Guide RNA and use thereof

Abstract

The present invention pertains to: 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.

Description

Guide RNA and its uses

[0001] The present invention relates to a novel form of guide RNA used in a Type I CRISPR-Cas system and its use.

[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 improving the breeding of crops and livestock, regenerative medicine, and gene therapy.

[0003] The CRISPR-Cas systems found in bacteria and archaea are divided into Class 1, which cleaves the target sequence using a complex of multiple proteins, and Class 2, which cleaves the target sequence using 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 human cultured 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.

[0005] International Publication No. 2018 / 225858

[0006] The CRISPR-Cas3 system described in the examples of Patent Document 1 uses a pre-crRNA (precursor crRNA) having full-length repeat sequences at both ends, which causes pre-crRNA processing in eukaryotic cells and successfully 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 has been made in light of these circumstances, and its purpose is to clarify the detailed structure of guide RNA necessary for the functioning of a type I CRISPR-Cas system in eukaryotic cells, and to establish a type I CRISPR-Cas system that utilizes a novel form of guide RNA.

[0008] As a result of extensive research conducted by the present inventors to achieve the above-mentioned object, they have found that, in order for the Type I CRISPR-Cas system to function as a complex, the 5' repeat sequence of the guide RNA needs to be cleaved by processing, whereas processing of the 3' repeat sequence is not necessary, 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, and nucleic acid detection and DNA editing were possible.

[0010] Furthermore, the present inventors have found that this mechanism is not limited to a particular subtype but is common to Type I CRISPR-Cas systems, and have completed 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 arranged on the 5' side of the spacer sequence, and either no repeat sequence is arranged on the 3' side of the spacer sequence or a sequence that is not cleaved by processing to form a cascade complex is arranged.

[0013] (2) 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 according to (1).

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

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

[0021] 1 shows the results of an evaluation of in vitro cleavage activity (CONAN assay) using a Type I-E CRISPR-Cas system utilizing various forms of crRNA (pre-crRNA, crRNA with a mature 5'-end repeat sequence or a mature 3'-end repeat sequence, and mature crRNA). 2 shows the results of an evaluation of in vitro cleavage activity (CONAN assay) using a Type I-E CRISPR-Cas system utilizing a truncated crRNA lacking the 3'-end repeat sequence. 3'-end repeat sequence mature crRNA was used as a positive control. 4 shows the results of an evaluation of genome editing activity using a Type I-E CRISPR-Cas system utilizing a truncated crRNA lacking the 3'-end repeat sequence. 5 shows the results of an evaluation of genome editing activity using a Type I-C CRISPR-Cas system utilizing a truncated crRNA lacking the 3'-end repeat sequence. 6 shows the results of an evaluation of genome editing activity using a Type I-C CRISPR-Cas system utilizing a truncated crRNA lacking the 3'-end repeat sequence. Pre-crRNA was used as a positive control. This figure shows the results of evaluating genome editing activity by the Type I-D CRISPR-Cas system, which utilizes a truncated crRNA lacking the 3' repeat sequence. Pre-crRNA was used as a positive control. This figure shows an overview of prime editing by the Type I-E CRISPR-Cas system, which utilizes a truncated crRNA lacking the 3' repeat sequence, and the results thereof. This figure shows the results of target DNA detection by the Type I-E CRISPR-Cas system, which utilizes a truncated crRNA lacking the 3' repeat sequence.

[0022] <Guide RNA and Type I CRISPR-Cas System Comprising the Guide RNA> 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 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) Unraveling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology, Vol. 12 (No. 7), pp. 479-492; Jackson RN et al. (2014) Fitting CRISPR-associated Cas3 into 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 a Type I CRISPR-Cas system is used 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 a 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, in the Type IA CRISPR-Cas system derived from Pyrococcus furiosus, it typically consists of the 30-base sequence set forth in SEQ ID NO: 1, and in Synechocystis sp. The type I-B CRISPR-Cas system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 2, which has a chain length of 36 bases; the type I-C CRISPR-Cas system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 3, which has a chain length of 32 bases; the type I-D CRISPR-Cas system derived from Microcystis aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 4, which has a chain length of 37 bases; the type I-E 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 I-F CRISPR-Cas system derived from Pseudomonas aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 6, which has a chain length of 28 bases; and the type I-G CRISPR-Cas system derived from Thioalkalivibrio sulfidiphilus typically consists of the nucleotide sequence set forth in SEQ ID NO: In the CRISPR-Cas system, it typically consists of the base sequence set forth in SEQ ID NO: 7, which has a chain length of 36 bases.

