TracrRNA unit and genome editing method

Abstract

The present invention addresses the problem of providing a novel tracrRNA unit and a genome editing method using the same. The problem is solved by a tracrRNA unit that is used in a CRISPR-Cas9 genome editing system and is composed of a first single-stranded RNA and a second single-stranded RNA, the first single-stranded RNA and the second single-stranded RNA are discontinuous, the first single-stranded RNA has at least a first part and a second part, the first part and the second part do not overlap, the first part has a sequence complementary to a part of crRNA, the second part has a sequence complementary to a part of crRNA, the first part has a sequence complementary to a part of crRNA, and the second part has a sequence complementary to a part of crRNA. The second part has a sequence complementary to the second single-stranded RNA.

Description

Technical Field

[0001] This specification discloses a tracrRNA unit and a genome editing method. Background Art

[0002] The Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system is an immune system against viruses and plasmids found in bacteria and archaea (Non-patent document 1). The system is currently used as a method for deleting, replacing or inserting nucleotide sequences in target regions of genomic DNA of various organisms in vitro (Patent Documents 1 and 2).

[0003] In bacteria and archaea, CRISPR RNA (crRNA) having a nucleotide sequence complementary to the nucleotide sequence of the target region of genomic DNA and tracrRNA having a nucleotide sequence complementary to crRNA are different RNA chains, which hybridize through complementary nucleotide sequences to form a crRNA:tracrRNA double strand and guide Cas9. On the other hand, in general in vitro, as described in Non-Patent Document 1, Patent Document 1 and Patent Document 2, a single guide RNA (sgRNA) in which crRNA and tracrRNA are combined in the form of a single RNA chain with a linker loop sequence is used instead of a crRNA:tracrRNA double strand.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2013 / 176772

[0007] Patent Document 2: International Publication No. 2014 / 093661

[0008] Non-patent literature

[0009] Non-patent document 1: M Jinek, et al.: SCIENCE, 28Jun 2012, Vol 337, Issue 6096, pp. 816-821

[0010] Non-patent document 2: H Nishimasu, et al.: Cell, 27 Feb 2014, Vol 156, pp. 935-949 Summary of the invention

[0011] Problem that the invention aims to solve

[0012] like Figure 1 As shown in (A), tracrRNA forms three hairpin loops in its RNA chain. The three hairpin loops are called the first stem loop, the second stem loop, and the third stem loop from the nucleotide sequence close to the crRNA hybridization. Regarding tracrRNA, as described in non-patent literature 2 above, it is known that even if the joint between the first stem loop and the second stem loop is partially defective, it can function as tracrRNA, but it is completely unclear whether the function of tracrRNA can be maintained when the other parts are improved.

[0013] The object of the present invention is to provide a novel tracrRNA unit and a genome editing method using the same.

[0014] Solutions for solving problems

[0015] Item 1.

[0016] A tracrRNA unit, which is a tracrRNA unit for a CRISPR-Cas9 genome editing system, and is composed of a first single-stranded RNA and a second single-stranded RNA,

[0017] The first single-stranded RNA and the second single-stranded RNA are not continuous.

[0018] The first single-stranded RNA has at least a first portion and a second portion,

[0019] Part 1 and part 2 do not overlap, part 1 has a sequence complementary to a portion of crRNA, and part 2 has a sequence complementary to the second single-stranded RNA.

[0020] Item 2.

[0021] A tracrRNA unit, which is a tracrRNA unit for a CRISPR-Cas9 genome editing system, and is composed of a first single-stranded RNA, a second single-stranded RNA, and a third single-stranded RNA,

[0022] The first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are not continuous.

[0023] The first single-stranded RNA has at least a first portion and a second portion,

[0024] Part 1 and part 2 do not overlap, part 1 has a sequence complementary to a portion of crRNA, and part 2 has a sequence complementary to a second single-stranded RNA,

[0025] The second single-stranded RNA has at least the first portion,

[0026] The first portion of the second single-stranded RNA has a sequence complementary to the third single-stranded RNA.

[0027] Item 3.

[0028] The tracrRNA unit according to item 1 or 2, wherein the crRNA and the first single-stranded RNA are linked in this order.

[0029] Item 4.

[0030] The tracrRNA unit according to item 2, wherein the crRNA, the first single-stranded RNA, and the second single-stranded RNA are linked in this order.

[0031] Item 5.

[0032] The tracrRNA unit according to item 1, wherein at least one of the first single-stranded RNA and the second single-stranded RNA has a length of 39 nucleotides or less.

[0033] Item 6.

[0034] The tracrRNA unit according to item 2, wherein at least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA has a length of 39 nucleotides or less.

[0035] Item 7.

[0036] The tracrRNA unit according to item 2, wherein the first single-stranded RNA and the second single-stranded RNA are discontinuous, and the second single-stranded RNA and the third single-stranded RNA are continuous.

[0037] Item 8.

[0038] A genome editing method using:

[0039] The tracrRNA unit according to any one of items 1 to 7,

[0040] crRNA, and

[0041] Cas9.

[0042] Effects of the Invention

[0043] A new tracrRNA unit and a genome editing method using the same can be provided. Since the new tracrRNA unit has one short RNA chain, the cost of chemically synthesized RNA chains can be reduced. In addition, by splitting the tracrRNA, the number of 5' and 3' ends of the RNA chain increases, and the number and type of functional motifs given to the tracrRNA can be increased, thereby increasing the application in donor sharing (Tethering) and conventional nucleic acid modification design. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The structure of a conventional tracrRNA and the outline of the tracrRNA unit of the present invention are shown. (A) The structure of a conventional crRNA:tracrRNA double strand is shown. (B) The structure of the tracrRNA unit of the first embodiment of the present invention is shown. a is the segmentation form of the first stem loop. b is the segmentation form of the second stem loop. (C) The structure of the tracrRNA unit of the second embodiment of the present invention is shown.

[0045] Figure 2 The sequence of a conventional tracrRNA is shown.

