WO2021085580A1 - Polynucleotide and use for same - Google Patents

Polynucleotide and use for same Download PDF

Info

Publication number
WO2021085580A1
WO2021085580A1 PCT/JP2020/040738 JP2020040738W WO2021085580A1 WO 2021085580 A1 WO2021085580 A1 WO 2021085580A1 JP 2020040738 W JP2020040738 W JP 2020040738W WO 2021085580 A1 WO2021085580 A1 WO 2021085580A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
sequence
cre
encoding
recombinant
Prior art date
Application number
PCT/JP2020/040738
Other languages
French (fr)
Japanese (ja)
Inventor
雄二 恒川
文雄 松崎
Original Assignee
国立研究開発法人理化学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立研究開発法人理化学研究所 filed Critical 国立研究開発法人理化学研究所
Publication of WO2021085580A1 publication Critical patent/WO2021085580A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • the present invention relates to polynucleotides, vectors, gene recombination kits, Cre modified proteins or fusion proteins thereof, and gene recombination methods.
  • recombinase a gene recombination technology using a recombinant enzyme
  • An example of such a gene recombination technique is a Cre / loxP system using Cre recombinase and loxP sequences.
  • the recombinant enzyme and a promoter sequence that functions specifically at a site are used in combination to form a site-specific group rather than the whole body. Recombination can occur (see, for example, Non-Patent Documents 1 and 2).
  • the present inventors have assembled even in cells in which the recombinant enzyme should not be expressed when a donor DNA containing a polynucleotide encoding a recombinant enzyme is introduced into the cell.
  • the present inventors translated the transcript after the polynucleotide encoding the recombinant enzyme contained in the donor DNA before being knocked in was transcribed non-specifically to the cell type. I faced a new challenge of In the present specification, this phenomenon is referred to as "leak".
  • This phenomenon of "leakage” can be a particular problem when a large amount of donor DNA is used to introduce the recombinant enzyme into cells.
  • the de novo knock-in method developed by the present inventors (Tsunekawa Y et al. (2016) "Developing a de novo targeted knock-in method based on in utero electroporation into the mammal brain," Development, Vol.143 (No) .17), pp.3216-3222.)
  • the HITI method (Suzuki K, Tsunekawa Y, et al. (2016) "In vivo genome editing via CRISPR / Cas9 mediated mammal-independent targeted integration," Nature, Vol.540 (Issue 7331), pp.144-149.) Can be a particular problem.
  • One aspect of the present invention is to provide a polynucleotide capable of reducing the occurrence of leaks.
  • the present inventors have found that the occurrence of leaks can be reduced by controlling the step of translation among the two steps of transcription and translation involved in the leak, and complete the present invention. I arrived. More specifically, we found that the occurrence of leaks can be reduced by substituting at least some of the translation initiation codons present in the polynucleotide encoding the recombinant enzyme with untranslation initiation codons. We have found and completed the present invention.
  • the present invention includes the following configurations. ⁇ 1> The polynucleotide shown in any of the following (G1) to (G4): (G1) (i) In a polynucleotide encoding a recombinant enzyme, a protein consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) a protein having recombinant activity.
  • the polynucleotide encoding (G2) (i) At least one translation initiation in a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme.
  • a polynucleotide consisting of a polynucleotide whose codon has been replaced with an untranslated start codon and (ii) encoding a protein having recombinant activity; (G3) (i) Consists of a polynucleotide in which at least one start codon is replaced with a non-translation start codon in a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme.
  • a polynucleotide encoding a protein having recombinant activity (G4) (i) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is untranslated.
  • a polynucleotide consisting of a polynucleotide substituted with a codon and (ii) encoding a protein having recombinant activity (ii) The polynucleotide according to ⁇ 1>, wherein the translation start codon is ATG, CTG, TTG, GTG, or ATA.
  • ⁇ 3> The polynucleotide according to ⁇ 1> or ⁇ 2>, wherein the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof.
  • ⁇ 4> A vector containing the polynucleotide according to any one of ⁇ 1> to ⁇ 3>.
  • ⁇ 5> The vector according to ⁇ 4>, further comprising a splicing acceptor sequence on the 5'end side of the polynucleotide.
  • ⁇ 6> A genetic recombination kit comprising the polynucleotide according to any one of ⁇ 1> to ⁇ 3> or the vector according to ⁇ 4> or ⁇ 5>.
  • a genetic recombination method comprising the step of introducing the polynucleotide according to any one of ⁇ 1> to ⁇ 3> or the vector according to ⁇ 4> or ⁇ 5> into a cell or a non-human subject.
  • a polynucleotide capable of reducing the occurrence of leaks is provided.
  • FIG. 1 It is a schematic diagram comparing the conventional donor DNA and the donor DNA in one embodiment of the present invention. It is a schematic diagram explaining the vector which concerns on one Embodiment of this invention and the method of using it. It is a schematic diagram which shows the composition of the donor DNA used in Example 1.
