.

[0184] vector

[0185] As used herein, "vector" refers to any material capable of transporting genetic material into a cell, unless otherwise specified. For example, a vector may be, but is not limited to, a DNA molecule containing the target genetic material, such as a nucleic acid encoding an effector protein of the CRISPR / Cas system and / or a nucleic acid encoding a guide RNA. The term encompasses all meanings that would be understood by those skilled in the art and may be appropriately interpreted depending on the context.

[0186]

[0187] Limitations of prior art

[0188] The challenge of discovering a CRISPR / Cas12a system capable of eukaryotic genome editing.

[0189] Currently, various bacterial CRISPR / Cas systems are being studied and continuously discovered. However, CRISPR / Cas systems are fundamentally derived from the prokaryotic immune system. Therefore, while they function well in prokaryotes, they do not function well in eukaryotic cells due to various factors. Many studies have been conducted to utilize the CRISPR / Cas system for eukaryotic gene editing (e.g., studies utilizing NLSs linked to Cas proteins). However, even with this research, it is not possible to ensure that all CRISPR / Cas systems function in a eukaryotic environment.

[0190] Even if a novel CRISPR / Cas system is discovered and its composition is determined, it is impossible to predict whether the system can edit eukaryotic genes based solely on information about its composition. Therefore, to determine whether a CRISPR / Cas system can be utilized for eukaryotic genome editing, verification through actual experiments is essential.

[0191] CRISPR / Cas12a system comprising the Cas12a protein disclosed in KR 2023-0111399 A

[0192] 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.

[0193]

[0194] A novel CRISPR / Cas12a system capable of editing eukaryotic genomes

[0195] Overview of a Novel CRISPR / Cas12a System Capable of Eukaryotic Genome Editing

[0196] This specification discloses a novel CRISPR / Cas12a system capable of eukaryotic genome editing. The novel CRISPR / Cas12a system comprises a novel Cas12a protein or a variant thereof; and a programmable guide RNA. The Cas12a protein or variant thereof may be linked to one or more nuclear localization signals. The inventors of this specification, based on the CRISPR / Cas12a system disclosed in the above-mentioned prior document 1, 1) found the direct repeat of the 'correct' guide RNA that actually works, 2) selected a novel CRISPR / Cas12a system that can be used for eukaryotic genome editing among the disclosed CRISPR / Cas12a systems, 3) identified a part of the Cas12a protein included in the above-mentioned novel CRISPR / Cas12a system that interacts with the PAM and designed additional mutations at this position, and 4) developed CRISPR / Cas12a systems that can edit a desired site in a eukaryotic genome through actual experiments. Some of the CRISPR / Cas12a systems disclosed in this specification include the Cas12a protein disclosed in the above-mentioned prior document 1, but the eukaryotic genome editing activity of which has not been found. Some of the CRISPR / Cas12a systems disclosed in this specification include variants of the above-mentioned Cas12a protein. The components of the CRISPR / Cas12a system operate as an RNA-protein complex, the CRISPR / Cas12a complex. The CRISPR / Cas12a system is utilized in slightly different forms, depending on the needs: the CRISPR / Cas12a complex, the CRISPR / Cas12a component expression vector, and the CRISPR / Cas12a composition. Below, novel Cas12a proteins, their variants, and programmable guide RNAs are described in more detail, based on the CRISPR / Cas12a complex.

[0197] CRISPR / Cas12a Complex Assembly #1 - Novel Cas12a Protein

[0198] This specification discloses a novel Cas12a protein. Specifically, a Cas12a protein comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 2 is disclosed.

[0199] CRISPR / Cas12a Complex Construction #2 - Novel Cas12a Protein Variants

[0200] This specification discloses a previously unpublished variant of the Cas12a protein capable of eukaryotic genome editing. This variant was discovered by introducing mutations that enhance PAM interaction function, based on the novel Cas12a protein described above. It is well known that Cas proteins recognize and interact with the PAM sequence, and that this is crucial for "target-specific activity" in conjunction with the guide domain of a guide RNA. The inventors of this specification, inspired by previous research, sought to 1) identify the site of PAM interaction and 2) introduce mutations at that site to obtain a Cas12a protein variant with enhanced PAM interaction function. However, not all mutations described in the prior literature resulted in increased gene editing efficiency. Through rigorous experimentation, the inventors of this specification identified "usable" variants that enhance nucleobase editing activity, enhance activity only for specific targets, and exhibit nucleobase editing activity on a variety of targets. The variants are described in detail in the section ‘Novel Cas12a protein variants’.

[0201] CRISPR / Cas12a Complex Configuration #3 - Nuclear Localization Signal

[0202] To use the CRISPR / Cas12a complex for eukaryotic genome editing, the complex must be able to translocate into the eukaryotic nucleus. To translocate the CRISPR / Cas12a complex into the eukaryotic nucleus, a nuclear localization signal (NLS) can be linked to the Cas12a protein. The nuclear localization signal is not limited in type or configuration as long as it delivers the complex to the nucleus of a eukaryotic cell, and any known structure can be used. For example, the nuclear localization signal can be SV40. One or more nuclear localization signals can be linked to the Cas12a protein, and the nuclear localization signals can be linked directly to the Cas12a protein or via an amino acid linker. The specific configuration of the Cas12a protein linked to the nuclear localization signal is described in detail in the 'Possible Embodiments of the Invention' section.

[0203] CRISPR / Cas12a Complex Construction #4 - Programmable Guide RNA

[0204] The CRISPR / Cas12a complex comprises a programmable guide RNA. The programmable guide RNA comprises 1) a scaffold, which is a portion that interacts critically with the Cas12a protein, and 2) a guide domain designed to match the target site. The CRISPR / Cas12a system is known to operate solely with crRNA, without tracrRNA. The scaffold of the programmable guide RNA essentially comprises the direct repeat (DR) portion of the crRNA. The inventors of this specification 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 by re-specifying this portion, they invented a CRISPR / Cas12a system that actually works. The scaffold may further include additional components that contribute to improving gene editing efficiency in addition to the direct repeat portion. The guide domain can target a target sequence and is appropriately designed to match the target nucleic acid to be edited. The specific definitions and relationships of the guide domain sequence, target nucleic acid, target site, target sequence, etc., and their relationships are described in more detail in the relevant sections.

[0205] CRISPR / Cas12a System Feature #1 - Differences from Previous Literature

[0206] As mentioned above, the above prior document 1 (Korean Publication No. KR 2023-0111399 A) discloses a novel ortholog of the Cas12a protein. The above prior document 1 discloses the amino acid sequence of the novel Cas12a protein, the PAM sequence recognized by the novel Cas12a protein, the direct repeat sequence for the novel Cas12a protein, etc. However, the above prior document 1 only tested the intracellular gene editing activity of a very small portion of the novel CRISPR / Cas12a systems, and for the majority of the novel CRISPR / Cas12a systems, it did not provide any evidence as to whether the systems are capable of gene editing in eukaryotic cells. On the other hand, the inventors of this specification re-examined the data on the novel Cas12a protein and guide RNA therefor disclosed in the above-mentioned prior art 1, and through additional research, found that the direct repeat sequence in the above-mentioned guide RNA was incorrectly specified, and re-specified it as the correct sequence. In addition, the inventors of this specification demonstrated through experiments that eukaryotic genes can be edited using the Cas12a protein having the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 3 among the novel Cas12a proteins disclosed in the above-mentioned prior art 1. Furthermore, the inventors of this specification clearly specified a configuration for editing eukaryotic genes using the above-mentioned Cas12a protein, thereby completing the invention of a novel CRISPR / Cas12a system.

[0207] CRISPR / Cas12a System Feature #2 - Novel Guide RNA Specificity

[0208] The inventors of this specification re-examined the data on the novel Cas12a protein and its guide RNA disclosed in the above-mentioned prior art document 1, and through further research, discovered that the direct repeat sequence in the guide RNA was incorrectly specified. The inventors re-specified the direct repeat sequence and, through experiments, discovered that the Cas12a protein having an amino acid sequence selected from SEQ ID NOs: 1 to 3 and the guide RNA operating therewith possess gene editing activity in eukaryotic cells. Specifically, the sequences of the Cas12a protein and the direct repeat that can interact with it to form a complex, as discovered by the inventors, are as follows:

[0209] Cas12a protein of SEQ ID NO: 1 and direct repeat of SEQ ID NO: 33 (or a similar sequence);

[0210] Cas12a protein of SEQ ID NO: 2 and direct repeat of SEQ ID NO: 34 (or a similar sequence); and

[0211] The Cas12a protein of SEQ ID NO: 3 and the direct repeat of SEQ ID NO: 35 (or a similar sequence).

[0212] The Cas12a protein of the above combination and the guide RNA containing the direct repeat can interact to form a complex, which has target-specific nucleic acid cleavage activity.

[0213] CRISPR / Cas12a System Feature #3 - Development of Novel Cas12a Protein Variants

[0214] This specification discloses a novel Cas12a protein variant having a previously unknown configuration. The Cas12a protein variant is prepared by 1) introducing a mutation into a site interacting with PAM (PAM recognition site mutation), 2) introducing a mutation that further increases nucleic acid cleavage efficiency (Ultra Nuclease mutation), or 3) appropriately combining the above mutations, based on the sequence of the novel Cas12a protein discovered above. The inventors of this specification also revealed that the above Cas12a protein variants can be used for eukaryotic genome editing, and some of them exhibit improved gene editing efficiency than the novel Cas12a protein.

[0215]

[0216] Novel Cas12a protein variants

[0217] Overview of novel Cas12a protein variants

[0218] The inventors of this specification disclose novel Cas12a protein variants. The Cas12a protein variants are prepared by 1) introducing mutations into the PAM-interacting site (PAM recognition site mutation), 2) introducing mutations that further enhance nucleic acid cleavage efficiency (Ultra Nuclease mutation), or 3) appropriately combining the above mutations, based on the sequence of the novel Cas12a protein discovered above. For example, the Cas12a protein variants include any one of the amino acid sequences of SEQ ID NO: 264 to SEQ ID NO: 279. Each mutation was inspired by prior literature. Specifically, the novel Cas12a protein was structurally analyzed using a simulation model, and the positions of the mutations were identified by comparing them with the amino acid sequence of the Cas12a protein that disclosed the mutations. As a result of confirming actual gene editing using each mutation or combination of each mutation, it was found that not all mutations disclosed in the prior literature improved gene editing efficiency. Through careful analysis, the inventors of this specification have identified variants that can be used for eukaryotic genome editing because they show better overall efficiency than the wild-type Cas12a protein, better editing efficiency for specific targets, or higher editing efficiency than the wild-type Cas12a protein.

