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

Abstract

To provide crRNA that enables highly efficient genome editing using Type I CRISPR-Cas system and is highly stable within cells.SOLUTION: A crRNA comprises: a spacer sequence containing 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; and is circular.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to crRNA, a Type I CRISPR-Cas system, a method for editing target DNA, a method for producing cells with edited target DNA, 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 containing the same, and a method for editing target DNA using these, a method for producing cells with edited target DNA, and a method for detecting target DNA, as well as a kit for use in these methods. [Background technology]

[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 crop and livestock breeding, 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, all classified as Class 2, have been developed as genome editing tools. In particular, the CRISPR-Cas9 system contains the Cas9 protein and guide RNA (comprising a crRNA that specifically recognizes and binds to a sequence near the target region on the target DNA, and a tracrRNA that forms a complex with the Cas9 protein). 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 circularizing the guide RNA to improve its stability in vitro and in Escherichia coli.

[0004] While all of the above systems are classified as Class 2, recently, it has been discovered that Type I CRISPR-Cas systems, which belong to Class 1, can be used as genome editing tools in eukaryotic cells (WO 2018 / 225858 (Patent Document 1)). This Type I CRISPR-Cas system comprises a Cas protein with nuclease activity (typically the Cas3 protein, or the Cas10 protein in Type ID), a cascade consisting of multiple Cas proteins (Cascade proteins), and crRNA. It has been shown to specifically cleave target regions even in eukaryotic cells, such as cultured human cells, and to efficiently introduce deletion mutations over a wide range of several hundred to several kb that includes the target region. Furthermore, compared to the CRISPR-Cas9 system, the sequence recognized and bound by the crRNA (the crRNA recognition sequence) is longer and more specific, making it less susceptible to nonspecific cleavage and considered to be safer.

[0005] Previous studies have reported that genome editing in eukaryotic cells using the Type I CRISPR-Cas system requires the use of precursor crRNAs that retain full-length repeat sequences at both ends. When such precursor crRNAs are introduced into eukaryotic cells, they are processed by Cas proteins (Cas5 and Cas6 proteins) to form mature crRNAs. The mature crRNAs and Cascade proteins then form a complex (Cascade complex) near the target region, and the Cas proteins, which have nuclease activity, cleave the target region, enabling genome editing. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 225858 [Non-patent literature]

[0007] [Non-Patent Document 1] Li Liu et al.,ACS Synth.Biol.,2023,12,p350-359 Summary of the Invention [Problem to be solved by the invention]

[0008] However, while the usefulness of the Type I CRISPR-Cas system has only recently been discovered, research into the system has yet to progress sufficiently, and even more efficient genome editing is needed. Furthermore, the crRNA precursors required for genome editing in eukaryotic cells using the Type I CRISPR-Cas system are short RNAs of approximately 90 bases, and are therefore easily degraded within the cell. Therefore, a significant amount of crRNA precursors must be introduced into cells to ensure sufficient genome editing efficiency.

[0009] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technologies, and aims to provide a 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, methods for detecting target DNA, and kits for use in these methods. [Means for solving the problem]

[0010] As a result of intensive research to achieve the above-mentioned objective, 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-strand DNA cleavage activity and achieves excellent genome editing efficiency compared to when the conventional linear crRNA precursor (linear pre-crRNA) is used.

[0011] Furthermore, we found that circularization enables genome editing in eukaryotic cells, not only when the crRNA is constructed from a circularized crRNA precursor, but also, surprisingly, when the mature crRNA is circularized, a process that previously failed to demonstrate genome editing activity when directly introduced into eukaryotic cells. We also found that the ability to form Cascade complexes (e.g., complex formation rate) is equivalent to that achieved with linear pre-crRNA, even after circularization. Furthermore, we found that the superior genome editing efficiency described above can be achieved with a lower amount of Cas protein than with linear pre-crRNA. When circularized crRNA is constructed from a circularized mature crRNA, i.e., consisting of one spacer sequence and one repeat sequence, the RNA length can be made shorter (typically approximately 61 nt) compared to the crRNA precursor (typically approximately 90 nt), thereby reducing the risk of introducing errors during synthesis and potentially reducing synthesis costs. Furthermore, circularization significantly alters the morphology, making it easier to separate impurities, such as incomplete crRNA, from the crRNA that cannot contribute to genome editing.

[0012] Furthermore, the inventors discovered that by making the crRNA circular, it exhibits significantly higher stability in cells than not only conventional linear pre-crRNA precursors but also linear pre-crRNA precursors whose ends have been modified to increase their stability in 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 is provided based on these findings in the following aspects. [1] a spacer sequence comprising 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. and It is cyclic, crRNA. [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, The following (A)~(C): (A) Cas protein with nuclease activity (B) Cascade proteins (C) crRNA described in [1] or [2] Including, the system. [4] A method for editing target DNA, [3] contacting the Type I CRISPR-Cas system described in [3] with target DNA and editing the target DNA. A method comprising: [5] A method for producing cells with edited target DNA, [3] A step of introducing or expressing the Type I CRISPR-Cas system according to [3] into a cell, contacting it with a target DNA, and editing the target DNA. A method comprising: [6] The method according to [5], wherein the cell is a eukaryotic cell. [7] A method for detecting target DNA in a sample, [3] contacting a sample with the Type I CRISPR-Cas system and detecting target DNA in the sample. A method comprising: [8] A kit for use in the method according to any one of [4] to [7], The following (A')~(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; Including, The expression vector for the crRNA 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. kit. [Effects of the Invention]