  • FIG. It is a figure which shows the result of Example 1.
  • FIG. It is a schematic diagram showing Cre1 to Cre6 (wild type Cre and a partial peptide thereof) produced in Example 2. It is a figure which shows the result of Example 2.
  • FIG. It is a figure which shows the result of Example 3.
  • FIG. It is a schematic diagram which shows the composition of the donor DNA used in Example 4 and knockin sight of the donor DNA. It is a figure which shows the result of Example 4.
  • Example 7 It is a schematic diagram which shows the composition of the donor DNA used in Example 5 and knockin sight of the donor DNA. It is a figure which shows the result of Example 5. It is a schematic diagram which shows the composition of the donor DNA used in Example 6 and knockin sight of the donor DNA. It is a figure which shows the result of Example 6. It is the result of Example 7, and is the figure which shows the EGFP expression rate of a negative control. It is the result of Example 7, and is the figure which shows the EGFP expression rate of a positive control. It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used the wild type Cre. It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used Opti Cre2. It is a figure which shows the result of Example 7. It is a figure which shows the result of Example 8.
  • polynucleotide is DNA
  • T thymine
  • U uracil
  • the upper panel of FIG. 1 represents the donor DNA 10 in the prior art.
  • the donor DNA 10 contains a polynucleotide 1 encoding a recombinant enzyme.
  • the polynucleotide 1 encoding the recombinant enzyme contains a translation initiation codon 5 (such as ATG).
  • the recombinant enzyme is released from the donor DNA 10 (polynucleotide 1 encoding the recombinant enzyme) even when the donor DNA 10 is not knocked in. It will be expressed. This phenomenon is referred to herein as a "leak".
  • the lower panel of FIG. 1 represents the donor DNA 10a in one embodiment of the present invention.
  • the donor DNA 10a contains the modified polynucleotide 1a. Specifically, in the modified polynucleotide 1a, at least one translation initiation codon 5 is replaced with the untranslation initiation codon 5a in the polynucleotide 1 encoding the recombinant enzyme. According to this configuration, leaks can be reduced at the translation level rather than at the transcription level (see Examples 1 and 3 for the effectiveness of the leak reduction strategy at the translation level).
  • reducing leakage means, for example, that the donor DNA 10a in one embodiment of the present invention is a recombinant enzyme derived from a donor DNA that has not been knocked in, rather than the donor DNA 10 of the prior art. It represents a state in which the expression level is reduced.
  • the expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state is 50% or less of the expression level of the recombinant enzyme derived from the donor DNA 10 of the prior art in the non-knocked state. 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 1% or less, or 0%.
  • the expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state and the expression level of the recombinant enzyme derived from the donor DNA 10 in the non-knocked state were measured by, for example, the method used in the examples. obtain. Specifically, it can be measured by introducing donor DNA10 or donor DNA10a into the cell without Cas9 (or its expression vector) and measuring the expression level of the recombinant enzyme. Methods for quantifying and comparing the expression levels of recombinant enzymes are well known in the art.
  • the modified polynucleotide 1a encodes a protein having recombinant activity. That is, the translated product of the modified polynucleotide 1a has recombinant activity.
  • the recombinant activity of the translated product of the modified polynucleotide 1a is 50% or more, 60% or more, 70% of the recombinant activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme. Above, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • the recombination activity of the translation product of the modified polynucleotide 1a may be 100% or more of the recombination activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme.
  • Whether or not the translated product of the modified polynucleotide 1a has recombinant activity can be confirmed by an assay common in the art (see Example 2 for a specific example). .. Alternatively, it is possible to estimate whether or not the translated product of the modified polynucleotide 1a has recombination activity by three-dimensional structural analysis. Further, a method for quantifying and comparing recombinant activity is a technique well known in the art (see Examples 7 and 8 for specific examples).
  • polynucleotide in the present specification includes RNA and DNA.
  • examples of polynucleotides in the form of RNA include mRNA.
  • examples of polynucleotides in the form of DNA include various DNA fragments, cDNAs, genomic DNAs.
  • RNA and DNA can have any structure (such as double-stranded or single-stranded).
  • the polynucleotide according to one embodiment of the present invention is the polynucleotide shown in any of the following (G1) to (G4).
  • G1 (I) In a polynucleotide encoding a recombinant enzyme, a polynucleotide consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) encoding a protein having recombinant activity. Polynucleotide that is.
  • G2 In a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme, at least one translation start codon is not present.
  • G3 In a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is substituted with a non-translation initiation codon, and (i) ii) A polynucleotide encoding a protein having recombinant activity.
  • G4 In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is replaced with a non-translation initiation codon.
  • Ii A polynucleotide encoding a protein having recombinant activity.
  • (G1) is intended as "a polynucleotide in which one or more translation initiation codons contained in the polynucleotide are replaced with non-translation initiation codons from a polynucleotide encoding a wild-type recombinant enzyme".
