Guiding RNA and use thereof

Abstract

The present invention relates to a guide RNA in which a repetitive sequence cleaved by a process for forming a cascade reaction complex is disposed on the 5'side of a spacer sequence, and a repetitive sequence is not disposed on the 3 'side of the spacer sequence, or a sequence cleaved not by a process for forming a cascade reaction complex is disposed, and a type I CRISPR-Cas system using the guide RNA.

Description

Technical Field

[0001] This invention relates to novel forms of guide RNA used in type I CRISPR-Cas systems and their utilization. Background Technology

[0002] Genome editing technology is a technique that specifically cuts the genomic DNA sequence in animal or plant cells, utilizing their inherent repair mechanisms to freely rewrite it into any sequence. Its applications worldwide extend beyond biological science research to include crop and livestock breeding, regenerative medicine, and gene therapy.

[0003] The CRISPR-Cas systems possessed by bacteria and archaea are divided into two categories: one that cleaves target sequences through a complex of multiple proteins, and another that cleaves target sequences through a single protein. To date, CRISPR-Cas9, CRISPR-Cas12 (Cpf1), and CRISPR-Cas13, developed as genome editing tools, are all classified into category two.

[0004] On the other hand, it has recently been discovered that CRISPR-Cas3, belonging to type I CRISPR (Patent Document 1), can be used as a genome editing tool for eukaryotic cells. It has been determined that this CRISPR-Cas3 system can efficiently introduce large-scale deletion mutations of hundreds to several kb around the target sequence in eukaryotic cells such as human cultured cells. Furthermore, compared to CRISPR-Cas9, it is considered to have higher safety because the target recognition sequence in the guide RNA (crRNA) is longer and less prone to non-specific cleavage.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2018 / 225858 Summary of the Invention

[0008] The problem that the invention aims to solve

[0009] In the CRISPR-Cas3 system described in the embodiments of Patent Document 1 above, a precursor crRNA with full-length repetitive sequences at both ends is used, thereby successfully enabling the processing of the precursor crRNA in eukaryotic cells to form a functional genome editing complex. However, the processing of the precursor crRNA has not been fully studied, and the structure of the guide RNA required for the formation of the functional genome editing complex has not been fully elucidated.

[0010] The present invention is made in view of the following situation, and its purpose is to clarify the detailed structure of the guide RNA required for the function of the type I CRISPR-Cas system in eukaryotic cells, and to establish a type I CRISPR-Cas system utilizing a novel form of guide RNA.

[0011] Methods for solving problems

[0012] To achieve the above objectives, the inventors have conducted repeated and in-depth studies, and the results have clarified that in order for the type I CRISPR-Cas system to function as a complex, the 5' side repeat sequence of the guide RNA needs to be cleaved by processing. On the other hand, the processing of the 3' side repeat sequence is not necessary, and a functional complex can be formed even when using the cleaved repeat sequence produced by processing.

[0013] The inventors further analyzed the mechanism and determined that even when using shortened crRNA that completely eliminates the 3' side repeat sequence, a functional complex is still formed, enabling nucleic acid detection and DNA editing.

[0014] Furthermore, the inventors discovered that this mechanism is not limited to a specific subtype, but is universal in type I CRISPR-Cas systems, thus completing this invention.

[0015] This invention relates to guide RNAs of type I CRISPR-Cas systems and their utilization thereof, which have lost processing sensitivity on the 3' side of the spacer sequence due to deletion or modification of the 3' side repeat sequence, and more specifically includes the following types.

[0016] (1) A type I CRISPR-Cas system with a repeating sequence configured on the 5' side of the spacer sequence that can be cleaved by processing for forming a cascade reaction complex, and with no repeating sequence configured on the 3' side of the spacer sequence, or with a sequence that is not cleaved by processing for forming a cascade reaction complex.

[0017] (2) DNA encoding the guide RNA described in (1).

[0018] (3) An expression vector containing the DNA described in (2).

[0019] (4) A type I CRISPR-Cas system containing the guide RNA described in (1).

[0020] (5) A method for manufacturing a sample in which the target DNA has been edited, comprising contacting the CRISPR-Cas system described in (4) with the sample containing the target DNA.

[0021] (6) According to the method described in (5), wherein the sample is a cell.

[0022] (7) According to the method described in (6), the cell is a eukaryotic cell.

[0023] (8) A method for detecting target DNA in a sample, comprising contacting the CRISPR-Cas system described in (4) with the sample.