[0219] PAM Recognition Site Mutations #1 - Overview

[0220] This specification discloses a variant of a novel Cas12a protein having a PAM recognition site mutation introduced. Specifically, the mutation is related to a position that interacts with the PAM. For example, a PAM recognition site mutation for the amino acid sequence of SEQ ID NO: 1 is a mutation that replaces each of positions E155, G531, K537, or any combination thereof with a different amino acid. For another example, a PAM recognition site mutation for the amino acid sequence of SEQ ID NO: 3 is a mutation that replaces each of positions Q190, S579, Y585, or any combination thereof with a different amino acid. Here, the mutant amino acid is preferably arginine, which has a strong (+) charge, but is not limited thereto. The inventors of this specification, inspired by prior literature, additionally introduced a PAM recognition site mutation into the novel Cas12a protein to enhance target nucleic acid editing activity. Through careful experimentation, the inventors identified mutations that enhance gene editing activity, mutations that enhance activity only for specific targets, and mutations that are 'usable' because they exhibit a certain level of gene editing activity on a variety of targets.

[0221] PAM Recognition Site Mutation #2 - How Mutations Improve Gene Editing Efficiency

[0222] It is already well known that Cas proteins recognize and interact with the PAM sequence portion and are important for exhibiting 'target-specific activity' together with the guide domain of the guide RNA. 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: According to (30742127; PMCID: PMC6401248.), when specific residues interacting with the PAM sequence portion of the Cas12a protein are replaced with residues having stronger activity, the function of recognizing the target nucleic acid is improved. Specifically, the prior literature identified three positions interacting with PAM in the AsCas12a protein, and revealed that when all of these were replaced with arginine having a strong (+) charge, both gene editing activity and base editing activity increased. The inventors of this specification got an idea from the above study and tried to improve the target nucleic acid editing activity by additionally introducing mutations in the PAM binding portion into the novel Cas12a protein.Specifically, we identified key residues interacting with the PAM sequence through structural analysis, introduced "PAM binding site mutations" by replacing these residues with strongly positively charged amino acids, and examined whether these mutations affected gene editing efficiency. Experimental results revealed that not all PAM binding site mutations resulted in enhanced gene editing activity. Below, we describe the structural prediction method and the process of identifying the PAM binding site introduction site in more detail.

[0223] PAM Recognition Site Mutation #3 - Structure Prediction Using AlphaFold

[0224] 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. 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. The inventors of this specification predicted that substituting these key residues with appropriate amino acids would allow for stronger binding to the PAM sequence, ultimately contributing to improved gene editing efficiency. The inventors of this specification attempted to improve gene editing efficiency by substituting each of the above key residues with arginine, which has a strong (+) charge. Specifically, for each position derived above, we constructed novel Cas12a proteins containing mutations in all possible combinations (mutations at one position, two positions, or all three positions). Furthermore, we constructed CRISPR / Cas12a systems containing each of the above Cas12a proteins, and measured their respective gene editing activities.

[0225] Ultra Nuclease Mutation #1 - Overview

[0226] This specification discloses a novel Cas12a protein with an Ultra Nuclease mutation. Specifically, the mutation is associated with the DNA binding activity of the Cas12a protein or with the function of stabilizing the CRISPR / Cas12a complex. For example, an Ultra Nuclease mutation in the amino acid sequence of SEQ ID NO: 1 refers to a mutation that substitutes a different amino acid at each of positions N526, E791, or any combination thereof. Preferably, the mutant amino acid is arginine for N526, and leucine for E791, but is not limited thereto. The inventors of this specification, inspired by prior literature, additionally introduced an Ultra Nuclease mutation into the novel Cas12a protein to enhance target nucleic acid editing activity. Through meticulous experimentation, the inventors identified mutations that enhance gene editing activity, mutations that enhance activity only for specific targets, and "usable" mutations that exhibit a certain level of gene editing activity on various targets.

[0227] Ultra Nuclease Mutation #2 - How Mutations Improve Gene Editing Efficiency

[0228] The inventors of this specification referred to prior 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) to discover additional mutations. The above prior literature is a study 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 in prior literature 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 novel Cas12a protein would increase the gene editing efficiency. Through sequence alignment and structural analysis, the inventors found an amino acid position in the novel Cas12a corresponding to the mutation position of AsCas12a introduced in the previous study, and designed an Ultra Nuclease mutation that substituted it with arginine or leucine. Thereafter, the Cas12a protein was manufactured by introducing the Ultra Nuclease mutation into the novel Cas12a protein, and a CRISPR / Cas12a system containing the same was manufactured to confirm the eukaryotic genome editing efficiency.

[0229] Any combination of each mutation

[0230] The inventors of this specification predicted that the above PAM recognition site mutation and the above Ultra Nuclease mutation could produce synergy when combined because 1) the mutation introduction sites do not overlap and 2) each site performs a different function. Therefore, the above PAM mutation and the Ultra Nuclease mutation were randomly combined to produce Cas12a protein variants, and a CRISPR / cas12a system containing the same was produced to confirm the efficiency of eukaryotic genome editing.

[0231] Not all transformations are associated with increased editing efficiency.

[0232] Experimental results showed that not all mutations improved gene editing efficiency (see Experimental Examples). Through rigorous experiments, the inventors identified and specified 1) variants that exhibited higher gene editing efficiency than the novel Cas12a protein, 2) variants that exhibited good efficiency only for specific sequences, and 3) variants that exhibited a certain level of gene editing efficiency and were thus usable. For example, Cas12a proteins comprising amino acid sequences selected from among SEQ ID NOs: 264, 269 to 270, and 275 to 277 exhibited higher gene editing efficiency than Cas12a proteins comprising amino acid sequences selected from among SEQ ID NOs: 1 to 3. As another example, Cas12a proteins comprising amino acid sequences selected from SEQ ID NOs: 265 to 268, SEQ ID NOs: 271 to 274, and SEQ ID NOs: 278 to 279 exhibited gene editing effects at a sufficiently usable level when incorporated into the CRISPR / Cas12a system.

[0233]

[0234] programmable guide RNA

[0235] Overview of Programmable Guide RNA

[0236] The CRISPR / Cas12a system disclosed herein comprises a programmable guide RNA (or a nucleic acid encoding the same). The programmable guide RNA can interact with the Cas12a protein to form an RNA-protein complex. The programmable guide RNA 1) interacts with the Cas12a protein to form a complex, and 2) guides the RNA-protein complex to a target sequence near a PAM sequence, thereby enabling gene editing to occur at the intended site. Among these, the portion that interacts with the 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.

[0237] scaffold

[0238] The programmable guide RNA comprises a scaffold. The scaffold can interact with and bind to the Cas12a protein, allowing the programmable guide RNA to form a complex with the Cas12a protein. The scaffold comprises a direct repeat of the crRNA. The inventors of this specification specifically disclose the direct repeat that actually binds to and forms a complex with the Cas12a protein disclosed in this specification. For example, when the Cas12a protein comprises SEQ ID NO: 1 or a variant amino acid sequence of SEQ ID NO: 1, the direct repeat comprises the nucleic acid sequence of SEQ ID NO: 33. In another example, when the Cas12a protein comprises SEQ ID NO: 3 or a variant amino acid sequence of SEQ ID NO: 3, the direct repeat comprises the nucleic acid sequence of SEQ ID NO: 35. In addition to the direct repeat, the scaffold may further comprise an additional RNA domain that imparts structural stability to the programmable guide RNA. For example, the above scaffold may further include a stem-loop. The specific configuration of the above scaffold, etc., is described in the 'Possible Embodiments of the Invention' section.

[0239] Guide domain

[0240] The above programmable guide RNA comprises a guide domain. The guide domain targets a target nucleic acid and guides the RNA-protein complex to bind to the target nucleic acid. The guide domain is designed with a sequence related to the target nucleic acid and enables the CRISPR / Cas12a complex to edit the nucleic acid 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 Cas12a protein 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 CRISPR / Cas12a complex stably binds to that location and can function. The specific structure of the guide domain, etc., is described in the "Possible Embodiments of the Invention" section.

[0241] Structure of programmable guide RNA and additional RNA sequences

[0242] 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.

[0243]

[0244] CRISPR / Cas12a system

[0245] Overview of the CRISPR / Cas12a System

[0246] This specification discloses a CRISPR / Cas12a system. The entity that actually edits the nucleotide is a CRISPR / Cas12a complex, which is a combination of the Cas12a protein described above and a programmable guide RNA. With the advancement of biotechnology, it is also possible to deliver nucleic acids encoding proteins and RNAs to cells, and to express each protein and RNA within the cells to form a CRISPR / Cas12a complex. Therefore, the term "CRISPR / Cas12a system" used in this specification encompasses 1) a CRISPR / Cas12a complex, 2) a vector capable of expressing each component of the CRISPR / Cas12a complex (a CRISPR / Cas12a vector), and 3) a CRISPR / Cas12a composition.

[0247] CRISPR / Cas12a complex

[0248] The above CRISPR / Cas12a system may refer to a CRISPR / Cas12a complex. The CRISPR / Cas12a complex refers to an RNA-protein complex formed by combining the Cas12a protein described above, or a Cas12a protein linked to one or more nuclear localization signals, and a programmable guide RNA. The CRISPR / Cas12a complex actually recognizes the target nucleic acid and edits the nucleobases at the intended target site.

[0249] CRISPR / Cas12a vector

[0250] The above CRISPR / Cas12a system may refer to a CRISPR / Cas12a vector. The above CRISPR / Cas12a vector refers to a vector capable of expressing each component of the CRISPR / Cas12a complex. The above CRISPR / Cas12a vector includes a nucleic acid encoding the Cas12a protein described above, or a Cas12a protein linked to one or more nuclear localization signals, and a nucleic acid encoding the above programmable guide RNA. The above CRISPR / Cas12a vector is configured to express the Cas12a protein and the programmable guide RNA when delivered to a cell. When the above CRISPR / Cas12a vector is delivered into a cell, the Cas12a protein and the programmable guide RNA are expressed in the cell, and they combine to form a CRISPR / Cas12a complex and function as intended. In addition to the nucleic acids encoding each component, the above CRISPR / Cas12a vector includes components for proper expression within the cell. The above 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] CRISPR / Cas12a composition

[0252] The above base editing system may refer to a CRISPR / Cas12a composition. The above CRISPR / Cas12a composition includes a CRISPR / Cas12a protein or a nucleic acid encoding the CRISPR / Cas12a protein; and a programmable guide RNA or a nucleic acid encoding the programmable guide RNA. The above CRISPR / Cas12a composition encompasses the CRISPR / Cas12a complex, CRISPR / Cas12a vector, and various other implementation technologies described above. For example, the above CRISPR / Cas12a composition may include an mRNA encoding the CRISPR / Cas12a protein and a programmable guide RNA.

