Crrna, type i crispr-cas system, method for editing target DNA, method for producing cells in which target DNA has been edited, method for detecting target DNA, and kit

Abstract

Provided is crRNA that is circular and includes a spacer sequence which includes a sequence that specifically recognizes and binds to a crRNA recognition sequence in target DNA, and a repeat sequence which can be cleaved by at least one Cas protein selected from the group consisting of Cas9 proteins and Cas9 proteins.

Description

crRNA, Type I CRISPR-Cas system, method for editing target DNA, method for producing cells with edited target DNA, method for detecting target DNA, and kit

[0001] The present invention relates to crRNA, a Type I CRISPR-Cas system, a method for editing target DNA, a method for producing cells in which target DNA has been edited, a method for detecting target DNA, and a kit. More specifically, the present invention relates to a novel form of crRNA, a Type I CRISPR-Cas system comprising the same, and a method for editing target DNA using these, a method for producing cells in which target DNA has been edited, and a method for detecting target DNA, as well as kits for use in these methods.

[0002] Genome editing technology specifically cuts genomic DNA sequences within animal or plant cells and uses their inherent repair mechanisms to freely rewrite them into any sequence. Its use is expanding worldwide, not only in the field of bioscience research, but also in the fields of improved breeding of agricultural crops and livestock, regenerative medicine, and gene therapy.

[0003] CRISPR-Cas systems found in bacteria and archaea are divided into class 1, which cleaves the target region using a complex (cascade) of multiple proteins, and class 2, which cleaves using a single protein. To date, CRISPR-Cas9 systems, CRISPR-Cas12 (Cpf1) systems, and CRISPR-Cas13 systems, which are classified as class 2, have been developed as genome editing tools. In particular, the CRISPR-Cas9 system is a system that includes the Cas9 protein and guide RNA (consisting of crRNA that specifically recognizes and binds to a sequence near the target region on the target DNA, and tracrRNA that forms a complex with the Cas9 protein), and various studies have been conducted on these Cas9 proteins and guide RNAs. For example, Non-Patent Document 1 (Li Liu et al., ACS Synth. Biol., 2023, 12, pp. 350-359) describes that guide RNA is circularized to improve its stability in vitro and in Escherichia coli.

[0004] All of the above systems are classified as Class 2, but recently, it has been discovered that the Type I CRISPR-Cas system, which belongs to Class 1, can be used as a genome editing tool for eukaryotic cells (WO 2018 / 225858 (Patent Document 1)). This Type I CRISPR-Cas system is a system that includes a Cas protein with nuclease activity (typically a Cas3 protein, or in Type I-D, a Cas10 protein), a cascade composed of multiple Cas proteins (cascade proteins), and crRNA. It has been found that it can specifically cleave target regions even in eukaryotic cells such as human cultured cells, and can introduce deletion mutations of a wide range of several hundred to several kb containing the target region with high efficiency. In addition, compared to the above-mentioned CRISPR-Cas9 system, the sequence recognized and bound by crRNA (crRNA recognition sequence) is long and highly specific, making it less likely to cause nonspecific cleavage and considered to be highly safe.

[0005] It has been reported that genome editing techniques in eukaryotic cells using the Type I CRISPR-Cas system require the use of a crRNA precursor (precursor crRNA) that retains full-length repeat sequences at both ends. When such a crRNA precursor is introduced into a eukaryotic cell, it is processed by Cas proteins (Cas5 protein, Cas6 protein) within the cell to become mature crRNA. This mature crRNA and Cascade protein form a complex (Cascade complex) near the target region, and the Cas protein, which has nuclease activity, cleaves the target region, enabling genome editing.

[0006] International Publication No. 2018 / 225858

[0007] Li Liu et al. , ACS Synth. Biol. , 2023, 12, p350-359

[0008] However, research into the Type I CRISPR-Cas system, whose usefulness has only recently been discovered, has not yet progressed sufficiently, and even more efficient genome editing is required. Furthermore, the above-mentioned crRNA precursor required for genome editing in eukaryotic cells using the Type I CRISPR-Cas system is a short RNA of about 90 bases, and is therefore easily degraded within the cell. Therefore, there was also the problem that a large amount of crRNA precursor needed to be introduced into the cell to ensure sufficient genome editing efficiency.

[0009] The present invention has been made in view of the problems associated with the above-mentioned conventional technologies, and aims to provide crRNA that enables highly efficient genome editing using a Type I CRISPR-Cas system and is highly stable within cells, a Type I CRISPR-Cas system containing the same, methods for editing target DNA using these, methods for producing cells in which target DNA has been edited, and methods for detecting target DNA, as well as kits for use in these methods.

[0010] As a result of intensive research to achieve the above-mentioned object, the inventors have found that by circularizing the crRNA contained in the Type I CRISPR-Cas system to form a novel form of circular crRNA, it exhibits significantly higher single-stranded DNA cleavage activity than when a conventional linear crRNA precursor (linear pre-crRNA) is used, and excellent genome editing efficiency can be achieved.

[0011] Furthermore, circularization not only enables genome editing in eukaryotic cells when the crRNA is circularized from a crRNA precursor, but also surprisingly enables genome editing in eukaryotic cells when the mature crRNA is circularized, which previously could not be confirmed as having genome editing activity when directly introduced into eukaryotic cells. It has also been discovered that even when circularized, the ability to form a cascade complex (complex formation rate, etc.) is equivalent to that when linear pre-crRNA is used, and that the above-mentioned excellent genome editing efficiency can be achieved with a smaller amount of Cas protein than with linear pre-crRNA. When the circular crRNA is circularized from a mature crRNA, i.e., consisting of one spacer sequence and one repeat sequence, the RNA length can be made shorter (typically about 61 nt) than the crRNA precursor (typically about 90 nt), thereby reducing the risk of introducing error sequences during synthesis and also enabling lower synthesis costs. In addition, because circularization significantly changes the morphology, it also facilitates separation from impurities such as incomplete crRNA that cannot contribute to genome editing.

[0012] Furthermore, the inventors discovered that by making the crRNA circular, it exhibits significantly higher stability within cells than not only conventional linear pre-crRNA precursors but also linear pre-crRNA precursors whose ends have been modified to increase stability within cells, and that the above-mentioned excellent genome editing efficiency can be maintained for a sufficient period of time, leading to the completion of the present invention.

[0013] The present invention provides the following aspects based on this finding: [1] A circular crRNA comprising: a spacer sequence including a sequence that specifically recognizes and binds to a crRNA recognition sequence on a target DNA; and a repeat sequence that can be cleaved by at least one Cas protein selected from the group consisting of Cas5 protein and Cas6 protein. [2] The crRNA of claim 1, consisting of one spacer sequence and one repeat sequence. [3] A Type I CRISPR-Cas system for editing target DNA, comprising the following (A) to (C): (A) a Cas protein having nuclease activity, (B) a Cascade protein, and (C) the crRNA of [1] or [2]. [4] A method for editing target DNA, comprising the step of contacting target DNA with the Type I CRISPR-Cas system of [3] and editing the target DNA. [5] A method for producing a cell in which target DNA has been edited, comprising the steps of introducing or expressing the Type I CRISPR-Cas system according to [3] into a cell, contacting the cell with target DNA, and editing the target DNA. [6] The method according to [5], wherein the cell is a eukaryotic cell. [7] A method for detecting target DNA in a sample, comprising the steps of contacting the Type I CRISPR-Cas system according to [3] with a sample and detecting the target DNA in the sample.[8] A kit for use in the method according to any one of [4] to [7], comprising the following (A') to (C'): (A') at least one selected from the group consisting of a Cas protein having nuclease activity, an expression vector for the Cas protein, and a polynucleotide encoding the Cas protein; (B') at least one selected from the group consisting of a Cascade protein, an expression vector for the Cascade protein, and a polynucleotide encoding the Cascade protein; (C') at least one selected from the group consisting of the crRNA according to [1] or [2], an expression vector for the crRNA, and a polynucleotide encoding the crRNA; wherein the crRNA expression vector is at least one selected from the group consisting of (i) a vector comprising a polynucleotide encoding the spacer sequence and the repeat sequence, and (ii) a vector comprising an insertion site for the polynucleotide encoding the spacer sequence and a polynucleotide encoding the repeat sequence.

[0014] According to the present invention, it is possible to provide a Type I CRISPR-Cas system that enables highly efficient genome editing and has high stability within cells, a Type I CRISPR-Cas system containing the same, methods for editing target DNA using the system, methods for producing cells in which target DNA has been edited, and methods for detecting target DNA, as well as kits for use in these methods.