[0046] Figure 3 One embodiment of a tracrRNA unit is shown.

[0047] Figure 4 One embodiment of a tracrRNA unit is shown.

[0048] Figure 5 One embodiment of a tracrRNA unit is shown.

[0049] Figure 6 One embodiment of a tracrRNA unit is shown.

[0050] Figure 7 The results of an in vitro cleavage experiment targeting the killifish slc45a2 gene are shown.

[0051] Figure 8 The results of observation of the eye of a killifish embryo obtained by injecting tracrRNA composed of the first single-stranded RNA and the second single-stranded RNA, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA together with Cas9 and crRNA are shown.

[0052] Fig. 9 The results of HMA analysis using a killifish embryo lysate obtained by injecting tracrRNA composed of the first single-stranded RNA and the second single-stranded RNA, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA together with Cas9 and crRNA are shown.

[0053] Figure 10-1 The results of mutation analysis using Sanger sequencing and TIDE are shown.

[0054] Figure 10-2 The results of mutation analysis using Sanger sequencing and TIDE are shown.

[0055] Figure 10-3 The results of mutation analysis using Sanger sequencing and TIDE are shown.

[0056] Figure 10-4 The results of mutation analysis using Sanger sequencing and TIDE are shown.

[0057] Figure 10-5 The results of mutation analysis using Sanger sequencing and TIDE are shown. DETAILED DESCRIPTION

[0058] 1. tracrRNA unit

[0059] In this specification, the tracrRNA unit refers to a complex in which tracrRNA is composed of two or more single-stranded RNAs and maintains the function as tracrRNA. The labeling of nucleotides follows the labeling of general RNA or DNA.

[0060] In addition, the term "linked" as used herein means: linking into a single polynucleotide chain, and preferably means linking two polynucleotide chains via a phosphodiester bond.

[0061] In the present specification, "having a ... sequence" means comprising a ... sequence or consisting of a ... sequence.

[0062] 1-1. First Embodiment

[0063] The first embodiment relates to Figure 1 The tracrRNA unit is a tracrRNA unit that divides the tracrRNA into two at the first stem-loop or the second stem-loop shown in (B). Figure 1 In a of (B), tracrRNA is split at the first stem-loop portion. Figure 1 In b of (B), tracrRNA is split at the second stem-loop portion.

[0064] More specifically, the tracrRNA unit can be composed of a first single-stranded RNA ( Figure 1 (B) "#1") and the second single-stranded RNA ( Figure 1 The first single-stranded RNA and the second single-stranded RNA are not continuous. The first single-stranded RNA has at least a first part and a second part, and the first part and the second part do not overlap. The first part has a sequence complementary to a part of the crRNA, and the second part has a sequence complementary to the second single-stranded RNA.

[0065] Figure 2The nucleotide sequence (sequence number 1) of the conventional tracrRNA is shown. In the conventional tracrRNA, nucleotide numbers (nt No.) 1 to 12 are complementary sequences of the first crRNA, nt No. 17 to 21 are complementary sequences of the second crRNA, nt No. 28 to 29 are loops of the first stem loop, nt No. 35 to 39 are joints, nt No. 45 to 48 are loops of the second stem loop, and nt No. 60 to 62 are loops of the third stem loop. The sequences before and after each loop are hybridized to form a hairpin loop. For example, the first stem loop is formed by hybridization of at least nt No. 25 to 27 and nt No. 33, 32, and 30. The second stem loop is formed by hybridization of at least nt No. 41 to 44 and nt No. 52 to 49. The third stem loop is formed by hybridization of at least nt No. 54 to 59 and nt No. 68 to 63.

[0066] When tracrRNA is split at the first stem-loop portion, the split may be performed between nt No. 27 and 28, between nt No. 28 and 29, or between nt No. 29 and 30 of SEQ ID NO: 1. Alternatively, the split may be performed by deleting nt No. 28, nt No. 29, or nt No. 28 and 29.

[0067] When the tracrRNA is split at the second stem-loop portion, the split may be performed between nt No. 44 and 45, between nt No. 45 and 46, between nt No. 46 and 47, between nt No. 47 and 48, or between nt No. 48 and 49 of SEQ ID NO: 1. Alternatively, the split may be performed by deleting one or more nucleotides among nt Nos. 45 to 48.

[0068] The first single-stranded RNA and / or the second single-stranded RNA may use RNA having a segmented nucleotide sequence in its sequence. Alternatively, an additional sequence of about 1 to 10 nucleotides may be added to one or both of the 5' end or the 3' end of the first single-stranded RNA and / or the second single-stranded RNA.

[0069] In addition, a portion of the first single-stranded RNA and / or the second single-stranded RNA may be missing as long as it satisfies the function of having a tracrRNA when constructing the tracrRNA unit. In this case, it is preferred that the length of at least one of the first single-stranded RNA and the second single-stranded RNA is 39 nucleotides or less. When the first single-stranded RNA and the second single-stranded RNA include an additional sequence, it is preferred that the length of at least one of the first single-stranded RNA and the second single-stranded RNA, including the additional sequence, is 39 nucleotides or less.

[0070] Here, the portion of the first portion of the first single-stranded RNA that is a sequence complementary to a portion of the crRNA may have Figure 2 The complementary sequence of the 1st crRNA and the complementary sequence of the 2nd crRNA shown in the figure is a part of at least one of them. In addition, the part of the second part of the first single-stranded RNA as a sequence complementary to the second single-stranded RNA can also include a part of the sequence before and after the loop portion of the 1st stem-loop or a part of the sequence before and after the loop portion of the 2nd stem-loop.

[0071] For sequence number 1, as described in non-patent document 2, even if nt Nos. 35 to 39, part or all of the linker, preferably nt Nos. 36 to 39 are missing, the function of tracrRNA can be maintained. Therefore, it is preferred to exclude the embodiment in which only nt Nos. 36 to 39 are missing, preferably only the linker is missing.