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

[0015] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of the structure of the spacer sequence and repeat sequence contained in the circular crRNA of the present invention. [Figure 2] (a) Denaturing polyacrylamide gel electrophoresis images of Seq_1 and Seq_2 (without ligase: Ligase(-) and with ligase: Ligase(+) respectively) obtained in Test Example 1(2), and (b) denaturing polyacrylamide gel electrophoresis images 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). [Figure 3]FIG. 1 is a schematic diagram showing the synthesis method of circular mature-crRNA (a) and circular pre-crRNA (b) in Test Example 2(1). [Figure 4] Electrophoretic images confirming the synthesis of circular mature-crRNA and circular pre-crRNA in Test Example 2(1). [Figure 5] 1 shows the change in signal intensity (RFU / min) when using circular mature-crRNA or linear pre-crRNA in the presence (Activator+) or absence (Activator-) of double-stranded DNA fragments at a Cas6 protein concentration of 7.2 μM in the in vitro test of Test Example 2(2). [Figure 6] 1 shows the change in signal intensity (RFU / min) in the in vitro test of Test Example 2(2) when using circular mature-crRNA or linear pre-crRNA with 3.6 μM Cas6 protein and with (Activator+) or without (Activator-) double-stranded DNA fragments. [Figure 7] (a) is a schematic conceptual diagram showing the circular mature-crRNA (circular crRNA, Example), linear pre-crRNA (PC), linear crRNA-1, and linear crRNA-2 used in Test Example 3(1), and (b) is an electrophoretic image of PCR products obtained by introducing these crRNAs into K562 cells expressing Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) and using the recovered total genomic DNA as a template. [Figure 8] 1 is a graph showing the genome editing efficiency (Edited (%), average of two tests) calculated from a comparison of band densities in the electrophoretic image of Test Example 3(1). [Figure 9]Electrophoretic images of PCR products obtained using the total genomic DNA recovered from K562 cells expressing the Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) after introducing the circular pre-crRNA (circular crRNA, Example), linear pre-crRNA (PC), and pMax_GFP plasmid obtained in Test Example 3(2) into the cells. [Figure 10] FIG. 1 is a schematic conceptual diagram showing 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 recovery (end points of each arrow) in Test Example 4. [Figure 11] Electrophoresis images obtained in Test Example 4 for each combination 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). [Figure 12] 1 is a graph showing the genome editing efficiency (Edited (%)) calculated from a comparison of band intensities in the electrophoretic image of Test Example 4. [Figure 13] This is a schematic diagram showing the locations of representative nucleotide sequences in which deletions were confirmed by long-read sequencing of PCR products in Test Example 4, when circular mature-crRNA (Circ) was used and doxycycline was added simultaneously with electroporation (0 h). DETAILED DESCRIPTION OF THE INVENTION

[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、タイプI CRISPR-Casシステム> The present invention provides a spacer sequence comprising 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. and It is cyclic, The present invention also provides a Type I CRISPR-Cas system for editing target DNA, comprising: The following (A)~(C): (A) Cas protein with nuclease activity (B) Cascade proteins (C) Circular crRNA of the present invention The present invention provides a system including:

[0018] (Target DNA) In the present invention, DNA containing a target region to be edited is referred to as "target DNA." The target DNA of the present invention is double-stranded DNA, and for the purpose of illustrating its correspondence with the Type I CRISPR-Cas system of the present invention, at least one strand of the target DNA is defined as having a structure containing, from the 5' end, a PAM sequence and the target region, and the complementary sequence that forms a complementary strand with the target region is contained in the crRNA recognition sequence to which the crRNA spacer sequence specifically recognizes and binds, or the crRNA recognition sequence is contained in the complementary sequence, or the complementary sequence and the crRNA recognition sequence at least partially overlap with 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 CRISPR-Cas systems) with nuclease activity. However, this does not exclude the possibility that a region adjacent to the target region may also be edited. The length of the target region according to the present invention is preferably 20 to 50 bases, more preferably 28 to 37 bases.

[0020] In the present invention, the "crRNA recognition sequence" refers to a sequence that the spacer sequence recognizes and binds to. The length of the crRNA recognition sequence of 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 (e.g., preferably about 20 bases), thereby increasing sequence specificity for the target DNA.

[0021] A "PAM (protospacer adjacent motif) sequence" is a sequence recognized by the Cas proteins that make up the cascade of a Type I CRISPR-Cas system. Its length varies depending on the type of Type I CRISPR-Cas system, but typically consists of 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. For example, when the Cas proteins that make up the cascade are those that make up a Type IB CRISPR-Cas system or a Type IF CRISPR-Cas system, the PAM sequences recognized by these proteins include 5'-CCA, 5'-CC, and 5'-ACN. When the Cas proteins that make up a Type IE CRISPR-Cas system are those that make up the same system, the PAM sequences recognized by these proteins include 5'-AAG, 5'-AGG, and 5'-GAG. However, these PAM sequences can be altered by modifying the Cas proteins (e.g., by introducing mutations), thereby expanding the range of target regions. In the present invention, the PAM sequence is located 5' of the target region, and the crRNA recognition sequence is determined depending on the positions 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. 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, genes of viruses that infect a host, bacteria, or protozoa, etc.). DNA present outside a cell may be DNA derived from a cell or DNA amplified and synthesized outside the 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 by comprising a spacer 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, and is circular.

