Method for editing nucleic acid sequence and method for producing nucleic acid sequence

Abstract

To provide a method for editing nucleic acid sequences and method for producing nucleic acid sequences, in which a nucleic acid fragment that is knocked-in does not require a long homology arm.SOLUTION: Provided is a method for editing a nucleic acid sequence, comprising: associating a guide RNA including a guide sequence and a protein binding sequence with a CRISPR enzyme and a double-stranded target nucleic acid; cleaving the target nucleic acid with the CRISPR enzyme to form a cut portion having a first overhanging end; and inserting a nucleic acid fragment having at both ends second overhanging ends including a sequence complementary to the base sequence of the first overhanging end, into the cut portion of the target nucleic acid, wherein the guide sequence includes a sequence complementary to the target nucleic acid, the protein binding sequence is a region that associates with the CRISPR enzyme, and the nucleic acid fragment has a double strand sandwiched between the second overhanging ends.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for editing a nucleic acid sequence and a method for producing a nucleic acid sequence. [Background technology]

[0002] The CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) system has attracted attention as a next-generation genome editing technology. In particular, the method using Cas9 as an RNA-guided DNA endonuclease has been actively researched.

[0003] Patent Document 1 discloses genome editing using Cas9. In the CRISPR / Cas9 system, Cas9 and a guide RNA having a sequence homologous to a target DNA sequence are co-expressed to cleave the target DNA. Thus, the CRISPR / Cas9 system has the advantage that a guide RNA having a sequence homologous to the target DNA sequence is synthesized and genome editing is possible using a single protein, Cas9. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014 / 093661 Summary of the Invention [Problem to be solved by the invention]

[0005] When Cas9 cleaves the target double-stranded DNA, the cleavage site becomes blunt-ended. Therefore, when gene knock-in is performed at the cleavage site, the nucleic acid fragment to be knocked in (hereinafter also referred to as knock-in donor) requires a long homology arm that is homologous to the sequence adjacent to the cleavage site of the target double-stranded DNA. For example, when the knock-in donor is an oligo DNA fragment of about 1 to several tens of bp, a homology arm of 40 bases or more is generally used. When knocking in a gene in which the knock-in donor is about several hundred bp or more, a homology arm of about 500 bp to 2 kbp is generally used.

[0006] An object of the present invention is to provide a method for editing a nucleic acid sequence and a method for producing a nucleic acid sequence in which the knocked-in nucleic acid fragment does not require a long homology arm. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A method for editing a nucleic acid sequence, comprising: associating a guide RNA including a guide sequence and a protein binding sequence with a CRISPR enzyme and a double-stranded target nucleic acid; cleaving the target nucleic acid with the CRISPR enzyme to form a cleavage portion having a first overhanging end; and inserting a double-stranded nucleic acid fragment having second overhanging ends at both ends, the second overhanging ends comprising a sequence complementary to the base sequence of the first overhanging end, into the cleavage portion of the target nucleic acid, wherein the guide sequence includes a sequence complementary to the target nucleic acid, the protein binding sequence is a region that associates with the CRISPR enzyme, and the nucleic acid fragment has a double strand sandwiched between the second overhanging ends. [2] The nucleic acid sequence editing method of [1], wherein the second overhanging end further comprises a base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end, and the base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end comprises a base sequence complementary to the target nucleic acid adjacent to the 3' end of the first overhanging end. [3] The nucleic acid sequence editing method described in [2], wherein the base sequence adjacent to the 5' end of the sequence complementary to the first cohesive end in the second cohesive end is 5 to 20 bases. [4] The nucleic acid sequence editing method according to any one of [1] to [3], wherein the CRISPR enzyme is Cas12j8, Cas12a or Cas9 nickase. [5] The method for editing a nucleic acid sequence according to any one of [1] to [4], wherein the target nucleic acid is a vector or genomic DNA of 0.5 to 20 kbp or more. [6] The method for editing a nucleic acid sequence according to any one of [1] to [5], wherein the nucleic acid fragment is 10 bp to 20 kbp. [7] The method for editing a nucleic acid sequence described in any one of [1] to [6], further comprising transfecting a vector containing a sequence encoding the guide RNA, a vector containing a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell. [8] The method for editing a nucleic acid sequence according to any one of [1] to [6], further comprising transfecting a vector comprising a sequence encoding the guide RNA and a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell. [9] The nucleic acid sequence editing method described in [7], wherein the cell is a type selected from the group consisting of human fetal kidney cells, CHO cells, and Cos7 cells.

[10] The nucleic acid sequence editing method described in [8], wherein the cell is a type selected from the group consisting of human fetal kidney cells, CHO cells, and Cos7 cells.