[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 so that such cleavage does not occur. In a typical processing process, the repeat sequence forms a loop structure, and this loop structure 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 I-A, between the 28th and 29th bases of the repeat sequence in Type I-B, between the 19th and 20th bases of the repeat sequence in Type I-C, between the 31st and 32nd bases of the repeat sequence in Type I-D, between the 21st and 22nd bases of the repeat sequence in Type I-E, between the 20th and 21st bases of the repeat sequence in Type I-F, and between the 28th and 29th bases of the repeat sequence in Type I-G. 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 no cleavage due to processing occurs on the 3' side of the spacer sequence. Modifications are not particularly limited, and examples include 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 the RNA aptamer sequence, recognition protein, and recognition compound, please refer to the description in the section "Method for detecting target DNA" below.

[0031] The "spacer sequence" in the present invention is a sequence designed as a sequence complementary to the target DNA. The target DNA may be endogenous DNA 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, etc. that infect hosts.

[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 the expression vector of the guide RNA. The type of expression vector is not particularly limited, and a vector capable of expressing the guide RNA in its usage 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.). The expression vector may contain a promoter sequence for inducing transcription or a sequence for enhancing transcription (e.g., an enhancer sequence).

[0034] Expression vectors can be prepared by known techniques. These techniques include those described in the operating manuals included with 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] The Class 1 Type I CRISPR-Cas system containing the guide RNA of the present invention typically contains a Cas3 protein and a Cascade protein as components other than the guide RNA. However, when the purpose is not to cleave the target DNA (for example, when binding to an expression control region to suppress transcription), it can also be used as a system that does not contain a component having nuclease activity (typically, a Cas3 protein, but in Type I-D, Cas10d).

[0036] Typically, type I-A includes Cas3-HD, Cas3-HEL, Cas5, Cas7, Cas8, and Cas11 as cascade proteins; type I-B includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 as cascade proteins; type I-C includes Cas3, Cas5, Cas7, Cas8, and Cas11 as cascade proteins; and type I-D includes C as cascade proteins. Type I-E includes Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11 as cascade proteins; Type I-F includes Cas2-3, Cas5, Cas6, Cas7, and Cas8 as cascade proteins; and Type I-G includes 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 I-B and Type I-C can exhibit DNA editing activity even when they do not contain Cas11, albeit at a lower level compared to when they contain Cas11, and the inventors have confirmed that this is also true for Type I-D). Thus, the Type I CRISPR-Cas system of the present invention also includes systems that do not contain Cas11.

[0037] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add a nuclear localization signal to promote the localization of the Cas3 protein or Cascade protein to the nucleus. 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 and 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 loaded into one (same) vector, or all or part of them can be loaded into separate vectors. Furthermore, the DNA encoding each protein can be expressed as a single protein by linking it via DNA encoding an amino acid sequence (such as 2A peptide) that is cleaved by intracellular protease, and then separated into each protein by the action of the protease for use. Expression vectors can be prepared by known techniques, as in the case of the guide RNA described above.

[0040] <Method for producing a sample in which target DNA has been edited> The present invention also provides a method for producing a sample in which target DNA has been edited, which comprises 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 aspects of DNA editing is the cleavage of target DNA by a component having nuclease activity in the Type I CRISPR-Cas system (typically the Cas3 protein, but in Type I-D, Cas10d), followed by the introduction of mutations via DNA repair in the cell. As shown in the Examples of the present application, this aspect also makes it possible to generate large deletions in the target DNA.

[0043] In another embodiment of DNA editing, the Cas3 protein or Cascade protein can be fused to a heterologous protein having the desired activity to form a chimeric protein, depending on the purpose. In this embodiment, various editing operations can be performed on the target DNA depending on the activity of the heterologous protein to be fused. Examples of the activities of the heterologous protein to be fused include, but are not limited to, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity (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 in which the nuclease activity of the Cas3 protein (in the case of type ID, the Cas10d protein) is partially or completely deleted can be used.