  • (G2) to (G4) refer to "one or more start codons contained in the polynucleotide from a variant or mutant polynucleotide of the polynucleotide encoding the wild-type recombinant enzyme”. It is intended as a "polynucleotide substituted with an untranslated start codon".
  • the polynucleotide encodes a protein having recombinant activity is determined by inserting the polynucleotide into a desired expression vector and then using the expression vector as a desired host. It can be confirmed by introducing and detecting whether or not gene recombination (for example, homologous recombination, non-homologous recombination) occurs in the host. If the gene has been recombined in the host, the polynucleotide can be determined to be "a polynucleotide encoding a protein having recombination activity". The detection of gene recombination may be carried out according to a known method (see, for example, Examples).
  • "several bases” are, for example, 50, 45, 40, 35, 30, 25, 20, 19, 19, 18, 17, 16, 15 There are 14, 14, 12, 11, 10, 10, 9, 8, 7, 6, 5, 4, 3, and 2 bases.
  • sequence identity is intended to be the proportion of the same number of bases.
  • sequence identity of the base sequence can be determined using, for example, BLASTN (Altschul SF (1990) "Basic local alignment search tool", Journal of Molecular Biology, Vol.215 (Issue 3), pp.403). -410).
  • Additions or deletions may be allowed to optimally align the base sequences to be compared.
  • the above-mentioned sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably. Is 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • hybridizing under stringent conditions means that hybridization is performed at 50 to 60 ° C. for 16 hours in a hybridization solution having a salt concentration of 6 ⁇ SSC to obtain a salt of 0.1 ⁇ SSC.
  • the condition for hybridization after washing in a solution of a concentration is sodium chloride: 150 mM and sodium citrate: 15 mM.
  • the translation initiation codon substituted with the untranslation initiation codon is not particularly limited.
  • Examples of translation start codons include ATG, CTG, TTG, GTG, ATA and the like. Of these, ATG is a typical translation initiation codon that is common to a wide range of species. On the other hand, CTG, TTG, GTG and ATA are atypical translation initiation codons that have been reported to function as translation initiation codons. Therefore, in the polynucleotide according to one embodiment of the present invention, it is preferable that one or more of the ATGs are replaced with non-translation initiation codons.
  • one or more of the ATGs are replaced with untranslated initiation codons, and one or more of the atypical translation initiation codons are untranslated initiation codons. It is more preferable that it is replaced with a codon.
  • the non-translation start codon in which the translation start codon is replaced is not particularly limited.
  • the methionine encoded by ATG is not encoded by codons other than ATG. Therefore, when ATG is replaced with an untranslated start codon, the ATG encoding methionine is inevitably replaced with a codon encoding an amino acid other than methionine.
  • the ATG is preferably replaced with a codon encoding leucine (TTA, TTG, CTT, CTC, CTA or CTG). This is because methionine and leucine have similar properties as amino acids (hydrophilic / hydrophobic, isoelectric point, three-dimensional structure, etc.). Which codon encoding leucine is replaced with ATG can be appropriately determined depending on, for example, the codon usage frequency of the organism into which the polynucleotide is introduced.
  • CTG, TTG, GTG and ATA can be replaced with non-translation start codons without changing the encoded amino acids.
  • CTG is replaced with CTC.
  • TTG is replaced with TTA.
  • GTG is replaced by GTA.
  • ATA is replaced with ATT.
  • the position of the translation initiation codon to be replaced with the non-translation initiation codon is not particularly limited as long as the desired translation product has recombination activity.
  • the position of the translation initiation codon substituted with the non-translation initiation codon is, for example, the amino terminal of the recombinant enzyme, the region on the amino terminal side of the recombinant enzyme, and the like.
  • the position may correspond to the region on the amino-terminal side of the central amino acid of the recombinant enzyme and the region on the amino-terminal side of the catalytic domain of the recombinant enzyme.
  • the number of translation initiation codons to be replaced with non-translation initiation codons is not particularly limited as long as the desired translation product has recombinant activity. For example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the number of translation initiation codons replaced by untranslation initiation codons increases.
  • the upper limit of the number of translation initiation codons to be replaced with the untranslation initiation codon is not particularly limited, and may be 10, 20, 30, 40, 50, or 100.
  • the ratio of translation initiation codons replaced with non-translation initiation codons is not particularly limited as long as the translation product has recombinant activity. For example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the proportion of translation initiation codons replaced by untranslation initiation codons increases.
  • the upper limit of the ratio of translation initiation codons substituted with untranslated initiation codons is not particularly limited, and is 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less. , 80% or less, 90% or less, or 100% or less.
  • the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof (eg, a codon variant obtained by codon-optimizing a wild-type nucleotide sequence).
  • the recombinant enzyme is Cre, FLP, or Dre. More preferably, the recombinant enzyme is Cre.