[0025] A "spacer sequence" is a sequence originally 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-stranded structure need not be completely complementary. In the present invention, the sequence complementarity between the 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 depending on 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] A "repeat sequence" is a sequence that repeats via a spacer sequence in the CRISPR structure of the bacterial genome from which the Type I CRISPR-Cas system is derived, forming a stem-loop structure. The wild-type repeat sequence varies depending on the type of Type I CRISPR-Cas system and the species of the bacterium from which it is derived. For example, a Type IA CRISPR-Cas system derived from Pyrococcus furiosus typically comprises the 30-base nucleotide sequence set forth in SEQ ID NO: 1, a Type IB CRISPR-Cas system derived from Synechocystis sp. typically comprises the 36-base nucleotide sequence set forth in SEQ ID NO: 2, a Type IC CRISPR-Cas system derived from Neisseria lactamica typically comprises the 32-base nucleotide sequence set forth in SEQ ID NO: 3, and a Type I CRISPR-Cas system derived from Microcystis aeruginosa typically comprises the 32-base nucleotide sequence set forth in SEQ ID NO: 4. The type D CRISPR-Cas system typically consists of the nucleotide sequence set forth in SEQ ID NO: 4, which has a chain length of 37 bases; the type IE CRISPR-Cas system derived from Escherichia coli typically consists of the nucleotide sequence set forth in SEQ ID NO: 5, which has a chain length of 29 bases; the type IF 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 the type IG 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, like the typical wild-type repeat sequences described above, the repeat sequences of the present invention may be appropriately modified as long as they are cleavable by at least one Cas protein selected from the group consisting of Cas5 and Cas6 proteins 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 of the typical wild-type repeat sequences described above, in which one or more bases (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) have been substituted, deleted, and / or inserted.

[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 shown in the nucleotide sequence of SEQ ID NO: 1 in Type IA, between the 28th and 29th bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 2 in Type IB, between the 19th and 20th bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 3 in Type IC, between the 31st and 32nd bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 4 in Type ID, between the 21st and 22nd bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 5 in Type IE, between the 20th and 21st bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 6 in Type IF, and between the 28th and 29th bases of the repeat sequence shown in the nucleotide sequence of SEQ ID NO: 7 in Type IG. 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 (duplex formation ability with the crRNA recognition sequence, cascade complex formation ability with Cas proteins, target region cleavage activity, etc.). The additional sequence is preferably, for example, 8 bases or less, more preferably 6 bases or less, if 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, if 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 linearized as described below, i.e., a sequence that is added to the 5' or 3' end of one unit of the mature crRNA structure when linearized, such an additional sequence may include, for example, a sequence for circularization in expression using the expression vector described below (SEQ ID NO: 17, 18, etc.), the RNA aptamer sequence described below, an RNA sequence for knock-in donor (e.g., for prime editing), etc., and is not particularly limited, but preferably has 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 mature crRNA structure. That is, the circular crRNA of the present invention may be a circularized mature crRNA, a circularized crRNA precursor in which a repeat sequence or other sequence is further added to the mature crRNA, a circularized 5' mature crRNA or a circularized 3' mature crRNA in which the 5' or 3' end of the crRNA precursor has been removed in the same manner as the mature crRNA, a circularized RNA in which the above-mentioned additional sequence is further added to each of the crRNAs, or a circularized RNA containing one or more of the above crRNAs in multiple combinations. By containing multiple mature crRNA structures (i.e., sets of spacer sequences and repeat sequences), each can target multiple corresponding regions.

[0033] A typical structural unit of the mature crRNA is a "5' handle sequence-spacer sequence-3' handle sequence" structure. For example, in the case of type IE, the "5' handle sequence" typically consists of eight bases, from the 22nd to the 29th, of the repeat sequence represented by the nucleotide sequence of SEQ ID NO: 5, and is held by the Cas5 protein. For example, in the case of type IE, the "3' handle sequence" typically consists of 21 bases, from the 1st to the 21st, of the repeat sequence represented by the nucleotide sequence of 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 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] The structure of the crRNA precursor typically includes a "leader sequence-repeat sequence-spacer sequence-repeat sequence (LRSR structure)" or 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 an example of the structure of the spacer sequence and repeat sequence contained in the circular crRNA of the present invention, typical mature crRNA, crRNA precursor (RSR structure), 5' mature crRNA, and 3' mature crRNA, as well as the cleavage site by Cas protein, are shown in the schematic diagram of Figure 1. However, as mentioned 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 crRNAs, which previously could not be directly introduced into eukaryotic cells to demonstrate genome editing activity, circularization of the mature crRNA can achieve significantly superior genome editing efficiency compared to crRNA precursors, which previously demonstrated genome editing activity in eukaryotic cells. In addition, from the perspectives of reducing the risk of introducing error sequences during synthesis and reducing synthesis costs, the circular crRNA of the present invention preferably has a circular structure in which one structural unit of the mature crRNA is circularized, i.e., it is composed 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, but 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 methods such as those 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]. Alternatively, the circular crRNA can be obtained by circularizing crRNA expressed from a polynucleotide or expression vector using the self-ligation system described in, for example, [Nature Biotechnology 40, 2022, pp. 1388-1393]. Therefore, the circular crRNA of the present invention may be in the form of the circular crRNA (RNA), a polynucleotide encoding the circular crRNA, or an expression vector containing the polynucleotide.

[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 non-nucleic acid component such as a sugar chain. Such polynucleotides can be prepared by known methods or methods similar thereto, for example, by artificial synthesis.

[0040] When the circular crRNA of the present invention is in the form of a polynucleotide encoding it or an expression vector, these can include, 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] The expression vector for the circular crRNA is preferably one that can stably express the encoded crRNA without being integrated into the host genome. Various commonly used vectors can be used as base vectors for such expression vectors, and can be appropriately selected depending on the cells to be introduced and the introduction method. 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 poly(A) addition sequence).

[0043] The expression vector can be prepared by known methods or methods based thereon. For example, in addition to the method described in the operation manual attached to the 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 comprehensive manuals.