[0024] The effects of the invention

[0025] This invention clarifies that in a type I CRISPR-Cas system, the system functions even after the 3' side repeat sequence is deleted or modified. Consequently, various applications become possible, such as nucleic acid detection / epigenome editing utilizing a combination of guide RNA with an added RNA aptamer sequence and an RNA aptamer recognition molecule; knock-in of guide RNA utilizing a added donor sequence; and lead editing of guide RNA utilizing a sequence with added primer binding sites and a template sequence of reverse transcriptase. Attached Figure Description

[0026] Figure 1 This is a graph showing the results of the evaluation (CONAN assay) of in vitro cleavage activity generated by the IE-type CRISPR-Cas system utilizing various forms of crRNA (precursor crRNA, crRNA with 5' side repeats or 3' side repeats as mature forms, and mature crRNA).

[0027] Figure 2 This is a graph showing the results of an evaluation (CONAN assay) of the in vitro cleavage activity generated by an IE-type CRISPR-Cas system utilizing a shortened crRNA with a missing 3' side repeat sequence. As a positive control, crRNA with a mature 3' side repeat sequence was used.

[0028] Figure 3 This is a graph showing the results of an evaluation of genome editing activity generated by an IE-type CRISPR-Cas system utilizing shortened crRNA with a missing 3' side repeat sequence. Precursor crRNA was used as a positive control.

[0029] Figure 4 This is a graph showing the results of an evaluation of genome editing activity generated by an IC-type CRISPR-Cas system utilizing shortened crRNA with a missing 3' side repeat sequence. Precursor crRNA was used as a positive control.

[0030] Figure 5This is a graph showing the results of an evaluation of genome editing activity generated by an ID-type CRISPR-Cas system utilizing shortened crRNA with a missing 3' side repeat sequence. Precursor crRNA was used as a positive control.

[0031] Figure 6 This is a diagram showing the outline and results of the lead editing produced by the IE-type CRISPR-Cas system, which utilizes a shortened crRNA with a missing 3' side repeat sequence.

[0032] Figure 7 This is a graph showing the results of detection of target DNA generated by an IE-type CRISPR-Cas system that utilizes a shortened crRNA with a missing 3' side repeat sequence. Detailed Implementation

[0033] <Guide RNA, and the type I CRISPR-Cas system containing the guide RNA>

[0034] This invention provides a novel form of guide RNA in the type I CRISPR-Cas system.

[0035] Class 1 CRISPR-Cas systems are classified into types I and III. Type I is further classified into six types: IA, IB, IC, ID, IE, and IF, as well as type IG, which is a subtype of type IB (e.g., see [van der Oost J et al. (2014) Unravelling 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]).

[0036] In a type I CRISPR-Cas system, the guide RNA forms a complex with the Cas3 protein and the cascade protein, which acts on the target DNA. In the use of a type I CRISPR-Cas system in eukaryotic cells, the guide RNA has traditionally been a precursor crRNA (crRNA with repetitive sequences cleaved by processing flanking the spacer sequence) (Patent Document 1 above). However, the inventors have discovered that cleavage of the repetitive sequences on the 3' side of the spacer sequence is not essential in the processing (hereinafter, sometimes simply referred to as the "processing") for forming the cascade complex.

[0037] Therefore, the guide RNA of the present invention is characterized by having a repeating sequence that is cleaved during processing on the 5' side of the spacer sequence, and having no repeating sequence or a sequence that is not cleaved during processing on the 3' side of the spacer sequence. That is, the guide RNA of the present invention typically has a structure of "repeat sequence cleaved during processing - spacer sequence" or "repeat sequence cleaved during processing - spacer sequence - sequence not cleaved during processing".

[0038] In this invention, "repetitive sequences" are sequences that appear repeatedly in the CRISPR structure of bacterial genomes derived from type I CRISPR-Cas lines, mediated by spacer sequences.

[0039] The wild-type repetitive sequences vary depending on the subtype of the type I CRISPR-Cas system and the species of bacteria from which they originate. For example, in the type IA CRISPR-Cas system derived from *Pyrococcus furiosus*, they typically consist of the sequence described in sequence number 1, which has a chain length of 30 bases. In the type IB CRISPR-Cas system derived from *Synechocystis* sp., they typically consist of the sequence described in sequence number 2, which has a chain length of 36 bases. In *Neisseria lactosa*... In the CRISPR-Cas system of type IC derived from *Lactamica*, the sequence typically consists of the sequence described in sequence number 3, which has a chain length of 32 bases. In the CRISPR-Cas system of type ID derived from *Microcystis aeruginosa*, the sequence typically consists of the sequence described in sequence number 4, which has a chain length of 37 bases. In the CRISPR-Cas system of type IE derived from *Escherichia coli*, the sequence typically consists of the sequence described in sequence number 5, which has a chain length of 29 bases. In the CRISPR-Cas system of type IF derived from *Pseudomonas aeruginosa*, the sequence typically consists of the sequence described in sequence number 6, which has a chain length of 28 bases. In the CRISPR-Cas system of type IG derived from *Thioalkalivibrio sulfidiphilus*, the sequence typically consists of the sequence described in sequence number 7, which has a chain length of 36 bases.