  • recombinant enzymes are known recombinant enzymes, and the base sequences encoding them are also known.
  • the base sequence encoding the above recombinant enzyme is as follows. Cre: (SEQ ID NO: 1) FLP: (SEQ ID NO: 2) Dre: (SEQ ID NO: 3) Tre: (SEQ ID NO: 4).
  • Examples of the above-mentioned modified enzyme of the recombinant enzyme include VCre, SCre, FLPO, FLPE, DreE and the like. These variants are known variants, and the base sequences encoding them are also known. Specific nucleotide sequences are as follows: SEQ ID NO: 25 (VCre), SEQ ID NO: 26 (SCre), SEQ ID NO: 27 (FLPO), SEQ ID NO: 28 (FLPE).
  • the polynucleotide according to one embodiment of the present invention may be one or more selected from the group consisting of the following (G1') to (G4').
  • G1' (I) In the polynucleotide consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 4, at least one translation initiation codon is substituted with a non-translation initiation codon, and (ii) set.
  • the vector according to one aspect of the present invention contains the above-mentioned polynucleotide. By introducing such a vector into cells, leakage derived from the vector (for example, donor DNA) can be reduced.
  • vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, virus-derived vectors (bacterial plasmids, bacteriophage, yeast episomes, etc.), yeast chromosomal elements, viruses (vaculovirus, etc.).
  • examples include papovavirus, vaccinia virus, adenovirus, adeno-associated virus, tripoxvirus, pseudomad dog disease virus, herpesvirus, lentivirus, retrovirus, etc.), and vectors derived from these combinations (cosmid, phagemid, etc.).
  • the plasmid vector is preferable because of its high versatility.
  • a plasmid vector is preferable in that a large amount of donor DNA can be introduced into the cell.
  • the vector contains a splicing acceptor sequence (SA sequence) on the 5'end side of a polynucleotide sequence in which the translation initiation codon is replaced with a nontranslation initiation codon.
  • the vector has a cleavage site (such as a gRNA recognition sequence), a buffering polynucleotide, and a splicing acceptor sequence (such as a gRNA recognition sequence) on the 5'end of a polynucleotide sequence in which the translation initiation codon has been replaced with a non-translation initiation codon.
  • SA sequence is included in this order.
  • the composition of the vector is such that a cleavage site (gRNA recognition sequence, etc.), a polynucleotide for buffering, and a splicing acceptor sequence are arranged on the 5'terminal side of the modified polynucleotide 1a.
  • the splicing acceptor sequence When producing the above-mentioned mature mRNA without using the splicing acceptor sequence, it is necessary to knock in the modified polynucleotide 1a immediately after exon E2. On the other hand, if the splicing acceptor sequence is used, it is not necessary to knock in the modified polynucleotide 1a immediately after the exon E2. Instead, the modified polynucleotide 1a may be knocked in anywhere in the intron I2. Therefore, the use of the splicing acceptor sequence has an advantage that the number of knock-in sites of the modified polynucleotide 1a can be increased. This advantage is, in other words, the advantage of facilitating the design of gRNAs.
  • indel may occur at the boundary region between the polynucleotide 1a and the exon E2. If indel occurs at the boundary region between polynucleotide 1a and exon E2, the amino acid sequence encoded by exon E2 may change, or a frame shift may occur during translation of polynucleotide 1a.
  • the insertion site should be intron I1 (intron between the first exon and the second exon). It is preferable to set it.
  • the SA sequence may be a sequence unique to the knock-in site or a more versatile sequence (see Examples 4 and 5 for details).
  • the vector according to one embodiment of the present invention may contain an arbitrary sequence in addition to the modified polynucleotide 1a.
  • the modified polynucleotide 1a may contain a polynucleotide sequence that expresses another protein (such as Cas9).
  • kit ⁇ One aspect of the present invention is a genetically modified kit comprising the above-mentioned polynucleotide or the above-mentioned vector.
  • kit means any combination of reagents and the like used for any purpose. This use may be for medical use or for experimental use. Specifically, the application may be a genome editing application.
  • the kit comprises reagents and / or ancillary substances used for genetic recombination.
  • reagents and / or ancillary substances include nucleases (such as Cas), gRNAs, TALENs, ZFNs.
  • the kit may include one or more containment vessels (boxes, bottles, dishes, etc.) for storing reagents and / or ancillary substances.
  • the kit may include two or more containers, each container containing a portion of the components of the kit. At this time, the user may receive each container collectively or individually.
  • Protein (modified recombinant enzyme) One aspect of the present invention is a protein (modified recombinant enzyme) which is a translation product of the above-mentioned polynucleotide (modified polynucleotide 1a).
  • modified polynucleotide 1a modified polynucleotide 1a
  • the protein is a recombinant enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 10, 11 or 12.
  • These recombinant enzymes are translation products of "Opti Cre2", “Opti FLP” and “Opti Dre” in Examples described later.