[0072] In the present invention, for example, even if nt Nos. 1 to 8 of SEQ ID NO: 1 are deleted, it can still function as tracrRNA or as a tracrRNA unit.

[0073] In this embodiment, the first single-stranded RNA can be linked to the crRNA described later.

[0074] In addition, when a part of the nucleotide sequence of tracrRNA is missing, the first single-stranded RNA and the second single-stranded RNA can be linked in sequence from the 5' side to the 3' side to constitute the tracrRNA unit.

[0075] The first single-stranded RNA and the second single-stranded RNA are preferably introduced into cells, individuals or tissues after annealing between complementary sequences to form a tracrRNA unit. Annealing is not particularly limited, for example, by heating at about 90°C to 98°C for about 3 minutes to about 10 minutes in a thermal cycler, etc., and then slowly lowering to room temperature (for example, lowering to 25°C at 0.1°C per second). In addition, during annealing, crRNA can be added and annealed to form a guide RNA unit.

[0076] 1-2. Second Embodiment

[0077] The second embodiment relates to Figure 1 The first stem-loop and the second stem-loop shown in (C) divide the tracrRNA into three tracrRNA units. Figure 1 In (C), tracrRNA is divided at the first stem-loop portion and the second stem-loop portion.

[0078] More specifically, the tracrRNA unit can be composed of a first single-stranded RNA ( Figure 1 (C) as "#1", the second single-stranded RNA ( Figure 1 (C) "#2") and the third single-stranded RNA ( Figure 1 (C) (shown as "#3" in "C"). The first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are not continuous. The first single-stranded RNA has at least a first part and a second part, and the first part and the second part do not overlap. The first part has a sequence complementary to a part of the crRNA, and the second part has a sequence complementary to the second single-stranded RNA. The second single-stranded RNA has at least a first part, and the first part of the second single-stranded RNA has a sequence complementary to the third single-stranded RNA. Here, in the second single-stranded RNA, the part complementary to the second part of the first single-stranded RNA and the first part of the second single-stranded RNA do not overlap.

[0079] The division of the first stem-loop portion and the second stem-loop portion is the same as that of the first embodiment, and the above description is incorporated herein.

[0080] When tracrRNA is split at the third stem-loop portion, the split may be performed between nt Nos. 59 and 60, between nt Nos. 60 and 61, between nt Nos. 61 and 62, or between nt Nos. 62 and 63 of SEQ ID NO: 1. Alternatively, the split may be performed by deleting one or more nucleotides among nt Nos. 60 to 62.

[0081] At least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA may use an RNA having a divided nucleotide sequence in its sequence. Alternatively, at least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA may have an additional sequence of about 1 to 10 nucleotides at one or both of the 5' end or the 3' end.

[0082] A portion of at least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA may be missing as long as it has the function of tracrRNA when constructing the tracrRNA unit. In this case, it is preferred that the length of at least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA is 39 nucleotides or less, including the additional sequence.

[0083] Here, the portion of the first portion of the first single-stranded RNA that is a sequence complementary to a portion of the crRNA may have Figure 2The complementary sequence of the 1st crRNA and the complementary sequence of the 2nd crRNA shown in the figure is a part of at least one of them. In addition, the part of the 2nd part of the 1st single-stranded RNA as a sequence complementary to the 2nd single-stranded RNA may also include a part of the sequence before and after the loop of the 1st stem-loop or a part of the sequence before and after the loop of the 2nd stem-loop. And then, the part of the 2nd single-stranded RNA as a sequence complementary to the 3rd single-stranded RNA may also include a part of the sequence before and after the loop of the 3rd stem-loop.

[0084] Regarding sequence number 1, as described in non-patent document 2, even if nt Nos. 36 to 39 and part or all of the linker are missing, the function of tracrRNA can be maintained. Therefore, it is preferable to exclude the embodiment in which only the linker is missing.

[0085] In the present invention, for example, even if nt Nos. 1 to 8 of SEQ ID NO: 1 are deleted, it can still function as tracrRNA or as a tracrRNA unit.

[0086] In this embodiment, the first single-stranded RNA can be connected to crRNA. The crRNA includes a nucleotide sequence complementary to the target genome sequence (preferably 20 nucleotides) and a nucleotide sequence complementary to the first single-stranded RNA. Furthermore, the first single-stranded RNA connected to crRNA can be connected to the second single-stranded RNA.

[0087] In addition, when a part of the nucleotide sequence of tracrRNA is missing, the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA can be linked in sequence from the 5' side to the 3' side to constitute the tracrRNA unit.

[0088] The first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are preferably introduced into cells, individuals, or tissues after annealing between complementary sequences to form a tracrRNA unit. Annealing is not particularly limited, for example, by heating at about 90°C to 98°C for about 3 to 10 minutes in a thermal cycler, etc., and then slowly lowering to room temperature (for example, lowering to 25°C at 0.1°C per second). In addition, during annealing, crRNA can be added and annealed to form a guide RNA unit.

[0089] 2. Chimeric RNA formed by connecting crRNA and tracrRNA units

[0090] The third embodiment relates to a chimeric RNA formed by connecting crRNA and the single-stranded RNA constituting the tracrRNA unit described in 1. wherein a portion of the nucleotide sequence shown in SEQ ID NO: 1 is missing in the chimeric RNA.

[0091] 3. DNA fragments encoding individual RNAs constituting the tracrRNA unit or encoding chimeric RNAs

[0092] The fourth embodiment relates to a DNA fragment encoding each of the single-stranded RNAs described in 1. above or the chimeric RNA described in 2. above.

[0093] DNA fragments can be single-stranded or double-stranded.

[0094] In addition, the DNA fragment can be integrated into a vector. The vector is not limited as long as it can transcribe the above-mentioned single-stranded RNA or chimeric RNA from the DNA fragment. The DNA fragment is inserted downstream of a promoter nucleotide sequence such as H1, 7SK and U6 promoters. The vector can exemplify a plasmid vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, and the like.