[0040] In this invention, the repeat sequences on the 5' side of the spacer sequence (hereinafter, sometimes simply referred to as "5' side repeat sequences") are typically wild-type repeat sequences, but mutations (addition, deletion, substitution, and / or insertion of bases) can exist as long as they are cleaved during processing in the same way as wild-type repeat sequences. On the other hand, the repeat sequences on the 3' side of the spacer sequence (hereinafter, sometimes simply referred to as "3' side repeat sequences") were discovered by the inventors to be cleaved during processing. Therefore, the guide RNA of this invention has the characteristic that no repeat sequences that are cleaved during processing are placed on the 3' side of the spacer sequence. The 3' side of the spacer sequence may not have repeat sequences, or sequences that are not cleaved during processing may be added.

[0041] The sequence not cleaved during processing can be any sequence unrelated to the processing, or it can be a repeating sequence modified in a way that prevents cleavage. In typical processing, the repeating sequence forms a loop structure, which is cleaved by a specific Cas (e.g., Cas6, Cas5, etc.). The cleavage site is typically between the 22nd and 23rd bases of the repeating sequence in type IA, between the 28th and 29th bases in type IB, between the 19th and 20th bases in type IC, between the 31st and 32nd bases in type ID, between the 21st and 22nd bases in type IE, between the 20th and 21st bases in type IF, and between the 28th and 29th bases in type IG. Therefore, examples of modified repetitive sequences include, for instance, repetitive sequences that have been shortened to exclude the cleavage site, and repetitive sequences in which the bases surrounding the cleavage site have been modified.

[0042] The guide RNA of this invention is not cleaved at the 3' side of the spacer sequence, thus making it suitable for various modifications at the 3' side. Modifications are not particularly limited, but examples include markers, tags, donor sequences, and lead editing sequences (sequences of primer binding sites and template sequences for reverse transcriptase). When using this invention for lead editing, as shown in the embodiments of this application, the lead editing sequence may be further supplemented with an RNA aptamer sequence. Furthermore, in this case, the reverse transcriptase may be fused with a recognition protein or bound with a recognition compound. For further information on RNA aptamer sequences, recognition proteins, and recognition compounds, please refer to the description in "Methods for Detecting Target DNA" described later.

[0043] The "spacer sequence" in this invention is a sequence designed as a complement to the target DNA. The target DNA can be either endogenous or exogenous DNA. Examples of endogenous DNA include genomic DNA from chromosomes, mitochondria, and chloroplasts. Examples of exogenous DNA include reporter genes, biomarker genes, and genes from viruses, bacteria, protozoa, etc., that infect a host.

[0044] The guide RNA of this invention can be used as artificially synthesized RNA, or as RNA expressed from DNA.

[0045] The expression vector for guide RNA serves as the base vector, and various commonly used vectors can be used. The type of expression vector is not particularly limited; a vector suitable for expressing guide RNA in its environment (e.g., host cells) can be selected. Examples of expression vectors include, for instance, plasmid vectors, phage vectors, viral vectors, retroviral vectors, chromosome vectors, episome vectors, and virus-derived vectors (bacterial plasmids, phages, yeast episomes, etc.), yeast chromosome elements and viruses (baculoviruses, papillomaviruses, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, herpesviruses, lentiviruses, retroviruses, etc.), and vectors derived from combinations thereof (plastic particles, phage particles, etc.). Expression vectors may also contain promoter sequences for inducing transcription and sequences for enhancing transcription (e.g., enhancer sequences).

[0046] Expression vectors can be prepared using well-known methods. In addition to the methods described in the instruction manual accompanying the vector preparation kit, various guidelines can be cited as examples. For instance, [Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001] is a general guideline.

[0047] The CRISPR-Cas system of type I, which includes the guide RNA of the present invention, typically contains a Cas3 protein and a cascade reaction protein as components other than the guide RNA. However, it can also be used as a system that does not contain components with nuclease activity (typically a Cas3 protein, but Cas10d in type I) when the purpose is not to cleave the target DNA (e.g., to inhibit transcription by binding to the expression control region).