[0044] Examples of Cas3 protein mutants include, for example, in Type I-E, a mutant of HD domain H74A (dnCas3), a mutant of K320N in SF2 domain motif 1 (dhCas3), and a double mutant of S483A / T485A in SF2 domain motif 3 (dh2Cas3). For example, by using a fusion protein of a mutant in which part or all of the nuclease activity of Cas3 has been eliminated and a deaminase as a component of the Type I CRISPR-Cas system of the present invention, precise genome editing can be performed by substituting bases without causing deletions at the target site. Techniques for applying deaminase to the CRISPR-Cas system 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 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, too, a mutant lacking part or all of the nuclease activity of the Cas3 protein (or the Cas10d protein in the case of Type I-D) can be used. Techniques for applying transcriptional regulatory proteins to the CRISPR-Cas system are 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 in which target DNA is edited 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 individual animals, cells constituting organs and tissues removed from animals, and cultured cells derived from animal tissues. Specific examples include germ cells such as oocytes and sperm; embryonic cells at various stages of embryonic development (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, hepatocytes, 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. Particularly preferred are pronuclear stage fertilized eggs. Oocytes can be thawed from 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 produce non-human individuals from cells. When producing non-human individuals from cells in animals, germ cells or pluripotent stem cells are usually 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. If necessary, oocytes can be cultured under appropriate conditions until implantation. The transplantation and culture of oocytes can be carried out according to known techniques (Nagy A. et al., Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2003). From the obtained non-human individual, offspring or clones in which the desired DNA has been edited 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 isolated 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 technical field 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 method, liposome method, DEAE-dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, transduction, and infection with a viral vector. Such 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, which comprises 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 on the 3' side of the spacer sequence, the target DNA can be detected by using a guide RNA labeled on the 3' side.

[0058] The label is not particularly limited as long as it is detectable, and examples thereof include RNA aptamer sequences / recognition proteins such as MS2 / MCP, PP7 / PCP, boxB / λN22P, and Pepper / tDeg, and RNA aptamer sequences / recognition compounds such as MS2 / MCP, PP7 / PCP, boxB / λN22P, and Pepper / tDeg. When using an RNA aptamer sequence / recognition protein, 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, thereby detecting the target DNA using fluorescence as an indicator (Yang LZ et al., Cell Insight 1 (2022) 100044). Furthermore, when an RNA aptamer sequence / recognition compound is used, the RNA aptamer sequence is added to a guide RNA, and a recognition compound is bound to the RNA aptamer sequence, whereby the target DNA can be detected using color development as an indicator.

[0059] In these detections, in order to avoid degradation of the target DNA by the nuclease activity of the Type I CRISPR-Cas system, it is preferable to use a system in which the nuclease activity has been eliminated, for example, a system that utilizes a Cas3 protein in which the nuclease activity has been eliminated (Cas10d in Type I-D), or a system in which the Cas3 protein (Cas10d in Type I-D) has been excluded.

[0060] Another aspect 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 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 bound 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, target DNA in a test sample can be detected by using as an indicator 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.

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

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

[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 at various crRNA lengths. This detection system utilized the Escherichia coli-derived Type I-E CRISPR. Cas3 protein was expressed and purified using insect cells Sf9. Specifically, a His-tagged Cas3 gene with a nuclear localization signal (bpNLS) was incorporated into a baculovirus gene expression vector, and then baculovirus was produced using the Escherichia coli strain DH10bac. The produced baculovirus was infected into insect cells Sf9 to express the Cas3 protein. After that, the total protein was collected and the Cas3 protein was purified using a nickel column and gel filtration.

[0069] Cascade components were also expressed and purified using insect cells Sf9. Specifically, a baculovirus gene expression vector was created in which the genes for Cas5, Cas6, Cas7, Cas8, and Cas11, each with a His-tagged nuclear localization signal (bpNLS) attached, were linked via a 2A peptide sequence. Proteins were expressed in the same manner as above, and the Cas3 protein was purified by nickel column and gel filtration.

[0070] The crRNAs were obtained by synthesizing double-stranded DNA fragments encoding each gene, transcribing them in vitro, and then column purifying them. In this experiment, pre-crRNA (SEQ ID NO: 8), mature crRNA (SEQ ID NO: 9), 5' mature crRNA (crRNA with only the 5' repeat sequence as mature), and 3' mature crRNA (crRNA with only the 3' repeat sequence as mature) were used.