[0044] (Cas protein group) In addition to the circular crRNA (C) of the present invention, the Type I CRISPR-Cas system of the present invention also includes a Cas protein group consisting of multiple Cas proteins as a component. The Cas protein group includes (A) a Cas protein with nuclease activity (sometimes referred to herein as "nCas protein") and (B) other Cas proteins (sometimes referred to herein as "Cascade proteins"). However, when the purpose is not to cleave target DNA (e.g., 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 omitted from the Type I CRISPR-Cas system of the present invention, or the nCas protein may be a mutant lacking part or all of its nuclease activity.

[0045] Cas proteins with nuclease activity (nCas proteins) are typically Cas3 proteins (sometimes referred to simply as "Cas3" herein, and the same applies to other Cas proteins), but in Type ID, they are Cas10 proteins. In Type IA, Cas3-HD and Cas3-HEL complex together to form Cas3, while in Type IF, 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 proteins of the present invention also preferably have helicase activity. By cooperating with the circular crRNA of the present invention and the Cascade protein, nCas proteins can cleave the target region of the target DNA at at least one site (preferably multiple sites). Among the Cas proteins listed in each of the following Type I systems, Cas proteins other than nCas proteins (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 (e.g., 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 Type I of Class 1 among CRISPR-Cas systems. Type I systems are further classified into several types, and are currently classified into six types: Type IA, Type IB, Type IC, Type ID, Type IE, and Type IF, as well as Type IG, a subtype of Type IB (see, for example, [van der Oost J et al. (2014) Unravelling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiologym, Vol. 12 (No. 7), pp. 479-492] and [Jackson RN et al. (2014) Fitting CRISPR-associated Cas3 into the Helicase Family Tree, Current Opinion in Structural Biology, Vol. 24, pp. 106-114]).

[0047] The Cas proteins contained in each Type I system typically include: Type IA: Cas3-HD, Cas3-HEL, Cas5, Cas6, Cas7, Cas8, and Cas11; Type IB: Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; Type IC: Cas3, Cas5, Cas7, Cas8, and Cas11; Type ID: Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; Type IE: Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; Type IF: Cas2-3, Cas5, Cas6, Cas7, and Cas8; and Type IG: Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11. However, even when Cas11 is excluded from the configuration of a Type I CRISPR-Cas system, each system exhibits DNA editing activity (for example, it is known that Type IB and Type IC exhibit DNA editing activity even without Cas11, albeit at a lower level compared to when Cas11 is included, and the same is true for Type ID). Therefore, the Type I CRISPR-Cas system of the present invention also includes systems that do not contain Cas11.

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

[0049] The amino acid sequences of the Cas proteins constituting the Type I CRISPR-Cas system can be obtained, for example, from public databases (e.g., Genbank). Furthermore, the Cas proteins may be modified (e.g., by substitution, deletion, insertion, and / or addition of amino acid residues) based on the amino acid sequences of these known Cas proteins, as long as the modifications do not inhibit the effects of the present invention (e.g., cascade complex formation ability (preferably including the ability to cleave the repeat sequence of the crRNA) and nuclease activity). The ability of the Cas proteins to form cascade complexes 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 of the unmodified version (e.g., a protein consisting of a known amino acid sequence) (e.g., Test Example 2(2) below).

[0050] Furthermore, if necessary, functional molecules may be added to each Cas protein constituting the Type I CRISPR-Cas system. Examples of such functional molecules include nuclear localization signals (e.g., those described in [Wu et al., 2009, Biophysical Journal, Vol. 96 (Issue 9), pp. 3840-3849]) that promote translocation into the nucleus of eukaryotic cells, localization signals that promote localization to mitochondria or chloroplasts, tags for easy purification (e.g., HN tag, His tag, FLAG tag, glutathione-S-transferase (GST) tag), and reporter proteins for easy detection (e.g., fluorescent proteins such as green fluorescent protein (GFP) and chemiluminescent proteins such as luciferase). These functional molecules may be added singly or in combination. The functional molecules may be added, for example, to the N-terminus and / or C-terminus of each Cas protein.

[0051] In the type IE CRISPR-Cas system of 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 optimized) 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 International Publication No. 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 IE 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 preform a complex consisting of two or more of them and then introduce this into cells. Examples of such combinations include a complex consisting of two Cas11 molecules; a complex consisting of six Cas7 molecules; a complex consisting of one Cas8 molecule, one Cas5 molecule, and one Cas6 molecule; a complex consisting of one Cas3 molecule, one Cas8 molecule, two Cas11 molecules, six Cas7 molecules, one Cas5 molecule, and one Cas6 molecule; and combinations of one or more of these. Furthermore, in this case, it is also preferable to preform a complex (cascade) of these complexes (cascades) with circular crRNA (cascade complex) and then introduce this into cells. Such complexes can be prepared by known methods or methods similar thereto, for example, by the method described above [Kazuto Yoshimi et al., Nature Communications, 2022, 13:4917].

[0054] When the Cas proteins constituting the Cas protein group are in the form of polynucleotides encoding them, the polynucleotides may consist of DNA alone, or may consist of RNA, GNA, LNA, BNA, PNA, TNA, etc., or a mixture of these. Furthermore, they may be modified with components other than nucleic acids, such as sugar chains. Such polynucleotides can be prepared by known methods or methods similar thereto, for example, by artificial synthesis.

