Guide RNA and use thereof

Abstract

To establish a type-I CRISPR-Cas system using a guide RNA of a novel form.SOLUTION: Provided are a guide RNA having, on the 5' side of a spacer sequence, a repeat sequence that is cleaved through processing for forming a cascade complex, and not having a repeat sequence or having a sequence that is not cleaved through processing for forming a cascade complex, on the 3' side of the spacer sequence; and a type-I CRISPR-Cas system using the guide RNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel form of guide RNA used in Type I CRISPR-Cas systems and uses thereof. [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 bioscience research but also in crop and livestock breeding, regenerative medicine, and gene therapy.

[0003] The CRISPR-Cas systems found in bacteria and archaea are divided into class 1, in which the target sequence is cleaved by a complex of multiple proteins, and class 2, in which the target sequence is cleaved by a single protein. CRISPR-Cas9, CRISPR-Cas12 (Cpf1), and CRISPR-Cas13, which have been developed as genome editing tools to date, are all classified as class 2.

[0004] Recently, it has been discovered that CRISPR-Cas3, a type I CRISPR belonging to class 1, can be used as a genome editing tool in eukaryotic cells (Patent Document 1). It has been found that this CRISPR-Cas3 system can efficiently introduce extensive deletion mutations of several hundred to several kb around the target sequence in eukaryotic cells, such as cultured human cells. Furthermore, compared to CRISPR-Cas9, the target recognition sequence in the guide RNA (crRNA) is longer, making nonspecific cleavage less likely to occur, and therefore it is considered to be safer. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 225858 Summary of the Invention [Problem to be solved by the invention]

[0006] The CRISPR-Cas3 system described in the examples of Patent Document 1 uses a pre-crRNA (precursor crRNA) with full-length repeat sequences at both ends, which successfully causes pre-crRNA processing in eukaryotic cells and forms a functional genome editing complex. However, the pre-crRNA processing process has not been fully investigated, and the structure of the guide RNA required for the formation of a functional genome editing complex has not been fully elucidated.

[0007] The present invention was made in light of these circumstances, and its purpose is to clarify the detailed structure of guide RNAs required for the functioning of type I CRISPR-Cas systems in eukaryotic cells and to establish type I CRISPR-Cas systems that utilize novel forms of guide RNAs. [Means for solving the problem]

[0008] As a result of extensive research conducted by the inventors to achieve the above-mentioned objective, they discovered that for the Type I CRISPR-Cas system to function as a complex, the 5' repeat sequence of the guide RNA must be cleaved by processing, whereas processing of the 3' repeat sequence is not required, and a functional complex can be formed even when the repeat sequence after cleavage by processing is used.

[0009] The inventors further analyzed this mechanism and found that even when a truncated crRNA in which the 3' repeat sequence was completely deleted was used, a functional complex was formed, enabling nucleic acid detection and DNA editing.

[0010] Furthermore, the present inventors discovered that this mechanism is not limited to a specific subtype but is common to Type I CRISPR-Cas systems, leading to the completion of the present invention.

[0011] The present invention relates to a guide RNA for a Type I CRISPR-Cas system in which the 3' repeat sequence has been deleted or modified, thereby losing sensitivity to processing on the 3' side of the spacer sequence, and uses thereof, and more specifically, includes the following aspects.

[0012] (1) A guide RNA for a Type I CRISPR-Cas system, in which a repeat sequence that is cleaved by processing to form a cascade complex is located on the 5' side of the spacer sequence, and either no repeat sequence is located on the 3' side of the spacer sequence or a sequence that is not cleaved by processing to form a cascade complex is located on the 3' side of the spacer sequence.

[0013] (2) A DNA encoding the guide RNA described in (1).

[0014] (3) An expression vector containing the DNA described in (2).

[0015] (4) A type I CRISPR-Cas system comprising the guide RNA described in (1).

[0016] (5) A method for producing a sample in which target DNA has been edited, comprising contacting the CRISPR-Cas system described in (4) with a sample containing target DNA.

[0017] (6) The method according to (5), wherein the sample is a cell.

[0018] (7) The method according to (6), wherein the cell is a eukaryotic cell.