[11] A method for producing a nucleic acid sequence, comprising: associating a guide RNA comprising a guide sequence and a protein binding sequence with a CRISPR enzyme and a double-stranded target nucleic acid; cleaving the target nucleic acid with the CRISPR enzyme to form a cleaved portion having a first overhanging end; and inserting a double-stranded nucleic acid fragment having a second overhanging end comprising a sequence complementary to a base sequence of the first overhanging end into the cleaved surface of the target nucleic acid to obtain a nucleic acid sequence, wherein the guide sequence comprises a sequence complementary to the target nucleic acid, the protein binding sequence is a region that associates with the CRISPR enzyme, and the nucleic acid fragment has a double strand sandwiched between the second overhanging ends.

[12] The method for producing a nucleic acid sequence described in

[11] , wherein the second overhanging end further comprises a base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end, and the base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end comprises a base sequence complementary to the target nucleic acid adjacent to the 3' end of the first overhanging end.

[13] The method for producing a nucleic acid sequence according to

[11] or

[12] , wherein the base sequence adjacent to the 5'-end of the sequence complementary to the first cohesive end in the second cohesive end is 5 to 20 bases long.

[14] The method for producing a nucleic acid sequence described in any one of

[11] to

[13] , wherein the CRISPR enzyme is Cas12j8, Cas12a or Cas9 nickase.

[15] The method for producing a nucleic acid sequence according to any one of

[11] to

[14] , wherein the target nucleic acid is a vector or genomic DNA having a base pair of 0.5 to 20 kbp or more.

[16] The method for producing a nucleic acid sequence according to any one of

[11] to

[15] , wherein the nucleic acid fragment is 10 bp to 20 kbp.

[17] The method for producing a nucleic acid sequence described in any one of

[11] to

[16] , further comprising transfecting a vector containing a sequence encoding the guide RNA, a vector containing a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

[18] The method for producing a nucleic acid sequence described in any one of

[11] to

[16] , further comprising transfecting a vector containing a sequence encoding the guide RNA and a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

[19] The method for producing a nucleic acid sequence described in

[17] , wherein the cell is one selected from the group consisting of a human fetal kidney cell, a CHO cell, and a Cos7 cell.

[20] The method for producing a nucleic acid sequence described in

[18] , wherein the cell is one selected from the group consisting of human fetal kidney cells, CHO cells and Cos7 cells. Effect of the Invention

[0008] According to the above aspect, it is possible to provide a nucleic acid sequence editing method and a nucleic acid sequence production method in which the nucleic acid fragment to be knocked in does not require a long homology arm. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram illustrating a nucleic acid sequence editing method and a nucleic acid sequence manufacturing method according to one embodiment. [Diagram 2] FIG. 1 is a schematic diagram illustrating a nucleic acid sequence editing method and a nucleic acid sequence manufacturing method according to one embodiment. [Diagram 3] FIG. 2 is a schematic diagram showing a portion of a nucleic acid fragment according to another embodiment. [Figure 4] FIG. 1 is a schematic diagram showing a target vector according to Experimental Example 1. [Diagram 5] 1 is a graph showing the fluorescence intensities measured in Examples 1-1 to 1-2 and Comparative Examples 1-1 to 1-3. [Figure 6] FIG. 1 is a schematic diagram showing a part of the sequence of a target nucleic acid according to Experimental Example 2. [Figure 7] Fluorescence microscope images of cells observed in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] <Definition> "Nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or polymers thereof in either single- or double-stranded form. "Nucleic acid sequence" refers to a polymer of DNA or RNA in either single- or double-stranded form.

[0011] The term "gene" refers to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" refers to a segment of DNA involved in producing or encoding a non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., small guide RNA), or microRNA.

[0012] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.

[0013] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against exogenous nucleic acids. CRISPR / Cas systems are classified into classes 1 and 2, which are further classified into types I through VI.

[0014] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, artificial chromosome or cosmid, to which another DNA segment, or "insert," may be attached so as to bring about replication of the attached segment in a cell.

[0015] When a numerical range is stated, for example, as "1 to 10," the numerical range is defined as including the lower limit of 1 and the upper limit of 10.

[0016] First Embodiment (Method for editing nucleic acid sequence and method for producing nucleic acid sequence) Hereinafter, the embodiments will be described in detail with reference to the drawings. The nucleic acid sequence editing method in this embodiment includes the steps of: associating a guide RNA including a guide sequence and a protein binding sequence, a CRISPR enzyme, and a double-stranded target nucleic acid; cleaving the target nucleic acid with the CRISPR enzyme to form a cleavage portion having a first overhanging end; and inserting a double-stranded nucleic acid fragment having second overhanging ends at both ends, the second overhanging ends comprising a sequence complementary to the base sequence of the first overhanging end, into the cleavage portion of the target nucleic acid, wherein the guide sequence includes a sequence complementary to the target nucleic acid, the protein binding sequence is a region that associates with the CRISPR enzyme, and the nucleic acid fragment has a double strand sandwiched between the second overhanging ends.