[0095] 4. Genome editing methods using tracrRNA units

[0096] The fifth embodiment relates to a genome editing method using the tracrRNA unit described in 1. In addition, the fifth embodiment relates to a genome editing method using the chimeric RNA described in 2. Genome editing may include methods such as replacement, deletion and insertion of nucleotide sequences of the target genome, or gene expression control and epigenomic editing.

[0097] Here, "use" means: using the tracrRNA unit and / or chimeric RNA for genome editing in cells. That is, introducing the tracrRNA unit and / or chimeric RNA into cells in the form of RNA or introducing the DNA fragment described in 3. above into cells in the form of a vector.

[0098] In a preferred embodiment, the tracrRNA unit described in 1. above is used together with crRNA and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme.

[0099] The crRNA comprises a nucleotide sequence (preferably 20 nucleotides) complementary to the nucleotide sequence of the target genome (hereinafter also referred to as the "target sequence") and a nucleotide sequence complementary to all or part of the first part of the above-mentioned first single-stranded RNA.

[0100] The method for selecting the target sequence is well known. The target sequence can be selected using publicly known design tools such as optimized CRISPR design tools (Massachusetts Institute of Technology, ZhangLab's webpage (http: / / crispr.mit.edu / )), E-CRISP (http: / / www.e-crisp.org / E-CRISP / (German Cancer Research Center)), ZiFiT Targeter (http: / / zifit.partners.org / ZiFit / (Zing Finger Consortium)), Cas9design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (University of Tokyo)), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Huazhong Agricultural University)), CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul University)).

[0101] The tracrRNA unit, crRNA, CRISPR enzyme and their encoding nucleic acid can be introduced by microinjection, electroporation, lipofection, etc.

[0102] The tracrRNA unit and crRNA can be injected in a range of about 0.05 pg to 25 pg, preferably 0.25 pg to 10 pg, and more preferably 0.5 pg to 2.5 pg, respectively.

[0103] When the CRISPR enzyme is introduced in the form of a protein or a nucleic acid encoding the protein, it can be injected in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg per cell.

[0104] The tracrRNA unit, crRNA, CRISPR enzyme, or nucleic acid encoding them can be administered systemically or locally to an individual using a delivery system such as liposomes. In the case of systemic administration, the tracrRNA unit, crRNA, CRISPR enzyme, or nucleic acid encoding them can be administered at a rate of 0.1 to 1000 mg / day per kg of individual body weight. In the case of local administration, the tracrRNA unit, crRNA, CRISPR enzyme, or nucleic acid encoding them can be administered at a rate of 100 mg / day per 1 cm 2 The tracrRNA unit, crRNA, CRISPR enzyme or its encoding nucleic acid is administered in a manner that achieves 0.01-100 mg / day in the target tissue.

[0105] In addition, it can also be changed to a method of using the tracrRNA unit and crRNA as independent units. As a chimeric RNA, the tracrRNA unit and crRNA can be connected and used. The chimeric RNA can be injected in a range of about 0.1pg to 50pg per cell, preferably 0.5pg to 20pg, and more preferably 1pg to 5pg. In addition, in the case of systemic administration of chimeric RNA, it can be administered in a manner of 0.1 to 1000mg / day per 1kg of individual body weight. In the case of local administration, it can be administered in a range of 1cm 2 The chimeric RNA is administered in such a manner that 0.01 to 100 mg / day is achieved in the target tissue.

[0106] The tracrRNA unit, crRNA or chimeric RNA can also be used in the following manner: a vector carrying the DNA fragment encoding these RNAs described in 3. above is introduced into a cell, an individual or a tissue, so that the RNA encoded by the DNA fragment is expressed after the introduction and used.

[0107] When the vector is a plasmid vector, the total amount of DNA per cell can be introduced in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg. When the plasmid vector is administered systemically, it can be administered in a manner that the total amount of DNA per kg of individual body weight reaches 0.1 to 1000 mg / day. When it is administered topically, it can be administered in a range of about 10 to 20 mg / day per 1 cm 2 The plasmid vector is administered to the target tissue so that the total amount of DNA reaches 0.01 to 100 mg / day. When a plasmid vector carrying DNA encoding a CRISPR enzyme is used, this is also included in the total amount of DNA.

[0108] When the vector is a viral vector, the total amount of virus carrying the target nucleotide sequence per cell can be calculated as 10 2 ~10 6 When the virus is administered systemically, the total amount of virus can reach 10 per kg of individual body weight. 10 ~10 18 vg / day. In the case of local administration, it can be administered at 1 cm 2 The total amount of virus in the target tissue reaches 10 9 ~10 16 When a virus carrying DNA encoding a CRISPR enzyme is used, it is also included in the total amount of the above-mentioned virus.

[0109] Furthermore, in this embodiment, donor oligo DNA such as single-stranded oligomers (ssODNs) can be co-introduced with tracrRNA units, crRNA, CRISPR enzymes or their encoding nucleic acids. ssODNs can be designed according to known methods.

[0110] As CRISPR enzymes, Cas9 derived from Streptococcus pyogenes, Staphylococcus aureus, Francisella novicida, Campylobacter jejuni, Neisseria meningitidis, Streptococcus thermophilus, Clostridium cellulolyticum, Treponema denticola, Brevibacillus laterosporus, Actinobacillus succinogenes, etc. can be exemplified. In addition, the literature: Supplementary Data of Gasiunas et al. (2020), Nat Commun, 11_5512 can also be cited. Lpn, Khu, Ain, Cgl, Esp1, Esp2, Fma, Lce, Lrh, Lsp1, Lsp2, Pac, Tba, Tde, Tpu, Vpa, Efa, Eit, Lan, Lmo described in 2 , Sag1, Sag2, Sdy, Seq1, Seq2, Sga, Smu, Sra, Bni, Ece, Edo, Fho, Mga, Mse, Sgo, Sma2, Ssa, Ssi, Ssu, Sth1A, Tsp, Bok, Cco, Cpe, Dde, Ghc2, Ghy3, Ghy4, Gsp, Kki, Nme2, Nsp, Tmo, Nsa, Jpa, Rsp, Bbo, Cca2, Cme2, Cme3, C me4, Csa, Ghc1, Ghe, Ghh1, Ghh2, Ghy1, Msc, Sdo, Spa, Cca1, Cga, Cme1, Ffr, Ghy2, Orh, Phi, Psp, Wvi and other Cas9.