[0019] (8) A method for detecting target DNA in a sample, comprising contacting the CRISPR-Cas system described in (4) with the sample. [Effects of the Invention]

[0020] The present invention has demonstrated that the Type I CRISPR-Cas system functions even when the 3' repeat sequence is deleted or modified, enabling a variety of applications, such as nucleic acid detection and epigenome editing using a combination of a guide RNA with an RNA aptamer sequence and an RNA aptamer recognition molecule, knock-in using a guide RNA with a donor sequence, and prime editing using a guide RNA with a primer binding site sequence and a reverse transcriptase template sequence. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows the results of the in vitro cleavage activity evaluation (CONAN assay) of the Type IE CRISPR-Cas system using various forms of crRNA (pre-crRNA, crRNA with mature 5' or 3' repeat sequences, and mature crRNA). [Figure 2] This figure shows the results of an in vitro CONAN assay evaluating the cleavage activity of the Type IE CRISPR-Cas system using a truncated crRNA lacking the 3' repeat sequence. As a positive control, a crRNA with a mature 3' repeat sequence was used. [Figure 3] This figure shows the results of evaluating the genome editing activity of the Type IE CRISPR-Cas system using a truncated crRNA lacking the 3' repeat sequence, with pre-crRNA used as a positive control. [Figure 4] This figure shows the results of evaluating the genome editing activity of the Type IC CRISPR-Cas system using a truncated crRNA lacking the 3' repeat sequence. Pre-crRNA was used as a positive control. [Figure 5] This figure shows the results of evaluating the genome editing activity of the Type ID CRISPR-Cas system using a truncated crRNA lacking the 3' repeat sequence. Pre-crRNA was used as a positive control. [Figure 6]This figure shows an overview of prime editing by the type IE CRISPR-Cas system using a truncated crRNA lacking the 3' repeat sequence, and its results. [Figure 7] Figure 1 shows the results of target DNA detection using the Type IE CRISPR-Cas system, which utilizes a truncated crRNA lacking the 3' repeat sequence. DETAILED DESCRIPTION OF THE INVENTION

[0022] <Guide RNAs and Type I CRISPR-Cas systems containing them> The present invention provides a novel form of guide RNA in a Type I CRISPR-Cas system.

[0023] Class 1 CRISPR-Cas systems are classified into Type I and Type III. Type I is further subdivided 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]).

[0024] In the Type I CRISPR-Cas system, a guide RNA forms a complex with a Cas3 protein and a Cascade protein, and the complex acts on target DNA. When using a Type I CRISPR-Cas system in eukaryotic cells, a pre-crRNA (a crRNA in which repeat sequences that are cleaved by processing are arranged on both sides of a spacer sequence) has typically been used as the guide RNA (see Patent Document 1 above). However, the present inventors have discovered that cleavage of the repeat sequence 3' of the spacer sequence is not essential in the processing process for forming the Cascade complex (hereinafter, sometimes simply referred to as the "processing process").

[0025] Therefore, the guide RNA of the present invention is characterized in that a repeat sequence that is cleaved during processing is located on the 5' side of the spacer sequence, and either no repeat sequence or a sequence that is not cleaved during processing is located on the 3' side of the spacer sequence. That is, the guide RNA of the present invention typically has a structure of "repeat sequence that is cleaved during processing-spacer sequence" or a structure of "repeat sequence that is cleaved during processing-spacer sequence-sequence that is not cleaved during processing."

[0026] The "repeat sequence" in the present invention is a sequence that is repeated via a spacer sequence in the CRISPR structure of the bacterial genome from which the Type I CRISPR-Cas system is derived.

[0027] The wild-type repeat sequence varies depending on the subtype of the Type I CRISPR-Cas system and the type of bacteria from which it is derived. For example, a Type IA CRISPR-Cas system derived from Pyrococcus furiosus typically comprises the 30-base sequence set forth in SEQ ID NO: 1; a Type IB CRISPR-Cas system derived from Synechocystis sp. typically comprises the 36-base sequence set forth in SEQ ID NO: 2; a Type IC CRISPR-Cas system derived from Neisseria lactamica typically comprises the 32-base sequence set forth in SEQ ID NO: 3; a Type ID CRISPR-Cas system derived from Microcystis aeruginosa typically comprises the 37-base sequence set forth in SEQ ID NO: 4; a Type IE CRISPR-Cas system derived from Escherichia coli typically comprises the 29-base sequence set forth in SEQ ID NO: 5; and a Type IF CRISPR-Cas system derived from Pseudomonas aeruginosa typically comprises the 36-base sequence set forth in SEQ ID NO: 6. In the CRISPR-Cas system, it typically consists of the 28-base sequence set forth in SEQ ID NO: 6, and in the Type IG CRISPR-Cas system derived from Thioalkalivibrio sulfidiphilus, it typically consists of the 36-base sequence set forth in SEQ ID NO: 7.