  • the protein may be a fusion protein with another protein. Since the method for producing the fusion protein is well known in the art, the description thereof will be omitted.
  • the genetic recombination method includes the step of introducing the above-mentioned polynucleotide or the above-mentioned vector into a cell or a subject (for example, a human subject or a non-human subject).
  • introducing into a cell is intended to introduce, for example, a polynucleotide or vector into a cell in vitro.
  • introducing into a subject is intended to introduce, for example, a polynucleotide or vector into cells constituting a living body in vivo.
  • the subject is not human.
  • the subject is a non-human mammal.
  • non-human mammals include cloven-hoofed animals (boars, wild boars, pigs, sheep, goats, etc.), cloven-hoofed animals (horses, etc.), carnivores (mouse, rats, hamsters, squirrels, etc.) Etc.), meats (dogs, cats, ferrets, etc.).
  • the non-human mammals described above include wild animals in addition to livestock or companion animals (pets).
  • the method for introducing a polynucleotide or vector into a cell or a subject is not particularly limited.
  • a method for introducing a vector into a cell or a subject an appropriate method depending on the vector is widely known in the art.
  • Methods for introducing a polynucleotide as a DNA molecule (or RNA molecule) into a cell or subject include electroporation, microinjection, sonoporation, laser irradiation, and cationic substances (cationic polymers, cationic lipids, etc.). Transfection using complexation with calcium phosphate, etc.) can be mentioned.
  • the gene recombination method according to one embodiment of the present invention is preferably combined with an introduction form that introduces a large amount of unmodified DNA into a cell.
  • introduction forms include electroporation, virus, microinjection, lipid complex transfection and the like.
  • the protein encoded by the modified polynucleotide 1a is a Cre modified protein.
  • the modified polynucleotide 1a has one or more of the wild-type Cre translation initiation codons ATG replaced with non-translation initiation codons (preferably leucine-encoding codons). More preferably, the modified polynucleotide 1a contains one or more of the translation initiation codons (ATGs) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild Cre. , Is replaced with an untranslated start codon (preferably a codon encoding leucine).
  • ATGs translation initiation codons
  • the modified polynucleotide 1a has a translation initiation codon (ATG) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild-type Cre, which is a non-translation initiation codon (ATG). Preferably, it is substituted with a codon encoding leucine).
  • ATG translation initiation codon
  • ATG non-translation initiation codon
  • ATG non-translation initiation codon
  • it is substituted with a codon encoding leucine.
  • An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 5. This base sequence corresponds to "Opti Cre" in the examples.
  • the modified polynucleotide 1a is one in which one or more of the atypical translation initiation codons of wild Cre is substituted with the untranslation initiation codon in addition to the substitution of the translation initiation codon ATG described above. is there.
  • the atypical translation start codon to non-translation start codon substitutions are (i) CTG to CTC substitution, (ii) TTG to TTA substitution, (iii) GTG to GTA substitution, Or (iv) substitution from ATA to ATT.
  • the atypical translation initiation codon substituted for the untranslation initiation codon is within 30 bp, within 60 bp, within 90 bp, within 120 bp, within 180 bp, 210 bp from the 5'end of the polynucleotide encoding the wild-type Cre.
  • An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 6. This base sequence corresponds to "Opti Cre2" in the examples.
  • Cre-modified protein which is a translation product of the modified polynucleotide 1a of the embodiment described in this item, is also a preferred embodiment of the present invention.
  • amino acid sequence of the Cre modified protein include the amino acid sequence set forth in SEQ ID NO: 10. This amino acid sequence is a translation of "Opti Cre” or "Opti Cre 2" in the Examples. It has been suggested that this translation product may have reduced toxicity found in wild-type Cre (see Example 7 for details).
  • pLeaklessIII vector developed in 2016 development
  • Infusion manufactured by TAKARA
  • TAKARA TAKARA
  • Recombinant enzyme Wild-type Cre, Cre (Opti Cre) in which the translation start codon (ATG) was replaced with the untranslated start codon, and Cre (Opti Cre2) in which the atypical translation start codon was also replaced with the untranslated start codon were used.
  • the codon (ATG) corresponding to the 1st, 28th, 30th, 58th, and 77th methionine residues of wild-type Cre is encoded by the leucine residue. Substituted with codons (TTA, TTG, CTT, CTC, CTA or CTG).
  • Opti Cre2 the atypical translation initiation codon contained in Opti Cre was also replaced with the non-translation initiation codon. Specifically, CTG, which exists from the 5'end to 600 bp, was replaced with CTC, TTG was replaced with TTA, GTG was replaced with GTA, and ATA was replaced with ATT.
  • CTC which exists from the 5'end to 600 bp
  • TTG was replaced with TTA
  • GTG was replaced with GTA
  • ATA was replaced with ATT.
  • the amino acid encoded by the codon before substitution and the amino acid encoded by the codon after substitution are the same amino acid.