[0253]

[0254] Eukaryotic genome editing methods

[0255] Overview of Eukaryotic Genome Editing Methods

[0256] This specification discloses a method for editing a eukaryotic genome. The method comprises delivering the CRISPR / Cas12a system described above into a eukaryotic cell. The CRISPR / Cas12a system can target a predetermined target nucleic acid within the eukaryotic genome and edit the target nucleic acid or a portion adjacent thereto.

[0257] CRISPR / Cas12a system for eukaryotic genome editing

[0258] The above eukaryotic genome editing method utilizes the CRISPR / Cas12a system for eukaryotic genome editing. The CRISPR / Cas12a system is designed for eukaryotic genome editing and comprises the following components: a Cas12a protein linked to one or more nuclear localization signals; and a programmable guide RNA that targets a predetermined target nucleic acid within the eukaryotic genome. As described above, the CRISPR / Cas12a system can be implemented in various forms, as detailed in the preceding paragraph.

[0259] Cells targeted for genome editing

[0260] In the above method, the cell targeted for genome editing is a eukaryotic cell. The term "eukaryotic cell" encompasses various cell types and is not limited to any other cell types, as long as the CRISPR / Cas12a system can function. For example, the cell may be a plant cell, a non-human animal cell, or a human cell.

[0261] CRISPR / Cas12a system delivery method

[0262] The above eukaryotic genome editing method is performed by delivering the CRISPR / Cas12a system for eukaryotic genome editing to a eukaryotic cell. The above delivery process is performed to cause the CRISPR / Cas12a complex to contact or induce contact with the cellular genome within the target cell. As long as the above purpose is achieved, the delivery method is not otherwise limited. A person skilled in the art can deliver the CRISPR / Cas12a system for eukaryotic genome editing to a eukaryotic cell using a known method. The delivery method may vary appropriately depending on the specific implementation of the CRISPR / Cas12a system. For example, when the CRISPR / Cas12a system is a CRISPR / Cas12a complex, the above delivery process may be a process of packaging the CRISPR / Cas12a complex into a lipid nanoparticle and delivering it.

[0263] Environment for performing genome editing methods

[0264] The above genome editing method can be performed in any environment, as long as the CRISPR / Cas12a system for eukaryotic genome editing can be delivered to the cell. For example, it can be performed in vitro, in vivo, or ex vivo.

[0265]

[0266] Methods for editing nucleic acids in the cytoplasm

[0267] Overview of intracytoplasmic nucleic acid editing methods

[0268] This specification discloses a method for editing nucleic acids contained in the cytoplasm. The method for editing nucleic acids within the cytoplasm comprises delivering the CRISPR / Cas12a system described above into a cell. The CRISPR / Cas12a system can target nucleic acids within the cell and edit the nucleic acids or adjacent portions thereof.

[0269] Does not contain nuclear location signals

[0270] The method for editing nucleic acids within the cytoplasm is similar to the eukaryotic genome editing method described above. However, the structure of the CRISPR / Cas12a system used differs. Specifically, the CRISPR / Cas12a system used in the cytoplasmic nucleic acid editing method does not contain a nuclear localization signal. That is, the Cas12a protein included in the CRISPR / Cas12a system is not linked to a nuclear localization signal. This is because the purpose of this method is to allow the CRISPR / Cas12a system to operate in the cytoplasm, not the cell nucleus.

[0271]

[0272] Multiple target nucleic acid editing methods

[0273] Overview of Multiple Target Nucleic Acid Editing Methods

[0274] This specification discloses a method for editing two or more target nucleic acids. The method comprises delivering into a cell a vector construct encoding a Cas12a protein or a nucleic acid encoding the same; and a plurality of guide RNAs. When the method is performed, 1) the plurality of guide RNAs encoded in the vector construct are expressed in the cell, 2) each of the expressed guide RNAs binds to the Cas12a protein (or a Cas12a protein expressed from the encoding nucleic acid) to form a CRISPR / Cas12a complex, and 3) each guide RNA is induced to edit a target nucleic acid.

[0275] Vector construct encoding multiple guide RNAs

[0276] The above method uses a vector construct encoding multiple guide RNAs. The construct satisfies the following characteristics:

[0277] 1) Containing a nucleic acid encoding two or more guide RNAs.

[0278] 2) The nucleic acids encoding each guide RNA are directly connected without components such as the T6 terminator.

[0279] 3) Nucleic acids encoding two or more guide RNAs are operably linked to a single promoter.

[0280] The CRISPR / Cas12a complex is known to operate solely with crRNA, without the involvement of tracrRNA. Furthermore, it is known that guide RNAs are transcribed from the nucleic acid or gene encoding them, and undergo a maturation process in which they are independently cleaved without the involvement of tracrRNA. Applying this fact, a construct encoding multiple guide RNAs can be designed, as described above. The above vector construct contains nucleic acids encoding two or more guide RNAs, without the need to separate each encoding nucleic acid with additional components such as spacers or terminators. Furthermore, even if only one promoter is operably linked to nucleic acids encoding two or more guide RNAs and transcribed, each guide RNA is separated during the maturation process and expressed independently.

[0281] Method for editing multiple target nucleic acids using a vector construct encoding multiple guide RNAs

[0282] The above method is performed by delivering a vector construct encoding a plurality of guide RNAs; and a Cas12a protein or a nucleic acid encoding the Cas12a protein to a cell. The Cas12a protein may or may not be linked to a nuclear localization signal depending on whether the target nucleic acid is located in the nucleus or the cytoplasm. When the above method is performed, 1) the plurality of guide RNAs encoded in the above vector construct are expressed in the cell, 2) each of the expressed guide RNAs binds to the Cas12a protein (or the Cas12a protein expressed from the encoding nucleic acid) to form a CRISPR / Cas12a complex, and 3) each guide RNA is induced to edit the target nucleic acid. The above delivery method is not limited as long as the above vector construct and the Cas12a protein or the nucleic acid encoding the same can be delivered into the cell so that the above process can be performed. For example, it can be performed by appropriately selecting a known method.

[0283]

[0284] Possible embodiments of the invention

[0285] Novel Cas12a protein

[0286] Example 1: Novel Cas12a protein

[0287] A Cas12a protein comprising a sequence selected from the following:

[0288] ; ; and .

[0289] Novel Cas12a protein variants

[0290] Example 2, variants of novel Cas12a proteins

[0291] 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 an amino acid sequence selected from the following (hereinafter referred to as a reference amino acid sequence):

[0292] Sequence number 1 to sequence number 3.

[0293] Example 3, Basis for Transformation

[0294] In Example 2, the variant of the Cas12a protein is compared with the Cas12a protein of the reference amino acid sequence,

[0295] Improved ability to bind to the Protospacer Adjacent Motif (PAM);

[0296] or improved ability to bind to DNA;

[0297] The ability to stably maintain the CRISPR / Cas12a complex has been improved; or

[0298] It has improvements that are a random combination of the above.

[0299] Example 4, novel Cas12a variants, variant locations

[0300] In any one of Examples 2 to 3, the variant of the Cas12a protein comprises a mutation selected from the following:

[0301] Based on the amino acid sequence of SEQ ID NO: 1, E155R, G531R, K537R, S1137A, N526R, E791L, or any combination thereof;

[0302] Based on the amino acid sequence of SEQ ID NO: 2, Q190R, S579R, Y585R, or any combination thereof.

[0303] Example 5, novel Cas12a variant, sequence

[0304] In any one of Examples 2 to 4, the variant of the Cas12a protein comprises an amino acid sequence selected from the following:

[0305] ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

[0306] Nuclear Localization Signal (NLS)

[0307] Example 6, nuclear localization signal

[0308] Nuclear Localization Signal (NLS).

[0309] Example 7, nuclear localization signal, sequence

[0310] In Example 6, 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:

[0311] 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).

[0312] Example 8, Nuclear localization signal, meaning of combinations

[0313] In Example 7, '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:

[0314] 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).

[0315] Cas12a protein linked to a nuclear localization signal

[0316] Example 9: Cas12a protein linked to a nuclear localization signal

[0317] Cas12a protein with a nuclear localization signal, including:

[0318] The Cas12a protein of any one of Example 1, or a variant of the Cas12a protein of any one of Examples 2 to 5; and

[0319] One or more nuclear localization signals of any one of Examples 6 to 8.

[0320] Example 10, Connection Relationship

[0321] In Example 9, the Cas12a protein or a variant of the Cas12a protein is directly linked to the one or more nuclear localization signals or is linked via an amino acid sequence of SEQ ID NO: 95 to SEQ ID NO: 115.

[0322] Example 11, structural formula

[0323] Cas12a protein with nuclear localization signal of the following structure:

[0324] [NLS1] - [Linker1] - [Cas12a] - [Linker2] - [NLS2]

[0325] Here, the NLS1 is a nuclear localization signal of any one of Examples 6 to 8, or is absent,

[0326] The above NLS2 is a nuclear localization signal of any one of Examples 6 to 8, or is absent,

[0327] The above Linker1 is any one linker selected from SEQ ID NO: 67 to SEQ ID NO: 115, or is absent,

[0328] The above Linker2 is any one linker selected from SEQ ID NO: 67 to SEQ ID NO: 115, or is absent,

[0329] The above Cas12a is a variant of the Cas12a protein of any one of Example 1, or the Cas12a protein of any one of Examples 2 to 5,

[0330] The Cas12a protein linked to the nuclear localization signal comprises at least one nuclear localization signal.

[0331] Example 12, sequence

[0332] In any one of Examples 9 to 11, the Cas12a protein linked to the nuclear localization signal comprises an amino acid sequence selected from SEQ ID NO: 280 to SEQ ID NO: 298.

[0333] programmable guide RNA

[0334] Example 13, Programmable Guide RNA

[0335] Programmable guide RNAs containing:

[0336] scaffold,

[0337] 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 2 to 5 to form a complex; and

[0338] Guide domain,

[0339] Here, the guide domain is designed to target a target nucleic acid.

[0340] Example 14, Scaffold

[0341] In Example 13, the scaffold comprises a direct repeat (DR),

[0342] The above direct repeat portion comprises a nucleic acid sequence selected from among UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33), AAAAUUUCUACUCUUGUAGAU (SEQ ID NO: 34), and UAAAUUUCUACUGUUGUAGAU (SEQ ID NO: 35).