[0048] Typically, in type IA, the cascade reaction proteins include Cas3-HD, Cas3-HEL, Cas5, Cas7, Cas8, and Cas11; in type IB, the cascade reaction proteins include Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; in type IC, the cascade reaction proteins include Cas3, Cas5, Cas7, Cas8, and Cas11; in type ID, the cascade reaction proteins include Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; in type IE, the cascade reaction proteins include Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; in type IF, the cascade reaction proteins include Cas2-3, Cas5, Cas6, Cas7, and Cas8; and in type IG, the cascade reaction proteins include Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11. However, regarding Cas11, it should be understood that DNA editing activity can still be exhibited even when it is excluded from the components of a type I CRISPR-Cas system (for example, it is known that in types IB and IC, DNA editing activity can be exhibited even at a lower level compared to the case containing Cas11, and the inventors have confirmed that the same is true in type ID). Therefore, the type I CRISPR-Cas system of the present invention also includes systems that do not contain Cas11.

[0049] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add nuclear transfer signals to promote the localization of Cas3 proteins and cascade reaction proteins to the nucleus. These nuclear transfer signals can be added to the N-terminus and / or C-terminus of each protein. Similarly, for example, when targeting the mitochondrial genome and chloroplast genome, it is preferable to add transfer signals that promote their localization.

[0050] In the type I CRISPR-Cas system of this invention, Cas3 and the cascade reaction can be in the form of a protein, a polynucleotide (DNA, RNA) encoding the protein, or a vector expressing the protein. In the form of a polynucleotide, modifications to the base sequence suitable for expression in the host cell can also be performed (e.g., codon optimization).

[0051] In the construction of expression vectors, the DNA encoding Cas3 proteins and cascade reaction proteins can be designed to be carried in a single vector, or all or part of it can be carried in separate vectors. Alternatively, the DNA encoding each protein can be linked to DNA encoding an amino acid sequence (such as a 2A peptide) that will be cleaved by intracellular proteases, thereby expressing it as a protein. The protein can then be separated into individual proteins by the action of proteases for use. Expression vectors can be prepared using known methods, similar to those used for guide RNA.

[0052] <Methods for manufacturing samples with edited target DNA>

[0053] The present invention further provides a method for manufacturing a sample in which the target DNA has been edited, comprising contacting a CRISPR-Cas system containing the above-described guide RNA with a sample containing the target DNA.

[0054] The "DNA editing" in this invention includes DNA cutting at target sites, introduction of DNA mutations (base deletion, insertion, substitution), modification of DNA bases, control of DNA expression, and operations consisting of combinations thereof. DNA editing can be performed in vitro or in vivo, depending on the purpose. Additionally, it can be performed intracellularly or in cell-free systems.

[0055] One of the main modes of DNA editing involves the cleavage of target DNA by means of a component with nuclease activity in a type I CRISPR-Cas system (typically the Cas3 protein, but in type ID, Cas10d), followed by the introduction of mutations for intracellular DNA repair. As illustrated in the embodiments of this application, in this mode, large-scale deletions of the target DNA can also be induced.

[0056] In other forms of DNA editing, depending on the purpose, the Cas3 protein or cascade reaction protein can be fused with a heterologous protein having the desired activity, and used as a chimeric protein. In this form, various types of editing can be performed on the target DNA depending on the activity of the fused heterologous protein. The activities of the fused heterologous protein include, for example, 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 in the case of using the present invention for lead editing), ligase activity, photoreactivation activity, and glycosylation activity, but are not limited to these. In this case, a mutant in which some or all of the nuclease activity of the Cas3 protein (Cas10d protein in the case of ID type) is missing can be used.

[0057] As mutants of the Cas3 protein, for example in type IE, mutants of the HD domain H74A (dnCas3), the SF2 domain motif 1 K320N mutant (dhCas3), and the SF2 domain motif 3 S483A / T485A double mutant (dh2Cas3) can be used. For example, by using a mutant that causes partial or complete loss of Cas3 nuclease activity as a fusion protein with a deaminase as a component of the type I CRISPR-Cas system of the present invention, base substitutions can be performed without deletion at the target site, thereby enabling precise genome editing. The method of applying deaminases to the CRISPR-Cas system is well known (Nishida K. et al., Targeted nucleotide editing using hybridprokaryotic and vertebrate adaptive immune systems, Science, DOI: 10. 1126 / science. aaf8729, (2016)), and can be applied in the type I CRISPR-Cas system of the present invention.

[0058] Furthermore, by fusing Cas3 proteins or cascade reaction proteins with desired transcriptional regulatory proteins to utilize them as chimeric proteins, transcription of genes at target sites can be regulated. Examples of transcriptional regulatory proteins include, but are not limited to, photoinducible transcriptional regulators, small molecule / drug-responsive transcriptional regulators, transcription factors, and transcriptional repressors. In this case, mutants that partially or completely lack the nuclease activity of the Cas3 protein (Cas10d protein in the ID type) can also be used. Methods for applying transcriptional regulatory proteins to the CRISPR-Cas system are well known to those skilled in the art.