[0015]

[0023] FIG. 1 is a schematic diagram showing one embodiment of the configuration of the spacer sequence and repeat sequence contained in the circular crRNA of the present invention. (a) is a denaturing polyacrylamide gel electrophoresis image of Seq_1 and Seq_2 (without ligase added: Ligase(-) and with ligase added: Ligase(+)) obtained in Test Example 1(2), and (b) is a denaturing polyacrylamide gel electrophoresis image of the raw material (Seq_2 before cyclization: lane 1), the mixture after the cyclization reaction (lane 2), and the cyclized product after isolation and purification (lane 3). (b) is a schematic conceptual diagram showing the synthesis method of circular mature-crRNA (a) and circular pre-crRNA (b) in Test Example 2(1). (c) is an electrophoresis image confirming the synthesis of circular mature-crRNA and circular pre-crRNA in Test Example 2(1). In the in vitro test of Test Example 2 (2), the amount of Cas6 protein: 7.2 μM, with double-stranded DNA fragments (Activator +) or without double-stranded DNA fragments (Activator -), when using circular mature-crRNA or linear pre-crRNA, is a graph showing the change in signal intensity (RFU / min). In the in vitro test of Test Example 2 (2), the amount of Cas6 protein: 3.6 μM, with double-stranded DNA fragments (Activator +) or without double-stranded DNA fragments (Activator -), when using circular mature-crRNA or linear pre-crRNA, is a graph showing the change in signal intensity (RFU / min). Circular mature-crRNA (circular crRNA, Example), linear pre-crRNA (PC), linear crRNA-1, and linear crRNA-2 used in Test Example 3 (1) are shown in a schematic conceptual diagram (a), and these crRNAs were introduced into K562 cells expressing the Cas protein group (Cas3, Cas5, Cas6, Cas7, Cas8, Cas11), and the recovered total genomic DNA was used as a template. Electrophoretic image (b) of the PCR product obtained. Genome editing efficiency (Edited (%), average of 2 tests) calculated from a comparison of band densities in the electrophoretic image of Test Example 3 (1).Circular pre-crRNA (circular crRNA, Example), linear pre-crRNA (PC), and pMax_GFP plasmid obtained in Test Example 3 (2) were introduced into K562 cells expressing the Cas protein group (Cas3, Cas5, Cas6, Cas7, Cas8, Cas11), and the recovered total genomic DNA was used as a template to obtain an electrophoretic image of the PCR product. A schematic conceptual diagram showing the timing of each electroporation (transfection), the timing of doxycycline addition (+Dox: -6h, 0h, 12h, 24h), and the timing of cell recovery (end point of each arrow) in Test Example 4. Electrophoretic images obtained in Test Example 4 show the combinations of crRNA (Circ: circular mature-crRNA, Line: linear crRNA-2, Mod: terminally modified linear pre-crRNA, +: linear pre-crRNA) introduced into cells and the timing of doxycycline addition (-6 h, 0 h, 12 h, 24 h). A graph showing the genome editing efficiency (Edited (%)) calculated from a comparison of the band densities in the electrophoretic images of Test Example 4. A schematic conceptual diagram showing the location of a representative nucleotide sequence in which deletion was confirmed by long-read sequence analysis of the PCR product in Test Example 4 when circular mature-crRNA (Circ) was used and doxycycline was added simultaneously with electroporation (0 h).

[0016] The present invention will be described in more detail below by taking preferred embodiments as examples, but the present invention is not limited thereto.

[0017] <crRNA, Type I CRISPR-Cas System> The present invention provides a circular crRNA (sometimes referred to herein as "circular crRNA") that includes: a spacer sequence including a sequence that specifically recognizes and binds to a crRNA recognition sequence on target DNA; and a repeat sequence that can be cleaved by at least one Cas protein selected from the group consisting of Cas5 protein and Cas6 protein. The present invention also provides a Type I CRISPR-Cas system for editing target DNA, comprising the following (A) to (C): (A) a Cas protein having nuclease activity, (B) a Cascade protein, and (C) the circular crRNA of the present invention.

[0018] (Target DNA) In the present invention, DNA containing a target region to be subjected to DNA editing is referred to as "target DNA." The target DNA according to the present invention is double-stranded DNA, and in order to show its correspondence with the Type I CRISPR-Cas system of the present invention, for convenience, at least one strand has a structure containing, from the 5' end, a PAM sequence and the target region, and a complementary sequence that forms a strand complementary to the target region is contained in a crRNA recognition sequence that is specifically recognized and bound by a spacer sequence of crRNA, or the crRNA recognition sequence is contained in the complementary sequence, or the complementary sequence and the crRNA recognition sequence at least partially overlap each other.

[0019] In the present invention, the term "target region" refers to a region that undergoes single-strand cleavage by a Cas protein (typically a Cas3 protein, or a Cas10 protein in Type I-D) having nuclease activity in a Type I CRISPR-Cas system. However, this does not preclude editing of nearby regions other than the target region. The length of the target region according to the present invention is preferably 20 to 50 bases, and more preferably 28 to 37 bases.

[0020] In the present invention, a "crRNA recognition sequence" is a sequence that is recognized and bound by the spacer sequence. The length of the crRNA recognition sequence according to the present invention is preferably 10 to 70 bases, more preferably 20 to 50 bases, even more preferably 25 to 40 bases, and typically 32 to 37 bases. Because the present invention uses a Type I CRISPR-Cas system, the length of the crRNA recognition sequence can be made longer than when the CRISPR-Cas9 system is used (for example, preferably about 20 bases), thereby enabling increased sequence specificity for the target DNA.

[0021] The "PAM (protospacer adjacent motif) sequence" is a sequence recognized by the Cas proteins that constitute the cascade of the Type I CRISPR-Cas system, and its length varies depending on the type of Type I CRISPR-Cas system, but is typically 2 to 5 bases adjacent to the complementary sequence of the crRNA recognition sequence. The nucleotide sequence of the PAM sequence also varies depending on the type of Type I CRISPR-Cas system. Typically, for example, when the Cas proteins constituting the cascade are those constituting a Type I-B CRISPR-Cas system or a Type I-F CRISPR-Cas system, the PAM sequences recognized by these proteins include 5'-CCA, 5'-CC, 5'-ACN, etc.; when the Cas proteins constituting the Type I-E CRISPR-Cas system are those constituting a Type I-E CRISPR-Cas system, the PAM sequences recognized by these proteins include 5'-AAG, 5'-AGG, 5'-GAG, etc. However, these PAM sequences can also be changed by modifying the Cas proteins (e.g., by introducing a mutation), thereby expanding the range of target regions available. In the present invention, the PAM sequence is located on the 5' side of the target region, and the crRNA recognition sequence is determined depending on the position of the PAM sequence and the target region.

[0022] The nucleotide sequence of such target DNA is not particularly limited, and by designing the Type I CRISPR-Cas3 system of the present invention to match that sequence, it can become the target of DNA editing of the present invention.

[0023] The target DNA according to the present invention can be DNA present within a cell (endogenous DNA) or DNA present outside a cell, depending on the purpose. The DNA present within a cell may be endogenous DNA or exogenous DNA. Examples of endogenous DNA include genomic DNA in chromosomes, mitochondria, or chloroplasts, and examples of exogenous DNA include DNA introduced into a cell (reporter genes, marker genes, viruses that infect a host, genes of bacteria or protozoa, etc.). DNA present outside a cell may be DNA derived from a cell or DNA amplified and synthesized outside a cell.

[0024] (crRNA) The circular crRNA of the present invention is useful as a crRNA (CRISPR RNA) constituting a Type I CRISPR-Cas system. The circular crRNA of the present invention is characterized in that it is circular and comprises a spacer sequence including a sequence that specifically recognizes and binds to the crRNA recognition sequence on the target DNA, and a repeat sequence that can be cleaved by at least one Cas protein selected from the group consisting of Cas5 protein and Cas6 protein.

[0025] A "spacer sequence" is originally a sequence derived from exogenous DNA incorporated during the adaptation process into the CRISPR structure of the bacterial genome from which the Type I CRISPR-Cas system is derived. However, the spacer sequence of the present invention is a sequence designed to specifically recognize and bind to the crRNA recognition sequence. Such a spacer sequence is preferably a complementary sequence to the crRNA recognition sequence, but any nucleotide sequence capable of hybridizing with the crRNA recognition sequence to form a double strand need not be completely complementary. In the present invention, the sequence complementarity between such a spacer sequence and the crRNA recognition sequence is preferably 80% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). Those skilled in the art can appropriately calculate the sequence complementarity using known methods (e.g., BLAST (NCBI)).

[0026] The spacer sequence can be designed to correspond to the crRNA recognition sequence according to the target region of interest, and its length is preferably 10 to 70 bases, more preferably 20 to 50 bases, even more preferably 25 to 40 bases, and typically 32 to 37 bases.

[0027] The "repeat sequence" is a sequence that is repeated via a spacer sequence and forms a stem-loop structure in the CRISPR structure of the bacterial genome from which the Type I CRISPR-Cas system is derived. The wild-type repeat sequence varies depending on the type of Type I CRISPR-Cas system and the species of the derived bacterium. For example, in the Type I-A CRISPR-Cas system derived from Pyrococcus furiosus, it typically consists of the nucleotide sequence set forth in SEQ ID NO: 1, which has a chain length of 30 bases, and in the Type I-A CRISPR-Cas system derived from Synechocystis sp. The type I-B CRISPR-Cas system derived from Neisseria lactamica typically comprises the nucleotide sequence set forth in SEQ ID NO: 2 having a chain length of 36 bases, the type I-C CRISPR-Cas system derived from Neisseria lactamica typically comprises the nucleotide sequence set forth in SEQ ID NO: 3 having a chain length of 32 bases, the type I-D CRISPR-Cas system derived from Microcystis aeruginosa typically comprises the nucleotide sequence set forth in SEQ ID NO: 4 having a chain length of 37 bases, and the type I-D CRISPR-Cas system derived from Escherichia coli typically comprises the nucleotide sequence set forth in SEQ ID NO: 5 having a chain length of 37 bases. A type I-E CRISPR-Cas system typically consists of the nucleotide sequence set forth in SEQ ID NO: 5, which has a chain length of 29 bases; a type I-F CRISPR-Cas system derived from Pseudomonas aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 6, which has a chain length of 28 bases; and a type I-G CRISPR-Cas system derived from Thioalkalivibrio sulfidiphilus typically consists of the nucleotide sequence set forth in SEQ ID NO: 7, which has a chain length of 36 bases.

[0028] However, the repeat sequences of the present invention may be appropriately modified, as with the above-described typical wild-type repeat sequences, as long as they are cleavable by at least one Cas protein selected from the group consisting of Cas5 protein and Cas6 protein and are capable of forming a cascade complex with the Cas proteins. For example, the repeat sequences of the present invention may each independently be those in which one or more (e.g., 8 or less, 7 or less, 6 or less, preferably 5 or less, 4 or less, 3 or less, or 2 or less) bases have been substituted, deleted, and / or inserted in the above-described typical wild-type repeat sequences.