[0111] Furthermore, Cas9 may have an amino acid substitution and / or be combined with an effector molecule. As Cas9 with amino acid substitution, high specificity mutations such as the R691A mutation (also called HiFiCas9) of Streptococcus pyogenes Cas9 (also called SpCas9), nuclease inactivation mutations such as the D10A and H840A mutations (dCas9) of SpCas9, etc. can be cited. As effector molecules, Rad51 proteins for promoting homologous recombination repair and CtIP proteins for promoting micro-homologous end binding can be cited. Effector molecules can be combined with Cas9 via a linker. In addition, as Cas9 with an H840A mutation, a reverse transcriptase can be combined as an effector.

[0112] In this specification, "Cas9" may include wild-type Cas9 from various biological sources as described above, mutant Cas9 with amino acid substitutions, mutant Cas9 combined with effector molecules, and the like.

[0113] CRISPR enzymes can be introduced into cells, individuals or tissues in the form of proteins or nucleic acids encoding proteins.

[0114] The organism to which the genome editing method of the present embodiment is applied is not particularly limited. The organism may be a prokaryotic cell organism, or a eukaryotic cell organism, or a cultured cell derived from a eukaryotic cell organism. As eukaryotic cell organisms, for example, fish, reptiles, amphibians, birds, mammals, insects, crustaceans, arthropods, mollusks, echinoderms, cnidarians, annular animals, nematodes and other animal cell organisms, plants, algae and other plant cell organisms, fungi, protists and the like can be cited.

[0115] 5. Genome Editing Kit

[0116] The fourth embodiment relates to a genome editing kit comprising the tracrRNA unit, crRNA, and CRISPR enzyme or its encoding nucleic acid described in 1. above. For the explanation of each term, the explanation of 1. and 4. above can be cited here.

[0117] The fifth embodiment relates to a genome editing kit comprising the chimeric RNA and CRISPR enzyme or nucleic acid encoding the same as described in 2. above. For the description of each term, the description of 2. and 4. above can be cited here.

[0118] The sixth embodiment relates to a genome editing kit comprising a DNA fragment encoding each RNA constituting the tracrRNA unit described in 3. above or encoding a chimeric RNA, and a CRISPR enzyme or a nucleic acid encoding the same. For the description of each term, the description of 3. and 4. above can be cited here.

[0119] 6. Modifications

[0120] Each RNA strand and chimeric RNA constituting the tracrRNA unit disclosed in this specification may have a functional motif at the 5' and / or 3' end. As the functional motif, for example, biotin labeling, nuclease resistance modification, fluorescent labeling, protein binding motifs, etc. can be cited.

[0121] [Example]

[0122] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples.

[0123] 1. Design of tools for genome editing experiments

[0124] (1) crRNA

[0125] In this specification, when the base pair nucleotides formed with the target DNA are 20 nucleotides, "N" is set to an arbitrary nucleotide, and the nucleotide sequence of crRNA is 5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUG-3' (sequence number 2). In this embodiment, the target gene is slc45a2 of killifish, the target sequence containing PAM is 5'-ACGGAGCGCACTGGAAGCTGAGG-3' (sequence number 3), and the nucleotide sequence of crRNA is

[0126] 5'-ACGGAGCGCACUGGAAGCUGGUUUUAGAGCUAUGCUGUUUUG-3' (SEQ ID NO: 4). RNA synthesis was carried out by Fasmac.

[0127] (2) Conventional tracrRNA

[0128] In this specification, the nucleotide sequence of the conventional tracrRNA is Figure 2 In the sequence 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 1), nt Nos. 24 to 34 are referred to as "first stem-loop", nt Nos. 35 to 39 are referred to as "linker", and nt Nos. 40 to 53 are referred to as "second stem-loop". RNA synthesis was performed by Fasmac.

[0129] (3) Shortening of the 5' side sequence in tracrRNA

[0130] nt No. 1 to 8 were deleted from the conventional tracrRNA described in (2) above. The shortened nucleotide sequence was 5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGCU-3' (SEQ ID NO: 5). RNA synthesis was performed by Fasmac.

[0131] (4) Shortening of the linker in tracrRNA

[0132] nt No. 36 to 39 were deleted from the conventional tracrRNA described in (2) above. The shortened nucleotide sequence was 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUAACUUGAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 6). RNA synthesis was performed by Fasmac.

[0133] (5) Segmentation of the first stem-loop in tracrRNA

[0134] The conventional tracrRNA described in (2) above is divided into nt No. 1 to 28 and nt No. 29 to 69, and 5'-CCGG-3' is assigned to the 3' side of the first single-stranded RNA located at the 5' side, and 5'-CCGG-3' is assigned to the 5' side of the second single-stranded RNA located at the 3' side. The sequence of the unit is shown in Figure 3 a. The nucleotide sequence of the first single-stranded RNA is 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUCCGG-3' (SEQ ID NO: 7), and the nucleotide sequence of the second single-stranded RNA is 5'-CCGGAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 8). It is predicted that by forming Watson-Crick base pairs, Figure 3 nt No. 25, 26, 27, 28, 29, 30, 31, 32 of the first single-stranded RNA shown in sequence No. 7 in a will be Figure 3 It is predicted that nt No. 9, 8, 6, 5, 4, 3, 2, and 1 of the second single-stranded RNA shown in sequence No. 8 in a interact with each other by forming a mismatch base pair. Figure 3 The nt No. 24 of the first single-stranded RNA shown in sequence No. 7 in a will be Figure 3 a interacts with nt No. 10 of the second single-stranded RNA shown in sequence No. 8. The first "a" of sequence No. 7 interacts with Figure 2 The first position "a" of the serial number 1 shown in the figure corresponds to the fifth position "a" of the serial number 8. Figure 2The RNA is expressed as "a" at position 29 of SEQ ID NO: 1. RNA synthesis was performed by Fasmac.