[0059] In this invention, by adding donor DNA as a component of a type I CRISPR-Cas system, a desired base sequence can be knocked into the target DNA region. The donor DNA typically has homologous arms on both sides of the desired base sequence to be knocked in, and the desired base sequence is inserted into the target DNA region through homologous recombination repair mechanisms, etc.

[0060] The "sample" used in target DNA editing is not particularly restricted as long as it contains target DNA. It can be a cellular sample (cells, tissues, or biological organisms) or a cell-free sample.

[0061] Cells that edit target DNA include prokaryotic and eukaryotic cells. Prokaryotic cells include bacteria and archaea, while eukaryotic cells include animal cells, plant cells, algal cells, and fungal cells.

[0062] Examples of animal cells include, for example, mammalian cells, as well as cells from fish, birds, reptiles, amphibians, and insects. Animal cells include, for example, cells that constitute an individual animal, cells that constitute organs / tissues removed from an animal, and cultured cells derived from animal tissues. Specifically, examples include, for example, germ cells such as oocytes and sperm; embryonic cells at various stages of the embryo (e.g., single-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, morula embryos, etc.); stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, brain cells, and kidney cells. As for the oocytes used in the creation of genome-edited animals, oocytes from between fertilization and after fertilization can be used, but oocytes from after fertilization, i.e., zygotes, are preferred. Zygotes from pronuclear embryos are particularly preferred. Oocytes can be used after thawing from cryopreservation.

[0063] Mammals are a concept that includes both humans and non-human mammals. Examples of non-human mammals include even-toed ungulates such as cattle, wild boars, pigs, sheep, and goats; odd-toed ungulates such as horses; rodents such as mice, rats, guinea pigs, hamsters, and squirrels; lagomorphs such as rabbits; and carnivorous animals such as dogs, cats, and ferrets. These non-human mammals can be domesticated animals or companion animals (pets), or they can be wild animals.

[0064] Known methods can be used as a means of creating non-human individuals from cells. In animals, germ cells or pluripotent stem cells are typically used when creating non-human individuals from cells. For example, molecules constituting the type I CRISPR-Cas system of this invention are introduced into oocytes, and the resulting oocytes are then transplanted into the uterus of a non-human female mammal in a pseudopregnant state, resulting in offspring. Transplantation can be performed using fertilized eggs from single-cell, two-cell, four-cell, eight-cell, sixteen-cell, or morula stages. Oocytes can be cultured under appropriate conditions until transplantation, as needed. Oocyte transplantation and culture can be performed based on known methods (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, desired DNA-edited offspring or clones can also be obtained.

[0065] Examples of plant cells include, for example, the cells of cereals, oil crops, forage crops, fruits, and vegetables. Plant cells include, for example, the cells that constitute an individual plant, the cells that constitute organs / tissues separated from a plant, and cultured cells derived from plant tissues. Examples of plant organs / tissues include, for example, leaves, stems, shoot tips (growing points), roots, tubers, and callus tissue. Examples of plants include rice, corn, bananas, peanuts, sunflowers, tomatoes, Arabidopsis thaliana, tobacco, wheat, barley, potatoes, soybeans, cotton, and carnations, as well as their propagation materials (e.g., seeds, tubers, rhizomes, etc.).

[0066] In plants, the totipotency of somatic cells has been known since ancient times, and methods for regenerating plant bodies from plant cells have been established in a wide variety of plants. Therefore, for example, by introducing molecules constituting the type I CRISPR-Cas system of this invention into plant cells, and regenerating plant bodies from the resulting plant cells, it is possible to obtain desired DNA-edited plant bodies. From the obtained plant bodies, desired DNA-edited offspring, clones, or propagation material can also be obtained. As a method for obtaining individuals by redifferentiating plant tissues through tissue culture, methods already established in this art can be utilized (…). (Transformation Procedures [Plant Section]) Edited by Yutaka Tabei / Chemical Writers pp.340-347 (2012)).

[0067] The method for introducing the type I CRISPR-Cas system of this invention into cells is not limited. Examples include electroporation, calcium phosphate method, liposome method, DEAE-dextrose method, microinjection, cationic lipid-mediated transfection, electroporation, transduction, and infection using viral vectors. Such methods are described in several standard laboratory manuals, such as "Leonard G. Davis et al., Basic methods in molecular biology, New York: Elsevier, 1986".

[0068] <Methods for detecting target DNA>

[0069] The present invention further provides a method for detecting target DNA in a sample, comprising contacting a CRISPR-Cas system containing the above-described guide RNA with the sample.

[0070] As the "sample" in the detection method of the present invention, the desired sample (hereinafter referred to as the "test sample") for detecting the target DNA can be used. The test sample can be a cellular sample or a cell-free sample, and examples include biological tissues, cells, cell lysates, body fluids (urine, saliva, serum, plasma, whole blood, etc.), or samples containing purified or synthetic DNA.