[0055] When each Cas protein constituting the Cas protein group is in the form of an expression vector encoding it, the expression vector and its construction method can be the same as 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, a single expression vector may contain multiple polynucleotides encoding each of the Cas proteins constituting the Cas protein group. 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 a system in which all polynucleotides encoding each Cas protein constituting the Cas protein group are incorporated into a single (same) expression vector. It is also possible to further design a system in which all or part of the polynucleotides encoding each Cas protein constituting the Cas protein group are incorporated into separate expression vectors. For example, it is possible to design a system in which polynucleotides encoding the Cascade proteins are incorporated into a single (same) expression vector and polynucleotides encoding Cas3 are incorporated into a separate expression vector. From the viewpoint of expression efficiency, etc., a method in which polynucleotides encoding each of the Cas proteins constituting the Cas protein group are respectively loaded into six different expression vectors is preferred. Alternatively, 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 (e.g., 2A peptide) that is cleaved by intracellular protease may be inserted between the multiple polynucleotides.

[0056] <Method for editing target DNA> The present invention is a method for editing target DNA, comprising: contacting the Type I CRISPR-Cas system of the present invention with target DNA and editing the target DNA; 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, attracting the Cascade protein to the target DNA and forming a Cascade complex consisting of the crRNA and the Cascade. Furthermore, the nCas protein is also attracted 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. Preferably, the circular crRNA of the present invention is cleaved by the Cas5 or Cas6 protein constituting the Cascade protein to generate a mature crRNA prior to binding to the crRNA recognition sequence, and the mature crRNA then binds to the crRNA recognition sequence.

[0058] Cleavage of the target region can efficiently induce base substitutions or deletions in the target region through the self-repair mechanism of single-stranded DNA, or through the self-repair mechanism of double-stranded DNA breaks 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 induce large-scale deletions (knockouts) in the region containing the target region or in a region adjacent to it. Furthermore, for example, when a base substitution occurs in the target region, various mutations can be introduced within the cell, for example, by repairing the base on the opposite strand of the substituted strand to pair with the substituted base due to a mismatch in double-stranded DNA, or by replacing the base with another base during repair, or by deleting or inserting one or more bases. Furthermore, by further contacting the target DNA with a knock-in fragment that can be introduced into the target region or a donor DNA for homologous recombination repair, it is possible to introduce (knock in) a desired nucleotide sequence at the break site. Such knock-in techniques can be performed using conventional methods or methods based thereon, as appropriate. 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 possessing a desired activity, depending on the purpose. In this embodiment, various types of editing of target DNA can be performed depending on the activity of the fused heterologous protein. Examples of activities of the fused heterologous protein 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, the nCas protein may be a mutant lacking part or all of its nuclease activity.

[0060] Examples of nCas protein mutants include, for example, in Type IE, a Cas3 HD domain H74A mutant (dnCas3), a K320N mutant in SF2 domain motif 1 (dhCas3), and a double S483A / T485A mutant in SF2 domain motif 3 (dh2Cas3). Furthermore, by using a fusion protein of a deaminase with a mutant lacking part or all of the nuclease activity of the nCas protein as a component of the Type I CRISPR-Cas system of the present invention, more precise DNA editing can be achieved by substituting bases without causing deletions in the target region. Methods for applying deaminases to CRISPR-Cas systems 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 such methods 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 to form a 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, the nCas protein can be a mutant lacking part or all of its nuclease activity. Techniques for applying transcriptional regulatory proteins to the CRISPR-Cas system are well 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 "inside a cell" 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 (such as cells of mammals, fish, birds, reptiles, amphibians, and insects), plant cells, algae cells, and yeast. Examples of prokaryotic cells include Escherichia coli, Salmonella, Bacillus subtilis, lactic acid bacteria, and extreme thermophiles.

[0063] Examples of "animal cells" include 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, 2-cell, 4-cell, 8-cell, 16-cell, and morula stages); 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, liver cells, pancreatic cells, brain cells, and kidney cells. Pre- and post-fertilization oocytes can be used as the oocytes used 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, calli, etc.

[0066] Furthermore, the "cell-free system" used in the editing method of the present invention refers to a system that does not contain living cells (eukaryotic cells or prokaryotic cells). The cell-free system of the present invention is not particularly limited as long as it 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 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, the method of introducing or expressing the Type I CRISPR-Cas system into cells 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 buffers such as phosphate buffer, Tris buffer, Good's buffer, and borate buffer are preferred.

[0068] <Method for producing cells with targeted DNA editing> The present invention provides a method for producing a cell with edited target DNA, comprising: a step of introducing or expressing the Type I CRISPR-Cas system of the present invention into a cell, contacting it with a target DNA, and editing the target DNA. Also provided is a method (sometimes simply referred to herein as a "production method") comprising the steps of: (a) preparing a CRISPR-Cas system comprising: (i) a CRISPR-Cas system that is capable of expressing a CRISPR-Cas gene; (ii) a CRISPR-Cas system that is capable of expressing a CRISPR-Cas gene; (iii) a CRISPR-Cas system that is capable of expressing a CRISPR-Cas gene; (iv) a CRISPR-Cas system that is capable of expressing a CRISPR-Cas gene; (v) a CRISPR-Cas system that is capable of expressing a CRISPR-Cas gene; (vi ...

[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 in cells, and are preferably eukaryotic cells, 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, the Type I CRISPR-Cas system is contacted with the target DNA in a cell by introducing the Cas proteins and circular crRNA that constitute 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 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 that constitute the Type I CRISPR-Cas system is introduced into the cell independently 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) that expresses the protein or RNA. The forms of the Cas proteins and circular crRNA that constitute the Type I CRISPR-Cas system, the polynucleotides encoding them, and the expression vectors that express 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 cell type, etc. Examples of such methods include electroporation, microinjection, particle gun technology, calcium phosphate technology, polyethyleneimine (PEI) technology, liposome technology (lipofection), DEAE-dextran technology, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium ion therapy, lithium acetate technology, spheroplast technology, and heat shock methods (calcium chloride method, rubidium chloride method). Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, New York: Elsevier, 1986.