  • the nucleotide sequence encoding the wild-type Cre is SEQ ID NO: 1, and the amino acid sequence of the wild-type Cre is SEQ ID NO: 9.
  • the nucleotide sequence encoding Opti Cre is SEQ ID NO: 5, and the amino acid sequence of Opti Cre is SEQ ID NO: 10.
  • the nucleotide sequence encoding Opti Cre2 is the same as SEQ ID NO: 6, and the amino acid sequence of Opti Cre2 is the same as the amino acid sequence of Opti Cre.
  • Humanized Cas9 (hCas9) was used for the introduction of the recombinant enzyme.
  • the nucleotide sequence encoding hCas9 is SEQ ID NO: 13.
  • mCherry red fluorescent protein
  • flox-STOP-EGFP sequence a sequence constructed so that EGFP is expressed by the expression of Cre
  • the base sequence encoding mChery is SEQ ID NO: 14.
  • the nucleotide sequence encoding the flox-STOP-EGFP sequence is SEQ ID NO: 15.
  • the CAG promoter was used as the promoter sequence for non-specific gene expression.
  • a CAX promoter sequence modified from the CAG promoter was used as a promoter sequence for site-specific expression of a gene in a nerve cell.
  • the promoter regions of the CAG promoter and the CAX promoter are the same, and the SEQ ID NO: 24.
  • SA sequence The SA sequence of mouse Tubb3 gene 4th exon (SEQ ID NO: 16) or the SA sequence of human immunoglobulin (chimeric intron; SEQ ID NO: 17) was used.
  • the plasmid was introduced into the cerebral cortex of a mouse (or ferret) fetal by in utero electroporation.
  • Example 1 Leakage of wild-type Cre gene
  • the presence or absence of leaks from the donor DNA was examined using donor DNA containing wild-type Cre (Cr that did not replace the translation initiation codon). Specifically, wild-type Cre was knocked in to the Tubb3 locus specifically expressed in nerve cells. The HITI method was adopted for gene transfer. Since the HITI method does not require a sequence such as a homology arm, the influence of sequences other than Cre on the leak can be minimized.
  • the specific composition of the donor DNA containing the wild-type Cre is shown in FIG.
  • a gRNA recognition sequence SEQ ID NO: 18
  • SEQ ID NO: 18 a gRNA recognition sequence existing at the 3'end of the Tubb3 gene is arranged at both ends of the base sequence encoding the wild-type Cre.
  • mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
  • -PCAX-hCas9 expression vector of hCas9
  • PCAG-mCherry-gRNA expression vector of mCherry and gRNA
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • Cre Donner DNA
  • Example 2 Relationship between methionine contained in wild-type Cre and recombinant activity
  • the following vector was introduced into the cerebral cortex of a mouse fetal on the 14th day of embryonic development.
  • -PCAG-Cre-1-6 expression vector of any of Cre1 to Cre6
  • PCAG-mChery expression vector of mCherry
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence.
  • Example 3 Leakage of Opti Cre gene
  • Opti Cre was introduced into the intron and expressed using donor DNA containing the splicing acceptor sequence (SA sequence) and Opti Cre. Specifically, a donor vector in which the SA sequence (SA sequence of the 4th exon of the mouse Tubb3 gene), the base sequence encoding Opti Cre, and the 3'UTR region of the mouse Tubb3 gene are arranged in order from the 5'end side. (See the upper panel in FIG. 8 for a more detailed configuration). This donor DNA was introduced into the intron region of the mouse Tubb3 gene by the HITI method (see the lower panel of FIG. 8).
  • the gRNA recognition sequence used is SEQ ID NO: 19.
  • mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
  • -PCAX-hCas9 expression vector of hCas9
  • PgRNA expression vector of gRNA
  • PCAG-mCherry expression vector of mCherry
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • PCAG hyper piggybase expression vector of hyper piggybase
  • Donner DNA containing SA sequence and Opti Cre.
  • the brain was excised to prepare a section of the cerebral cortex. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
  • Opti Cre can be expressed site-specifically by the gene trap method when a donor DNA in which the SA sequence is arranged on the 5'terminal side of Opti Cre is used.
  • Example 5 Donor DNA having SA sequence (No. 2)
  • an endogenous SA sequence unique to the target site for gene transfer was used.
  • Opti Cre was used with a more versatile endogenous SA sequence.
  • the SA sequence (chimeric intron) of human immunoglobulin was used as the SA sequence (see FIG. 10 for the specific composition of the donor DNA). The experiment was carried out in the same manner as in Example 4 except that the SA sequence was changed. The results are shown in FIG.
  • EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. This suggests that Opti Cre can be expressed site-specifically by the gene trap method even when a non-endogenous SA sequence is used.
  • Example 6 Application to a non-mouse model and a model using HDR
  • HDR Homology Directed Repair
  • Example 6 a modified Cre (Opti Cre2) in which the atypical translation start codon was substituted in addition to ATG was used.