[0028] In the present invention, the repeat sequence on the 5' side of the spacer sequence (hereinafter sometimes simply referred to as the "5' repeat sequence") is typically a wild-type repeat sequence, but like the wild-type repeat sequence, it may contain mutations (addition, deletion, substitution, and / or insertion of bases) as long as it is cleaved during the processing process. On the other hand, the present inventors have discovered that the repeat sequence on the 3' side of the spacer sequence (hereinafter sometimes simply referred to as the "3' repeat sequence") is not necessarily cleaved during the processing process. Therefore, the guide RNA of the present invention is characterized in that a repeat sequence that is cleaved during the processing process is not located on the 3' side of the spacer sequence. The 3' side of the spacer sequence may not have a repeat sequence, or a sequence that is not cleaved during the processing process may be added.

[0029] The sequence that is not cleaved during the processing process may be any sequence unrelated to processing, or may be a repeat sequence modified to prevent such cleavage. In a typical processing process, the repeat sequence forms a loop structure, which is cleaved by the action of a specific Cas (e.g., Cas6, Cas5, etc.). The cleavage site is typically between the 22nd and 23rd bases of the repeat sequence in Type IA, between the 28th and 29th bases of the repeat sequence in Type IB, between the 19th and 20th bases of the repeat sequence in Type IC, between the 31st and 32nd bases of the repeat sequence in Type ID, between the 21st and 22nd bases of the repeat sequence in Type IE, between the 20th and 21st bases of the repeat sequence in Type IF, and between the 28th and 29th bases of the repeat sequence in Type IG. Therefore, examples of modified repeat sequences include repeat sequences that have been shortened so as not to include the cleavage site, and repeat sequences in which the bases around the cleavage site have been modified.

[0030] The guide RNA of the present invention is suitable for various modifications to the 3' side because it does not undergo cleavage due to processing on the 3' side of the spacer sequence. Modifications are not particularly limited, but include, for example, labels, tags, donor sequences, and prime editing sequences (primer binding site sequences and reverse transcriptase template sequences). When the present invention is used for prime editing, as shown in the examples of the present application, an RNA aptamer sequence may be further added to the prime editing sequence. In this case, the reverse transcriptase may be fused with a recognition protein or bound to a recognition compound. For details about RNA aptamer sequences, recognition proteins, and recognition compounds, please refer to the description in the section "Method for detecting target DNA" below.

[0031] The "spacer sequence" of the present invention is a sequence designed to be complementary to the target DNA. The target DNA may be endogenous or exogenous DNA. Examples of endogenous DNA include genomic DNA in chromosomes, mitochondria, and chloroplasts. Examples of exogenous DNA include reporter genes, marker genes, and genes of viruses, bacteria, protozoa, and the like that infect the host.

[0032] The guide RNA of the present invention can be used as an artificially synthesized RNA, or as an RNA expressed from DNA.

[0033] Various commonly used vectors can be used as base vectors for guide RNA expression vectors. The type of expression vector is not particularly limited; a vector capable of expressing guide RNA in its intended environment (e.g., in host cells) can be appropriately selected. Examples of expression 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 combinations thereof (cosmids, phagemids, etc.). Expression vectors may contain promoter sequences for inducing transcription or sequences for enhancing transcription (e.g., enhancer sequences).

[0034] Expression vectors can be prepared by known techniques. These techniques include those described in the operating manuals that accompany vector preparation kits, as well as those described in various manuals. For example, Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual, 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001, is a comprehensive manual.

[0035] Class 1 Type I CRISPR-Cas systems containing the guide RNA of the present invention typically contain a Cas3 protein and a Cascade protein as components other than the guide RNA. However, if the purpose is not to cleave target DNA (for example, when binding to an expression control region to suppress transcription), the system can also be used as a system that does not contain a component with nuclease activity (typically a Cas3 protein, but in Type ID, Cas10d).