[0072] When the Type I CRISPR-Cas system is introduced into or expressed in a cell, it 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, resulting in a cell with edited target DNA.

[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 the 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, and 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, with mice being particularly preferred. 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] Any known method can be used as an appropriate method for producing a non-human individual from cells in which the target DNA has been edited. 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 oocytes, and the resulting oocytes are implanted into the uterus of a pseudopregnant female non-human mammal, after which offspring can be obtained. 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 the resulting plant cells. Furthermore, offspring or clones in which the desired DNA has been edited can also be obtained from the resulting non-human individual.

[0075] Confirmation of the presence or absence of targeted 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: contacting the Type I CRISPR-Cas system of the present invention with a sample and detecting target DNA in the sample 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 sample desired to detect the target DNA, 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 label attached to a circular crRNA is used. For example, a circular crRNA in which a label is attached to the 3' end of the spacer sequence is circularized can be used to detect target DNA.

[0079] The labeling substance is not particularly limited as long as it is detectable, and examples 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 using the RNA aptamer sequence / recognition protein, 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, allowing target DNA to be detected using fluorescence as an indicator (see, for example, [Yang LZ et al., Cell Insight 1 (2022) 100044]). When using the RNA aptamer sequence / recognition compound, the RNA aptamer sequence is added to crRNA, and a recognition compound is bound to the RNA aptamer sequence, allowing target DNA to be detected using color development as an indicator.

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

[0082] Another embodiment of the detection method of the present invention utilizes 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, indiscriminately cleaving surrounding single-stranded DNA (ssDNA). This phenomenon can be utilized to detect target DNA (WO 2021 / 149829). More specifically, for example, a reaction system containing a sample for detecting target DNA and a Type I CRISPR-Cas system is mixed with single-stranded probe DNA, the cleavage of which can be detected with a labeling substance, to detect target DNA in the sample. The signal generated by the labeling substance upon cleavage of the single-stranded probe DNA can be used as an indicator to detect target DNA in the sample. When using this detection principle, there is no need 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 by 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')~(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 omitted or the nCas protein may be a mutant lacking all or part of its nuclease activity.

[0089] The above (A') to (C'), including their preferred embodiments, are as described above for 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, a polynucleotide encoding the protein or RNA, or an expression vector containing the polynucleotide and expressing the protein or RNA. 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 is (i) a vector comprising a polynucleotide encoding the spacer sequence and the repeat sequence; However, in order for the user to design a spacer sequence according to the target region, (ii) a vector comprising an insertion site for a polynucleotide encoding the spacer sequence and a polynucleotide encoding the repeat sequence. It may also be possible to use the following.

[0091] In the kit of the present invention, the combination of (A') to (C') is not particularly limited. It may be a combination containing each of (A') to (C') individually, or a premix of two or more of (A') to (C'). For example, (B') may be in a different form for each Cas protein constituting the Cascade protein, or in the form of a complex or composition containing two or more Cas proteins, or in the form of polynucleotides encoding two or more Cas proteins, or in the form of an expression vector containing the polynucleotides. 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 polynucleotides encoding two or more Cas proteins, or in the form of an expression vector containing the polynucleotides. Furthermore, for example, when (A') and (B') are introduced into cells as proteins and (C') is introduced into cells as RNA, two or more of these may be premixed in the form of a complex. When (A') to (C') are introduced into cells as expression vectors, they may be premixed 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, reagents for cell isolation, media for cell culture, dilution buffers, reconstitution solutions, wash buffers, nucleic acid introduction reagents, protein introduction reagents, control reagents, labeling substances, probe molecules, etc. The kits may also include instructions for carrying out the editing methods, production methods, and detection methods 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.). [Example]

[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'-phosphorylated oligoribonucleotides Two 61-nt 5'-phosphorylated oligoribonucleotide sequences (Seq_1 / nucleotide sequence number: 9, Seq_2 / nucleotide sequence number: 14) were synthesized using a phosphoramidite reagent (Chemgene) and a CPG solid 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 were carried out by heating at 65°C for 30 minutes in a 1:1 mixture of 40% aqueous methylamine and 30% aqueous ammonia. The reaction solution was filtered and dried under reduced pressure. The residue was then dissolved in 1 M tetrabutylammonium fluoride (THF) and incubated overnight at room temperature. Next, the mixture was desalted using a NAP-25 column (Merck) and then subjected to alcohol precipitation to obtain a crude oligonucleotide product.

[0096] From the resulting crude product, the desired full-length phosphorylated form was isolated by reverse phase HPLC under the following conditions: System: Prominence HPLC (Shimadzu Corporation, liquid delivery unit: LC-20AD, detection unit: SPD-M40, etc.); Column: YMC TriartBio C4, 250×10mm ID (S-5μm, 30nm); Solution A: 100 mM TEAA (pH 7.0) buffer containing 5% acetonitrile; Solution B: acetonitrile; Column equilibration conditions: 0% solution B; Gradient elution conditions: 5–25% solution B / 0–20 min; Column temperature: 50°C; Flow rate: 3mL / 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 phosphate-protecting groups were removed by precipitation at RT for 10 minutes. The target oligonucleotide contained in the reaction mixture 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; Waters); Solution A: 8.6mM triethylamine-100mM 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: 0.3mL / min; Detection wavelength: 260 nm As a result, the calculated molecular weight of Seq_1 was 19,755.8, whereas the measured value was 19,757.2 (+1.4), and the calculated molecular weight of Seq_2 was 19,755.8, whereas the measured value was 19,757.3 (+1.5).