  • the target of gene transfer was the Pax6 gene specifically expressed in undifferentiated cells (the gRNA recognition sequence used was SEQ ID NO: 20).
  • a vector containing mCherry and flox-STOP-EGFP sequences contained LR sequences (SEQ ID NOs: 21 and 22), and
  • hyper piggybase which is a type of transposon, was also introduced into the genome. This allows the mCherry and flox-STOP-EGFP sequences to be randomly integrated into the genome. Therefore, it is possible to prevent the proportion of cells incorporating the mCherry and flox-STOP-EGFP sequences from being reduced by cell division.
  • the following vector was introduced into the cerebral cortex of a ferret fetal on the 30th day of embryonic development. Further, a ferret into which an expression vector other than the expression vector of hCas9 was introduced was used as a negative control.
  • -PCAX-hCas9 expression vector of hCas9
  • -PLR5 CAG-mCherry mCherry expression vector; has LR sequence
  • gRNA expression vector gRNA expression vector
  • PLR5 CAG-flox-STOP-EGFP reporter vector containing fluorx-STOP-EGFP sequence; having LR sequence
  • HDR Cre Donor Donator DNA of Opti Cre2
  • PCAX hyper piggybase expression vector of hyper piggybase
  • Example 7 Comparison of activity between OptiCre2 and wild-type Cre.
  • the recombinant activity of OptiCre2 was compared with the recombinant activity of wild-type Cre by an assay using cultured cells.
  • 293T cells were seeded on a 6-well plate and the following vector was introduced using lipofectamine 2000.
  • -PCAG-Cre or pCAG optiCre2 Expression vector of Cre or OptiCre2, respectively
  • -CAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • ATG was placed on the 5'end side of the base sequence encoding OptiCre2 so that OptiCre2 could be translated normally.
  • pCAG-Cre or pCAG optiCre2 was made into a dilution series and injected into wells at gradual concentrations (1 ⁇ g / well, 100 ng / well, 10 ng / well, 1 ng / well, and 10 pg / well).
  • the ratio of EGFP-expressing cells to all cells was measured by FACS. This measurement was standardized by dividing by the percentage of all cells expressing EGFP in positive control.
  • a positive control cells into which pCAG-EGFP (EGFP introduction vector) was introduced were used (1 ⁇ g / well).
  • a negative control untransfected cells were used.
  • the measurement results by FACS are shown in FIGS. 14 to 17, and the results after standardization are shown in FIGS.
  • OptiCre2 has a recombination activity equivalent to that of wild-type Cre.
  • Opti Cre2 may reduce the toxicity of wild-type Cre. That is, it was suggested that Opti Cre2 is a variant that not only reduces the leakage from the donor DNA but also reduces the toxicity of the wild-type Cre.
  • Example 8 Modified activity of FLP and Dre
  • variants of FLP and Dre which are enzymes frequently used other than Cre, were prepared and their activities were examined. Specifically, similarly to OptiCre2, a variant in which the translation initiation codon contained in the wild-type FLP and the wild-type Dre was replaced with the non-translation initiation codon was prepared. These variants are referred to as Opti FLP and Opti Dre, respectively.
  • the specific sequence is SEQ ID NO: 7 for the base sequence of Opti FLP, SEQ ID NO: 11 for the amino acid sequence of Opti FLP, SEQ ID NO: 8 for the base sequence of Opti Dre, and SEQ ID NO: 12 for the amino acid sequence of Opti Dre.
  • the present invention can be used, for example, in the field of biotechnology (for example, genome editing).
  • Polynucleotide encoding a recombinant enzyme 1a Modified polynucleotide (polynucleotide according to one embodiment of the present invention) 5: Translation start codon 5a: Non-translation start codon

Abstract

The purpose of one embodiment of the present invention is to provide a polynucleotide that can reduce leakage. The polynucleotide (the modified polynucleotide 1a) according to one embodiment of the present invention: (i) is a polynucleotide (1a) that is produced by replacing at least one translation initiation codon (5) of a polynucleotide (1) that codes for a recombination enzyme (or a variant of the polynucleotide (1) that codes for a recombination enzyme) with a non–translation initiation codon (5a); and (ii) codes for a protein that has recombination activity.

Description

ポリヌクレオチドおよびその利用Polynucleotides and their uses
　本発明は、ポリヌクレオチド、ベクター、遺伝子組み換えキット、Ｃｒｅ改変タンパク質もしくはその融合タンパク質、および遺伝子組換え方法に関する。 The present invention relates to polynucleotides, vectors, gene recombination kits, Cre modified proteins or fusion proteins thereof, and gene recombination methods.