[0135] (6) Segmentation of the second stem-loop in tracrRNA

[0136] The conventional tracrRNA described in (2) above is divided into nt No. 1 to 44 and nt No. 49 to 69, and 5'-GGCC-3' is assigned to the 3' side of the first single-stranded RNA located at the 5' side, and 5'-GGCC-3' is assigned to the 5' side of the second single-stranded RNA located at the 3' side. The sequence of the unit is shown in Figure 3 b. Figure 3 The nucleotide sequence of the first single-stranded RNA shown in sequence number 9 in b is 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GGCC-3' (sequence number 9), Figure 3 The nucleotide sequence of the second single-stranded RNA shown in sequence number 10 in b is 5'-GGCCAAGUGGCACCGAGUCGGUGCU-3' (sequence number 10). It is predicted that nt No. 41, 42, 43, 44, 45, 46, 47, 48 of the first single-stranded RNA will interact with nt No. 8, 7, 6, 5, 4, 3, 2, 1 of the second single-stranded RNA by forming Watson-Crick base pairs, and it is predicted that nt No. 40 of the first single-stranded RNA will interact with nt No. 9 of the second single-stranded RNA by forming mismatch base pairs. The first "a" of sequence number 9 interacts with Figure 2 The first position "a" of the serial number 1 shown in the figure corresponds to the fifth position "a" of the serial number 10. Figure 2 The RNA is synthesized by Fasmac.

[0137] (7) Shortening of the 5' side sequence in tracrRNA, shortening of the linker, and splitting of the second stem-loop

[0138] From the conventional tracrRNA described in (2) above, nt No. 1 to 8 and nt No. 36 to 39 are deleted, and the tracrRNA is divided into nt No. 9 to 35, 40 to 44, and nt No. 49 to 69, and 5'-GGCC-3' is added to the 3' side of the first single-stranded RNA located at the 5' side, and 5'-GGCC-3' is added to the 5' side of the second single-stranded RNA located at the 3' side. The sequence of the unit is shown in Figure 4 a, the predicted structure of the unit is shown in Figure 4b. The nucleotide sequence of the first single-stranded RNA is 5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUAACUUGGCC-3' (SEQ ID NO: 11), and the nucleotide sequence of the second single-stranded RNA is 5'-GGCCAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 10). It is predicted that by forming Watson-Crick base pairs, Figure 4 ntNo.29,30,31,32,33,34,35,36 of the first single-stranded RNA shown in sequence No.11 in a will be Figure 4 It is predicted that nt No. 8, 7, 6, 5, 4, 3, 2, and 1 of the second single-stranded RNA shown in sequence No. 10 in a interact with each other by forming a mismatch base pair. Figure 4 The ntNo.28 of the first single-stranded RNA shown in sequence No.11 in a will be Figure 4 a) interacts with nt No. 9 of the second single-stranded RNA represented by sequence No. 10. The first position "u" of sequence No. 11 interacts with Figure 2 The 9th bit "u" of the serial number 1 shown in the figure corresponds to the 5th bit "a" of the serial number 10. Figure 2 The RNA is synthesized by Fasmac.

[0139] (8) Separation of the first stem-loop and the second stem-loop in tracrRNA

[0140] The conventional tracrRNA described in (2) above is divided into nt No. 1 to 28, nt No. 29 to 44, and nt No. 49 to 69, and 5'-CCGG-3' is assigned to the 3' side of the first single-stranded RNA located at the most 5' side, 5'-CCGG-3' is assigned to the 5' side of the second single-stranded RNA located in the middle, and 5'-GGCC-3' is assigned to the 3' side, and 5'-GGCC-3' is assigned to the 5' side of the third single-stranded RNA located at the most 3' side. The sequence of the unit is shown in Figure 5 a, the predicted structure of the unit is shown in Figure 5 b. The nucleotide sequence of the first single-stranded RNA is 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUCCGG-3' (SEQ ID NO: 7), the nucleotide sequence of the second single-stranded RNA is 5'-CCGGAGUCCGUUAUCAACUUGGCC-3' (SEQ ID NO: 12), and the nucleotide sequence of the third single-stranded RNA is 5'-GGCCAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 10). It is predicted that by forming Watson-Crick base pairs, Figure 5 nt No. 25, 26, 27, 28, 29, 30, 31, 32 of the first single-stranded RNA shown in sequence No. 7 in a will be Figure 5 The nt No. 9, 8, 6, 5, 4, 3, 2, and 1 of the second single-stranded RNA shown in sequence No. 12 in a interact with each other, and it is predicted Figure 5 The nt Nos. 17, 18, 19, 20, 21, 22, 23, and 24 of the second single-stranded RNA shown in sequence No. 12 in a are Figure 5 The ntNo.8, 7, 6, 5, 4, 3, 2, and 1 of the third single-stranded RNA shown in sequence No. 10 in a interact with each other. By forming mismatched base pairs, it is predicted that Figure 5 The nt No. 24 of the first single-stranded RNA shown in sequence No. 7 in a will be Figure 5 a interacts with nt No.10 of the second single-stranded RNA represented by sequence No.12, and is predicted Figure 5 The nt No.16 of the second single-stranded RNA shown in sequence No.12 in a will be Figure 5 The first position "a" of sequence number 7 interacts with nt No. 9 of the third single-stranded RNA shown in sequence number 10 in a. Figure 2 The first digit "a" of the serial number 1 shown in the figure corresponds to the fifth digit "a" of the serial number 12. Figure 2 The 29th position "a" of the serial number 1 shown in the figure corresponds to the 5th position "a" of the serial number 10. Figure 2 The RNA is synthesized by Fasmac.