[0036] Typically, type IA includes Cas3-HD, Cas3-HEL, Cas5, Cas7, Cas8, and Cas11 as cascade proteins; type IB includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 as cascade proteins; type IC includes Cas3, Cas5, Cas7, Cas8, and Cas11 as cascade proteins; and type ID includes Ca Type I CRISPR-Cas systems contain Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11 as cascade proteins; Type IE contains Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 as cascade proteins; Type IF contains Cas2-3, Cas5, Cas6, Cas7, and Cas8 as cascade proteins; and Type IG contains Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11 as cascade proteins. However, it should be understood that Cas11 can still exhibit DNA editing activity even when it is excluded from the components of the Type I CRISPR-Cas system (for example, it is known that Type IB and Type IC can exhibit DNA editing activity, albeit at a lower level than when Cas11 is included, even when Cas11 is not included, and the inventors have confirmed that this is also true for Type ID). Therefore, the Type I CRISPR-Cas system of the present invention also includes a system that does not contain Cas11.

[0037] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add a nuclear localization signal to promote nuclear localization of the Cas3 protein or Cascade protein. The nuclear localization signal can be added to the N-terminus and / or C-terminus of each protein. Similarly, when targeting, for example, the mitochondrial genome or chloroplast genome, it is preferable to add a localization signal that promotes localization thereto.

[0038] In the Type I CRISPR-Cas system of the present invention, Cas3 and Cascade may be in the form of a protein, a polynucleotide (DNA, RNA) encoding the protein, or a vector expressing the protein. In the polynucleotide form, the base sequence may be modified (e.g., codon optimization) to make it suitable for expression in a host cell.

[0039] In constructing an expression vector, the DNA encoding the Cas3 protein and the Cascade protein can be designed to be carried on a single (same) vector, or all or part of them can be carried on separate vectors. Alternatively, the DNA encoding each protein can be linked via DNA encoding an amino acid sequence (e.g., 2A peptide) that is cleaved by intracellular protease, allowing it to be expressed as a single protein, which can then be separated into individual proteins by the action of the protease for use. Expression vectors can be constructed by known techniques, as with the guide RNAs described above.

[0040] <Method for producing a sample with edited target DNA> The present invention also provides a method for producing a sample in which target DNA has been edited, comprising contacting a CRISPR-Cas system containing the above-mentioned guide RNA with a sample containing target DNA.

[0041] "DNA editing" in the present invention includes DNA cleavage at a target site, introduction of mutations into DNA (base deletion, insertion, or substitution), modification of DNA bases, control of DNA expression, and combinations of these. DNA editing may be performed in vitro or in vivo, depending on the purpose. It may also be performed in a cell or in a cell-free system.

[0042] One of the main modes of DNA editing in Type I CRISPR-Cas systems is the cleavage of target DNA by a component with nuclease activity (typically the Cas3 protein, but in Type ID, Cas10d), followed by the introduction of mutations via DNA repair in cells. As shown in the Examples of this application, this mode also allows for the generation of large deletions in target DNA.

[0043] In another embodiment of DNA editing, the Cas3 protein or Cascade protein can be fused to a heterologous protein with the desired activity to form a chimeric protein. In this embodiment, various editing operations can be performed on target DNA 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 (e.g., RNA-dependent DNA polymerase activity when the present invention is used for prime editing), ligase activity, photolyase activity, and glycosylase activity. In this case, a mutant lacking part or all of the nuclease activity of the Cas3 protein (or the Cas10d protein in the case of type ID) can be used.

[0044] Examples of Cas3 protein mutants that can be used in Type IE include an HD domain H74A mutant (dnCas3), an SF2 domain motif 1 K320N mutant (dhCas3), and an SF2 domain motif 3 S483A / T485A double mutant (dh2Cas3). For example, by incorporating a fusion protein of a deaminase with a mutant that partially or completely eliminates the nuclease activity of Cas3 into the Type I CRISPR-Cas system of the present invention, precise genome editing can be achieved by substituting bases without causing deletions at the target site. Methods for applying deaminase to CRISPR-Cas systems are known (Nishida K. et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI: 10.1126 / science.aaf8729, (2016)), and these methods can be applied to the Type I CRISPR-Cas system of the present invention.