[0343] Example 15: Meaning of targeting a target nucleic acid

[0344] In any one of Examples 13 to 14, the description that 'the guide domain targets the target nucleic acid' has the following meaning:

[0345] The target nucleic acid is a double-stranded DNA comprising a target strand and a non-target strand,

[0346] The above guide domain and the above target nucleic acid are in a relationship selected from the following:

[0347] 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;

[0348] The guide domain hybridizes or complementarily binds to all or part of the target strand;

[0349] 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

[0350] Any combination of the above relationships.

[0351] Example 16, length of guide domain

[0352] In any one of Examples 13 to 15, the guide domain has a length selected from the following:

[0353] 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

[0354] A length within the range of the two numbers described above, for example, 16nt to 23nt.

[0355] Example 17, including mismatches

[0356] In Example 15, the guide domain hybridizes or complementarily binds to all or part of the target strand,

[0357] 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:

[0358] having nucleic acid sequences that are completely complementary to each other;

[0359] The remaining nucleic acid sequences are complementary, excluding one, two, three, four, or five mismatches;

[0360] 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;

[0361] 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

[0362] Any combination of the above relationships.

[0363] Example 18, Guide Domain Sequence Homology

[0364] In Example 15, the nucleic acid sequence of the guide domain comprises a nucleic acid sequence selected from the following:

[0365] 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

[0366] 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.

[0367] Example 19, stem-loop inclusion possible

[0368] In any one of Examples 13 to 18, the scaffold comprises a stem-loop of SEQ ID NO: 49.

[0369] Example 20, Guide RNA Structure

[0370] In any one of Examples 13 to 19,

[0371] 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.

[0372] Here, the 3' end of the scaffold and the 5' end of the guide domain are connected directly or via an arbitrary RNA sequence.

[0373] Example 21, structural formula limitation

[0374] In any one of Examples 13 to 20, the programmable guide RNA has the following structure:

[0375] 5' - [scaffold] - [RNA linker] - [guide domain] - 3'

[0376] Here, the RNA linker is an arbitrary RNA sequence or is absent,

[0377] In the absence of the above RNA linker, the above structure is as follows:

[0378] 5' - [Scaffold] - [Guide Domain] - 3'.

[0379] Example 22, including additional domains

[0380] In Example 21, the programmable guide RNA further comprises one or more additional domains,

[0381] 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.

[0382] Example 23, with stem loop

[0383] In Example 22, the programmable guide RNA further comprises a stem-loop,

[0384] The above stem loop is connected to the 5' end of the above programmable guide RNA.

[0385] Example 24, stem loop sequence

[0386] In Example 23, the stem-loop comprises a nucleic acid sequence of SEQ ID NO: 49.

[0387] CRISPR / Cas12a complex

[0388] Example 25, CRISPR / Cas12a complex

[0389] CRISPR / Cas12a complex containing:

[0390] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5, or the Cas12a protein linked to the nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as the Cas12a protein); and

[0391] A programmable guide RNA of any one of Examples 13 to 24;

[0392] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0393] The above CRISPR / Cas12a complex has a function of cleaving a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0394] Hereinafter, the CRISPR / Cas12a complex is also referred to as an RNA-protein complex, or ribonucleoprotein (RNP).

[0395] Example 26, Cas12a protein and scaffold combination

[0396] In Example 25,

[0397] The scaffold of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0398] A scaffold comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a protein comprising the above amino acid sequence among Cas12a proteins linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0399] A scaffold comprising an amino acid sequence selected from SEQ ID NO. 3 and SEQ ID NO. 275 to SEQ ID NO. 279, or a protein comprising the above amino acid sequence among Cas12a proteins linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO. 35; or

[0400] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0401] CRISPR / Cas12a vector

[0402] Example 27, CRISPR / Cas12a vector

[0403] Expression vectors of CRISPR / Cas12a system components, including:

[0404] A nucleic acid encoding a Cas12a protein of any one of Example 1, a variant of a Cas12a protein of any one of Examples 2 to 5, or a Cas12a protein linked to a nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as a Cas12a protein); and

[0405] A nucleic acid encoding a programmable guide RNA of any one of Examples 13 to 24;

[0406] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0407] The above CRISPR / Cas12a complex has a function of cleaving a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0408] Example 28, Cas12a encoding DNA

[0409] In Example 27, the nucleic acid encoding the Cas12a protein comprises a nucleic acid sequence selected from among SEQ ID NO: 17 to SEQ ID NO: 19.

[0410] Example 29, including additional components

[0411] In any one of Examples 27 to 28, the component expression vector further comprises one or more selected from the following:

[0412] Promoter; enhancer; intron; polyadenylation signal; Kozak consensus sequence; internal ribosome entry site (IRES); splice acceptor; 2A sequence; and replication origin.

[0413] Example 30, Promoter Addition

[0414] In Example 29, the promoter is at least one selected from the following:

[0415] 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.

[0416] Example 31, adding a replication origin

[0417] In any one of Examples 29 to 30, the replication origin is at least one selected from the following:

[0418] 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.

[0419] Example 32, vector types

[0420] In any one of Examples 27 to 31, the vector is a viral vector, a non-viral vector, or any combination thereof.

[0421] Example 33, viral vector

[0422] In Example 32, the vector is a viral vector selected from the following:

[0423] Retrovirus; lentivirus; adenovirus; adeno-associated virus; vaccinia virus; poxvirus; herpes simplex virus; and any combination thereof.

[0424] Example 34, non-viral vector

[0425] In Example 32, the vector is a non-viral vector selected from the following:

[0426] Plasmid; phage; naked DNA; DNA complex; PCR amplicon; mRNA; and any combination thereof.

[0427] Example 35, plasmid

[0428] In Example 34, the plasmid is one selected from the following:

[0429] 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.

[0430] Example 36, Phage

[0431] In Example 34, the phage is one selected from the following:

[0432] λgt4λB; λ-Charon; λΔz1; M13; and any combination thereof.

[0433] Example 37, single vector

[0434] In any one of Examples 27 to 36, the vector is a single vector composed of one molecule.

[0435] Example 38, multiple vectors

[0436] In any one of Examples 27 to 37, the vector is a plurality of vectors composed of two or more molecules.

[0437] Example 39, each component contained in a different vector

[0438] In Example 38, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are contained in vectors of different molecules.

[0439] Example 40, DNA and RNA embodiment

[0440] In any one of Examples 27 to 39, the nucleic acid encoding the Cas12a protein and the nucleic acid encoding the programmable guide RNA are each independently DNA or RNA.

[0441] Example 41, combination of Cas12a protein and programmable guide RNA

[0442] In any one of Examples 27 to 40, the scaffold of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0443] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0444] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 275 to SEQ ID NO: 279, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 35; or

[0445] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0446] CRISPR / Cas12a composition

[0447] Example 42, CRISPR / Cas12a composition

[0448] A CRISPR / Cas12a composition comprising:

[0449] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5, or the Cas12a protein linked to the nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as the Cas12a protein), or a nucleic acid encoding the Cas12a protein; and

[0450] A programmable guide RNA of any one of Examples 13 to 24, or a nucleic acid encoding the guide RNA;

[0451] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0452] The above CRISPR / Cas12a complex has a function of cleaving a target nucleic acid targeted by the guide domain of the programmable guide RNA, or a nucleic acid adjacent thereto.

[0453] Example 43, including CRISPR / Cas12a complex

[0454] In Example 42, the CRISPR / Cas12a composition comprises a CRISPR / Cas12a complex of any one of Examples 25 to 26.

[0455] Example 44, including CRISPR / Cas12a vector

[0456] In Example 42, the CRISPR / Cas12a composition comprises an expression vector of a CRISPR / Cas12a system component of any one of Examples 27 to 41.

[0457] Example 45, including Cas-encoding mRNA

[0458] In Example 42, the CRISPR / Cas12a composition comprises an mRNA encoding the Cas12a protein and a programmable guide RNA.

[0459] Example 46, combination of Cas12a protein and programmable guide RNA

[0460] In any one of Examples 42 to 45, the scaffold of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0461] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0462] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 275 to SEQ ID NO: 279, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 35; or

[0463] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0464] Composition for editing eukaryotic genomes

[0465] Example 47, Composition for Eukaryotic Genome Editing

[0466] A composition for editing a eukaryotic genome comprising:

[0467] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5, or the Cas12a protein linked to the nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as the Cas12a protein), or a nucleic acid encoding the Cas12a protein; and

[0468] A programmable guide RNA of any one of Examples 13 to 24, or a nucleic acid encoding the guide RNA;

[0469] Here, the programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0470] The above CRISPR / Cas12a complex has a function of cleaving a target nucleic acid in a eukaryotic genome targeted by the guide domain of the above programmable guide RNA, or a nucleic acid adjacent thereto.

[0471] Example 48, including CRISPR / Cas12a complex

[0472] In Example 47, the CRISPR / Cas12a composition comprises the CRISPR / Cas12a complex of any one of Examples 25 to 26.

[0473] Example 49, including CRISPR / Cas12a vector

[0474] In Example 47, the CRISPR / Cas12a composition comprises an expression vector of a CRISPR / Cas12a system component of any one of Examples 27 to 41.

[0475] Example 50, including Cas encoding mRNA

[0476] In Example 47, the CRISPR / Cas12a composition comprises an mRNA encoding the Cas12a protein and a programmable guide RNA.

[0477] Example 51, combination of Cas12a protein and programmable guide RNA

[0478] In any one of Examples 47 to 50, the scaffold of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0479] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0480] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 275 to SEQ ID NO: 279, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 35; or

[0481] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0482] Eukaryotic genome editing methods

[0483] Example 52, Gene Editing Method #1 - Composition Delivery

[0484] Eukaryotic genome editing methods, including:

[0485] Delivering to the eukaryotic cell the CRISPR / Cas12a complex of any one of Examples 25 to 26, the CRISPR / Cas12a component expression vector of any one of Examples 27 to 41, the CRISPR / Cas12a composition of any one of Examples 42 to 46, or the eukaryotic gene editing composition of any one of Examples 47 to 51;

[0486] Here, the guide domain of the programmable guide RNA is designed to target a target nucleic acid within the eukaryotic genome.

[0487] Example 53, Alternative Expressions for Transmission

[0488] In Example 52, the expression 'deliver to a eukaryotic cell' is replaced with the following expression:

[0489] Introduce into a eukaryotic cell; administer into a eukaryotic cell; inject into a eukaryotic cell; transfect into a eukaryotic cell; transduct into a eukaryotic cell; or any combination of the above. #### Example 54, Gene Editing Method #2 - Complex Contact

[0490] Eukaryotic genome editing methods, including:

[0491] A process of contacting or inducing contact between the genome of the eukaryotic cell and the CRISPR / Cas12a complex of any one of Examples 25 to 26;

[0492] Here, the guide domain of the programmable guide RNA is designed to target a target nucleic acid within the eukaryotic genome.