[0017] The nucleic acid sequence editing method in this embodiment includes: associating a guide RNA including a guide sequence and a protein binding sequence with a CRISPR enzyme and a double-stranded target nucleic acid; cleaving the target nucleic acid with the CRISPR enzyme to form a cleavage portion having a first overhanging end; and inserting a double-stranded nucleic acid fragment having a second overhanging end including a sequence complementary to the base sequence of the first overhanging end into the cleavage surface of the target nucleic acid to obtain a nucleic acid sequence, wherein the guide sequence includes a sequence complementary to the target nucleic acid, the protein binding sequence is a region that associates with the CRISPR enzyme, and the nucleic acid fragment has a double strand sandwiched between the second overhanging ends.

[0018] 1-2 are schematic diagrams illustrating a nucleic acid sequence editing method and a nucleic acid sequence manufacturing method according to the present embodiment. A guide RNA 1 includes a guide sequence 11 and a protein binding sequence 12. The guide sequence 11 is a sequence complementary to a guide RNA recognition sequence 21, which is a part of a double-stranded target nucleic acid 2. The protein binding sequence 12 is a sequence with which a CRISPR enzyme 3 associates. The protein binding sequence 12 allows the CRISPR enzyme 3 to be recruited to the target nucleic acid 2. This causes the guide RNA 1, the CRISPR enzyme 3, and the target nucleic acid 2 to associate with each other.

[0019] The CRISPR enzyme 3 is an enzyme having RNA-guided DNA endonuclease activity. The CRISPR enzyme 3 applied to this embodiment is a nuclease that generates a protruding end in a target nucleic acid 2. The nuclease domain of the CRISPR enzyme 3 is activated by recognizing a PAM (Protospacer adjacent motif) sequence 4. The CRISPR enzyme 3 cleaves the target nucleic acid 2 at a cleavage site 5 near the PAM sequence 4 to form a first protruding end 22. In FIG. 1, an example is described in which Cas12j8 is used as the CRISPR enzyme 3 and a first protruding end 22 protruding by 9 bases is formed, but the present invention is not limited thereto.

[0020] 2 is a knock-in donor that is inserted, i.e., knocked-in, into the target nucleic acid 2. The nucleic acid fragment 6 includes a desired nucleic acid sequence to be introduced into the target nucleic acid 2 and a second overhanging end 61 that includes a sequence complementary to the first overhanging end 22.

[0021] The number of bases of the first overhanging end 22 and the second overhanging end 61 may be the same, or the number of bases of the second overhanging end 61 may be greater than that of the first overhanging end 22. In this embodiment, a case where the number of bases of the first overhanging end 22 and the second overhanging end 61 is the same will be described. In this embodiment, the number of bases of the first overhanging end 22 and the second overhanging end 61 depends on the cleavage position of the target nucleic acid 2 by the CRISPR enzyme 3. For example, when Cas12j8 is used as the CRISPR enzyme 3, the number of bases of the first overhanging end 22 and the second overhanging end 61 is 9. A case where the number of bases of the second overhanging end 61 is greater than that of the first overhanging end 22 will be described later as a modified example.

[0022] As shown in FIG. 2, the nucleic acid fragment 6 binds to the cleaved portion of the cleaved target nucleic acid 2, and the nucleic acid fragment 6 is knocked into the target nucleic acid 2. The binding of the nucleic acid fragment 6 to the cleaved target nucleic acid 2 is performed by incubation at any temperature. For example, when performed inside a cell, the binding is preferably performed at 0 to 45°C, and more preferably at 35 to 40°C. Since the nucleic acid fragment 6 has the second protruding end 61, the nucleic acid fragment 6 can bind to the cleaved portion of the cleaved target nucleic acid 2 even by incubation at 0 to 45°C.

[0023] In this way, by using the CRISPR enzyme 3 that forms the first cohesive end 22 and the nucleic acid fragment 6 that includes the second cohesive end 61 that includes a sequence complementary to the first cohesive end 22, accurate knock-in is possible even if the nucleic acid fragment to be knocked in does not have a long homology arm. This allows editing and production of a desired nucleic acid sequence.

[0024] As described above, the guide RNA 1 includes the guide sequence 11 and the protein binding sequence 12. The guide sequence 11 is a sequence complementary to the base sequence of the desired knock-in position in the target nucleic acid 2, that is, the guide RNA sequence 21. Therefore, by synthesizing the guide RNA 1 including the guide sequence complementary to the base sequence of the desired knock-in position in the target nucleic acid 2, the nucleic acid fragment 6 can be knocked in at the desired position.