[0029] When the repeat sequence is cleaved by the Cas protein, typically, the repeat sequence is cleaved by the Cas6 protein in types IA, B, and D to G, and by the Cas5 protein in type IC, at the stem-loop structure formed by the repeat sequence. The cleavage site is typically between the 22nd and 23rd bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 1 in Type I-A, between the 28th and 29th bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 2 in Type I-B, between the 19th and 20th bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 3 in Type I-C, between the 31st and 32nd bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 4 in Type I-D, between the 21st and 22nd bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 5 in Type I-E, between the 20th and 21st bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 6 in Type I-F, and between the 28th and 29th bases of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 7 in Type I-G. Therefore, examples of modified repeat sequences include repeat sequences in which a portion other than the cleavage site has been shortened (deleted), and repeat sequences in which bases other than the cleavage site have been modified (substituted and / or inserted) (see, for example, [Ki Hyun Nam et al., Structure 20, September 5, 2012, pp. 1574-1584]).

[0030] The length of the repeat sequence according to the present invention varies depending on the type of CRISPR-Cas system and the modification content as described above, but is preferably 10 to 45 bases, more preferably 20 to 40 bases, and typically 28 to 37 bases.

[0031] Furthermore, the circular crRNA of the present invention may further contain any nucleotide sequence (additional sequence) other than the repeat sequence and spacer sequence, as long as it does not inhibit the effects of the present invention (such as the ability to form a double strand with the crRNA recognition sequence, the ability to form a cascade complex with the Cas proteins, and the activity of cleaving the target region). The additional sequence is preferably, for example, 8 bases or less, and more preferably 6 bases or less, when it is a sequence contained between the repeat sequence and the spacer sequence in one structural unit of the mature crRNA obtained when it is linearized as described below. On the other hand, when the additional sequence is a sequence that is removed by cleavage of the repeat sequence by the Cas protein, or is not included in one unit of the mature crRNA structure obtained when the repeat sequence is linearized as described below, i.e., when the additional sequence is linearized and becomes a sequence added to the 5' end or 3' end of one unit of the mature crRNA structure, examples of such additional sequences include, for example, sequences for circularization in expression by the expression vectors described below (SEQ ID NOs: 17, 18, etc.), the RNA aptamer sequences described below, and RNA sequences for knock-in donors (e.g., for prime editing), and the like, and are not particularly limited, but preferably have a length of 150 bases or less.

[0032] The structure of the circular crRNA of the present invention is not particularly limited, as long as it contains at least one spacer sequence and one repeat sequence, and when linearized, contains at least one unit of mature crRNA structure. That is, the structure of the circular crRNA of the present invention may be a structure in which the mature crRNA is circularized, a structure in which a crRNA precursor having a structure in which a repeat sequence or the like is further added to the mature crRNA is circularized, a structure in which a 5' mature crRNA or a 3' mature crRNA in which the 5' or 3' side of the crRNA precursor has been removed in the same manner as the mature crRNA is circularized, a structure in which an RNA in which the above-mentioned additional sequence is further added to each of the crRNAs is circularized, or a structure in which an RNA containing one or more of the above crRNAs in multiple combinations is circularized. By containing multiple units of mature crRNA structure (i.e., a set of spacer sequence and repeat sequence), it is possible to target multiple corresponding regions.

[0033] One unit of the mature crRNA structure typically has a "5' handle sequence-spacer sequence-3' handle sequence" structure. For example, in the case of Type I-E, the "5' handle sequence" typically consists of 8 bases, from the 22nd to the 29th, of the repeat sequence represented by the nucleotide sequence set forth in SEQ ID NO: 5, and is held by the Cas5 protein. Furthermore, for example, in the case of Type I-E, the "3' handle sequence" typically consists of 21 bases, from the 1st to the 21st, of the repeat sequence represented by the nucleotide sequence set forth in SEQ ID NO: 5, and forms a stem-loop structure between the 6th and 21st bases, which is held by the Cas6 protein. Therefore, the chain length of the mature crRNA is usually 61 to 66 bases. However, some types of mature crRNA do not have a 3' handle sequence.

[0034] Typical examples of the structure of the crRNA precursor include a "leader sequence-repeat sequence-spacer sequence-repeat sequence (LRSR structure)" structure and a "repeat sequence-spacer sequence-repeat sequence (RSR structure)" structure. The leader sequence is an AT-rich sequence that functions as a promoter for expressing the crRNA precursor.

[0035] As one embodiment of the spacer sequence and repeat sequence configuration contained in the circular crRNA of the present invention, typical embodiments of mature crRNA, crRNA precursor (RSR structure), 5' mature crRNA, and 3' mature crRNA, as well as the cleavage site (Cut site) by Cas protein, are shown in the schematic diagram of Figure 1. However, as described above, each sequence may be appropriately modified, and the present invention is not limited to these.

[0036] Among the above, the present inventors have found that even for mature crRNA, which previously could not be confirmed to have genome editing activity when directly introduced into eukaryotic cells, by circularizing it, it is possible to achieve particularly superior genome editing efficiency compared to crRNA precursors, which previously could be confirmed to have genome editing activity in eukaryotic cells. In addition, from the viewpoints of reducing the risk of introducing error sequences during synthesis and reducing synthesis costs, it is preferable that the circular crRNA of the present invention has a structure in which one structural unit of the mature crRNA is circularized, i.e., a structure consisting of one spacer sequence and one repeat sequence.

[0037] The length of the circular crRNA of the present invention is determined by the combination of the spacer sequence, repeat sequence, and, if necessary, the additional sequence, and is not particularly limited and can be several kilobases. For example, it is preferably 35 to 400 bases, more preferably 40 to 210 bases, more preferably 45 to 140 bases, and even more preferably 50 to 70 bases.

[0038] Such circular crRNA can be artificially synthesized and prepared using any method known in the art. For example, it can be prepared by circularizing synthesized crRNA using the method described in [Naoko Abe et al., Methods Mol Biol., 2018:1724:pp. 181-192; Naoko Abe et al., Angew. Chem. Int. Ed., 2013,52(27),pp. 7004-7008; Naoko Abe et al., Sci Rep., 2015 Nov 10,5:16435, doi:10.1038 / srep16435]. The circular crRNA can also be obtained by circularizing crRNA expressed from a polynucleotide or an expression vector using, for example, the self-ligation system described in Nature Biotechnology 40, 2022, pp. 1388-1393. Therefore, the circular crRNA of the present invention may be in the form of a polynucleotide encoding the circular crRNA or an expression vector containing the polynucleotide, in addition to the circular crRNA (in the form of RNA).

[0039] When the circular crRNA is in the form of a polynucleotide encoding it, the polynucleotide may consist of DNA alone, or may consist of GNA, LNA, BNA, PNA, TNA, etc., or a mixture thereof. It may also be modified with a component other than nucleic acid, such as a sugar chain. Such polynucleotides can be prepared by known methods or methods similar thereto, and can be, for example, artificially synthesized.

[0040] When the circular crRNA of the present invention is in the form of a polynucleotide encoding it or an expression vector, these may contain, for example, the nucleotide sequence set forth in SEQ ID NO: 17 and the nucleotide sequence set forth in SEQ ID NO: 18 (see, for example, Nature Biotechnology 40, 2022, pp. 1388-1393), thereby allowing the expressed crRNA to be circularized by self-ligation to form the circular crRNA of the present invention.

[0041] Preferably, the expression vector for the circular crRNA is one that can stably express the encoded crRNA without being integrated into the host genome. Various commonly used vectors can be used as the base vector for such an expression vector, and can be appropriately selected depending on the cells to be introduced and the introduction method, etc. Examples of such base vectors include plasmid vectors, phage vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, and virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.); yeast chromosomal elements and viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, avian poxviruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, etc.); and vectors derived from a combination of two or more of these (cosmids, phagemids, etc.).

[0042] The expression vector preferably contains sites for transcription initiation and termination and a ribosome binding site in the transcription region. The expression vector also preferably contains one or more of a promoter sequence appropriate for the type of cell into which it is introduced, a sequence for enhancing transcription from DNA (e.g., an enhancer sequence), and a sequence for stabilizing the transcribed RNA (e.g., a polyA addition sequence).

[0043] The expression vector can be prepared by a known method or a method similar thereto. For example, in addition to the method described in the implementation manual attached to a vector preparation kit, methods described in various manuals, such as Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001, are also available.

[0044] (Cas Protein Group) The Type I CRISPR-Cas system of the present invention includes, in addition to the circular crRNA (C) of the present invention, a Cas protein group consisting of multiple Cas proteins as a component. The Cas protein group includes (A) a Cas protein having nuclease activity (hereinafter sometimes referred to as "nCas protein") and (B) other Cas proteins (hereinafter sometimes referred to as "Cascade proteins"). However, when the cleavage of target DNA is not the purpose (for example, when binding to an expression control region to suppress transcription or when labeling crRNA using the detection method of the present invention described below), the (A) nCas protein may be excluded from the Type I CRISPR-Cas system of the present invention, or the nCas protein may be a mutant in which part or all of its nuclease activity has been deleted.

[0045] A Cas protein having nuclease activity (nCas protein) is typically a Cas3 protein (sometimes referred to simply as "Cas3" herein; the same applies to other Cas proteins), but in Type I-D, it is a Cas10 protein. In Type I-A, Cas3-HD and Cas3-HEL complex together to form Cas3, while in Type I-F, Cas3 forms a complex with Cas2. In the present invention, "nuclease activity" preferably refers to single-stranded DNA cleavage activity, and "having nuclease activity" refers to the ability to cleave single-stranded DNA at at least one site. The nCas protein according to the present invention also preferably has helicase activity. The nCas protein can cleave a target region on a target DNA at least at one site (preferably multiple sites) by cooperating with the circular crRNA and the Cascade protein of the present invention. Among the Cas proteins listed in each of the following Type I systems, Cas proteins other than the nCas protein (i.e., cascade proteins in the present invention) typically form a cascade in the Type I CRISPR-Cas system, but do not necessarily form a cascade. Cas proteins involved in the formation of a cascade complex (for example, a Cas protein that cleaves circular crRNA) are included (see, for example, [Emmanuelle Charpentier et al., FEMS Microbiology Reviews, fuv23, 39, 2015, pp. 428-441]).