[0071] One aspect of the detection method of the present invention utilizes a label added to the guide RNA. Since the guide RNA of the present invention does not undergo processing-induced cleavage on the 3' side of the spacer sequence, target DNA can be detected using the guide RNA with a label added to the 3' side.

[0072] As a marker, there are no particular limitations as long as it can be detected, but examples include, for instance, 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.

[0073] In the case of using RNA aptamer sequences / recognition proteins, the RNA aptamer sequence is added to the guide RNA. By allowing the recognition protein, which is fused with fluorescent proteins such as GFP, to bind to the RNA aptamer sequence, the target DNA can be detected using fluorescence as an indicator (Yang LZ et al., Cell Insight 1 (2022) 100044). Alternatively, in the case of using RNA aptamer sequences / recognition compounds, the RNA aptamer sequence is added to the guide RNA. By allowing the RNA aptamer sequence to bind to the recognition compound, the target DNA can be detected using color development as an indicator.

[0074] In these detections, to avoid the degradation of target DNA by the nuclease activity of the type I CRISPR-Cas system, it is preferable to use a system that eliminates nuclease activity, for example, a system that uses the nuclease-inactivating Cas3 protein (Cas10d in type ID), thus excluding the Cas3 protein (Cas10d in type ID).

[0075] Another aspect of the detection method of the present invention is a method using single-stranded probe DNA (see Example 1). It is known that in a type I CRISPR-Cas system, if the target DNA in the sample is recognized and bound, the surrounding single-stranded DNA (ssDNA) will be indiscriminately cleaved, and this phenomenon can also be used to detect target DNA (International Publication No. 2021 / 149829).

[0076] Specifically, in the reaction system containing the sample and the type I CRISPR-Cas system for detecting target DNA, single-stranded probe DNA capable of detecting its cleavage is pre-mixed, thereby enabling the detection of target DNA in the sample using the signal generated by the 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 this invention itself.

[0077] There are no particular restrictions on the single-stranded probe DNA as long as it can be detected by its cleavage energy; it can be either straight or circular, but straight strands are preferred. The nucleic acids constituting the single-stranded probe DNA can contain more than one type of modification (e.g., base modification, backbone modification, sugar modification).

[0078] A preferred form of labeling for single-stranded probe DNA is the fluorochrome / quencher pair. In this form, the signal from the fluorochrome is reduced or eliminated when the fluorochrome / quencher pair is in proximity to the single-stranded probe DNA, but a sufficient signal from the fluorochrome is detected if the single-stranded probe DNA is cleaved and the fluorochrome separates from the quencher. Therefore, the target DNA in the test sample can be detected using the fluorescence signal generated by the single-stranded probe DNA cleaved by a type I CRISPR-Cas system that recognizes and binds to the target DNA as an indicator.

[0079] Another preferred approach to labeling single-stranded probe DNA is the donor / acceptor pair for fluorescence resonance energy transfer (FRET). In this approach, when the donor / acceptor pair is in proximity to the single-stranded probe DNA, excitation of the donor induces excitation and luminescence of the acceptor (i.e., FRET signal generation). However, if the single-stranded probe DNA is cleaved and the donor separates from the acceptor, the FRET signal decreases or disappears. Therefore, the decrease or disappearance of the FRET signal in the single-stranded probe DNA cleaved by a type I CRISPR-Cas system that recognizes and binds to the target DNA can be used as an indicator to detect the target DNA in the test sample.

[0080] In addition, for example, immunochromatography (lateral flow assay) can be used in the detection of single-stranded probe DNA.

[0081] Contact between the test sample, the type I CRISPR-Cas system, and the single-stranded probe DNA can be achieved, for example, by mixing them. In cases where the test sample contains cells, further steps may be included for introducing the CRISPR-Cas system and the single-stranded probe DNA into the cells within the test sample.

[0082] Example

[0083] The present invention will be further described in detail below through embodiments, but the present invention is not limited to the following embodiments.

[0084] [Example 1] In vitro DNA cleavage activity of various crRNA lengths

[0085] In this detection system, an IE-type CRISPR derived from *E. coli* was utilized. The Cas3 protein was expressed and purified using insect Sf9 cells. Specifically, the Cas3 gene carrying a His-tagged nuclear transfer signal (bpNLS) was integrated into a baculovirus gene expression vector, and then a baculovirus was generated using *E. coli* strain DH10bac. The generated baculovirus was used to infect Sf9 insect cells to express the Cas3 protein. The total protein was then recovered and purified by nickel column chromatography and gel filtration.