[0025] The protein binding sequence 12 varies depending on the organism and type of the CRISPR enzyme. For example, when the CRISPR enzyme is Cas12j8, the protein binding sequence is sequence 1 (SEQ ID NO: 1). In addition, when the CRISPR enzyme is Cas12j8, the protein binding sequence of Cas12j1 (SEQ ID NO: 2), the protein binding sequence of Cas12j2 (SEQ ID NO: 3), the protein binding sequence of Cas12j3 (SEQ ID NO: 4), the protein binding sequence of Cas12j4 (SEQ ID NO: 5), the protein binding sequence of Cas12j5 (SEQ ID NO: 6), the protein binding sequence of Cas12j7 (SEQ ID NO: 7), and the protein binding sequence of Cas12j9 (SEQ ID NO: 2) can also be used.

[0026] CRISPR enzyme 3 is a nuclease that cleaves target nucleic acid 2 and forms a cleavage portion having a protruding end. Examples of CRISPR enzyme 3 include bacteriophage-derived Cas12j18, Acidaminococcus sp.-derived Cas12a (AsCas12a), Francisella novicida-derived Cas12a (FnCas12a), and Streptococcus pyogenes-derived Cas9 (SpCas9) nickase. Cas12a and Cas12j are classified as class 2 V type. When target nucleic acid 2 is cleaved with Cas12a, a first protruding end 22 with a four-base protrusion is generated.

[0027] In the Cas9 nickase, one of the cleavage domains of wild-type Cas9 is inactivated, and the Cas9 nickase cleaves only one of the two strands of the target nucleic acid 2 to introduce a nick. When the Cas9 nickase is used as the CRISPR enzyme in the nucleic acid sequence editing method and the nucleic acid sequence production method of this embodiment, two types of guide RNAs containing different guide sequences are prepared so that protruding fragments with an appropriate number of bases are generated by cleavage of the target nucleic acid, and the Cas9 nickase, the two types of guide RNAs, and the target nucleic acid are associated to form a cleavage portion having a first protruding end 22.

[0028] When the CRISPR enzyme 3 is Cas9 nickase or Cas12a, the nucleic acid fragment 6 can be knocked in to the cleavage site having the first cohesive end 22, as in the case of Cas12j8.

[0029] The CRISPR enzyme 3 may have a mutation in part of the wild-type CRISPR enzyme, as long as the technical effects of the nucleic acid sequence editing method and nucleic acid sequence manufacturing method of this embodiment are not impaired.

[0030] The nucleic acid fragment 6 is a double-stranded DNA fragment including a second overhanging end 61 and an arbitrary sequence. The arbitrary sequence is not particularly limited and can be appropriately selected depending on the purpose of being knocked into the target nucleic acid 2. For example, the nucleic acid fragment 6 may include an arbitrary gene or a DNA sequence for SNP repair. The nucleic acid fragment 6 may be a vector such as an episomal vector. The length of the nucleic acid fragment 6 is not particularly limited and may be 10 bp to 20 kbp. In this specification, the length of the nucleic acid fragment 6 refers to the number of bases in the double-stranded portion not including the second overhanging end.

[0031] The nucleic acid fragment 6 may have a label moiety, if necessary. As used herein, the terms "label", "detectable label", or "label moiety" refer to any moiety that allows signal detection and may be broadly dependent on the specific properties in the analysis. Label moieties include both directly detectable labels and indirectly detectable labels. As an example of a directly detectable label, a fluorescent label may be any fluorescent label (e.g., fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, and ALEXAFLUOR® labels, etc.), fluorescent proteins (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any fluorescent derivatives thereof), etc.). In this embodiment, the method of detecting the label moiety may include spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, and chemical means.

[0032] The nucleic acid sequence editing method and the nucleic acid sequence production method of the present embodiment may be performed in a test tube, in a cell, or in a living body. When the nucleic acid sequence editing method and the nucleic acid sequence production method of the present embodiment are performed in a cell, the cell is not particularly limited, and for example, cells derived from mammals such as humans, non-mammals, plants, bacteria, yeast, etc. can be used. Specifically, fetal kidney cells derived from humans or other mammals, CHO cells derived from Chinese hamster ovaries, Cos7 cells derived from African green monkey kidneys, etc. can be used.

[0033] When performing the nucleic acid sequence editing method or the nucleic acid sequence production method of the present embodiment in a cell, it is preferable that the guide RNA1 and the CRISPR enzyme 3 are encoded in the same or different vectors and transfected into the cell. For example, different vectors in which the guide RNA1 and the CRISPR enzyme 3 are encoded may be transfected into the cell. Alternatively, a vector in which the guide RNA1 and the CRISPR enzyme 3 are encoded may be transfected into the cell. A known method may be appropriately adopted for transfecting the cell.