[0493] Example 55, limited delivery method

[0494] In any one of Examples 52 to 54, delivering the eukaryotic cell gene editing composition to the eukaryotic cell is performed by a method selected from the following:

[0495] 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.

[0496] Example 56, Relationship with PAM sequence

[0497] In any one of Examples 52 to 55,

[0498] The above target nucleic acid is double-stranded DNA,

[0499] The double-stranded DNA comprises a first strand (non-target strand) having a Protospacer Adjacent Motif (PAM) sequence and a protospacer sequence adjacent to the PAM, and a second strand (target strand) having a nucleic acid sequence complementary to the first strand,

[0500] The guide domain of the above programmable guide RNA has a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% matched, identical, equivalent, and / or homologous to the above protospacer sequence.

[0501] Example 57, PAM sequence

[0502] In Example 56,

[0503] The PAM sequence is 5'-TTTV-3' or 5'-TTTN-3', wherein V is A, C, or G, and N is A, C, G, or T.

[0504] Example 58, Eukaryotic cell types

[0505] In any one of Examples 52 to 57, the eukaryotic cell is selected from a plant cell, a non-human animal cell, and a human cell.

[0506] Example 59, human cells

[0507] In Example 58, the eukaryotic cell is a human iPS cell (induced Pluripotent Stem Cell).

[0508] Example 60, Isolated Cells

[0509] In any one of Examples 52 to 59, the eukaryotic cell is a cell isolated from an organism.

[0510] Example 61, Method Execution Environment

[0511] In any one of Examples 52 to 60, the method is performed in vivo, in vitro, and / or ex vivo.

[0512] Method for cleaving nucleic acids in the cytoplasm

[0513] Example 62, Method for cleaving nucleic acid in the cytoplasm of a eukaryotic cell

[0514] A method for cleaving nucleic acids in the cytoplasm of a eukaryotic cell, comprising:

[0515] Delivering the CRISPR / Cas12a composition to the eukaryotic cell;

[0516] wherein the CRISPR / Cas12a composition comprises:

[0517] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5 (hereinafter collectively referred to as the Cas12a protein), or a nucleic acid encoding the Cas12a protein; and

[0518] A programmable guide RNA of any one of Examples 13 to 24, or a nucleic acid encoding the programmable guide RNA;

[0519] Here, the Cas12a protein does not contain a nuclear localization signal,

[0520] The above programmable guide RNA binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0521] The CRISPR / Cas12a complex targets nucleic acids in the eukaryotic cytoplasm using the guide domain of the programmable guide RNA.

[0522] Example 63, combination of Cas12a protein and guide RNA

[0523] In Example 62, the scaffold of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

[0524] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0525] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 275 to SEQ ID NO: 279, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 35; or

[0526] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0527] Example 64, Extending the Transmission Expression

[0528] In any one of Examples 62 to 63, the phrase 'deliver to a eukaryotic cell' is replaced with the following expression:

[0529] Introduce into a eukaryotic cell; administer into a eukaryotic cell; inject into a eukaryotic cell; transfect into a eukaryotic cell; transduct into a eukaryotic cell; or any combination of the above.

[0530] Multiplex vector payload

[0531] Example 65, vector structure

[0532] A multiplex vector structure (construct) containing:

[0533] promoter;

[0534] DNA encoding the first guide RNA; and

[0535] DNA encoding a second guide RNA;

[0536] Here, the first guide RNA is any one selected from Examples 13 to 24,

[0537] The second guide RNA is any one selected from Examples 13 to 24,

[0538] The DNA encoding the first guide RNA is operably linked to a promoter,

[0539] The 5' end of the DNA encoding the second guide RNA is directly linked to the 3' end of the first guide RNA,

[0540] The first guide domain and the second guide domain have different nucleic acid sequences.

[0541] Example 66, including 3 or more guide RNAs

[0542] A multiplex vector structure containing the following structures:

[0543] [Promoter] - [1st guide RNA encoding DNA] - [2nd guide RNA encoding DNA] - [Nth guide RNA encoding DNA]

[0544] Here, n is an integer greater than or equal to 3,

[0545] 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 of the programmable guide RNAs selected from Examples 13 to 24,

[0546] The above first guide RNA encoding DNA is operably linked to the above promoter,

[0547] The first guide RNA encoding DNA, the second guide RNA encoding DNA, and the nth guide RNA encoding DNA are directly connected.

[0548] Method for cleaving multiple target nucleic acids

[0549] Example 67, Method for Cleaving Two or More Target Nucleic Acids

[0550] A method for cleaving two or more target nucleic acids within a eukaryotic genome, comprising:

[0551] Delivering a multiplex editing composition to said eukaryotic cell;

[0552] wherein the multiplex editing composition comprises:

[0553] Vector structure of Example 65; and

[0554] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5, the Cas12a protein linked to the nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as the Cas12a protein), or a nucleic acid encoding the Cas12a protein;

[0555] Here, the first guide RNA and the second guide RNA included in the vector structure each bind to the Cas12a protein to form a CRISPR / Cas12a complex,

[0556] As a result of performing the above method, the target nucleic acid targeted by the first guide RNA and the target nucleic acid targeted by the second guide RNA are edited.

[0557] Example 68, Method for Cleaving Three or More Target Nucleic Acids

[0558] A method for cleaving three or more target nucleic acids within a eukaryotic genome, comprising:

[0559] Delivering a multiplex editing composition to said eukaryotic cell;

[0560] wherein the multiplex editing composition comprises:

[0561] Vector structure of Example 66; and

[0562] The Cas12a protein of any one of Example 1, the variant of the Cas12a protein of any one of Examples 2 to 5, the Cas12a protein linked to the nuclear localization signal of any one of Examples 9 to 12 (hereinafter collectively referred to as the Cas12a protein), or a nucleic acid encoding the Cas12a protein;

[0563] Here, each guide RNA included in the vector structure binds to the Cas12a protein to form a CRISPR / Cas12a complex,

[0564] As a result of performing the above method, target nucleic acids greater than or equal to the number of types of guide RNAs included in the vector structure (i.e., n) are edited.

[0565] Example 69, regardless of transmission order

[0566] In any one of Examples 67 to 68, the vector construct and the Cas12a protein or the nucleic acid encoding the Cas12a protein are delivered to the eukaryotic cell simultaneously or in any order.

[0567] Example 70, Cas12a and guide combination

[0568] In any one of Examples 67 to 69, the scaffold of the Cas12a protein and each of the guide RNAs is a combination selected from the following:

[0569] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 33;

[0570] A scaffold comprising a protein comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 275 to SEQ ID NO: 279, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 35; or

[0571] A scaffold comprising a protein comprising the amino acid sequence of SEQ ID NO: 2, or a Cas12a protein linked to a nuclear localization signal, and a direct repeat sequence of SEQ ID NO: 34.

[0572] Example 71, Extending the Transmission Expression

[0573] In any one of Examples 67 to 70, the phrase 'deliver to a eukaryotic cell' is replaced with the following expression:

[0574] Introduce into a eukaryotic cell; administer into a eukaryotic cell; inject into a eukaryotic cell; transfect into a eukaryotic cell; transduct into a eukaryotic cell; or any combination of the above.

[0575]

[0576] Experimental example

[0577] 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.

[0578] Experimental Example 1. Experimental Method and Materials

[0579] Experimental Example 1.1. CRISPR Enzyme Expression Vector Production #1

[0580] 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.

[0581] Experimental Example 1.2. Guide RNA Preparation

[0582] 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.

[0583] Experimental Example 1.3. CRISPR Enzyme Production and Purification

[0584] 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.

[0585] Experimental Example 1.4. Verification of the Manufactured CRISPR Enzyme

[0586] 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.

[0587] Experimental Example 1.5. Preparation of CRISPR / Cas12a Complex

[0588] 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.

[0589] Experimental Example 1.6. Cell Culture

[0590] 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.

[0591] Experimental Example 1.7. Measurement of in vivo gene editing activity #1

[0592] 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.

[0593] Experimental Example 1.8. Measurement of in vivo gene editing activity #2

[0594] 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.

[0595] 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 / ).

[0596] Experimental Example 1.9. Plasmid Construction for Mammalian Cell Experiments

[0597] pCMV-dLbCpf1-BE-YE1 (Addgene, #154145) was modified to express the novel Cas12a protein using Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, NEB #E5520). The novel Cas12a sequence was optimized to 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 crRNA-encoding plasmid was constructed by Gibson assembly of PCR-amplified oligonucleotides into pLb-Cpf1-pGL3-U6-sgRNA (Addgene, #107682) using Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, NEB #E5520).

[0598] Experimental Example 1.10. Protein Purification

[0599] A novel Cas12a protein and a plasmid encoding the novel Cas12a protein were constructed according to Experimental Example 1.9. Each protein was constructed to be expressed by including His6 at the N-terminus. Rosetta expression cells (EMD Millipore) were transformed with the above plasmids. Selected transformants were cultured overnight in Luria-Bertani (LB) medium containing 100 μg / ml kanamycin at 37°C. Ten ml of the overnight cultured cells were inoculated into 400 ml LB medium containing 100 μg / ml kanamycin and incubated at 30°C until an OD600 of 0.5–0.6 was reached. The cell culture was cooled to 16°C for 1 h, supplemented with 0.5 mM IPTG, and incubated for 14–18 h. For protein purification, cells were harvested by centrifugation at 5000 g for 10 min at 4°C and lysed by sonication in 5 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM dithiothreitol (DTT), 10 mM imidazole, pH 8.0) supplemented with lysozyme (Sigma) and protease inhibitor (Roche complete, EDTA-free). The soluble lysate obtained after centrifugation at 18,000 g for 30 min at 4°C was incubated with Ni-NTA agarose resin (Qiagen) for 1 h at 4°C. The lysate / Ni-NTA mixture was applied to a column and washed with buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). The novel Cas12a protein (and its variants) were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The buffer of the eluted protein solution was exchanged with storage buffer (20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM DTT, 20% glycerol), and the protein was concentrated using a centrifugal filter device (Millipore).