[0045] Furthermore, gene transcription at a target site can be regulated by fusing the Cas3 protein or Cascade protein 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, a mutant lacking part or all of the nuclease activity of the Cas3 protein (or the Cas10d protein in the case of Type ID) can also be used. Techniques for applying transcriptional regulatory proteins to the CRISPR-Cas system are well known to those skilled in the art.

[0046] In the present invention, a desired nucleotide sequence can be knocked into a target DNA region by adding a donor DNA as a component of the Type I CRISPR-Cas system. The donor DNA typically has homology arms on both sides of the desired nucleotide sequence to be knocked in, and the desired nucleotide sequence is inserted into the target DNA region via a homologous recombination repair mechanism or the like.

[0047] The "sample" used for editing the target DNA is not particularly limited as long as it contains the target DNA. It may be a cellular sample (cell, tissue, or individual organism) or an acellular sample.

[0048] Cells for editing target DNA include prokaryotic and eukaryotic cells, including bacteria and archaea, and eukaryotic cells, including animal cells, plant cells, algae cells, and fungal cells.

[0049] 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 oocytes for creating genome-edited animals, but fertilized oocytes, i.e., fertilized eggs, are preferred. Pronuclear stage fertilized eggs are particularly preferred. Oocytes can be used by thawing cryopreserved oocytes.

[0050] 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.

[0051] Known methods can be used to generate non-human individuals from cells. When generating non-human individuals from cells in animals, germ cells or pluripotent stem cells are typically used. For example, molecules constituting the Type I CRISPR-Cas system of the present invention are introduced into oocytes, and the resulting oocytes are then implanted into the uterus of a pseudopregnant female non-human mammal, followed by the production of offspring. Implantation can be performed using fertilized eggs at the 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, or morula stage. Oocytes can be cultured under appropriate conditions until implantation, if necessary. Oocyte implantation and culture can be performed according to known techniques (Nagy A. et al., Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press, 2003). From the resulting non-human individuals, offspring or clones with desired DNA editing can also be obtained.

[0052] Examples of plant cells include cells of grains, oil crops, forage crops, fruits, and vegetables. Plant cells include, for example, cells constituting individual plants, cells constituting organs or tissues separated from plants, and cultured cells derived from plant tissue. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, and calli. Examples of plants include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation, as well as their propagation materials (e.g., seeds, tuberous roots, tubers, etc.).

[0053] It has long been known that somatic cells of plants possess totipotency, and methods for regenerating plants from plant cells have been established for various plants. Therefore, for example, by introducing molecules constituting the Type I CRISPR-Cas system of the present invention into plant cells and regenerating plants from the resulting plant cells, a plant with a desired DNA edited can be obtained. From the resulting plant, progeny, clones, or propagation materials with the desired DNA edited can also be obtained. Methods established in the art can be used to regenerate plant tissues by tissue culture to obtain individuals (Transformation Protocols [Plant Edition], edited by Yutaka Tabei, Kagaku Dojin, pp. 340-347 (2012)).

[0054] The method for introducing the Type I CRISPR-Cas system of the present invention into cells is not particularly limited. Examples include electroporation, calcium phosphate transfection, liposome transfection, DEAE-dextran transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, and infection with viral vectors. These methods are described in many standard laboratory manuals, such as "Leonard G. Davis et al., Basic Methods in Molecular Biology, New York: Elsevier, 1986."

[0055] <Method for detecting target DNA> The present invention also provides a method for detecting target DNA in a sample, comprising contacting the sample with a CRISPR-Cas system comprising the above-described guide RNA.

[0056] The "sample" in the detection method of the present invention can be any desired sample in which target DNA is to be detected (hereinafter referred to as a "test sample"). The test sample can be a cellular sample or an acellular sample, and examples include biological tissues, cells, cell lysates, body fluids (urine, saliva, serum, plasma, whole blood, etc.), or samples containing purified or synthesized DNA.

[0057] One embodiment of the detection method of the present invention is a method that utilizes a label attached to a guide RNA. Since the guide RNA of the present invention is not cleaved by processing at the 3' side of the spacer sequence, the target DNA can be detected by using a guide RNA labeled at the 3' side.