[0034] The target nucleic acid 2 may be a genome or a vector. When the target nucleic acid 2 is a vector, a sequence complementary to the guide sequence 11 of the guide RNA 1 is inserted into the vector. The vector may be 0.5 to 20 kbp.

[0035] As an example, the episomal vector pEB-Multi vector (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is a vector for rapidly obtaining a constitutive expression strain. The EBNA1 and OriP sequences encoded by pEB-Multi are essential for constitutive expression, but these sequences make the vector long at about 10 kbp.

[0036] When knocking in a gene of about 10 kbp into a long vector such as an episomal vector, the length of the produced vector will be about 20 kb. In general, it is extremely difficult to obtain a transformed strain with a plasmid of more than 10 kbp in normal E. coli, and a special E. coli strain such as XL-10Gold is required, but the transformation efficiency is not high.

[0037] On the other hand, according to the nucleic acid sequence editing method and nucleic acid sequence production method of the present embodiment, by transfecting an episomal vector, a nucleic acid fragment containing an arbitrary gene sequence, a vector expressing a CRISPR enzyme, and a guide RNA into an arbitrary cell, it is possible to knock in an arbitrary gene sequence into the episomal vector in the cell. Thereafter, by performing selection with an antibiotic or the like, a constitutive expression strain can be obtained. From the viewpoint of being able to produce such a large plasmid, the nucleic acid sequence editing method and nucleic acid sequence production method of the present embodiment are useful.

[0038] <Modification Example of the First Embodiment> In this modification example, the second protruding end 62 of the nucleic acid fragment 6' is different from the second protruding end 61 of the first embodiment. Since the rest is the same as in the first embodiment, the description is omitted.

[0039] FIG. 3 is a schematic diagram showing a part of the nucleic acid fragment 6' in this modification example. The second protruding end 62 of the nucleic acid fragment 6' of this modification example further includes a base sequence 63 complementary to the target nucleic acid 2 on the 5'-terminal side in addition to the sequence complementary to the first protruding end 22 as shown in FIG. 3. In the example of FIG. 3, a 9-base sequence complementary to the first protruding end 22 is adjacent, and a 11-base base sequence 63 complementary to the target nucleic acid 2 is further included in the second protruding end 62 on the 5'-terminal side thereof. That is, the base sequence 63 is a sequence complementary to the target nucleic acid 2 adjacent to the 3'-terminal side of the first protruding end 22.

[0040] When such a nucleic acid fragment 6' and the target nucleic acid 2 are combined, as shown in the lower part of FIG. 3, the base sequence 63 may invade between the double strands of the target nucleic acid 2 and form a flap 23 in a part of the target nucleic acid 2. The nucleic acid fragment 6' combined in this way is more stable than the nucleic acid fragment 6 of the first embodiment because the binding portion with the target nucleic acid 2 is longer. The flap 23 generated in this way can be cleaved by a flap endonuclease or the like.

[0041] As another aspect, since the second protruding ends 61 of the nucleic acid fragments 6 of the first embodiment are complementary to each other, polymerization of the nucleic acid fragments 6 may occur within the reaction system. Polymerization of the nucleic acid fragments 6 may reduce the substantial concentration of the nucleic acid fragments 6 in the reaction system, and may reduce the probability of binding between the target nucleic acid 2 and the nucleic acid fragment 6. On the other hand, since the base sequence 63 of the nucleic acid fragment 6' is not complementary, polymerization of the nucleic acid fragments 6' is unlikely to occur within the nucleic acid fragments 6'. Therefore, when the nucleic acid fragment 6' is used, it is considered that the decrease in the probability of binding between the target nucleic acid 2 and the nucleic acid fragment 6' can be suppressed.

[0042] The length of the second overhanging end 62 can be set arbitrarily, for example, preferably 9 to 30 bases, more preferably 11 to 25 bases. When the length of the second overhanging end 62 is 9 to 30 bases, the target nucleic acid 2 and the nucleic acid fragment 6' can be easily bound even in a cell. In another aspect, the TM value (melting temperature) of the second overhanging end 62 is preferably 37°C or higher, more preferably 42°C or higher. When the TM value of the second overhanging end 62 is 37°C or higher, the target nucleic acid 2 and the nucleic acid fragment 6 can be easily bound even in a cell. The length of the base sequence 63 can be set arbitrarily, for example, preferably 5 to 28 bases, more preferably 6 to 26 bases.

[0043] By using the nucleic acid fragment 6' of this modified example, even when a CRISPR enzyme with a short protruding end of the cleavage surface of only 4 bases, such as Cas12a, is used, the number of bases of the second protruding end 62 can be adjusted to, for example, 9 to 30 bases, thereby improving the probability of binding between the target nucleic acid 2 and the nucleic acid fragment 6'. EXAMPLES

[0044] The present invention will be specifically described below by way of examples, but the present invention is not limited to the description of the following examples.