[0086] The cascade reaction components were also expressed and purified using insect Sf9 cells. Specifically, the genes of Cas5, Cas6, Cas7, Cas8, and Cas11, respectively tagged with His-tagged nuclear transfer signals (bpNLS), were linked with 2A peptide sequences to prepare baculovirus gene expression vectors. The proteins were expressed in the same manner as above, and the Cas3 protein was purified by nickel column chromatography and gel filtration.

[0087] crRNAs are obtained by synthesizing double-stranded DNA fragments that encode them, followed by in vitro transcription and column purification. In this experiment, precursor crRNA (Sequence No. 8), mature crRNA (Sequence No. 9), 5' mature crRNA (crRNA with only the 5' side repeat sequence being mature), and 3' mature crRNA (crRNA with only the 3' side repeat sequence being mature) were used.

[0088] To investigate changes in the cleavage activity of the CRISPR-Cas3 lineage caused by crRNA length, non-specific cleavage (collateral cleavage activity) observed during target recognition was measured using a fluorescent quencher probe (56FAM-AAGGTCGGA-ZEN-GTCAACGGATTTGGTC-ABkFQ, IDT; the sequence between ZEN and ABkFQ is described in sequence number 10) (CONAN assay).

[0089] First, equal volumes of cascade reaction factor (0.8 μg / μL) and crRNA (250 ng / μL) were mixed and incubated at 37°C for 10 minutes in a complex formation buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2) to allow the cascade reaction complex to form. Next, Cas3 protein (final concentration 40 ng / μL), the cascade reaction complex (final concentration 32 ng / μL), a double-stranded DNA fragment containing the target sequence (mouse Tyr gene / sequence number 11) (final concentration 5 nM), and a fluorescence quencher probe (final concentration 1 μM) were mixed in a 10 μL system using a reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2, 1 mM ATP). The intensity of the FAM signal was measured every 30 seconds at 37°C using a real-time PCR device (CFX96 Touch DeepWell system, Bio-Rad Laboratories).

[0090] As a result, the 5' side repeat sequence showed fluorescence signals generated by collateral cleavage activity only in the precursor state, while the 3' side repeat sequence showed fluorescence signals in both the precursor and mature states. Figure 1 That is, the 5' side repeat sequence is necessary for the complexation and activation of CRISPR-Cas3.

[0091] Therefore, the possibility of further shortening the 3' side repeat sequence was investigated. The result was that even in the shortened crRNA (sequence number 12) state where the 3' side repeat sequence was absent, a fluorescent signal generated by collateral cleavage activity was detected. Figure 2 That is, the 21 bases on the 3' side remaining during the maturation process are not necessary for the complexation and activation of CRISPR-Cas3.

[0092] The results above indicate that the 5' side repeat sequence is necessary when cascade reaction factors form a complex with crRNA, while the 3' side repeat sequence is not necessary. Even shortened crRNAs with the complete loss of the 3' side repeat sequence show activity.

[0093] [Example 2] Determination of genome cleavage activity in human cultured cells K562 using IE-type shortened crRNA

[0094] In K562 cells induced by doxycycline to express Cas3 and cascade reaction constituents (Cas5, Cas6, Cas7, Cas8, Cas11), purified human EMX1 target crRNA (short form; 20 ng / μL) was introduced via electroporation using a 4D-Nucleofector System (Lonza). Cells were cultured in medium supplemented with 2 μg / ml doxycycline at 37°C and 5% CO2 for 2 days. Total cell count was then recovered, and the genome was extracted. PCR was performed using a primer set containing a 3.8 kb primer to amplify the PAM region of the EMX gene.

[0095] As a result, when using purified shortened crRNA (Sequence No. 14), a shorter band compared to the wild-type band was detected, similar to when using purified precursor crRNA (Sequence No. 13) and plasmid expression precursor crRNA. Figure 3 ).

[0096] To confirm the characteristics of genome editing, the PCR products were analyzed using a nanopore sequencer (Oxford Nanopore Technologies) for long-read sequencing. The results confirmed deletions of 562, 1619, and 2106 base pairs upstream of the 5' PAM spacer sequence in the target region. Figure 3 This demonstrates that shortened crRNAs can be used in genome editing of human cells.

[0097] [Example 3] Determination of genome cleavage activity in HEK293T human culture cells using IC-type shortened crRNA

[0098] To verify whether the same phenomenon occurs in other type I CRISPRs, the IC type system of Neisseria lactamica was used to investigate whether shortened crRNA introduces mutations into the human EMX1 gene.