[0058] The label is not particularly limited as long as it is detectable, but 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. When an RNA aptamer sequence / recognition protein is used, the RNA aptamer sequence is added to a guide RNA, 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 (Yang LZ et al., Cell Insight 1 (2022) 100044). When an RNA aptamer sequence / recognition compound is used, the RNA aptamer sequence is added to a guide RNA, and a recognition compound is bound to the RNA aptamer sequence, allowing target DNA to be detected using color development as an indicator.

[0059] 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 that uses a Cas3 protein (Cas10d in Type ID) in which nuclease activity has been eliminated, or a system in which the Cas3 protein (Cas10d in Type ID) has been excluded.

[0060] Another embodiment of the detection method of the present invention is a method that uses single-stranded probe DNA (see Example 1). In the Type I CRISPR-Cas system, when target DNA in a sample is recognized and bound, it is known to indiscriminately cleave surrounding single-stranded DNA (ssDNA), and it is also possible to detect target DNA by utilizing this phenomenon (WO 2021 / 149829).

[0061] Specifically, by mixing a single-stranded probe DNA whose cleavage can be detected with a sample containing the target DNA and a reaction system containing a Type I CRISPR-Cas system, the target DNA in the sample can be detected using the signal generated by cleavage of the single-stranded probe DNA as an indicator. When using this detection principle, it is not necessary to label the guide RNA of the present invention itself.

[0062] 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 contain one or more modifications (e.g., base modification, backbone modification, sugar modification).

[0063] One preferred embodiment of the label bound to the single-stranded probe DNA is a fluorescent dye / quencher pair. In this embodiment, when the fluorescent dye / quencher pair is bound to the single-stranded probe DNA and is in close proximity, 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 test 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.

[0064] Another preferred embodiment of the label attached to 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 the single-stranded probe DNA and is in close proximity, 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 and acceptor are separated, the FRET signal is reduced or eliminated. Therefore, the target DNA in the test 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.

[0065] Alternatively, for example, immunochromatography (lateral flow assay) can be used to detect single-stranded probe DNA.

[0066] The contact of the test sample, the Type I CRISPR-Cas system, and the single-stranded probe DNA can be achieved, for example, by mixing them. When the test sample contains cells, an additional operation can be included for introducing the CRISPR-Cas system and the single-stranded probe DNA into the cells in the test sample. [Example]

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0068] [Example 1] In vitro DNA cleavage activity of various crRNA lengths This detection system utilizes Escherichia coli-derived type IE CRISPR. Cas3 protein was expressed and purified using insect cells Sf9. Specifically, a His-tagged Cas3 gene with a nuclear localization signal (bpNLS) was inserted into a baculovirus gene expression vector, and then baculovirus was generated using the E. coli strain DH10bac. The resulting baculovirus was then used to infect Sf9 insect cells to express the Cas3 protein. The total protein was then recovered and purified using a nickel column and gel filtration.

[0069] Cascade components were also expressed and purified using insect cells Sf9. Specifically, the genes for Cas5, Cas6, Cas7, Cas8, and Cas11, each tagged with a His-tagged nuclear localization signal (bpNLS), were linked via a 2A peptide sequence to create a baculovirus gene expression vector. Proteins were expressed as described above, and the cascade components were purified by nickel column and gel filtration.

[0070] The crRNAs were synthesized from double-stranded DNA fragments encoding each gene, transcribed in vitro, and purified using a column. In this experiment, we used pre-crRNA (SEQ ID NO: 8), mature crRNA (SEQ ID NO: 9), 5' mature crRNA (only the 5' repeat sequence is mature crRNA), and 3' mature crRNA (only the 3' repeat sequence is mature crRNA).

[0071] To examine the effect of crRNA length on the cleavage activity of the CRISPR-Cas3 system, we performed a CONAN assay to measure nonspecific cleavage (collateral cleavage activity) observed during target recognition using a fluorescent quencher probe (56FAM-AAGGTCGGA-ZEN-GTCAACGGATTTGGTC-ABkFQ, IDT; the sequence between ZEN and ABkFQ is set forth in SEQ ID NO: 10).

[0072] Cascade factors (0.8 μg / μL) and crRNA (250 ng / μL) were mixed in equal amounts and incubated in complexation buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2) at 37 °C for 10 minutes to form the Cascade complex. Next, Cas3 protein (final concentration 40 ng / μL), Cascade complex (final concentration 32 ng / μL), double-stranded DNA fragment containing the target sequence (mouse Tyr gene / SEQ ID NO: 11) (final concentration 5 nM), and fluorescent quencher probe (final concentration 1 μM) were mixed in reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2, 1 mM ATP) to prepare a 10 μL system. The FAM signal intensity was measured over time every 30 seconds at 37°C using a real-time PCR device (CFX96 Touch Deep Well system, Bio-Rad Laboratories).