[0045] [Experimental Example 1] (Reagents and Materials) As the target vector, which is the target nucleic acid, an expression vector having a target sequence containing a guide RNA recognition sequence (SEQ ID NO: 9) and a PAM sequence (TTA) under a CAG promoter and a sequence encoding GFP was used, as shown in Figure 4. Note that although the guide RNA recognition sequence contains the start codon ATG, there is a frame shift with respect to the sequence encoding GFP, and therefore the target vector itself is designed not to express GFP.

[0046] The first knock-in oligonucleic acid corresponding to the nucleic acid fragment was designed to be a double-stranded oligonucleic acid having a 9-base overhang that matches the cleavage surface of Cas12j8. The first knock-in oligonucleic acid was prepared by purchasing a single-stranded oligonucleic acid (SEQ ID NO: 10) and a single-stranded oligonucleic acid (SEQ ID NO: 11) that partially contain complementary sequences from Sigma-Aldrich, and using the following method. First, the above-mentioned two types of single-stranded oligonucleic acids were mixed in equal amounts. The final concentrations of NaCl, Tris (pH 7.9 at 25°C), MgCl, and 10 mM were respectively mixed. 2 The two types of single-stranded oligonucleic acids described above were added to a buffer containing 1 mM DTT, and the mixture was kept at 90°C for 5 minutes. The mixture was then cooled to room temperature at a rate of 1°C / min to anneal the two types of single-stranded oligonucleic acids, thereby obtaining the first knock-in oligonucleic acid.

[0047] The second knock-in oligonucleic acid corresponding to the nucleic acid fragment was designed to be a double-stranded oligonucleic acid having a protruding portion of 20 bases in total, consisting of 9 bases that match the cleavage surface of Cas12j8 and 11 bases adjacent to it. The second knock-in oligonucleic acid was prepared by purchasing single-stranded oligonucleic acid (SEQ ID NO: 12) and single-stranded oligonucleic acid (SEQ ID NO: 13) containing partial sequences complementary to each other from Sigma-Aldrich, and preparing them in the same manner as the first knock-in oligonucleic acid.

[0048] The expression vector for Cas12j8 as a CRISPR enzyme was Cas12j-8-puro (Plasmid #194966) manufactured by Addgene. The guide RNA expression vector used was an expression vector containing a sequence (SEQ ID NO: 14) encoding the guide RNA sequence under the human U6 promoter.

[0049] (Introduction into cells) Example 1-1 HEK293T cells (40,000 cells) were transfected with a mixture of 30 ng of target vector, 30 ng of Cas12j8 expression vector, 20 ng of guide RNA expression vector, and 20 ng of first knock-in oligonucleotide using the transfection reagent PEImax (registered trademark, manufactured by Polysciences). The transfected cells were seeded on a 96-well plate (manufactured by Sanplatec, product number: 27993) and incubated at 37°C for 4 days. Four days after seeding, the fluorescence intensity was measured using a fluorescent plate reader (manufactured by Tecan, product number: infinite 200Pro).

[0050] Example 1-2 An experiment was carried out in the same manner as in Example 1-1, except that the second knock-in oligonucleic acid was used instead of the first knock-in oligonucleic acid.

[0051] Comparative Example 1-1 The experiment was performed in the same manner as in Example 1-1, except that only 30 ng of the target vector was transfected, and the Cas12j8 expression vector, the guide RNA expression vector, and the first knock-in oligonucleotide were not transfected.

[0052] Comparative Example 1-2 The experiment was performed in the same manner as in Example 1-1, except that a guide RNA expression vector containing a sequence (sequence number 15) encoding a guide RNA sequence different from that in the example was transfected, and the first knock-in oligonucleic acid was not transfected.

[0053] Comparative Example 1-3 The experiment was carried out in the same manner as in Example 1-1, except that the first knock-in oligonucleic acid was not transfected.

[0054] Fig. 5 is a graph showing the fluorescence intensities measured in Examples 1-1 to 1-2 and Comparative Examples 1-1 to 1-3. In Fig. 5, Com Ex. indicates Comparative Example, Ex. indicates Example, insert oligos indicates knock-in oligonucleic acid, 1st indicates the first knock-in oligonucleic acid, and 2nd indicates the second knock-in oligonucleic acid.

[0055] In Comparative Examples 1-1 and 1-2, no fluorescence was observed. In Comparative Example 1-3, fluorescence was observed. This is thought to be because a mutation (e.g., deletion or insertion) occurred at the cut surface of the target vector by Cas12j8 due to non-homologous end joining (also called NHEJ), and the ATG sequence in the target vector that was originally out of frame with the sequence encoding GFP happened to coincidentally match the frame of the sequence encoding GFP, resulting in the fluorescence being observed. In other words, the fluorescence observed in Comparative Example 1-3 is thought to be a measurement of the effect of NHEJ.