[0098] (2) Preparation of circular crRNA For Seq_1 and Seq_2, the ends were ligated and circularized using T4 RNA ligase 2 in the presence of 24-base oligodeoxyribonucleotides (for Seq_1 / nucleotide SEQ ID NO: 19, for Seq_2 / nucleotide SEQ ID NO: 20) complementary to the 12 bases at the 5' and 3' ends, respectively, to prepare circularized products. The reaction solution had the following composition: 1 μM Seq_1 or Seq_2, 2.5 μM oligodeoxyribonucleotide (for Seq_1 or Seq_2), 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2mM magnesium chloride, 1mM dithiothreitol, 400 μM ATP, 25ng / μL T4 RNA Ligase 2 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 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 mixture, vigorously stirred, and centrifuged. The supernatant was collected in a separate tube. Further chloroform was added to the collected supernatant and centrifuged in the same manner. RNA was recovered by alcohol precipitation, and 10 pmol of oligonucleotides were subjected to denaturing polyacrylamide gel electrophoresis (PAGE: 15% acrylamide, 7.5 M urea, 1x Tris-borate-EDTA (pH 8.3)). The gel was stained with SYBR Green II and visualized using a ChemiDoc MP Imager (Bio-Rad). Denaturing polyacrylamide gel electrophoresis images of the obtained 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 in Seq_2, indicating a higher cyclization efficiency. Therefore, we prepared and isolated a larger amount of Seq_2 cyclized product. 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, then an equal volume mixture of TE-saturated phenol and chloroform was added, vigorously stirred, and centrifuged. The supernatant was collected in a separate tube. Further chloroform was added to the collected supernatant, and the mixture was centrifuged in the same manner. The RNA was then recovered by alcohol precipitation. The mixture containing the cyclized product was then electrophoresed using preparative denaturing PAGE (containing 7.5 M urea and 20% formamide as denaturants). The target band in the gel after electrophoresis was visualized using UV shadowing. The band was excised, 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 1030 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 the effect of circular crRNA on the cleavage activity of the CRISPR-Cas system, we measured nonspecific single-strand DNA cleavage (collateral cleavage activity) observed upon recognition of target DNA containing the crRNA recognition sequence using a fluorescent quencher probe (56FAM-AAGGTCGGA-ZEN-GTCAACGGATTTGGTC-ABkFQ; IDT; nucleotide sequence number: 8).

[0101] (1) Preparation of each crRNA Circular crRNA was synthesized essentially as in Test Example 1. Specifically, 100 μM of a 5'-phosphorylated oligoribonucleotide (oligoRNA, nucleotide sequence number: 9) was mixed with 100 μM of an oligodeoxyribonucleotide (ligation oligo, nucleotide sequence number: 10) designed to span the circularization site (ligation site) at a molar ratio of 2:5 to a total volume of 4 μL. To this mixture, 2 μL of T4 Rnl 2 Reaction Buffer (NEW ENGLAND BioLabs) and 9 μL of nuclease-free water were added, and the mixture was heated at 90°C for 3 minutes and then gradually cooled to room temperature over 40 minutes to anneal the oligoRNA 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 II (New England BioLads) were added to the mixture and incubated at 37°C for 2 hours to produce circular mature-crRNA (61 nt in this example) by circularizing mature crRNA (mature-crRNA, nucleotide sequence: 11, 5' handle sequence (8 nt)-spacer sequence (32 nt)-3' handle sequence (21 nt)) (Figure 3(a)). Similarly, circular pre-crRNA (pre-crRNA, nucleotide sequence: 12) was also circularized to produce circular pre-crRNA (90 nt in this example) (Figure 3(b)). In Figure 3, "Cut site" indicates the site cleaved by Cas6 protein. The circularization of each crRNA was confirmed by measuring 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, which was linearized from circular mature-crRNA, were chemically synthesized at IDT. The linear crRNAs were prepared in two forms: one in which the circular mature-crRNA was cleaved within the repeat sequence (3' repeat sequence (10 nt) - spacer sequence (32 nt) - 5' repeat sequence (19 nt); nucleotide sequence number 13), and the other in which the circular mature-crRNA was cleaved within the spacer sequence (3' spacer sequence (12 nt) - repeat sequence (29 nt) - 5' spacer sequence (20 nt); nucleotide sequence number 14). Hereinafter, the non-circularized crRNA precursor is referred to as "linear pre-crRNA," and the linear crRNA represented by nucleotide sequence number 13 is referred to as "linear crRNA-1," and the linear crRNA represented by nucleotide sequence number 14 is referred to as "linear crRNA-2."

[0103] (2) In vitro testing First, equal amounts of the cascade proteins constituting the type IE CRISPR-Cas system cascade (Cas5, Cas6, Cas7, Cas8, and Cas11: 0.8 μg / μL of the type IE cascade) and 250 ng / μL of the circular mature-crRNA or linear pre-crRNA prepared in (1) above were mixed and incubated at 37°C for 30 minutes in a complex formation buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2) to form a cascade complex. Next, Cas3 protein (40 ng / μL), the Cascade complex (36 ng / μL), and a double-stranded DNA fragment containing the crRNA recognition sequence (Activator, nucleotide sequence number: 15; 5 nM) were mixed in a reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl, 10 μM CoCl, 1 mM ATP) to prepare a sample. In addition, 1 μL of the fluorescent quencher probe (10 μM) was prepared using 5 μL of nuclease-free water and 10× Alert Buffer (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). Using a real-time PCR device (CFX96 Touch Deep Well system, Bio-Rad Laboratories), the intensity (RFU) of the FAM signal generated by the decomposition of the fluorescent quencher probe was observed over time every 30 seconds at 37 ° C. The change in signal intensity (RFU / min: slope of the exponential rise phase, the same applies 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 Figure 5.