[0046] The Type I CRISPR-Cas system of the present invention is a Type I system that belongs to Class 1 Type I among CRISPR-Cas systems. Type I systems have been further classified into six types: Type IA, Type IB, Type IC, Type ID, Type IE, and Type I-F, as well as Type I-G, a subtype of Type IB (see, for example, van der Oost J et al. (2014) Unraveling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology, Vol. 12 (No. 7), pp. 479-492, Jackson RN et al. (2014) Fitting CRISPR-associated Cas3 into See "The Helicase Family Tree, Current Opinion in Structural Biology, Vol. 24, pp. 106-114."

[0047] The Cas protein group contained in each Type I system typically includes, in Type I-A, Cas3-HD, Cas3-HEL, Cas5, Cas6, Cas7, Cas8, and Cas11; in Type I-B, Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; in Type I-C, Cas3, Cas5, Cas7, Cas8, and Cas11; Type I-D includes Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; Type I-E includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; Type I-F includes Cas2-3, Cas5, Cas6, Cas7, and Cas8; and Type I-G includes Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11. However, even when Cas11 is excluded from the configuration of the Type I CRISPR-Cas system, each system exhibits DNA editing activity (for example, Type I-B and Type I-C are known to exhibit DNA editing activity, albeit at a lower level compared to when Cas11 is included, even when Cas11 is not included, and the same is true for Type I-D). Thus, the Type I CRISPR-Cas system of the present invention also includes a system that does not include Cas11.

[0048] The Type I CRISPR-Cas system of the present invention encompasses all seven types, Types I-A to I-G, but among these, from the viewpoint of suitability for genome editing in animal cells, those derived from Escherichia coli are preferred, and Type I-E is more preferred. Type I-E typically forms a cascade with one molecule of Cas8, two molecules of Cas11, six molecules of Cas7, one molecule of Cas5, and one molecule of Cas6, and one molecule of Cas3 cleaves the target region, with Cas6 preferably cleaving the repeat sequence of crRNA.

[0049] The amino acid sequence of each Cas protein constituting the Type I CRISPR-Cas system can be obtained, for example, from a public database (such as Genbank). Furthermore, the Cas proteins may be appropriately modified (e.g., by substitution, deletion, insertion, and / or addition of amino acid residues) based on the amino acid sequence of these known Cas proteins, as long as the effects of the present invention (such as cascade complex formation ability (preferably including the ability to cleave the repeat sequence of crRNA), nuclease activity, etc.) are not inhibited. The ability of each Cas protein to form a cascade complex and nuclease activity can be confirmed, for example, by using the circular crRNA of the present invention or a conventional crRNA precursor as the crRNA and demonstrating collateral cleavage activity equivalent to or greater than that (e.g., 50% or more, 80% or more) of that before modification (e.g., a protein consisting of a known amino acid sequence) (e.g., Test Example 2 (2) below).

[0050] Furthermore, each Cas protein constituting the Type I CRISPR-Cas system may further be added with a functional molecule, if necessary. Examples of the functional molecule include a nuclear localization signal for promoting translocation into the nucleus of a eukaryotic cell (e.g., those described in [Wu J et al., 2009, Biophysical journal, Vol. 96 (Issue 9), pp. 3840-3849], etc.), a localization signal for promoting localization to mitochondria or chloroplasts, a tag for facilitating purification (e.g., HN tag, His tag, FLAG tag, glutathione-S-transferase (GST) tag), and a reporter protein for facilitating detection (e.g., a fluorescent protein such as green fluorescent protein (GFP), a chemiluminescent protein such as luciferase). These may be used alone or in combination of two or more, but are not limited thereto. When the functional molecule is added, it can be added, for example, to the N-terminus and / or C-terminus of each Cas protein.

[0051] In the Type I CRISPR-Cas system according to the present invention, each Cas protein constituting the Cas protein group may be independently in the form of a protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide. In the polynucleotide form, the nucleotide sequence may be modified (e.g., codon optimization) to make it suitable for expression in a host cell.

[0052] The Cas proteins can be prepared by known methods or methods similar thereto. For example, a method for preparing Cas3 can be described in WO 2022 / 186063. Furthermore, the Cascade proteins can be prepared by the method described in [Kazuto Yoshimi et al., Nature Communications, 2022, 13:4917, https: / / doi.org / 10.1038 / s41467-022-32618-0]. Hereinafter, the Type I-E CRISPR-Cas system will be described as a representative example, but for other types of CRISPR-Cas systems, the Cas proteins constituting the system can be appropriately interpreted.

[0053] Furthermore, when introducing the Cas proteins constituting the Cas protein group into cells, it is preferable to form a complex consisting of two or more of them in advance and introduce this into the cells. Such combinations include, for example, a complex consisting of two molecules of Cas11; a complex consisting of six molecules of Cas7; a complex consisting of one molecule of Cas8, one molecule of Cas5, and one molecule of Cas6; a complex consisting of one molecule of Cas3, one molecule of Cas8, two molecules of Cas11, six molecules of Cas7, one molecule of Cas5, and one molecule of Cas6; and combinations of one or more of these. Furthermore, in this case, it is also preferable to form a complex (cascade complex) between these complexes (cascades) and circular crRNA and introduce this into cells. Such complexes can be prepared by known methods or methods similar thereto, for example, as described above [Kazuto Yoshimi et al. , Nature Communications, 2022, 13:4917].

[0054] When the Cas protein constituting the Cas protein group is in the form of a polynucleotide encoding it, the polynucleotide may consist of only DNA, or may consist of RNA, GNA, LNA, BNA, PNA, TNA, etc., or a mixture thereof. It may also be modified with a component other than nucleic acid, such as a sugar chain. Such polynucleotides can be prepared by known methods or methods similar thereto, and can be, for example, artificially synthesized.

[0055] When each Cas protein constituting the Cas protein group is in the form of an expression vector encoding it, examples of the expression vector and its preparation method include those similar to those listed for the expression vector for circular crRNA. In the expression vector, the polynucleotides encoding each Cas protein may be appropriately codon-optimized. Furthermore, the same expression vector may contain multiple polynucleotides encoding each of the multiple Cas proteins constituting the Cas protein group, and the number of polynucleotides is not particularly limited as long as the function of the Type I CRISPR-Cas system can be exerted in a host cell into which the expression vector is introduced. For example, it is possible to design all polynucleotides encoding each Cas protein constituting the Cas protein group to be incorporated into a single (same) expression vector. Furthermore, it is also possible to design all or part of the polynucleotides encoding each Cas protein constituting the Cas protein group to be incorporated into separate expression vectors. For example, it is possible to design polynucleotides encoding Cascade proteins to be incorporated into a single (same) expression vector and polynucleotides encoding Cas3 to be incorporated into a separate expression vector. From the viewpoint of expression efficiency, a method in which polynucleotides encoding each Cas protein constituting the Cas protein group are respectively loaded into six different expression vectors is preferred. Additionally, for purposes such as adjusting the expression level, multiple polynucleotides encoding the same Cas protein may be loaded into the same expression vector. For example, a design in which a polynucleotide encoding Cas3 is placed at two locations within one (same) type of expression vector is possible. Furthermore, when an expression vector contains multiple polynucleotides encoding multiple Cas proteins constituting the Cas protein group, a nucleotide sequence encoding an amino acid sequence (such as a 2A peptide) that is cleaved by intracellular proteases may be inserted between the multiple polynucleotides.

[0056] <Method for editing target DNA> The present invention provides a method for editing target DNA, comprising the steps of contacting the Type I CRISPR-Cas system of the present invention with target DNA and editing the target DNA (sometimes referred to herein simply as the "editing method"). In the editing method of the present invention, the Type I CRISPR-Cas system and target DNA are as described above, including preferred embodiments thereof.

[0057] In the editing method of the present invention, the Type I CRISPR-Cas system is contacted with the target DNA, and its nuclease activity cleaves the target region on the target DNA. When the Type I CRISPR-Cas system of the present invention, i.e., a combination of an nCas protein, a Cascade protein, and the circular crRNA of the present invention, is contacted with the target DNA, the spacer sequence of the circular crRNA recognizes and binds to the corresponding crRNA recognition sequence on the target DNA, and guides the Cascade protein to the target DNA, forming a Cascade complex consisting of the crRNA and Cascade. Furthermore, the nCas protein is also guided to the target region contained in the complementary strand of the crRNA recognition sequence, and its nuclease activity cleaves the exposed single-stranded DNA in the target region. In this case, it is preferable that the circular crRNA of the present invention is cleaved by the Cas5 protein or Cas6 protein constituting the cascade protein to generate a mature crRNA prior to binding to the crRNA recognition sequence, and that this mature crRNA binds to the crRNA recognition sequence.

[0058] As a result of cleavage of the target region, substitution or deletion of bases in the target region can be efficiently induced by the self-repair mechanism of single-stranded DNA, or by the self-repair mechanism of double-stranded DNA cleavage resulting from cleavage of the single-stranded DNA wound by the helicase activity of the nCas protein. According to the present invention, it is also possible to cause large-scale deletion (knockout) of the region containing the target region or a region nearby. Furthermore, for example, when base substitution occurs in the target region, various mutations can be introduced, for example, by repairing the base on the opposite strand of the strand where the substitution occurred so that it forms a pair with the substituted base due to a mismatch in double-stranded DNA within the cell, or by replacing it with another base during repair, or by causing the deletion or insertion of one or several dozen bases. Furthermore, by further contacting the target DNA with a knock-in fragment that can be introduced into the target region or donor DNA for homologous recombination repair, it is also possible to introduce (knock-in) the desired nucleotide sequence at the cleavage site. As such knock-in techniques, conventionally known methods or methods equivalent thereto can be appropriately employed. Therefore, editing of the target DNA according to the present invention includes the deletion of one or more bases, substitution with one or more other bases, or insertion of one or more bases in the target region, or a combination of these mutations.