[0600] Experimental Example 1.11. In vitro plasmid cleavage and analysis

[0601] To verify the nuclease function of the novel Cas12a, 1 μg of PCR amplification product containing the target sequence was incubated with the ribonucleoprotein complex at 37°C. Purified novel Cas12a (300 mM) was incubated with in vitro-transcribed crRNA (300 mM) for 15 minutes to generate the ribonucleoprotein complex, and then the ribonucleoprotein complex was incubated with the PCR amplification product for 8 hours. After this incubation, the cleaved amplification product was purified using the PCR Product Purification Kit (MG Med, MK12020) and subjected to agarose gel electrophoresis. Band intensities were measured using ImageJ software.

[0602] Experimental Example 1.12. Target Depth Sequencing

[0603] To analyze editing frequency, target regions were amplified through nested first-round PCR, second-round PCR, and third-round PCR using primers containing Tru-Seq HT Dual index and PrimeSTAR® GXL DNA Polymerase (TAKARA) to generate deep sequencing libraries. The libraries were sequenced using an Illumina iSeq paired-end sequencing system. Base editing and unwanted indel frequencies were expressed as the percentage of sequencing reads containing correct edits or indels among the total sequenced reads. The computer program used to analyze editing frequency is available on GitHub at prime_editor_analysis.

[0604] Experimental Example 2. Confirmation of the in vivo gene editing activity of the CRISPR / Cas12a system.

[0605] Experimental Example 2.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0606] 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.

[0607] The target nucleic acid sequence in the EMX1 gene is as follows: CTCATCTGTGCCCCTCCCTCCCTG (SEQ ID NO: 136)

[0608] The composition of each CRISPR / Cas12a complex used in the above experiments is shown in the following table:

[0609] 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

[0610] 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.

[0611] The specific experimental method is as follows:

[0612] 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.

[0613] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0614] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0615] 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.

[0616] Experimental Example 2.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0617] 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.

[0618] 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-

[0619] Looking at the above results, it can be seen that each Cas12a protein was expressed well according to Experimental Example 2.1.

[0620] Experimental Example 2.3. Results of in vivo gene editing activity experiments

[0621] The results of the T7E1 assay performed according to Experimental Example 2.1 are shown in Fig. 2.

[0622] 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.

[0623] To quantitatively confirm the results, NGS sequence analysis was performed and the results are shown in the following table:

[0624] 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%)

[0625] 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.

[0626] Furthermore, mutation analysis was performed on the results of the above indel introduction and is shown in Fig. 3.

[0627] 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.

[0628] For reference, the results of mutation analysis for LbCas12a used as a control group are shown in Figure 4.

[0629] Experimental Example 3. Confirmation of Gene Editing Activity According to Nuclear Localization Signal Composition

[0630] Experimental Example 3.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0631] 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.

[0632] As in Experimental Example 2, an in vivo gene editing activity experiment was performed targeting the EMX1 gene of immortalized HEK cells.

[0633] The target nucleic acid sequence in the EMX1 gene is as follows: CTCATCTGTGCCCCTCCCTCCCTG (SEQ ID NO: 136)

[0634] 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:

[0635] 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

[0636] The specific experimental method is as follows:

[0637] 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.

[0638] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0639] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0640] 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.

[0641] Experimental Example 3.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0642] 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.

[0643] Looking at the above results, it can be seen that each CRISPR enzyme was expressed well according to Experimental Example 3.1.

[0644] The molecular weight and concentration of each expression result are shown in the following table:

[0645] 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

[0646] 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.

[0647] 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.

[0648] Experimental Example 3.4. In Vivo Gene Editing Activity Experiment Results #2

[0649] 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.

[0650] 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.

[0651] Experimental Example 4. Confirmation of the in vivo gene editing activity of the CRISPR / Cas12a system #2

[0652] Experimental Example 4.1. CRISPR / Cas12a Structure Used in the Experiment and Method for Confirming Gene Editing Activity

[0653] 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.

[0654] The gene sequences and protospacer sequences used in the above experiments are shown in the following table:

[0655] 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)

[0656] The composition of each CRISPR / Cas12a complex used in the above experiments is shown in the following table:

[0657] 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

[0658] In the table above, the CRISPR enzyme sequence represents the sequence containing Cas12a and NLS.

[0659] 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.

[0660] The specific experimental method is as follows:

[0661] 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.

[0662] 2) Guide RNA was prepared according to Experimental Example 1.2.

[0663] 3) Using the CRISPR enzyme and guide RNA manufactured above, a CRISPR / Cas12a complex was manufactured according to Experimental Example 1.5.

[0664] 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.

[0665] Information on each primer used in the above experiment is shown in Figures 19 to 22:

[0666] Experimental Example 4.2. Results of CRISPR / Cas12a Production Used in the Experiment

[0667] 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.

[0668] Looking at the above results, it can be seen that each CRISPR enzyme was expressed well according to Experimental Example 4.1.

[0669] Experimental Example 4.3. In vitro gene editing activity experiment

[0670] To evaluate the in vitro cleavage activity of the CRISPR enzyme manufactured according to Experimental Example 4.1, the following experiment was conducted:

[0671] 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.

[0672] 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.

[0673] 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.

[0674] The results of the in vitro cleavage activity experiment of each CRISPR / Cas12a complex are shown in Figure 23.

[0675] Experimental results showed that most CRISPR / Cas12a complexes exhibited in vitro nucleic acid cleavage activity.

[0676] Experimental Example 4.4. In Vivo Gene Editing Activity Experiment #1 - Experimental Method

[0677] To confirm the in vivo gene editing activity of the CRISPR enzyme according to Experimental Example 4.1, the following experiment was conducted:

[0678] 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.

[0679] 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.

[0680] Experimental Example 4.5. In vivo gene editing activity experiment #2 - MTAP gene

[0681] 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:

[0682] 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%)

[0683] 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.

[0684] 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.

[0685] Experimental Example 4.6. In vivo gene editing activity experiment #3 - DYRK1A gene

[0686] 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:

[0687] 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%)

[0688] 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.

[0689] 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.

[0690] Experimental Example 4.7. In Vivo Gene Editing Activity Experiment #4 - Interpreting Experimental Results

[0691] 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.

[0692] Experimental Example 5. Eukaryotic Genome Editing Activity of the CRISPR / Cas12a System Containing Cas12a Protein Variants #1

[0693] Experimental Example 5.1. Exploring the Location of Mutations in the PAM Recognition Site

[0694] 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 mutation location of the PAM recognition site of the Cas12a protein discovered above.

[0695] Specifically, the structure of the CRISPR / Cas12a system containing the Cas12a protein of sequence number 1 bound to the target nucleic acid was analyzed using AlphaFold.

[0696] The analysis results are shown in Figures 19 to 21.

[0697] The analysis results revealed that positions E155, G531, and K537 in the amino acid sequence of sequence number 1 are positions that significantly interact with the PAM sequence.

[0698] Below, we measured the gene editing efficiency by actually introducing mutations into the locations indicated above.

[0699] Experimental Example 5.2. Experimental Method and Configuration Used in the Experiment

[0700] In Experimental Example 5.1, variants were prepared by substituting arginine for amino acids at specific positions and used in the experiment. All possible combinations of proteins were prepared by substituting arginine for each position. Specifically, plasmids encoding each variant Cas12a protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of co-operating with the corresponding Cas12a proteins were also prepared. The plasmid vectors were transfected into cells according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019). Subsequently, the cell genome was extracted and the gene editing activity was measured. The extracted cell genome was analyzed according to Experimental Example 1.12.

[0701] The composition of each CRISPR / Cas12a complex used in Experimental Example 5 is shown in the following table:

[0702] LabelChartLabelCas12a protein(SEQ ID NO)C-terminal NLSDirect repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 5.1218WT1KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70)UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)DYRK1A: 143MTAP: 140VEGFA: 419FANCF: 420CFTR: 421Example 5.2218WT + E155R264(Same as above)(Same as above)(Same as above)Example 5.3218WT + G531R265(Same as above)(Same as above)(Same as above)Example 5.4218WT+ E155R + G531R266(Same as above)(Same as above)(Same as above)Example 5.5218WT + E155R + K537R267(Same as above)(Same as above)(Same as above)Example 5.6218WT + K537R299(Same as above)(Same as above)(Same as above)Example 5.7218WT + G531R + K537R300(Same as above)(Same as above)(Same as above)Example 5.8218WT + E155R + G531R + K537R301(Same as above)(Same as above)(Same as above)ControlUNT----

[0703] Experimental Example 5.3. Experimental Results

[0704] The experimental results are shown in Figures 21 to 26. The quantitative data for the experimental results are shown in the following table:

[0705] LabelMTAPDYRK1AVEGFAFANCFCFTRsite3218WT26.0810.6021.020.428.9513.07(Example 5.1)34.829.9918.850.6011.0224.67 29.4013.6124.300.449.7318.36218WT+E155R33.6825.1137.171.4026.9713.10(Example 5.2)31.9939.9736.711.1723.8519.89 36.7532.3033.730.4821.4715.26218WT+G531R17.169.5328.850.4716.486.38(Example 5.3)20.947.5333.870.6111.0612.38 24.6711.8524.640.868.3917.55218WT+E155R+G531R5.655.4015.100.0613.5811.67(Example 5.4)11.2011.0616.520.186.3715.23 25.9011.9219.690.138.267.21218WT+E155R+K537R12.987.056.200.008.204.32(Example 5.5)18.017.2612.230.037.038.53 26.856.569.650.097.357.36218WT+K537R3.430.412.390.000.360.84(Example 5.6)5.050.274.820.100.131.02 6.850.324.020.000.431.25218WT+G531R+K537R5.570.458.320.001.066.66(Example 5.7)7.000.959.560.031.255.81 8.341.137.070.111.585.74218WT+E155R+G531R+K537R5.491.264.020.031.541.77(Example 5.8)7.872.156.030.164.232.62 7.051.796.290.132.392.53UNT0.260.040.510.030.100.07(Control)0.030.020.130.060.000.22 0.060.080.070.000.000.27

[0706] All figures represent the indel introduction rate as a percentage. Each experimental example was repeated three times. The experimental results showed that Examples 5.2 and 5.3 showed higher eukaryotic genome editing efficiency than the novel Cas12a protein (Example 5.3) without introducing mutations. Examples 5.4 and 5.5 did not show exceptional editing efficiency, but they showed usable editing efficiency.

[0707] Experimental Example 6. Eukaryotic Genome Editing Activity of the CRISPR / Cas12a System Containing Cas12a Protein Variants #2

[0708] Experimental Example 6.1. Exploring the Location of Mutations in the PAM Recognition Site

[0709] According to Experimental Example 5.1, the position that significantly interacts with PAM in the amino acid sequence of SEQ ID NO. 3 was found through AlphaFold structural analysis.