[0073] As a result, the 5' repeat sequence emitted a fluorescent signal due to collateral cleavage activity only in the precursor state, whereas the 3' repeat sequence emitted a fluorescent signal in both the precursor and mature states (Figure 1). Thus, the 5' repeat sequence was essential for CRISPR-Cas3 complex formation and activation.

[0074] Therefore, we next investigated whether the 3' repeat sequence could be further shortened. As a result, fluorescent signals due to collateral cleavage activity were detected even in the truncated crRNA (SEQ ID NO: 12) without the 3' repeat sequence (Figure 2). In other words, the 21 bases remaining in the 3' end during maturation were not essential for CRISPR-Cas3 complex formation and activation.

[0075] These results revealed that when cascade factors and crRNA form a complex, the 5' repeat sequence is essential, but the 3' repeat sequence is not, and that even truncated crRNAs that completely lack the 3' repeat sequence exhibit activity.

[0076] [Example 2] Measurement of genome cleavage activity in human cultured cells K562 using Type IE truncated crRNA Purified human EMX1-targeting crRNA (truncate; 20 ng / μL) was introduced into K562 cells expressing Cas3 and Cascade components (Cas5, Cas6, Cas7, Cas8, and Cas11) in a doxycycline-inducible manner using the 4D-Nucleofector System (Lonza) by electroporation. Cells were cultured in 2 μg / ml doxycycline-supplemented medium at 37°C and 5% CO2 for 2 days. The whole cells were then harvested, and the genome was extracted. PCR was performed using a primer set amplifying a 3.8-kb fragment containing the PAM region of the EMX1 gene.

[0077] As a result, when purified truncated crRNA (SEQ ID NO: 14) was used, a shorter band was detected compared to the wild-type band, as was the case with purified pre-crRNA (SEQ ID NO: 13) and plasmid-expressed pre-crRNA (Figure 3).

[0078] To characterize the genome editing, we performed long-read sequencing of the PCR products using a nanopore sequencer (Oxford Nanopore Technologies). The results showed deletions of 562, 1619, and 2106 base pairs upstream of the 5' PAM in the spacer sequence of the target region (Figure 3), confirming the feasibility of using truncated crRNA for genome editing in human cells.

[0079] [Example 3] Measurement of genome cleavage activity in human cultured cells HEK293T using Type IC truncated crRNA To verify whether a similar phenomenon occurs with other Type I CRISPRs, we used the Type I-C system of Neisseria lactamica to examine whether truncated crRNAs introduce mutations into the human EMX1 gene.

[0080] Plasmids expressing pre-crRNA (SEQ ID NO: 15) or truncated crRNA (SEQ ID NO: 16) were prepared and transfected into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) along with plasmids expressing five Cas (3, 5, 7, 8, 11) effectors and a puromycin resistance gene. Cells were cultured in medium supplemented with 1 μg / ml puromycin at 37°C and 5% CO2 for 2 days. Whole cells were harvested, and the genome was extracted. PCR was performed using a primer set amplifying a 3.8 kb fragment containing the PAM region of the EMX gene. Electrophoresis of the PCR products confirmed that the truncated crRNA, like the pre-crRNA, produced a shorter band than the wild-type band (Figure 4).

[0081] Furthermore, a band with a lower molecular weight than the wild-type band was excised, purified, and subjected to Sanger sequencing, revealing a 2835 base pair deletion (Figure 4), confirming that truncated crRNAs can be used for genome editing in human cells, even with Type IC CRISPR.

[0082] [Example 4] Measurement of genome cleavage activity in human cultured cells HEK293T using Type ID short form crRNA To verify whether a similar phenomenon occurs with other Type I CRISPRs, we used the Type I-D system of Microcystis aeruginosa to examine whether truncated crRNAs introduce mutations into the human EMX1 gene.