[0059] Another example of target DNA editing is a chimeric protein formed by fusing a Cas protein constituting a Cas protein group with a heterologous protein having a desired activity, depending on the purpose. In this embodiment, various editing operations can be performed on the target DNA depending on the activity of the heterologous protein to be fused. Examples of the activity of the heterologous protein to be fused include, but are not limited to, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, photolyase activity, and glycosylase activity. In this case, a mutant lacking part or all of its nuclease activity can also be used as the nCas protein.

[0060] Examples of nCas protein mutants include, for example, in Type I-E, a Cas3 HD domain H74A mutant (dnCas3), a K320N mutant of SF2 domain motif 1 (dhCas3), and a double S483A / T485A mutant of SF2 domain motif 3 (dh2Cas3). Furthermore, for example, by using a fusion protein of a mutant lacking part or all of the nuclease activity of the nCas protein and a deaminase as a component of the Type I CRISPR-Cas system of the present invention, it becomes possible to perform more precise DNA editing by substituting bases without causing deletions in the target region. Techniques for applying deaminase to the CRISPR-Cas system are known (see, for example, [Nishida K. et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI: 10.1126 / science.aaf8729, (2016)]), and can be applied to the Type I CRISPR-Cas system of the present invention.

[0061] Another example of target DNA editing is regulating gene transcription in the target region by fusing a Cas protein constituting the Cas protein group with a desired transcriptional regulatory protein and using the resulting chimeric protein. Examples of transcriptional regulatory proteins include, but are not limited to, light-inducible transcriptional regulators, small molecule / drug-responsive transcriptional regulators, transcription factors, and transcriptional repressors. In this case, too, a mutant lacking all or part of the nuclease activity of the nCas protein can be used. Techniques for applying transcriptional regulatory proteins to the CRISPR-Cas system are known to those skilled in the art.

[0062] The editing method of the present invention may be performed intracellularly or in a cell-free system. The "intracellular" where the editing method of the present invention is performed may be a eukaryotic cell or a prokaryotic cell, preferably a eukaryotic cell. Examples of eukaryotic cells include animal cells (e.g., cells of mammals, fish, birds, reptiles, amphibians, insects), plant cells, algae cells, and yeast. Examples of prokaryotic cells include Escherichia coli, Salmonella, Bacillus subtilis, lactic acid bacteria, and extreme thermophiles.

[0063] "Animal cells" include, for example, mammalian cells as well as cells from fish, birds, reptiles, amphibians, and insects. Animal cells include, for example, cells constituting an individual animal, cells constituting organs or tissues removed from an animal, and cultured cells derived from animal tissues. Specific examples include germ cells such as oocytes and sperm; germ cells of various stages of embryos (e.g., 1-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, morula embryos, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, and kidney cells. Pre- and post-fertilization oocytes can be used as oocytes for use in the method for producing a non-human individual described below, but post-fertilization oocytes, i.e., fertilized eggs, are preferred. Pronuclear stage embryos are particularly preferred. Oocytes that have been cryopreserved can be thawed and used.

[0064] The term "mammal" encompasses both humans and non-human mammals. Examples of non-human mammals include ungulates such as cattle, boars, pigs, sheep, and goats, perissodactyls such as horses, rodents such as mice, rats, guinea pigs, hamsters, and squirrels, lagomorphs such as rabbits, and carnivores such as dogs, cats, and ferrets. The non-human mammals may be livestock or companion animals (pets), or wild animals.

[0065] "Plant cells" include, for example, cells that constitute an individual plant, cells that constitute organs or tissues separated from a plant, cultured cells derived from plant tissue, etc. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, calluses, etc.

[0066] Furthermore, the "cell-free system" that serves as the field for the editing method of the present invention refers to a system that does not contain living cells (the eukaryotic cells or prokaryotic cells). The cell-free system according to the present invention is not particularly limited as long as it is a system that allows contact between the Type I CRISPR-Cas system and the target DNA, and examples thereof include a buffer solution; a cell lysate of the eukaryotic cells or prokaryotic cells; and a cell extract.

[0067] The method for contacting the Type I CRISPR-Cas system with the target DNA is not particularly limited. Intracellular contact includes, for example, a method of introducing or expressing the Type I CRISPR-Cas system into a cell containing the target DNA, as in the production method of the present invention described below. In a cell-free system, for example, a solution of target DNA may be mixed with a solution containing the Type I CRISPR-Cas system. The solvent for these solutions is not particularly limited, but is preferably, for example, a buffer solution such as phosphate buffer, Tris buffer, Good's buffer, or borate buffer.

[0068] <Method for producing cells in which target DNA has been edited> The present invention also provides a method for producing cells in which target DNA has been edited, comprising the steps of introducing or expressing the Type I CRISPR-Cas system of the present invention into a cell, contacting it with target DNA, and editing the target DNA (sometimes referred to herein simply as the "production method"). In the production method of the present invention, the Type I CRISPR-Cas system is as described above, including preferred embodiments thereof. In the production method of the present invention, the target DNA is also as described above, including preferred embodiments thereof, and the target DNA is DNA present in the cell (endogenous DNA and exogenous DNA, preferably genomic DNA).

[0069] The cells into which the Type I CRISPR-Cas system is introduced or expressed include the cells mentioned above when the editing method of the present invention is performed intracellularly, and are preferably eukaryotic cells, and preferably animal cells. According to the present invention, even when a crRNA consisting of a mature crRNA is used as the circular crRNA, excellent DNA editing efficiency can be achieved in eukaryotic cells.

[0070] In the production method of the present invention, contact between the Type I CRISPR-Cas system and the target DNA in a cell is achieved by introducing the Cas proteins and circular crRNA constituting the Type I CRISPR-Cas system into the cell in the form of protein or RNA, or by introducing them in the form of a polynucleotide encoding the protein or RNA or in the form of an expression vector containing the polynucleotide, thereby expressing them in the cell. Therefore, the Type I CRISPR-Cas system may be constructed such that each of the Cas proteins and circular crRNA constituting the system is independently introduced into the cell in the form of protein or RNA, or in the form of RNA or DNA (polynucleotide) encoding the protein or RNA, and expressed in the cell, or in the form of a vector (expression vector) expressing the protein or RNA, and expressed in the cell. The forms of the Cas proteins and circular crRNA constituting the Type I CRISPR-Cas system, the polynucleotides encoding them, and the expression vectors expressing them are as described above, including preferred embodiments thereof.

[0071] The method for introducing the Type I CRISPR-Cas system into cells is not particularly limited, and known methods for introducing proteins, DNA, RNA, or vectors into cells can be appropriately adopted depending on the type of cell, etc. Examples of such methods include electroporation, microinjection, particle gun, calcium phosphate method, polyethyleneimine (PEI) method, liposome method (lipofection), DEAE-dextran method, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium method, lithium acetate method, spheroplast method, heat shock method (calcium chloride method, rubidium chloride method), etc. Such methods are described in Davis et al. , Basic methods in molecular biology, New York: Elsevier, 1986].

[0072] When the Type I CRISPR-Cas system is introduced into a cell or expressed in a cell, the Type I CRISPR-Cas system comes into contact with the target DNA in the cell, and the editing of the target DNA described in the editing method of the present invention above causes cleavage and editing in the target region, making it possible to obtain a cell in which the target DNA has been edited.

[0073] The present invention also provides a method for producing a non-human individual containing cells in which the target DNA has been edited. This method includes a step of producing a non-human individual from cells obtained by the above-mentioned production method. Examples of the non-human individual include non-human animals and plants. Examples of the non-human animal include mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, monkeys, pigs, cows, goats, sheep), fish, birds, reptiles, amphibians, and insects. When producing a model animal, the mammal is preferably a rodent such as a mouse, rat, guinea pig, or hamster, and particularly preferably a mouse. Examples of the plant include grains, oilseed crops, forage crops, fruits, and vegetables. Specific examples of crops include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation.

[0074] As a method for producing a non-human individual from cells in which the target DNA has been edited, any known method can be used as appropriate. When producing a non-human individual from cells in an animal, germ cells or pluripotent stem cells are typically used. For example, the Type I CRISPR-Cas system is microinjected into an oocyte, and the resulting oocyte is implanted into the uterus of a pseudopregnant female non-human mammal, after which offspring can be obtained. In addition, it has long been known that somatic cells of plants possess totipotency. For example, a plant in which the desired DNA has been edited can be obtained by microinjecting the Type I CRISPR-Cas system into plant cells and regenerating a plant from the resulting plant cells. Furthermore, from the resulting non-human individual, progeny or clones in which the desired DNA has been edited can also be obtained.

[0075] Confirmation of the presence or absence of target DNA editing and determination of the genotype can be performed based on conventionally known techniques, such as PCR, sequencing, Southern blotting, etc.

[0076] <Method for detecting target DNA> The present invention also provides a method for detecting target DNA in a sample, comprising the steps of contacting the Type I CRISPR-Cas system of the present invention with a sample and detecting the target DNA in the sample (sometimes simply referred to as a "detection method" in this specification). In the detection method of the present invention, the Type I CRISPR-Cas system and target DNA are as described above, including preferred embodiments thereof.

[0077] The "sample" in the detection method of the present invention can be any desired sample in which the target DNA is to be detected, without any particular limitations. The sample may be a cell sample or an acellular sample, as long as the target DNA can be present in the sample, and examples thereof include biological tissues, cells, cell lysates, body fluids (urine, saliva, serum, plasma, whole blood, etc.), and samples containing purified or synthesized DNA.

[0078] In one embodiment of the detection method of the present invention, a labeling substance is added to a circular crRNA. For example, a target DNA can be detected by using a circular crRNA obtained by circularizing a crRNA in which a labeling substance is added to the 3' end of the spacer sequence.