[0056] In Example 1-1, it is considered that the first knock-in oligonucleic acid is knocked into the target vector, and the ATG sequence on the first knock-in oligonucleic acid is used as a start codon, thereby expressing GFP and observing fluorescence. However, as described above, it is considered that the fluorescence due to NHEJ observed in Comparative Example 1-3 is also included in the fluorescence intensity observed in Example 1-1. It is considered that at least the difference between the fluorescence intensity observed in Example 1-1 and the fluorescence intensity observed in Comparative Example 1-3 is the fluorescence intensity caused by the first knock-in oligonucleic acid being knocked into the target vector.

[0057] For the same reason, the difference between the fluorescence intensity observed in Example 1-2 and Comparative Example 1-3 is considered to be the fluorescence intensity caused by the second knock-in oligonucleic acid being knocked into the target vector. Also, it can be seen that the effect of increasing the fluorescence intensity by the second knock-in oligonucleic acid is significantly greater than that of Example 1-1, in which the first knock-in oligonucleic acid was used.

[0058] The reason for the increase in fluorescence intensity in Example 1-2 may be that a strand exchange reaction between the target vector, which is a target nucleic acid, and the second knock-in oligonucleic acid, which is a nucleic acid fragment, may have caused a portion of the double strand of the target vector to be peeled off, resulting in the binding of the second knock-in oligonucleic acid to the target vector, as shown in Figure 3. In this way, when a 20-base overhang (i.e., an overhanging end) is bound to the target vector, it is more stable than when a 9-base overhang is bound to the target vector, which is thought to have improved the knock-in efficiency.

[0059] Another reason for the increase in fluorescence intensity in Example 1-2 is that the polymerization of knock-in oligonucleic acids may have been inhibited. The 9-base overhanging portions of the first knock-in oligonucleic acid are complementary to each other, so the first knock-in oligonucleic acid may polymerize in cells. However, the 11-base portion of the second knock-in oligonucleic acid other than the cleavage surface of Cas12j8 is not complementary to each other, so this polymerization may have been inhibited. When knock-in oligonucleic acids polymerize with each other, the effective concentration of knock-in oligonucleic acid in cells is reduced, and the knock-in efficiency is thought to be reduced, but when the second knock-in oligonucleic acid is used, such a decrease in knock-in efficiency is thought to be suppressed.

[0060] [Experimental Example 2] The following experiment was carried out to create a giant plasmid using Cas12j8. The PAM sequence (TTT) and guide RNA recognition sequence (SEQ ID NO: 16) shown in Figure 6 were introduced into the multicloning site (MCS) of the episomal vector pEB-Multi-Hyg (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and a target nucleic acid was created with the guide RNA recognition sequence and the cleavage site by Cas12j8 as the target sequence.

[0061] The knock-in vector used as the nucleic acid fragment contained a PAM sequence (TTC), a guide RNA recognition sequence corresponding to the target sequence, a cleavage portion by Cas12j8, and a sequence encoding GFP, and lacked a promoter.

[0062] As a Cas12j8 and guide RNA expression vector, we used a vector from Addgene in which a sequence encoding a guide RNA (sequence number 17) was introduced under the U6 promoter of Cas12j-8-puro (plasmid #194966).

[0063] Example 2 Human fetal kidney cells HEK293T cells (40,000 cells) were transfected with a mixture of the above-mentioned target nucleic acid 40 ng, Cas12j8 and guide RNA expression vector 30 ng, and knock-in vector 30 ng using the transfection reagent PEImax (registered trademark, manufactured by Polysciences). The transfected cells were seeded on a 96-well plate (manufactured by Sanplatec, product number: 27993) and incubated at 37°C for 4 days. Four days after seeding, the fluorescence intensity was measured using a fluorescent plate reader (manufactured by Tecan, product number: infinite 200Pro).

[0064] As shown in Figure 7, successful knock-in of the knock-in vector into the target nucleic acid, that is, the episomal vector pEB-Multi-Hyg, was confirmed in approximately 10 cells in one microscope field.

[0065] The results shown in Example 2 indicate that a constitutive expression strain can be obtained by transfecting the target cells with the pEB-Multi vector, a vector incorporating a target sequence into the target gene, and Cas12j8 and a guide RNA expression vector, and then selecting with antibiotics or the like. [Explanation of symbols]

[0066] 1...guide RNA, 2...target nucleic acid, 3...CRISPR enzyme, 4...PAM sequence, 5...cleavage site, 6,6'...nucleic acid fragment, 11...guide sequence, 12...protein binding sequence, 21...guide RNA recognition sequence, 22...first protruding end, 23...flap, 61,62...second protruding end, 63...base sequence

Claims

1. Associating a guide RNA comprising a guide sequence and a protein binding sequence, a CRISPR enzyme, and a target nucleic acid that is double stranded; cleaving the target nucleic acid with the CRISPR enzyme to form a cleavage portion having a first overhanging end; and inserting a nucleic acid fragment having second cohesive ends at both ends, the second cohesive ends including a base sequence complementary to the base sequence of the first cohesive end, into the cleaved portion of the target nucleic acid, the guide sequence comprises a sequence complementary to the target nucleic acid; The protein binding sequence is a region that associates with the CRISPR enzyme, A method for editing a nucleic acid sequence, wherein the nucleic acid fragment has a double strand flanked by the second overhanging ends.