[0710] The analysis results are shown in Figures 27 and 28.

[0711] The analysis results revealed that positions Q190, S579, and Y585 in the amino acid sequence of sequence number 3 are positions that significantly interact with the PAM sequence.

[0712] Below, we measured the gene editing efficiency by actually introducing mutations into the locations indicated above.

[0713] Experimental Example 6.2. Experimental Method and Configuration Used in the Experiment

[0714] In Experimental Example 6.1, variants were prepared by substituting arginine for amino acids at specific positions and used in the experiment. All possible combinations of proteins were prepared by substituting arginine for each position. Specifically, plasmids encoding each variant Cas12a protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of cooperating with the corresponding Cas12a proteins were also prepared. The plasmid vectors were transfected into cells according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019). Subsequently, the cell genome was extracted and the gene editing activity was measured. The extracted cell genome was analyzed according to Experimental Example 1.12.

[0715] The composition of each CRISPR / Cas12a complex used in Experimental Example 6 is shown in the following table:

[0716] LabelChartLabelCas12a protein(SEQ ID NO)C-terminal NLSDirect repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 6.1236-WT-NLS63KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70)UAAAUUUCUACUGUUGUAGAU (SEQ ID NO: 36)DYRK1A: 143VEGFA: ?CFTR: ?Example 6.2236-WT (Q190R)-NLS6276(Same as above)(Same as above)(Same as above)Example 6.3236-WT (S579R)-NLS6277(Same as above)(Same as above)(Same as above)Example 6.4236-WT (Q190R + S579R)-NLS6275(Same as above)(Same as above)(Same as above)Example 6.5236-WT (Q190R + Y585R)-NLS6278(Same as above)(Same as above)(Same as above)Example 6.6236-WT (Q190R + S579R + Y585R)-NLS6279(Same as above)(Same as above)(Same as above)Example 6.7236-WT (Y585R)-NLS6305(Same as above)(Same as above)(Same as above)Example 6.8236-WT (S579R + Y585R)-NLS6306(Same as above)(Same as above)(Same as above)ControlUNT----

[0717] Experimental Example 6.3. Experimental Results

[0718] The experimental results are shown in Figures 29 to 32. The quantitative data for the experimental results are shown in the following table:

[0719] LabelDYRK1ACFTRVEGFA236-WT-NLS64.500.4714.67(Example 6.1)11.760.765.57 11.380.6818.96236-WT (Q190R)-NLS633.693.7331.69(Example 6.2)34.957.2931.28 33.277.8229.31236-WT (S579R)-NLS620.811.6930.74(Example 6.3)22.113.3435.76 19.142.9328.03236-WT (Q190R+S579R)-NLS637.4012.4338.69(Example 6.4)40.9813.1939.77 37.9412.8043.22236-WT (Q190R+Y585R)-NLS69.090.094.48(Example 6.5)12.150.064.56 9.710.144.55236-WT (Q190R+Y585R)-NLS69.090.094.48(Example 6.6)12.150.064.56 9.710.144.55236-WT (Q190R+S579R+Y585R)-NLS613.050.404.35(Example 6.7)17.150.538.70 24.850.977.48236-WT (Y585R)-NLS60.680.050.54(Example 6.8)0.890.030.59 0.510.020.41236-WT (S579R+Y585R)-NLS64.970.073.26(Example 6.9)6.200.163.52 6.860.153.74UNT0.04--(Control)0.03-- 0.03--

[0720] All figures represent the indel introduction rate as a percentage. Each experimental example was repeated three times. The experimental results showed that Examples 6.2 to 6.4 showed higher eukaryotic genome editing efficiency than the novel Cas12a protein (Example 6.1) without introducing mutations. Examples 6.5 to 6.6 did not show excellent editing efficiency, but they showed usable editing efficiency.

[0721] Experimental Example 7. Eukaryotic Genome Editing Activity of the CRISPR / Cas12a System Containing Cas12a Protein Variants #3

[0722] Experimental Example 7.1. Exploration of Ultra Nuclease Mutation Locations

[0723] 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 positions corresponding to M537 and F870 of the AsCas12a protein disclosed in the prior literature.

[0724] Additionally, with reference to previous 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), we sought to find mutation sites known to increase the duration of the R-loop and thus the efficiency of nucleic acid cleavage.

[0725] The analysis results are shown in Figures 33 to 37.

[0726] 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 sequence number 1.

[0727] Additionally, the position corresponding to the above R-loop mutation was identified as W354 in the amino acid sequence of sequence number 1.

[0728] Below, a variant of the Cas12a protein was prepared by combining the positions described above and the PAM recognition site mutation described in Experimental Example 5, and the gene editing efficiency was measured.

[0729] Experimental Example 7.2. Experimental Method and Configuration Used in the Experiment

[0730] In Experimental Example 7.1, mutants were prepared by substituting amino acids at specific positions with arginine and leucine, respectively, and used in the experiment. All possible combinations of proteins were prepared by substituting the appropriate amino acids at each mutation position. Specifically, plasmids encoding each mutant Cas12a protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of cooperating with the corresponding Cas12a proteins were also prepared. The corresponding plasmid vectors were transfected into cells according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019). Subsequently, the cell genome was extracted and the gene editing activity was measured. The extracted cell genome was analyzed according to Experimental Example 1.12.

[0731] The composition of each CRISPR / Cas12a complex used in Experimental Example 7 is shown in the following table:

[0732] LabelChart LabelCas12a protein(SEQ ID NO)C-terminal NLSDirect repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 7.1218-WT1KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70)UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)DYRK1A: 143MTAP: 140VEGFA: 419FANCF: 420CFTR: 421Site3: 422Example 7.2218-WT (E155R)264(Same as above)(Same as above)(Same as above)Example 7.3218-WT (E791L)269(Same as above)(Same as above)(Same as above)Example 7.4218-WT (E155R + E791L)270(Same as above)(Same as above)(Same as above)Example 7.5218-WT (E155R + N526R + E791L)271(Same as above)(Same as above)(Same as above)Example 7.6218-WT (N526R)272(Same as above)(Same as above)(Same as above)Example 7.7218-WT (N526R + E791L)273(Same as above)(Same as above)(Same as above)Example 7.8218-WT (E155R + N526R)274(Same as above)(Same as above)(Same as above)Example 7.9218-WT (W354A)302(Same as above)(Same as above)(Same as above)Example 7.10218-WT (E155A + W354A)303(Same as above)(Same as above)(Same as above)ControlUNT----.

[0733] Experimental Example 7.3. Experimental Results

[0734] The experimental results are shown in Figures 38 to 43. The quantitative data for the experimental results are shown in the following table:

[0735] LabelFANCFVEGFAsite3MTAPCFTRDYRK1A218-WT0.2829.1825.6040.1415.3210.27(Example 7.1)0.2728.7128.5241.6823.2412.67 0.4437.8728.7544.8212.2224.18218-WT (E155R)0.9247.0230.1341.2242.7043.61(Example 7.2)2.9350.7930.3449.6646.7951.24 2.9846.5342.2147.8340.7950.85218-WT (E791L)0.7248.2036.3238.0830.4214.16(Example 7.3)0.7451.1638.8130.6818.3420.89 0.8147.7840.2734.0636.0630.72218-WT (E155R+E791L)3.7341.6138.6338.3043.9729.82(Example 7.4)4.2450.1641.7344.7339.8340.37 2.5641.2743.9644.8249.1453.31218-WT (E155R+N526R+E791L)1.6522.2027.2230.3826.9732.34(Example 7.5)1.1333.7532.3441.6023.1037.15 3.1029.0232.3439.0324.0113.90218-WT (N526R)0.3015.7511.1524.701.081.55(Example 7.6)0.4119.4213.9731.442.133.11 0.2415.8214.2425.701.165.72218-WT (N526R+E791L)0.3327.4620.2032.762.542.02(Example 7.7)0.7626.1618.8528.051.997.42 0.7927.6421.4731.145.1611.51218-WT (E155R+N526R)0.3719.7724.2332.8414.0315.26(Example 7.8)0.7824.8529.3935.1913.6928.89 1.3327.2026.8146.2016.9828.53218-WT (W354A)0.0327.911.261.960.410.07(Example 7.9)0.1630.891.323.110.930.34 0.0031.361.453.570.330.23218-WT (E155R+W354A)0.0538.202.894.928.371.79(Example 7.10)0.0836.183.687.748.612.73 0.0044.002.738.5010.572.77UNT0.030.210.200.030.000.03(Control)0.030.030.170.070.000.00 0.030.000.130.070.040.05.

[0736] All figures represent the indel introduction rate as a percentage. Each experimental example was repeated three times. The experimental results showed that Examples 7.2 to 7.5 exhibited higher eukaryotic genome editing efficiency than the novel Cas12a protein (Example 7.1) without introducing mutations. Examples 7.6 to 7.8 did not show exceptional editing efficiency, but they were usable.

[0737] Experimental Example 8. Eukaryotic Genome Editing Activity of the CRISPR / Cas12a System Containing Cas12a Protein Variants #4

[0738] Experimental Example 8.1. Experimental Method and Configuration Used in the Experiment

[0739] In Experimental Example 5.1, a mutant in which an amino acid at a specific position is substituted with arginine, a mutant in which additional mutations were found by referring to a previous paper (Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV, Ishitani R, Zhang F, Nureki O. Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell. 2016 May 5;165(4):949-62. doi: 10.1016 / j.cell.2016.04.003. Epub 2016 Apr 21. PMID: 27114038; PMCID: PMC4899970.), and a wild-type Cas12a protein were used in the experiment with different nuclear localization signals.

[0740] Specifically, plasmids encoding each variant Cas12a protein were constructed according to Experimental Example 1.9, and guide RNA plasmids capable of co-operating with the corresponding Cas12a proteins were also prepared. The plasmid vectors were transfected into cells according to Experimental Example 1.6 using the Lipofectamine 2000 system (Thermo, 11668019). Subsequently, the cell genome was extracted and gene editing activity was measured. The extracted cell genome was analyzed according to Experimental Example 1.12.