[0079] The labeling substance is not particularly limited as long as it is detectable, and examples thereof include RNA aptamer sequence / recognition proteins such as MS2 / MCP, PP7 / PCP, boxB / λN22P, and Pepper / tDeg; and RNA aptamer sequence / recognition compounds such as MS2 / MCP, PP7 / PCP, boxB / λN22P, and Pepper / tDeg.

[0080] When the RNA aptamer sequence / recognition protein is used, the RNA aptamer sequence is added to crRNA, and a recognition protein fused with a fluorescent protein such as GFP is bound to the RNA aptamer sequence, thereby enabling detection of target DNA using fluorescence as an indicator (see, for example, [Yang LZ et al., Cell Insight 1 (2022) 100044]). Furthermore, when an RNA aptamer sequence / recognition compound is used, the RNA aptamer sequence is added to crRNA, and a recognition compound is bound to the RNA aptamer sequence, enabling detection of target DNA using color development as an indicator.

[0081] In these detections, in order to avoid degradation of the target DNA by the nuclease activity of the Type I CRISPR-Cas system, it is preferable to use a system in which the nuclease activity has been lost, for example, a system containing a mutant of the nCas protein in which the nuclease activity has been deleted, or a system in which the nCas protein has been excluded.

[0082] Another aspect of the detection method of the present invention is a method that uses single-stranded probe DNA (see Test Example 2 (2)). It is known that the Type I CRISPR-Cas system recognizes and binds to target DNA in a sample, and indiscriminately cleaves surrounding single-stranded DNA (ssDNA). This phenomenon can also be used to detect target DNA (WO 2021 / 149829). More specifically, for example, by mixing a single-stranded probe DNA, the cleavage of which can be detected by a labeling substance, as the ssDNA in a reaction system containing a sample for detecting target DNA and a Type I CRISPR-Cas system, the target DNA in the sample can be detected using the signal generated from the labeling substance upon cleavage of the single-stranded probe DNA as an indicator. When using this detection principle, it is not necessary to label the circular crRNA of the present invention itself.

[0083] The single-stranded probe DNA is not particularly limited as long as its cleavage is detectable, and may be linear or cyclic, but linear is preferred. The nucleic acid constituting the single-stranded probe DNA may be modified by one or more modifications (e.g., base modification, backbone modification, sugar modification).

[0084] One preferred embodiment of the labeling substance for detecting cleavage of the single-stranded probe DNA is a fluorescent dye / quencher pair. In this embodiment, when the fluorescent dye / quencher pair is bound to and in close proximity to the single-stranded probe DNA, the signal from the fluorescent dye is reduced or eliminated. However, when the single-stranded probe DNA is cleaved and the fluorescent dye is separated from the quencher, a sufficient signal from the fluorescent dye is detected. Therefore, the target DNA in the sample can be detected using as an indicator the fluorescent signal generated from the single-stranded probe DNA cleaved by the Type I CRISPR-Cas system that recognizes and binds to the target DNA.

[0085] Another preferred embodiment of the labeling substance for detecting cleavage of the single-stranded probe DNA is a donor / acceptor pair for fluorescence resonance energy transfer (FRET). In this embodiment, when the donor / acceptor pair is bound to and in close proximity to the single-stranded probe DNA, excitation of the donor causes excitation and emission of the acceptor (i.e., a FRET signal is generated). However, when the single-stranded probe DNA is cleaved and the donor is separated from the acceptor, the FRET signal is reduced or eliminated. Therefore, the target DNA in the sample can be detected using the reduction or elimination of the FRET signal in the single-stranded probe DNA cleaved by the Type I CRISPR-Cas system that recognizes and binds to the target DNA as an indicator.

[0086] Alternatively, for example, immunochromatography (lateral flow assay) can be used to detect the single-stranded probe DNA.

[0087] The sample, the Type I CRISPR-Cas system, and the single-stranded probe DNA can be contacted by, for example, mixing them. When the sample contains cells, a further operation for introducing the CRISPR-Cas system and the single-stranded probe DNA into the cells in the sample can be included.

[0088] <Kit> The present invention provides a kit for use in the editing method, production method, and detection method of the present invention, comprising the following (A') to (C'): (A') at least one selected from the group consisting of the nCas protein, an expression vector for the nCas protein, and a polynucleotide encoding the nCas protein, (B') at least one selected from the group consisting of the Cascade protein, an expression vector for the Cascade protein, and a polynucleotide encoding the Cascade protein, (C') at least one selected from the group consisting of the circular crRNA of the present invention, an expression vector for the circular crRNA, and a polynucleotide encoding the circular crRNA. However, as described above, depending on the purpose, (A') may be excluded, or the nCas protein may be a mutant in which part or all of the nuclease activity has been deleted.

[0089] The above (A') to (C'), including their preferred embodiments, are as described above in the circular crRNA and CRISPR-Cas3 system of the present invention. Each of these may independently be in the form of a protein or RNA, in the form of a polynucleotide encoding the protein or RNA, or in the form of an expression vector that expresses the protein or RNA and contains the polynucleotide. These forms, including their preferred embodiments, are as described above.

[0090] Furthermore, when (C') is in the form of an expression vector, the expression vector may be (i) a vector comprising a polynucleotide encoding the spacer sequence and the repeat sequence, but may also be (ii) a vector comprising an insertion site for the polynucleotide encoding the spacer sequence and a polynucleotide encoding the repeat sequence, so that the user can design the spacer sequence according to the target region.

[0091] In the kit of the present invention, the combination of forms of (A') to (C') is not particularly limited, and may be a combination containing each of (A') to (C') individually, or a pre-mixture of two or more of (A') to (C'). For example, (B') may be in a different form for each of the Cas proteins constituting the cascade protein, or in the form of a complex or composition containing two or more Cas proteins, or in the form of a polynucleotide encoding each of the two or more Cas proteins, or in the form of an expression vector containing the polynucleotide. Furthermore, (A') and (B') may be combined in the form of a complex or composition containing multiple Cas proteins constituting the Cas protein group, or in the form of a polynucleotide encoding each of the two or more Cas proteins, or in the form of an expression vector containing the polynucleotide. Furthermore, for example, when (A') and (B') are introduced into cells in the form of proteins and (C') is introduced into cells in the form of RNA, they may be in the form of a complex of two or more of these in advance, or when (A') to (C') are introduced into cells in the form of an expression vector, they may be in the form of an expression vector containing two or more polynucleotides.

[0092] The kits of the present invention may further include one or more additional reagents. Examples of such additional reagents include, but are not limited to, a reagent for separating cells, a medium for culturing cells, a dilution buffer, a reconstitution solution, a washing buffer, a nucleic acid introduction reagent, a protein introduction reagent, a control reagent, a labeling substance, a probe molecule, etc. The kits may also include instructions for carrying out the editing method, production method, and detection method of the present invention.

[0093] Each component included in the kit may be contained in a separate container, or two or more components may be contained in the same container in combination. Each component may be contained in a single-use amount in a container, or multiple doses may be contained in a single container. Furthermore, each component may be contained in a container in a dry form, or in a form dissolved in an appropriate solvent (e.g., a solvent containing a buffer, stabilizer, preservative, antiseptic, etc.).

[0094] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0095] (Test Example 1) Preparation of Circular crRNA (1) Preparation of 5'-Terminal Phosphorylated Oligoribonucleotide Two 61-nt 5'-terminal phosphorylated oligoribonucleotide sequences (Seq_1 / nucleotide SEQ ID NO: 9, Seq_2 / nucleotide SEQ ID NO: 14) were synthesized using a phosphoramidite reagent (Chemgene) and a CPG solid phase support (Link) according to the standard phosphoramidite method using an automated nucleic acid synthesizer (T-8-A20R8NC, Nippon Techno Service Co., Ltd.). The chemical phosphorylation reagent used was 2,2-dimethyl-1-(2-nitrophenyl)-1-propyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. After synthesis, cleavage from the solid support and deprotection reactions were carried out by heating at 65°C for 30 minutes using a 1:1 mixture of 40% aqueous methylamine and 30% aqueous ammonia. The reaction solution was filtered and dried under reduced pressure, and the residue was dissolved in 1 M tetrabutylammonium fluoride in THF and incubated overnight at room temperature. The oligonucleotide was then desalted using a NAP-25 column (Merck) and subjected to alcohol precipitation to obtain a crude oligonucleotide product.

[0096] The desired full-length phosphorylated form was isolated from the resulting crude product by reverse-phase HPLC under the following conditions: system: Prominence HPLC (Shimadzu Corporation, solution delivery unit: LC-20AD, detection unit: SPD-M40, etc.); column: YMC TriartBio C4, 250 x 10 mm ID (S-5 μm, 30 nm); solution A: 100 mM TEAA (pH 7.0) buffer containing 5% acetonitrile; solution B: acetonitrile; column equilibration condition: 0% solution B; gradient elution condition: 5 to 25% solution B / 0 to 20 minutes; column temperature: 50°C; flow rate: 3 mL / min; detection wavelength: 254 nm. The oligonucleotides were recovered from the eluate by alcohol precipitation, redissolved in ultrapure water, and irradiated with 365-nm light (4.0 mW / cm) using a 300 W xenon light source (MAX-350, Asahi Spectroscopy). ２ The protecting group of the phosphate group was removed by heating at 400°C for 10 minutes. The target oligonucleotide contained in the reaction solution was then recovered by alcohol precipitation.