2. the second cohesive end further comprises a base sequence adjacent to the 5'-end of the sequence complementary to the first cohesive end; The nucleic acid sequence editing method of claim 1, wherein the base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end includes a base sequence complementary to the target nucleic acid adjacent to the 3' end of the first overhanging end.

3. The nucleic acid sequence editing method according to claim 2, wherein the base sequence adjacent to the 5' end of the sequence complementary to the first overhanging end in the second overhanging end is 5 to 20 bases long.

4. The nucleic acid sequence editing method according to any one of claims 1 to 3, wherein the CRISPR enzyme is Cas12j8, Cas12a or Cas9 nickase.

5. The nucleic acid sequence editing method according to any one of claims 1 to 3, wherein the target nucleic acid is a vector or genomic DNA of 0.5 to 20 kbp or more.

6. The nucleic acid sequence editing method according to any one of claims 1 to 3, wherein the nucleic acid fragment is 10 bp to 20 kbp.

7. The nucleic acid sequence editing method according to any one of claims 1 to 3, further comprising transfecting a vector comprising a sequence encoding the guide RNA, a vector comprising a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

8. The nucleic acid sequence editing method according to any one of claims 1 to 3, further comprising transfecting a vector containing a sequence encoding the guide RNA and a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

9. The method for editing a nucleic acid sequence according to claim 7, wherein the cell is a type selected from the group consisting of human fetal kidney cells, CHO cells and Cos7 cells.

10. The method for editing a nucleic acid sequence according to claim 8, wherein the cell is a type selected from the group consisting of human fetal kidney cells, CHO cells and Cos7 cells.

11. Associating a guide RNA comprising a guide sequence and a protein binding sequence, a CRISPR enzyme, and a target nucleic acid that is double stranded; cleaving the target nucleic acid with the CRISPR enzyme to form a cleavage portion having a first overhanging end; and inserting a nucleic acid fragment having a second cohesive end including a sequence complementary to the base sequence of the first cohesive end into the cleaved surface of the target nucleic acid to obtain a nucleic acid sequence, the guide sequence comprises a sequence complementary to the target nucleic acid; The protein binding sequence is a region that associates with the CRISPR enzyme, The method for producing a nucleic acid sequence, wherein the nucleic acid fragment has a double strand flanked by the second overhanging ends.

12. the second cohesive end further comprises a base sequence adjacent to the 5'-end of the sequence complementary to the first cohesive end; The method for producing a nucleic acid sequence according to claim 11, wherein the base sequence adjacent to the 5'-end of the sequence complementary to the first protruding end includes a base sequence complementary to the target nucleic acid adjacent to the 3'-end of the first protruding end.

13. 13. The method for producing a nucleic acid sequence according to claim 12, wherein the base sequence adjacent to the 3'-end side of the sequence complementary to the first cohesive end in the second cohesive end is 5 to 20 bases long.

14. The method for producing a nucleic acid sequence according to any one of claims 11 to 13, wherein the CRISPR enzyme is Cas12j8, Cas12a or Cas9 nickase.

15. The method for producing a nucleic acid sequence according to any one of claims 11 to 13, wherein the target nucleic acid is a vector or genomic DNA of 0.5 to 20 kbp or more.

16. The method for producing a nucleic acid sequence according to any one of claims 11 to 13, wherein the nucleic acid fragment is 10 bp to 20 kbp.

17. The method for producing a nucleic acid sequence according to any one of claims 11 to 13, further comprising transfecting a vector comprising a sequence encoding the guide RNA, a vector comprising a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

18. The method for producing a nucleic acid sequence according to any one of claims 11 to 13, further comprising transfecting a vector comprising a sequence encoding the guide RNA and a sequence encoding the CRISPR enzyme, and the nucleic acid fragment into a cell.

19. 18. The method for producing a nucleic acid sequence according to claim 17, wherein the cell is a member selected from the group consisting of human fetal kidney cells, CHO cells and Cos7 cells.

20. 20. The method for producing a nucleic acid sequence according to claim 18, wherein the cell is a member selected from the group consisting of a human fetal kidney cell, a CHO cell and a Cos7 cell.

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