[0104] As shown in Figure 5, even when circular crRNA (mature circular-crRNA) was used, the collateral cleavage activity resulted in a change in signal intensity (RFU / min) equivalent to that observed with conventional linear pre-crRNA, confirming that the circular crRNA 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-strand DNA cleavage activity. Specifically, we performed the same procedure as above, except that the amount of Cas6 protein was adjusted to 3.6 μM, half the amount of 7.2 μM, and then measured the change in signal intensity (RFU / min). 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 the 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-strand 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 investigate whether circular crRNAs can be used for genome editing in eukaryotic cells, we examined the genome editing efficiency of each crRNA in human cultured cells K562, which contain doxycycline-inducible Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11).

[0108] (1) Circular mature-crRNA For the study, circular mature-crRNA (circular crRNA, Example), linear pre-crRNA (PC), linear crRNA-1, and linear crRNA-2 prepared in Test Example 2(1) above were used (Figure 7(a)). Circular mature-crRNA (circular crRNA), linear pre-crRNA (PC), and linear crRNA-1 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 medium supplemented with 2 μg / mL doxycycline for 6 hours prior to electroporation to maintain expression of the Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11). Each of the above crRNAs (20 ng / μL) was then introduced into the K562 cells by electroporation using the 4D-Nucleofector System (Lonza). After electroporation, the cells were cultured in medium supplemented with 2 μg / mL doxycycline at 37°C and 5% CO2 for 2 days. Genomic DNA was then extracted and subjected to PCR amplification using a primer set designed for a 3.8 kb (3825 bp) region containing the PAM sequence and target region of human EMX1. The PCR product was subjected to agarose gel electrophoresis. Each crRNA combination was tested in duplicate (rep1, rep2). The agarose gel electrophoresis image is shown in Figure 7(b). 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 (0.2 kb or more but less than 3.8 kb) due to base pair deletion (knockout) in the electrophoresis image. The average genome editing efficiency for each gene is shown in Figure 8.

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

[0111] As shown in Figure 7, when linear crRNAs (linear crRNA-1 and -2) linearized from circular mature-crRNA were used, no bands of 0.2 kb or more and less than 3.8 kb were observed, confirming no genome editing activity. On the other hand, as shown in Figures 7 and 9, when each circular crRNA (circular mature-crRNA (Figure 7) and circular pre-crRNA (Figure 9)) was used, base-pair-deleted bands of 0.2 kb or more and less than 3.8 kb were observed, as with the conventional linear pre-crRNA (PC). This confirmed that the circular crRNAs of the present invention are effective for genome editing in eukaryotic cells, and that the mature crRNAs, in particular, can only be used for genome editing after 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, the genome editing efficiency when using circular pre-crRNA 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, we delayed the induction of Cas protein expression after each crRNA introduction and measured genome editing efficiency. For this study, we used the circular mature-crRNA (Example, Circ) prepared in Test Example 2(1) above, linear crRNA-2 (Line), and a terminally modified linear pre-crRNA (Mod) in which the 5'-terminal and 3'-terminal 3 bases of the linear pre-crRNA were modified with 2'OMe and phosphorodithioate. The linear pre-crRNA prepared in Test Example 2(1) above was also used as a positive control (+).

[0113] First, K562 cells carrying doxycycline-inducible Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) were electroporated with each of the above crRNAs (20 ng / μL) using the 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). After 2 days of culture at 37°C and 5% CO2, all cells were harvested. Similarly to Test Example 3, crRNAs were introduced into K562 cells expressing Cas proteins (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) in 2 μg / mL doxycycline-containing medium starting 6 hours before electroporation. After 2 days of culture in 2 μg / mL doxycycline-containing medium at 37°C and 5% CO2, all cells were harvested. The timing of electroporation (transfection), doxycycline addition (+Dox: -6 h, 0 h, 12 h, 24 h), and cell harvest (end point of each arrow) are shown in the schematic diagram in Figure 10.

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

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

[0116] As shown in Figure 11, when circular mature-crRNA (Circ) was introduced into cells, a band shorter than 3.8 kb with a deleted base pair was observed 12 hours (12 h) or 24 hours (24 h) after the addition of doxycycline, i.e., the expression of Cas proteins, was observed, as was the case when terminally modified linear pre-crRNA (Mod) with enhanced intracellular stability was introduced into cells. 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 within the region containing the target region. These results confirm 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. [Industrial Applicability]

[0117] As described above, the present invention makes it possible to provide a Type I CRISPR-Cas system that enables highly efficient genome editing and is highly stable within cells, a Type I CRISPR-Cas system containing the crRNA, methods for editing target DNA using the system, methods for producing cells in which target DNA has been edited, methods for detecting target DNA, and 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 greatly contribute not only to the agricultural and industrial fields but also to the medical field such as gene therapy.

Claims

1. 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. and It is cyclic, crRNA.

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, The following (A) to (C): (A) Cas protein with nuclease activity (B) Cascade protein (C) The crRNA according to claim 1. Including, the system.

4. 1. A method for editing target DNA, comprising: A step of contacting the type I CRISPR-Cas system of claim 3 with a target DNA and editing the target DNA. A method comprising:

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

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

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

8. A kit for use in the method according to any one of claims 4 to 7, 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; Including, 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. kit.

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