[0097] The purity and molecular weight of the target oligonucleotides (Seq_1, Seq_2) were determined by LC-ESI-MS analysis under the following conditions: System: Agilent 1290 Infinity II equipped with 6530 LC / Q-TOF; Column: Acquity UPLC BEH C18 Column (130 Å, 1.7 μm, 2.1 mm × 50 mm; manufactured by Waters); Solution A: 8.6 mM triethylamine-100 mM hexafluoro-2-propanol buffer (pH 8.3); Solution B: methanol; Gradient elution conditions: 0-30% Solution B / 0-12.0 min, 90% Solution B / 12.1-15.0 min, 0% Solution B / 15.1-20.0 min; column temperature: 60°C; flow rate: 0.3 mL / min; detection wavelength: 260 nm. The results were as follows: Seq_1 had a calculated molecular weight of 19,755.8 and a measured molecular weight of 19,757.2 (+1.4), and Seq_2 had a calculated molecular weight of 19,755.8 and a measured molecular weight of 19,757.3 (+1.5).

[0098] (2) Preparation of circular crRNA For Seq_1 and Seq_2, both ends were ligated using T4 RNA ligase 2 in the presence of 24-base oligodeoxyribonucleotides complementary to the 12 bases at the 5' and 3' ends, respectively (for Seq_1 / nucleotide SEQ ID NO: 19, for Seq_2 / nucleotide SEQ ID NO: 20), to prepare circularized forms. The reaction solution had the following composition: 1 μM Seq_1 or Seq_2, 2.5 μM oligodeoxyribonucleotide (for Seq_1 or for Seq_2), 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM dithiothreitol, 400 μM ATP, and 25 ng / μL T4 RNA ligase. The solution before the addition of PEG8000 and ligase was heated at 90°C for 3 minutes and then slowly cooled to room temperature. PEG8000 and ligase were then added and the mixture was heated at 37°C for 1 hour. An equal volume mixture of TE-saturated phenol and chloroform was added to the reaction solution, which was then vigorously stirred and centrifuged. The supernatant was then collected in a separate tube. Chloroform was further added to the recovered supernatant and centrifuged in the same manner. RNA was then recovered by alcohol precipitation, and 10 pmol of the oligonucleotide was subjected to denaturing polyacrylamide gel electrophoresis (PAGE: 15% acrylamide, 7.5 M urea, 1x Tris-borate-EDTA (pH 8.3)). The gel after electrophoresis was stained with SYBR Green II and visualized using a ChemiDoc MP Imager (Bio-Rad). Denaturing polyacrylamide gel electrophoresis images of the resulting Seq_1 and Seq_2 (without ligase: Ligase(-) and with ligase: Ligase(+) respectively) are shown in Figure 2(a).

[0099] As shown in Figure 2(a), the target band (marked with an asterisk, arrow) was darker for Seq_2, indicating a higher cyclization efficiency. Therefore, a larger amount of cyclized Seq_2 was prepared and isolated / purified. Specifically, the reaction mixture [1 μM Seq_2, 2.5 μM oligodeoxyribonucleotide (for Seq_2), 10% PEG 8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM dithiothreitol, 400 μM ATP, 50 ng / μL T4 RNA ligase 2; reaction volume 5 mL] was heated at 37°C for 1 hour, after which an equal volume mixture of TE-saturated phenol and chloroform was added, followed by vigorously stirring and centrifugation. The supernatant was then collected in a separate tube. Further chloroform was added to the collected supernatant, followed by similar centrifugation, and the RNA was recovered by alcohol precipitation. The mixture containing the cyclized product was then electrophoresed on a preparative denaturing PAGE (containing 7.5 M urea and 20% formamide as denaturants). The target band in the electrophoretic gel was visualized using UV shadowing. The band was excised and crushed, and the RNA in the gel was extracted with ultrapure water. RNA was recovered from the extract by alcohol precipitation, and the cyclized product of Seq_2 was isolated and purified at 1,030 pmol with an isolation yield of 21%. Denaturing polyacrylamide gel electrophoresis images of the starting material (Seq_2 before cyclization: lane 1), the mixture after cyclization (lane 2), and the cyclized product after isolation and purification (lane 3) are shown in Figure 2(b).

[0100] (Test Example 2) Examination of in vitro DNA cleavage activity using circular crRNA To examine changes in the cleavage activity of the CRISPR-Cas system due to circular crRNA, a fluorescent quencher probe (56FAM-AAGGTCGGA-ZEN-GTCAACGGATTTGGTC-ABkFQ; manufactured by IDT; nucleotide sequence number: 8) was used to measure nonspecific single-stranded DNA cleavage (collateral cleavage activity) observed upon recognition of target DNA containing a crRNA recognition sequence.

[0101] (1) Preparation of each crRNA Circular crRNA was synthesized essentially in the same manner as in Test Example 1. That is, first, 100 μM of 5′-terminal phosphorylated oligoribonucleotide (oligoRNA, nucleotide SEQ ID NO: 9) and 100 μM of oligodeoxyribonucleotide (ligation oligo, nucleotide SEQ ID NO: 10) designed to span the circularization site (ligation site) were mixed at a molar ratio of 2:5 to a total volume of 4 μL. To this was added 2 μL of T4 Rnl 2 Reaction Buffer (NEW ENGLAND BioLabs) and 9 μL of nuclease-free water, and the mixture was heated at 90°C for 3 minutes and then gradually cooled to room temperature over 40 minutes to anneal the oligo RNA and ligation oligo. Next, 4 μL of Poly(ethylene glycol) (average mol wt: 8000, Sigma-Aldrich) prepared to 50% (w / v) with nuclease-free water and 1 μL of T4 RNA ligase 2 (NEW ENGLAND BioLads) were added to the mixture, and the mixture was incubated at 37°C for 2 hours to produce circular mature-crRNA (Example, 61 nt) in which mature crRNA (mature-crRNA, nucleotide sequence number: 11, 5' handle sequence (8 nt)-spacer sequence (32 nt)-3' handle sequence (21 nt)) was circularized (FIG. 3(a)). Similarly, a circular pre-crRNA (Example, 90 nt) was also prepared by circularizing a crRNA precursor (pre-crRNA, nucleotide sequence number: 12) (Figure 3(b)). In Figure 3, "Cut site" indicates the site cleaved by the Cas6 protein. Circularization of each crRNA was confirmed by its mobility in agarose gel electrophoresis. The agarose gel electrophoresis image is shown in Figure 4.

[0102] Non-circularized crRNA precursor (pre-crRNA, nucleotide sequence number: 12) and linear crRNA obtained by linearizing circular mature-crRNA were each prepared by chemical synthesis at IDT. The linear crRNA was prepared in two forms: one in which circular mature-crRNA was cleaved within the repeat sequence (repeat sequence 3' side (10 nt)-spacer sequence (32 nt)-repeat sequence 5' side (19 nt): nucleotide sequence number: 13), and one in which circular mature-crRNA was cleaved within the spacer sequence (spacer sequence 3' side (12 nt)-repeat sequence (29 nt)-spacer sequence 5' side (20 nt): nucleotide sequence number: 14). Hereinafter, the non-cyclized crRNA precursor will be referred to as "linear pre-crRNA," and among the linear crRNAs, the one represented by the nucleotide sequence number 13 will be referred to as "linear crRNA-1," and the one represented by the nucleotide sequence number 14 will be referred to as "linear crRNA-2."

[0103] (2) In vitro test First, the cascade proteins constituting the cascade of the Type I-E CRISPR-Cas system (Cas5, Cas6, Cas7, Cas8, Cas11: 0.8 μg / μL in terms of the amount of the Type I-E cascade) were mixed in equal amounts with 250 ng / μL of the circular mature-crRNA or linear pre-crRNA prepared in (1) above, and the mixture was diluted with a complex formation buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl ２ , 10 μM CoCl ２ The mixture was incubated at 37°C for 30 minutes in reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl) to form the cascade complex. ２ , 10 μM CoCl ２Cas3 protein (40 ng / μL), the Cascade complex (36 ng / μL), and a double-stranded DNA fragment containing a crRNA recognition sequence (Activator, nucleotide sequence number: 15; 5 nM) were mixed in a buffer containing 1 mM ATP (1 mM ATP, 1 mM ATP), and the mixture was used as a sample. Also, 1 μL of the fluorescent quencher probe (10 μM) was prepared using 5 μL of Nuclease-free water and 10 × Alert Buffer (manufactured by Thermo Fisher Scientific), and then, 20 μL of the sample and 5 μL of substrate solution were mixed (amount of Cas6 protein in the mixture: 7.2 μM), a real-time PCR device (CFX96 Touch Deep Well system, manufactured by Bio-Rad Laboratories) was used, and the intensity of the FAM signal (RFU) generated by decomposition of the fluorescent quencher probe was observed over time at 37 ° C., and the change in the signal intensity (RFU / min: slope of the exponential rise period, the same below) was determined (Activator +). The change in signal intensity (RFU / min) was also determined in the same manner except that the double-stranded DNA fragment (Activator) was not added (Activator-). The results are shown in FIG.

[0104] As shown in Figure 5, even when circular crRNA (circular mature-crRNA) was used, the same change in signal intensity (RFU / min) as that observed with conventional linear pre-crRNA was observed due to collateral cleavage activity, confirming that it has the same cascade complex formation ability (complex formation rate, etc.) and single-stranded DNA cleavage activity as the linear pre-crRNA.

[0105] Next, we investigated whether the use of circular crRNA could reduce the amount of Cas protein required for single-stranded DNA cleavage activity. That is, the amount of Cas6 protein was adjusted to 3.6 μM, half the amount of 7.2 μM, in the same manner as above, except that the change in signal intensity (RFU / min) was determined. The results are shown in Figure 6.

[0106] As shown in Figure 6, when the amount of Cas6 protein, which forms the cascade and cleaves crRNA, was reduced, the change in signal intensity decreased when linear pre-crRNA was used, whereas when circular crRNA (circular mature-crRNA) was used, no decrease in the change in signal intensity was observed, confirming that it exhibited significantly higher single-stranded DNA cleavage activity than when conventional linear pre-crRNA was used (p-value by two-sample T-test: p<0.001).