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

[0072] First, equal amounts of cascade factors (0.8 μg / μL) and crRNA (250 ng / μL) were mixed and then incubated in a complex formation buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl ２ , 10 μM CoCl ２ The mixture was incubated at 37°C for 10 minutes in reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl) to allow the formation of the cascade complex. ２ , 10 μM CoCl ２Cas3 protein (final concentration 40 ng / μL), Cascade complex (final concentration 32 ng / μL), double-stranded DNA fragment (final concentration 5 nM) containing the target sequence (mouse Tyr gene / SEQ ID NO: 11), and fluorescent quencher probe (final concentration 1 μM) were mixed in 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). In other words, 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, a fluorescent signal due to collateral cleavage activity was detected even in the truncated crRNA (SEQ ID NO: 12) lacking the 3' repeat sequence (Figure 2). In other words, the 21 bases remaining on the 3' side during maturation were not essential for CRISPR-Cas3 complex formation and activation.

[0075] These results demonstrate that when cascade factors and crRNA form a complex, the 5' repeat sequence is essential, while 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 I-E truncated crRNA Purified human EMX1 target crRNA (truncated; 20 ng / μL) was introduced into K562 cells expressing Cas3 and cascade components (Cas5, Cas6, Cas7, Cas8, Cas11) in a doxycycline-inducible manner by electroporation using a 4D-Nucleofector System (Lonza). The cells were placed in medium supplemented with 2 μg / ml doxycycline and incubated at 37°C, 5% CO ２ After culturing for 2 days at RT, all cells were collected, the genome was extracted, and PCR was performed using a primer set that amplifies a 3.8 kb fragment containing the PAM region of the EMX 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 applicability of truncated crRNA for genome editing in human cells.

[0079] Example 3 Measurement of genome cleavage activity in human cultured cells HEK293T using Type I-C 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 crRNA introduces 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) together with plasmids expressing five Cas (3, 5, 7, 8, 11) effectors and a puromycin resistance gene. The cells were placed in medium supplemented with 1 μg / ml of puromycin and incubated at 37°C, 5% CO ２ After culturing for 2 days at RT, all cells were harvested, the genome was extracted, and PCR was performed using a primer set amplifying a 3.8-kb segment containing the PAM region of the EMX gene. Electrophoresis of the PCR product confirmed that the truncated crRNA, like the pre-crRNA, formed a band shorter than the wild-type band (Figure 4).

[0081] Furthermore, a band lower in 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 Type I-C CRISPR truncated crRNA can also be used for genome editing in human cells.

[0082] Example 4 Measurement of genome cleavage activity in human cultured cells HEK293T using Type I-D 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 crRNA introduces 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 these were transfected into HEK293T cells together with plasmids expressing six Cas (3, 5, 6, 7, 10, 11) effectors and a puromycin resistance gene using Lipofectamine 2000 (Thermo Fisher Scientific). The cells were placed in medium supplemented with 1 μg / ml puromycin and incubated at 37°C, 5% CO ２After culturing for 2 days at RT, all cells were harvested, and the genome was extracted. PCR was performed using a primer set amplifying a 3.8-kb segment containing the PAM region of the EMX gene. Electrophoresis of the PCR product confirmed that the truncated crRNA, like the pre-crRNA, formed a band shorter 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). This confirms that truncated crRNAs can be used for genome editing in human cells, even with Type I-D CRISPR.

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

[0086] The termination codon of the GFP gene was selected as the target, and a plasmid encoding a modified pre-crRNA and six Cas(3,5-8,11) effectors was prepared. The Cas3 used here was either wild-type or a mutant (a double mutant of S483A / T485A in SF2 domain motif 3 (dh2Cas3)). 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, the cells were lysed, and luciferase activity was measured using the Nano Glo HiBiT Lytic Detection System (Figure 6). As a result, an increase in luciferase activity was observed when helicase-deficient Cas3 was used, suggesting that Hibit had been inserted into the GFP site (graph at the bottom right of Figure 6).

[0087] Example 6 Verification of GFP accumulation by modified crRNA with MS2 tandem sequence In the Type I-E CRISPR-Cas3 system, we verified whether a truncated crRNA (modified crRNA) with an MS2 tandem sequence added to the 3' end could be inserted in a target-specific manner.

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

[0089] The Type I 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 a label, making it suitable for use in a wide range of fields, including genome editing of animals and plants, as well as diagnosis by nucleic acid detection.

Claims

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

2. DNA encoding the guide RNA described in 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. A method for producing a sample in which a target DNA has been edited, comprising contacting the CRISPR-Cas system described in claim 4 with a sample containing the target DNA.

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

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

8. A method for detecting target DNA in a sample, comprising contacting the sample with the CRISPR-Cas system described in claim 4.

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