[0099] Plasmids expressing precursor crRNA (Sequence No. 15) or shortened crRNA (Sequence No. 16) were prepared and, together with plasmids expressing five Cas (3, 5, 7, 8, 11) effectors and a puromycin resistance gene, were introduced into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were added to medium supplemented with 1 μg / ml puromycin and cultured at 37°C and 5% CO2 for 2 days. Total cell count was recovered and genomic DNA was extracted. PCR was performed using a primer set containing a 3.8 kb primer amplification of the PAM region of the EMX gene. Electrophoresis of the PCR products confirmed that the shortened crRNA, like the precursor crRNA, formed a shorter band compared to the wild-type band. Figure 4 ).

[0100] In addition, the lower molecular weight band compared to the wild-type band was cut out and purified, and then subjected to Senggelin sequencing analysis, which confirmed the deletion of 2835 base pairs. Figure 4 This demonstrates that, for IC-type CRISPR, shortened crRNA can also be used in genome editing of human cells.

[0101] [Example 4] Determination of genome cleavage activity in HEK293T human culture cells using ID-type shortened crRNA

[0102] To verify whether the same phenomenon occurs in other type I CRISPR systems, the ID system of Microcystis aeruginosa was used to investigate whether shortened crRNA introduces mutations into the human EMX1 gene.

[0103] Plasmids encoding the precursor crRNA (SEQ ID NO. 17) and the shortened crRNA (SEQ ID NO. 18), respectively, were prepared and introduced into HEK293T cells along with plasmids expressing six Cas effectors (3, 5, 6, 7, 10, 11) and a puromycin resistance gene using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were added to a medium supplemented with 1 μg / ml puromycin and cultured at 37°C and 5% CO2 for 2 days. Total cell count was recovered and the genome was extracted. PCR was performed using a primer set containing a 3.8 kb primer amplification of the PAM region of the EMX gene. Electrophoresis of the PCR products confirmed that the shortened crRNA, like the precursor crRNA, formed a shorter band compared to the wild-type band. Figure 5 ).

[0104] In addition, the PCR product bands with a molecular weight smaller than that of the wild-type band were excised and purified, and then subjected to Senggelin sequencing analysis, which confirmed the deletion of 3131 base pairs and 2593 base pairs. Figure 5 This demonstrates that, for ID-type CRISPR, shortened crRNA can also be used in genome editing in human cells.

[0105] [Example 5] Validation using leader editing of modified crRNA with donor sequence

[0106] In the IE-type CRISPR-Cas3 system, a shortened crRNA (modified crRNA) with a HiBiT sequence and an MS2 sequence added to the 3' side was used to verify whether the HiBiT sequence could be inserted specifically for the target.

[0107] The stop codon region of the GFP gene was selected as the target to prepare plasmids encoding modified precursor crRNA and six Cas (3, 5-8, 11) effectors. Furthermore, wild-type or its mutant (the S483A / T485A double mutant of SF2 domain motif 3 (dh2Cas3)) was used for Cas3. 293T cells were transfected with plasmids encoding the aforementioned modified precursor crRNA, CAG-GFP expression plasmid, and MCP reverse transcriptase expression plasmid via liposomes, 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 The results showed an increase in luciferase activity when using helicase-deficient Cas3, suggesting that Hibit was inserted into the GFP site. Figure 6 (The bottom right bar chart).

[0108] [Example 6] Validation of GFP aggregation generated by modified crRNA with MS2 random sequence

[0109] This study verified whether target-specific insertion was possible using a shortened crRNA (modified crRNA) with an MS2 random sequence added to the 3' side in the IE-type CRISPR-Cas3 system.

[0110] Using the stop codon region of the GFP gene as a target, plasmids encoding a modified precursor crRNA and five Cas(5-8, 11) effectors were prepared. 293T cells were transfected with plasmids encoding the modified precursor crRNA and the MCP-EGFP (StayGold) expression plasmid via liposomes, and then cultured and passaged. Two days later, cell observation using a fluorescence microscope revealed GFP aggregation when using the modified crRNA, suggesting the ability to image the target sequence. Figure 7 ).

[0111] Industry availability

[0112] In the type I CRISPR-Cas system using the shortened crRNA of the present invention, guide RNAs with various modifications such as label addition can be used, making it suitable not only for genome editing of animals, plants, etc., but also for use in a wide range of fields such as diagnosis using nucleic acid detection.

Claims

1. A guide RNA for a type I CRISPR-Cas system, wherein the guide RNA has a repeating sequence configured on the 5' side of the spacer sequence that is cleaved during processing to form a cascade reaction complex, and no repeating sequence configured on the 3' side of the spacer sequence, or a sequence configured on the 3' side that is not cleaved during processing to form a cascade reaction complex.

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. A method for manufacturing a sample in which target DNA has been edited, comprising contacting the CRISPR-Cas system of claim 4 with a sample containing target DNA.

6. The method according to claim 5, wherein, The sample is cells.

7. The method according to claim 6, wherein, The cell is a eukaryotic cell.

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

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