[0107] (Test Example 3) Measurement of genome editing efficiency in human cultured cells using circular crRNA To examine whether circular crRNA can be used for genome editing in eukaryotic cells, the genome editing efficiency of each crRNA in cells was examined using human cultured cell K562, which contains a group of doxycycline-inducible Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11).

[0108] (1) Circular mature-crRNA For the study, the circular mature-crRNA (circular crRNA, Example) prepared in Test Example 2(1) above, linear pre-crRNA (PC), linear crRNA-1, and linear crRNA-2 were used ( FIG. 7( a)). Circular mature-crRNA (circular crRNA), linear pre-crRNA (PC), and linear crRNA-1 each contain a spacer sequence (nucleotide sequence number: 16) that recognizes the crRNA recognition sequence, which is designed to include the complementary strand of the target region located 3' to the PAM sequence (5'-AAG) on human EMX1.

[0109] First, K562 cells were cultured in 2 μg / mL doxycycline-supplemented medium for 6 hours before electroporation, maintaining the expression of the Cas protein group (Cas3, Cas5, Cas6, Cas7, Cas8, Cas11). Next, each of the above crRNAs (20 ng / μL) was introduced into the K562 cells by electroporation using a 4D-Nucleofector System (manufactured by Lonza). After electroporation, the cells were incubated in 2 μg / mL doxycycline-supplemented medium at 37°C, 5% CO ２After culturing for two days under these conditions, all cells were harvested, genomic DNA was extracted, and PCR amplification was performed using a primer set designed for a 3.8 kb (3,825 bp) region containing the PAM sequence and target region on human EMX1. The PCR product was subjected to agarose gel electrophoresis. The test was performed twice for each crRNA combination (rep 1, 2). The agarose gel electrophoresis image is shown in Figure 7(b). Furthermore, the genome editing efficiency (Edited (%)) when using each crRNA was calculated by comparing the density of the 3.8 kb band with the density of the shortened band (band of 0.2 kb or more but less than 3.8 kb) due to base pair deletion (knockout) in the electrophoresis image. The average of the two genome editing efficiencies is shown in Figure 8.

[0110] (2) Circular pre-crRNA For the study, the circular pre-crRNA (Example) prepared in (1) of Test Example 2 above, linear pre-crRNA (PC), and pMax_GFP plasmid (Addgene) were used as a negative control. Except for using these as crRNA, the PCR products were introduced into K562 cells expressing the Cas protein group (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) in the same manner as in (1) above, and the PCR products obtained using the recovered total genomic DNA as a template were subjected to agarose gel electrophoresis. The agarose gel electrophoresis image is shown in Figure 9.

[0111] As shown in Figure 7, when linear crRNA (linear crRNA-1, 2) obtained by linearizing circular mature-crRNA was used, no bands of 0.2 kb or more and less than 3.8 kb were observed, and genome editing activity was not confirmed. On the other hand, as shown in Figures 7 and 9, when each circular crRNA (circular mature-crRNA (Figure 7), circular pre-crRNA (Figure 9)) was used, a base pair-deleted band of 0.2 kb or more and less than 3.8 kb was observed, as in the case of using conventional linear pre-crRNA (PC). It was confirmed that the circular crRNA of the present invention is effective for genome editing in eukaryotic cells, and in particular, mature crRNA can only be used for genome editing by circularization. Furthermore, as shown in Figure 8, the average genome editing efficiency when using circular mature-crRNA was 52.7%, confirming genome editing activity that is approximately twice as efficient as when using conventional linear pre-crRNA (PC) (22.9%).Furthermore, as shown in Figure 9, when using circular pre-crRNA, the genome editing efficiency was also higher than when using linear pre-crRNA (PC), confirming that the circular crRNA of the present invention enables genome editing with particularly high efficiency.

[0112] (Test Example 4) Examination of the intracellular stability of circular crRNA by measuring genome editing efficiency in cultured human cells To examine the intracellular stability of circular crRNA, the expression induction period of the Cas protein group was delayed after introduction of each crRNA, and genome editing efficiency was measured. For the examination, circular mature-crRNA (Example, Circ) prepared in (1) of Test Example 2 above, linear crRNA-2 (Line), and terminally modified linear pre-crRNA (Mod) in which three bases at the 5' end and three bases at the 3' end of the linear pre-crRNA were modified with 2'OMe and phosphorodithioate were used. In addition, the linear pre-crRNA prepared in (1) of Test Example 2 above was used as a positive control (+).

[0113] First, each of the above crRNAs (20 ng / μL) was introduced into K562 cells carrying doxycycline-inducible Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) by electroporation using a 4D-Nucleofector System (Lonza). After electroporation, 2 μg / mL doxycycline was added at 0 hours (simultaneous with electroporation), 12 hours, or 24 hours to express the Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11). The cells were then incubated at 37°C, 5% CO ２ Further, similarly to Test Example 3, each crRNA was introduced into K562 cells that had been cultured in a medium supplemented with 2 μg / mL doxycycline for 6 hours prior to electroporation and in which the Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) were expressed, and the cells were incubated in a medium supplemented with 2 μg / mL doxycycline at 37° C., 5% CO ２ After culturing for 2 days under the conditions above, all cells were collected. The timing of each electroporation (transfection), the timing of doxycycline addition (+Dox: −6 h, 0 h, 12 h, 24 h), and the timing of cell collection (end points of each arrow) are shown in the schematic conceptual diagram of Figure 10.

[0114] Genomic DNA was extracted from each of the recovered cells and PCR-amplified using a primer set designed for a 3.8 kb region containing the PAM sequence and target region on human EMX1. The PCR product was subjected to agarose gel electrophoresis. Figure 11 shows the agarose gel electrophoresis images for each combination of the crRNA introduced into the cells and the timing of doxycycline addition (-6 h, 0 h, 12 h, 24 h). In addition, the genome editing efficiency (Edited (%)) for each combination was calculated by comparing the density of the 3.8 kb band in the electrophoresis image with the density of the knocked-out and shortened band (band of 0.2 kb or more and less than 3.8 kb). The results are shown in Figure 12.

[0115] Furthermore, to confirm the content of genome editing, circular mature-crRNA (Circ) was used, and the PCR product when doxycycline was added simultaneously with electroporation (0 h) was analyzed by long-read sequencing using a nanopore sequencer (Oxford Nanopore Technologies). A schematic diagram of the location of a representative nucleotide sequence confirmed to be deleted by sequence analysis is shown in Figure 13.

[0116] As shown in Figure 11, when circular mature-crRNA (Circ) was introduced into cells, terminally modified linear pre-crRNA (Mod) with enhanced intracellular stability was introduced into cells, as in the case of adding doxycycline, i.e., the expression of the Cas protein group, even 12 hours (12h) or 24 hours (24h) after introduction into the cells, a band shorter than 3.8 kb with a deleted base pair was observed. Furthermore, as shown in Figure 12, the genome editing efficiency when using circular mature-crRNA (Circ) was significantly higher than when using the terminally modified linear pre-crRNA (Mod), regardless of the timing of doxycycline addition. 13, when using circular mature-crRNA (Circ), deletions of 226 base pairs, 506 base pairs, 597 base pairs, and 1719 base pairs were detected as typical mutations within the region (3.8 kb) containing the PAM sequence and the target region, confirming that deletions of several hundred to several thousand base pairs occurred in the region containing the target region. These results confirmed that the circular crRNA of the present invention is particularly stable within cells, and that the excellent genome editing efficiency of circular crRNA can be maintained for a sufficient period of time.

[0117] As described above, according to the present invention, it is possible to provide a Type I CRISPR-Cas system that enables highly efficient genome editing and has high stability within cells, a Type I CRISPR-Cas system containing the same, methods for editing target DNA using these, methods for producing cells in which target DNA has been edited, and methods for detecting target DNA, as well as kits for use in these methods.

[0118] According to the present invention, the type I CRISPR-Cas system containing circular crRNA enables nucleic acid detection and highly efficient genome editing in eukaryotic cells and living organisms, which will make a significant contribution not only to the agricultural and industrial fields but also to the medical field, such as gene therapy.

Claims

1. A crRNA that is circular and comprises: a spacer sequence comprising a sequence that specifically recognizes and binds to a crRNA recognition sequence on a target DNA; and a repeat sequence that can be cleaved by at least one Cas protein selected from the group consisting of Cas5 protein and Cas6 protein.

2. The crRNA of claim 1, which consists of one spacer sequence and one repeat sequence.

3. A Type I CRISPR-Cas system for editing target DNA, comprising the following (A) to (C): (A) a Cas protein having nuclease activity; (B) a Cascade protein; and (C) the crRNA according to claim 1.

4. A method for editing target DNA, comprising the step of contacting the Type I CRISPR-Cas system according to claim 3 with target DNA and editing the target DNA.

5. A method for producing a cell in which target DNA has been edited, comprising the steps of introducing or expressing the Type I CRISPR-Cas system according to claim 3 into a cell, contacting the cell with target DNA, and editing the target DNA.

6. The method of claim 5, wherein the cell is a eukaryotic cell.

7. A method for detecting target DNA in a sample, comprising the steps of contacting the Type I CRISPR-Cas system of claim 3 with a sample and detecting the target DNA in the sample.

8. A kit for use in the method according to any one of claims 4 to 7, comprising the following (A') to (C'): (A') at least one selected from the group consisting of a Cas protein having nuclease activity, an expression vector for the Cas protein, and a polynucleotide encoding the Cas protein; (B') at least one selected from the group consisting of a Cascade protein, an expression vector for the Cascade protein, and a polynucleotide encoding the Cascade protein; (C') at least one selected from the group consisting of the crRNA according to claim 1, an expression vector for the crRNA, and a polynucleotide encoding the crRNA; wherein the crRNA expression vector is at least one selected from the group consisting of (i) a vector comprising a polynucleotide encoding the spacer sequence and the repeat sequence, and (ii) a vector comprising an insertion site for the polynucleotide encoding the spacer sequence and a polynucleotide encoding the repeat sequence.

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