[0141] (9) Shortening of the 5' side sequence in tracrRNA, splitting of the first stem-loop, and splitting of the second stem-loop

[0142] nt No. 1 to 8 were deleted from the conventional tracrRNA described in (2) above, and the tracrRNA was divided into nt No. 9 to 28, nt No. 29 to 44, and nt No. 49 to 69. 5'-CCGG-3' was assigned to the 3' side of the first single-stranded RNA located at the most 5' side, 5'-CCGG-3' was assigned to the 5' side of the second single-stranded RNA located in the middle, and 5'-GGCC-3' was assigned to the 3' side, and 5'-GGCC-3' was assigned to the 5' side of the third single-stranded RNA located at the most 3' side. The sequence of the unit is shown in Figure 6 a, the predicted structure of the unit is shown in Figure 6b. The nucleotide sequence of the first single-stranded RNA is 5'-UAGCAAGUUAAAAUAAGGCUCCGG-3' (SEQ ID NO: 13), the nucleotide sequence of the second single-stranded RNA is 5'-CCGGAGUCCGUUAUCAACUUGGCC-3' (SEQ ID NO: 12), and the nucleotide sequence of the third single-stranded RNA is 5'-GGCCAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 10). It is predicted that by forming Watson-Crick base pairs, Figure 6 The ntNo.17, 18, 19, 20, 21, 22, 23, 24 of the first single-stranded RNA shown in sequence No. 13 in a will be Figure 6 The nt No. 9, 8, 6, 5, 4, 3, 2, and 1 of the second single-stranded RNA shown in sequence No. 12 in a interact with each other, and it is predicted Figure 6 The nt Nos. 17, 18, 19, 20, 21, 22, 23, and 24 of the second single-stranded RNA shown in sequence No. 12 in a are Figure 6 The nt Nos. 8, 7, 6, 5, 4, 3, 2, and 1 of the third single-stranded RNA shown in sequence No. 10 in a interact with each other. It is predicted that by forming a mismatch base pair, Figure 6 The nt No. 16 of the second single-stranded RNA shown in sequence No. 13 in a will be Figure 6 a interacts with nt No.10 of the second single-stranded RNA represented by sequence No.12, and is predicted Figure 6 The nt No.16 of the second single-stranded RNA shown in sequence No.12 in a will be Figure 6 a interacts with nt No. 9 of the third single-stranded RNA shown in sequence No. 10. The first position "u" of sequence No. 13 interacts with Figure 2 The 9th bit "u" of the serial number 1 shown in the figure corresponds to the 5th bit "a" of the serial number 12. Figure 2 The 29th position "a" of the serial number 1 shown in the figure corresponds to the 5th position "a" of the serial number 10. Figure 2 The RNA is synthesized by Fasmac.

[0143] 2. In vitro cleavage assay

[0144] (1) Preparation of substrate DNA

[0145] PCR amplification of the target sequence around the slc45a2 locus was performed from the genomic DNA of the killifish using KOD FX (Toyobo Co., Ltd.) and the forward primer 5'-GTCACTCTGTATTGGGGCGCCTCCT-3' (SEQ ID NO. 14) and the reverse primer 5'-CTTGGCCTGATCTCAGGGCTGGATG-3' (SEQ ID NO. 15). The synthesis of the substrate was performed by Eurofins Genomics KK. The amplified product was purified with NucleoSpin Gel and PCR Clean-up (Takara Bio Co., Ltd.), and the DNA concentration was determined using NanoDrop (Thermo Fisher Scientific). Loading buffer was added to a solution containing 100 ng of DNA, and the bands were confirmed by electrophoresis in an agarose gel to which Midori Green (Japan Genetics Co., Ltd.) was added. The designed nucleotide length is 253 base pairs.

[0146] (2) Annealing of crRNA and tracrRNA units

[0147] The crRNA targeting the slc45a2 gene of killifish described in 1. (1) above at 40 to 200 ng / μL (final concentration) and the first single-stranded RNA and the second single-stranded RNA described in 1. (2) to (9) above, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are mixed in an equimolar amount. The number of moles of each single-stranded RNA constituting tracrRNA is calculated as follows based on the nucleotide length.

[0148] (crRNA concentration ng / μL)×(nucleotide length of each single-stranded RNA) / 42

[0149] The mixed solution was heated at 95°C for 5 minutes using a thermal cycler, then cooled to 25°C at a rate of 0.1°C per second, and stored at 4°C.

[0150] (3) In vitro cleavage test

[0151] To the RNA solution annealed in (2) above, add 0.5-2 μg / μL (final concentration) of Cas9 protein (Fasmac), 20 ng / μL (final concentration) of the substrate DNA prepared in (1) above, and H buffer (Takara Bio). Use a thermal cycler to react at 37°C for 60 minutes, then heat-treat at 80°C for 5 minutes, and store at 4°C.

[0152] (4) Agarose gel electrophoresis

[0153] RNaseA (NIPPON GENE CO., LTD) 1 μg / μL (final concentration) was added to the cleavage product of (3) above, and the reaction was carried out at 37°C for 30 minutes using a thermal cycler, followed by heat treatment at 80°C for 5 minutes, and then stored at 4°C. The loading buffer was added, and electrophoresis was performed on a 3% agarose gel to which Midori Green (Japan Genetics) was added, and the bands were confirmed by LED irradiation. The uncut substrate DNA was 253 base pairs, and the cut substrate DNA was predicted to be 144 base pairs and 109 base pairs.

[0154] (5) Results

[0155] Above Figure 7 The results of an in vitro cleavage test using the Cas9 protein derived from Streptococcus pyogenes and the annealed crRNA described in 1.(1) and the tracrRNA described in 1.(2) to (9) consisting of the first single-stranded RNA and the second single-stranded RNA, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are shown in. In each design, cleavage products of 114 base pairs and 109 base pairs were generated, confirming that they had DNA cleavage activity.