[0083] Plasmids expressing pre-crRNA (SEQ ID NO: 17) and truncated crRNA (SEQ ID NO: 18) were prepared and transfected into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) along with plasmids expressing six Cas (3, 5, 6, 7, 10, and 11) effectors and a puromycin resistance gene. Cells were cultured in medium containing 1 μg / ml puromycin at 37°C and 5% CO2 for 2 days. Whole cells were harvested, and the genome was extracted. PCR was performed using a primer set amplifying a 3.8 kb fragment containing the PAM region of the EMX gene. Electrophoresis of the PCR product confirmed that the truncated crRNA, like the pre-crRNA, produced a shorter band than the wild-type band (Figure 5).

[0084] Furthermore, PCR product bands with molecular weights smaller than the wild-type band were excised, purified, and subjected to Sanger sequencing, revealing deletions of 3131 and 2593 base pairs (Figure 5), confirming that Type ID CRISPR truncated crRNAs can be used for genome editing in human cells.

[0085] Example 5: Verification of prime editing by modified crRNA with donor sequence In the type IE CRISPR-Cas3 system, we verified whether the HiBiT sequence could be inserted specifically into the target using a truncated crRNA (modified crRNA) with a 33-base HiBiT sequence and MS2 sequence added to the 3' end.

[0086] We selected the termination codon of the GFP gene as the target and prepared plasmids encoding a modified pre-crRNA and six Cas(3,5-8,11) effectors. Here, we used wild-type Cas3 or its mutant (dh2Cas3, a double mutant with S483A / T485A in SF2 domain motif 3). 293T cells were lipofected with plasmids encoding the modified pre-crRNA, a CAG-GFP expression plasmid, and an MCP-Reverse Transcriptase expression plasmid, and then cultured and passaged. After 3 days, cells were lysed and luciferase activity was measured using the Nano Glo HiBiT Lytic Detection System (Figure 6). The results showed increased luciferase activity when helicase-deficient Cas3 was used, suggesting that HiBiT had been inserted into the GFP site (bottom right graph in Figure 6).

[0087] [Example 6] Verification of GFP accumulation by modified crRNA with MS2 tandem sequence In the type IE CRISPR-Cas3 system, we verified whether target-specific insertion was possible using a truncated crRNA (modified crRNA) with an MS2 tandem sequence added to the 3' end.

[0088] We selected the termination codon of the GFP gene as the target and prepared a plasmid encoding a modified pre-crRNA and five Cas(5-8,11) effectors. 293T cells were lipofected with the modified pre-crRNA and the MCP-EGFP (StayGold) expression plasmid, and then cultured and passaged. After two days, cells were observed under a fluorescence microscope. GFP accumulation was observed when the modified crRNA was used, suggesting that the target sequence could be imaged (Figure 7). [Industrial Applicability]

[0089] The type 1 CRISPR-Cas system using the truncated crRNA of the present invention can utilize guide RNAs that have been modified in various ways, such as by adding tags, making it suitable for use in a wide range of fields, including genome editing in animals and plants, as well as diagnosis through nucleic acid detection.

Claims

1. A guide RNA for a Type I CRISPR-Cas system, (a) a repeat sequence cleaved by a Cas6 protein or a Cas5 protein is arranged on the 5' side of the spacer sequence, and an arbitrary sequence other than the repeat sequence cleaved by a Cas6 protein or a Cas5 protein is arranged on the 3' side of the spacer sequence; (b) the spacer sequence is a sequence complementary to a target DNA in a eukaryotic cell; and (c) A guide RNA that, when present together with a Cas3 protein and a Cascade protein in a eukaryotic cell, forms a complex comprising the guide RNA, the Cas3 protein, and the Cascade protein via cleavage of a repeat sequence located on the 5' side of the spacer sequence by a Cas6 protein or a Cas5 protein.

2. DNA encoding the guide RNA of claim 1.

3. An expression vector comprising the DNA of claim 2.

4. A type I CRISPR-Cas system comprising the guide RNA of claim 1.

5. 10. A method for producing a sample in which target DNA has been edited, comprising contacting the CRISPR-Cas system of claim 4 with a sample containing target DNA, wherein the sample is a eukaryotic cell (excluding cells in a human individual, human germ cells, and human embryonic cells).

6. 10. A method for detecting target DNA in a sample, comprising contacting the CRISPR-Cas system of claim 4 with the sample, wherein the sample is a eukaryotic cell (excluding cells in a human individual).

Images