[0741] The composition of each CRISPR / Cas12a complex used in Experimental Example 8 is shown in the following table:

[0742] LabelChart LabelN-terminal NLSCas12a protein(SEQ ID NO)C-terminal NLSDirect repeat of guide RNAGuide domain of guide RNA(SEQ ID NO)Example 8.1218-WTPKKKRKV (SEQ ID NO: 67)1SEQ ID NO: 67UAAUUUCUACUAAGUGUAGAU (SEQ ID NO: 33)DYRK1A: 143MTAP: 140VEGFA: ?FANCF: ?CFTR: ?Site3: ?Example 8.2218-WT-NLS6-1KRPAATKKAGQAKKKKPAAKRVKLDGGGGSGGGGSGGGGSPAAKRVKLD (SEQ ID NO: 70)(Same as above)(Same as above)Example 8.3218-WT-S1137A-NLS6-286SEQ ID NO: 70(Same as above)(Same as above)Example 8.4218-WT-RR-NLS6-300SEQ ID NO: 70(Same as above)(Same as above)Example 8.5218-WT-RRRSEQ ID NO: 67301SEQ ID NO: 67(Same as above)(Same as above)Example 8.6218-WT-RRR-NLS6-301SEQ ID NO: 70(Same as above)(Same as above)Example 8.7218-WT-S1137A-RRR-NLS6-304SEQ ID NO: 70(Same as above)(Same as above)ControlUNT-----

[0743] 실험예 8.2. 실험 결과

[0744] 실험 결과는 도 44 내지 도 49에 나타내었다.

[0745] Experimental results showed that Examples 8.1 and 8.2 exhibited high eukaryotic genome editing efficiency. Example 8.3, while not exceptionally efficient, exhibited usable editing efficiency.

[0746] Experimental Example 9. Confirmation of Multiple Target Nucleic Acid Cleavage Activity Using Multiplex Vector Loads

[0747] Experimental Example 9.1. Experimental Overview and Examples Used in the Experiment

[0748] If the novel CRISPR / Cas12a system disclosed in this specification has the characteristics of known CRISPR / Cas12a, the nucleic acid encoding the above guide RNA will be capable of self-maturation without including any other components such as terminators.

[0749] Furthermore, even if DNA encoding multiple guide RNAs is linked together without any spacers or terminators to induce transcription, each guide RNA will mature without any problems.

[0750] The process of self-maturation of multiple guide RNA sequences linked to a single promoter is schematically illustrated in Figure 50.

[0751] Applying these features, it is very easy to design a vector structure that induces simultaneous editing of multiple target nucleic acids.

[0752] Referring to Experimental Example 1, a vector construct containing the encoding nucleic acid in the following table was prepared:

[0753] PromoterDirect Repeat #1Guide Domain #1Direct Repeat #2Guide Domain #2Direct Repeat #3Guide Domain #3GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC (SEQ ID NO: 423)TAATTTCTACTAAGTGTAGAT (SEQ ID NO: 50)CTCTCAAGACCCACAATCCAGGC (SEQ ID NO: 415)SEQ ID NO: 50GCCCCAATAATCCCCACATGTCA (SEQ ID NO: 139)SEQ ID NO: 50GAAGCACATCAAGGACATTCTAA (SEQ ID NO: 142)

[0754] A vector construct containing DNA sequences sequentially connected from the 5' end to the 3' end was prepared according to the configuration of the table above. The vector construct prepared above was delivered to a cell together with a nucleic acid encoding a Cas12a protein containing the amino acid of sequence number 1 prepared according to Experimental Example 1.9, and it was confirmed whether the target sequence gene of each guide RNA was edited.

[0755] Experimental Example 9.2. Experimental Results

[0756] The experimental results of Experimental Example 9.1 are shown in Fig. 51.

[0757] As a result of the experiment, it was confirmed that when the vector structure of Experimental Example 9.1 and the Cas12a protein of sequence number 1 were delivered together to cells, indels were introduced in all multiple targets as intended.

[0758] This means that using a vector construct comprising multiple guide RNAs disclosed in this specification and a Cas12a protein, multiple target nucleic acids within a eukaryotic genome can be edited.

[0759] Experimental Example 10. Primer sequences used in Experimental Examples 2 to 10.

[0760] The primer sequences used in Experimental Examples 2 to 8 are shown in the following table:

[0761] 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

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768] The novel CRISPR / Cas12a system and eukaryotic genome editing method disclosed in this specification enable editing of target nucleic acids within the eukaryotic genome. Therefore, the novel CRISPR / Cas12a system can be utilized in a variety of contexts requiring eukaryotic genome editing.

Claims

1. A method for editing a target nucleic acid in a eukaryotic genome, comprising: A process for delivering a composition for eukaryotic genome editing to a eukaryotic cell, Herein, the composition for editing the eukaryotic genome comprises: a Cas12a protein having one or more nuclear localization signals (NLS) linked thereto, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked thereto; and a guide RNA, or a nucleic acid encoding said guide RNA; Here, the guide RNA comprises a guide domain and a scaffold; Here, the guide domain targets the target nucleic acid, The above Cas12a protein and the above scaffold are a combination selected from the following: A scaffold comprising a Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, and a nucleic acid sequence of SEQ ID NO: 33; or A scaffold comprising a Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 275 to SEQ ID NO: 279, and a nucleic acid sequence of SEQ ID NO:

35.

2. In the method for editing a target nucleic acid in the eukaryotic genome of paragraph 1, The Cas12a protein linked to one or more nuclear localization signals has the following structure: [NLS1] - [Cas12a] - [NLS2] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94, The above Cas12a is the Cas12a protein of claim 1, The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94, At least one of the above NLS1 or NLS2 is present.

3. In the method for editing a target nucleic acid in the eukaryotic genome of paragraph 2, In the structure of the Cas12a protein linked to one or more nuclear localization signals, The above NLS1 is SEQ ID NO: 67, and the above NLS2 is SEQ ID NO: 67; or The above NLS~1 is absent, and the above NLS2 is sequence number 70.

4. In any one of the methods for editing a target nucleic acid in a eukaryotic genome of paragraphs 1 to 3, The eukaryotic genome editing composition comprises a CRISPR / Cas12a complex in which the Cas12a protein linked to the at least one nuclear localization signal and the guide RNA are bound.

5. In any one of the methods for editing a target nucleic acid in a eukaryotic genome of paragraph 1 to embodiment 56, The eukaryotic genome editing composition comprises a nucleic acid encoding a Cas12a protein linked to at least one nuclear localization signal and a nucleic acid encoding the guide RNA.

6. A composition for editing a eukaryotic genome, comprising: a Cas12a protein having one or more nuclear localization signals linked thereto, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked thereto; and a guide RNA or a nucleic acid encoding said guide RNA, Here, the guide RNA comprises a scaffold and a guide domain, The 3' end of the above scaffold and the 5' end of the above guide domain are connected; Here, the guide RNA binds to the Cas12a protein to form a complex, The guide domain of the above guide RNA can target a predetermined target nucleic acid, The above Cas12a protein and the above scaffold are a combination selected from the following: A Cas12a protein comprising an amino acid sequence of SEQ ID NO: 1, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 33; A Cas12a protein comprising the amino acid sequence of SEQ ID NO: 3, and a scaffold comprising the nucleic acid sequence of SEQ ID NO: 35; or A Cas12a protein comprising an amino acid sequence of SEQ ID NO: 2, and a scaffold comprising a nucleic acid sequence of SEQ ID NO:

34.

7. In the composition for editing the eukaryotic genome of Article 6, The composition comprises a complex in which the Cas12a protein and the guide RNA are bound.

8. In the composition for editing the eukaryotic genome of Article 6, The composition comprises a nucleic acid encoding the Cas12a protein and a nucleic acid encoding the guide RNA.

9. In any one of the eukaryotic genome editing compositions of clauses 6 to 8, The Cas12a protein linked to one or more nuclear localization signals has the following structure: [NLS1] - [Cas12a] - [NLS2] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94, The above Cas12a is the Cas12a protein of claim 1, The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94, At least one of the above NLS1 or NLS2 is present.

10. A CRISPR / Cas12a composition comprising: Cas12a protein, or a nucleic acid encoding said Cas12a protein; and a guide RNA or a nucleic acid encoding said guide RNA, Here, the guide RNA comprises a scaffold and a guide domain, The 3' end of the above scaffold and the 5' end of the above guide domain are connected; Here, the guide RNA binds to the Cas12a protein to form a complex, The guide domain of the above guide RNA can target a predetermined target nucleic acid, The above Cas12a protein and the above scaffold are a combination selected from the following: A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 264 to SEQ ID NO: 274, and a scaffold comprising a nucleic acid sequence of SEQ ID NO: 33; A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 275 to SEQ ID NO: 279, and a scaffold comprising a nucleic acid sequence of SEQ ID NO:

35.

11. In the CRISPR / Cas12a composition of Article 10, The composition comprises a complex in which the Cas12a protein and the guide RNA are bound.

12. In the CRISPR / Cas12a composition of Article 10, The composition comprises a nucleic acid encoding the Cas12a protein and a nucleic acid encoding the guide RNA.

13. In any one of the CRISPR / Cas12a compositions of claims 10 to 12, The above Cas12a protein further comprises one or more nuclear localization signals, The above Cas12a protein has the following structure: [NLS1] - [Cas12a] - [NLS2] Here, the NLS1 comprises or is absent an amino acid sequence selected from among SEQ ID NO: 67 to SEQ ID NO: 94, The above Cas12a is the Cas12a protein of claim 1, The above NLS2 comprises or is absent an amino acid sequence selected from SEQ ID NO: 67 to SEQ ID NO: 94, At least one of the above NLS1 or NLS2 is present.

14. A method for editing two or more target nucleic acids in a eukaryotic genome, comprising: (a) delivering a Cas12a protein having one or more nuclear localization signals linked to said eukaryotic cell, or a nucleic acid encoding a Cas12a protein having one or more nuclear localization signals linked to said eukaryotic cell; (b) delivering the vector construct to the eukaryotic cell; Here, the vector structure comprises DNA encoding a first guide RNA and DNA encoding a second guide RNA, The first guide RNA is composed of a first guide domain and a first scaffold, The first guide domain targets a first target nucleic acid in the eukaryotic genome, The second guide RNA is composed of a second guide domain and a second scaffold, The second guide domain targets a second target nucleic acid within the eukaryotic genome, The DNA encoding the first guide RNA is operably linked to a promoter, The DNA encoding the second guide RNA is directly linked to the 3' end of the first guide RNA; Here, the above processes (a) and (b) may be performed simultaneously or in any order, The Cas12a protein, the first scaffold, and the second scaffold are a combination selected from the following: A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 264 to SEQ ID NO: 274, a first scaffold comprising a nucleic acid sequence of SEQ ID NO: 33, and a second scaffold comprising a nucleic acid sequence of SEQ ID NO: 33; or A Cas12a protein comprising an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 275 to SEQ ID NO: 279, a first scaffold comprising a nucleic acid sequence of SEQ ID NO: 35, and a second scaffold comprising a nucleic acid sequence of SEQ ID NO: 35.

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