[0156] 3. Introducing mutations into killifish fertilized eggs

[0157] (1) Obtaining fertilized killifish eggs

[0158] Sexually mature male and female killifish are allowed to coexist in a tank, and fertilized eggs are collected just after spawning.

[0159] (2) Microinjection for injecting CRISPR solution into cells

[0160] The method described in (1) above was used to obtain fertilized eggs of killifish. The crRNA targeting the slc45a2 locus of killifish was annealed with the tracrRNA consisting of the first single-stranded RNA and the second single-stranded RNA, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA, and Cas9 protein (Fasmac) 0.5-2 μg / μL (final concentration), GFP mRNA 50ng / μL (final concentration), crRNA 40-160ng / μL (final concentration), tracrRNA 60-240ng / μL (final concentration) were added, and 1-5nL was introduced by microinjection per fertilized egg, transferred to feeding water added with a small amount of methylene blue, and maintained at 26°C. The next day, embryos with GFP fluorescent signals were selected as eggs in which the CRISPR solution was correctly injected into the cells.

[0161] (3) Analysis of phenotypes using genome editing

[0162] Four days after microinjection, the eyes of the killifish embryos were observed using a stereomicroscope (Olympus). When a mutation is introduced into the slc45a2 gene, melanocytes are not formed, so it is expected that part or all of the eyes lack melanin. The embryos that have been observed are maintained until the analysis described in the next section.

[0163] (4) Preparation of Killifish Broken Liquid

[0164] The fertilized eggs obtained in (3) above were transferred to a feeding water prepared by adding a small amount of methylene blue to 1× killifish ECM (0.1% NaCl, 0.003% KCl, 0.004% CaCl2, 0.016% MgSO4) and maintained in an incubator at 26°C. After 5 to 10 days, 25 μL of an alkaline buffer solution (25 mM NaOH, 0.2 mM EDTA) was added, and the egg membrane was pierced with a 200 μL pipette tip, and heat-treated at 95°C for 5 minutes using a thermal cycler. After neutralization by adding an equal amount (25 μL) of 40 mM Tris-HCl (pH 8.0), the eggs were stored at 4°C as genomic DNA samples.

[0165] (5) Mutation analysis based on heteroduplex mobility assay (HMA)

[0166] 5 to 10 days after microinjection, the killifish embryos were disrupted using the method described in (4) above. The region described in 2. (3) above was amplified by PCR and electrophoresed using MultiNA (Shimadzu Corporation). When a mutation is introduced into the target region, heteroduplexes are generated during the annealing process of PCR. It is predicted that band shifts will occur due to differences in mobility due to differences in physical properties. When mutations are introduced more strongly, a variety of mutations will occur, so it is predicted that band shifts will be detected more strongly.

[0167] (6) Mutation analysis based on Sanger sequencing

[0168] The amplified product generated by PCR described in (3) above was used to obtain the waveform of the nucleotide sequence by Sanger sequencing. The Sanger sequencing was performed by Eurofins Genomics KK. The sequence and frequency of the mutant allele were estimated using TIDE (Netherlands Cancer Institute).

[0169] (7) Results

[0170] Figure 8The results of observation of the eyes of killifish embryos injected with Cas9 protein and annealed crRNA described in 1.(1) above and tracrRNA composed of the first single-stranded RNA and the second single-stranded RNA described in 1.(5) to (9) above, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are shown. In all multiple embryos, the lack of melanin in the whole or part of the eye was observed.

[0171] Fig. 9 The results of HMA analysis of killifish embryo lysate injected with Cas9 protein and annealed crRNA described in 1.(1) above and tracrRNA composed of the first single-stranded RNA and the second single-stranded RNA described in 1.(5) to (9) above, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are shown. Band shift due to mutation introduction was observed in all multiple embryos.

[0172] Figure 10-1 to Figure 10-5 The results of mutation analysis based on Sanger sequencing and TIDE using killifish embryo lysate injected with Cas9 protein and annealed crRNA described in 1.(1) above and tracrRNA composed of the first single-stranded RNA and the second single-stranded RNA described in 1.(5) to (9) above, or the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are shown. The presence of mutant alleles with base deletion or insertion was observed in all multiple embryos.

[0173] The above results indicate that even when tracrRNA is fragmented and a portion of the nucleotide sequence is deleted, the function of tracrRNA is maintained.

Claims

1. A tracrRNA unit, which is a tracrRNA unit for a CRISPR-Cas9 genome editing system, consisting of a first single-stranded RNA and a second single-stranded RNA, The first single-stranded RNA and the second single-stranded RNA are not continuous. The first single-stranded RNA has at least a first portion and a second portion, Part 1 and part 2 do not overlap, part 1 has a sequence complementary to a portion of crRNA, and part 2 has a sequence complementary to the second single-stranded RNA.

2. A tracrRNA unit, which is a tracrRNA unit for a CRISPR-Cas9 genome editing system, and is composed of a first single-stranded RNA, a second single-stranded RNA, and a third single-stranded RNA, The first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA are not continuous. The first single-stranded RNA has at least a first portion and a second portion, Part 1 and part 2 do not overlap, part 1 has a sequence complementary to a portion of crRNA, and part 2 has a sequence complementary to a second single-stranded RNA, The second single-stranded RNA has at least the first portion, The first portion of the second single-stranded RNA has a sequence complementary to the third single-stranded RNA.

3. The tracrRNA unit according to claim 1 or 2, wherein The crRNA and the first single-stranded RNA are linked in this order.

4. The tracrRNA unit according to claim 2, wherein The crRNA, the first single-stranded RNA, and the second single-stranded RNA are linked in this order.

5. The tracrRNA unit according to claim 1, wherein At least one of the first single-stranded RNA and the second single-stranded RNA has a length of 39 nucleotides or less.

6. The tracrRNA unit according to claim 2, wherein At least one of the first single-stranded RNA, the second single-stranded RNA, and the third single-stranded RNA has a length of 39 nucleotides or less.

7. A genome editing method, using: The tracrRNA unit according to any one of claims 1 to 6, crRNA, and Cas9.

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