　生命科学分野で用いられる遺伝子組換え技術の中に、組換え酵素（レコンビナーゼ）を利用した遺伝子組換え技術がある。このような遺伝子組換え技術の一例として、ＣｒｅレコンビナーゼおよびｌｏｘＰ配列を利用した、Ｃｒｅ／ｌｏｘＰシステムが挙げられる。このような組換え酵素を利用する技術では、組換え酵素と、部位（例えば、臓器、組織など）特異的に機能するプロモーター配列とを組合せて用いることによって、全身ではなく、部位特異的に組換えを発生させることができる（例えば、非特許文献１、２を参照）。 Among the gene recombination technologies used in the field of life science, there is a gene recombination technology using a recombinant enzyme (recombinase). An example of such a gene recombination technique is a Cre / loxP system using Cre recombinase and loxP sequences. In a technique using such a recombinant enzyme, the recombinant enzyme and a promoter sequence that functions specifically at a site (for example, an organ, a tissue, etc.) are used in combination to form a site-specific group rather than the whole body. Recombination can occur (see, for example, Non-Patent Documents 1 and 2).
　本発明者らは、ゲノム編集技術の開発過程において、組換え酵素をコードしたポリヌクレオチドを含むドナーＤＮＡを細胞へ導入した場合に、本来ならば組換え酵素が発現しないはずの細胞においても、組換え酵素が発現してしまうという、新規な課題に直面した。さらに研究を進めたところ、本発明者らは、ノックインされる前のドナーＤＮＡに含まれる組換え酵素をコードしたポリヌクレオチドが、細胞種非特異的に転写された後、当該転写産物が翻訳されるという、新規な課題に直面した。本明細書では、この現象を「リーク」と表現する。 In the process of developing genome editing technology, the present inventors have assembled even in cells in which the recombinant enzyme should not be expressed when a donor DNA containing a polynucleotide encoding a recombinant enzyme is introduced into the cell. We faced a new challenge of expressing a recombinant enzyme. As a result of further research, the present inventors translated the transcript after the polynucleotide encoding the recombinant enzyme contained in the donor DNA before being knocked in was transcribed non-specifically to the cell type. I faced a new challenge of In the present specification, this phenomenon is referred to as "leak".
　この「リーク」という現象は、組換え酵素を細胞へ導入するために、大量のドナーＤＮＡを用いる場合に、特に問題となりうる。例えば、本発明者らが開発したde novoノックイン法（Tsunekawa Y et al. (2016) "Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain," Development, Vol.143(No.17), pp.3216-3222.）や、ＨＩＴＩ法（Suzuki K, Tsunekawa Y, et al. (2016) "In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration," Nature, Vol.540(Issue 7631), pp.144-149.）においては、特に問題となりうる。 This phenomenon of "leakage" can be a particular problem when a large amount of donor DNA is used to introduce the recombinant enzyme into cells. For example, the de novo knock-in method developed by the present inventors (Tsunekawa Y et al. (2016) "Developing a de novo targeted knock-in method based on in utero electroporation into the mammal brain," Development, Vol.143 (No) .17), pp.3216-3222.) And the HITI method (Suzuki K, Tsunekawa Y, et al. (2016) "In vivo genome editing via CRISPR / Cas9 mediated mammal-independent targeted integration," Nature, Vol.540 (Issue 7331), pp.144-149.) Can be a particular problem.
　本発明の一態様は、リークの発生を低減できるポリヌクレオチドを提供することを目的とする。 One aspect of the present invention is to provide a polynucleotide capable of reducing the occurrence of leaks.
　本発明者らは、鋭意検討した結果、リークに関与する転写および翻訳という２つのステップのうち、特に翻訳というステップを制御することによって、リークの発生を低減できることを見出し、本発明を完成させるに至った。より具体的に、本発明者らは、組換え酵素をコードしているポリヌクレオチド中に存在する翻訳開始コドンの少なくとも一部を非翻訳開始コドンに置換することによって、リークの発生を低減できることを見出し、本発明を完成させるに至った。 As a result of diligent studies, the present inventors have found that the occurrence of leaks can be reduced by controlling the step of translation among the two steps of transcription and translation involved in the leak, and complete the present invention. I arrived. More specifically, we found that the occurrence of leaks can be reduced by substituting at least some of the translation initiation codons present in the polynucleotide encoding the recombinant enzyme with untranslation initiation codons. We have found and completed the present invention.
　本発明には、以下の構成が含まれる。
＜１＞
　以下の（Ｇ１）～（Ｇ４）の何れかに示される、ポリヌクレオチド：
　（Ｇ１）（i）組換え酵素をコードしているポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド；
　（Ｇ２）（i）組換え酵素をコードしているポリヌクレオチドの、１または数個の塩基が置換、欠失、挿入および／または付加された塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド；
　（Ｇ３）（i）組換え酵素をコードしているポリヌクレオチドと配列同一性が９０％以上のポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド；
　（Ｇ４）（i）組換え酵素をコードしているポリヌクレオチドと相補的なＤＮＡ配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。
＜２＞
　上記翻訳開始コドンは、ＡＴＧ、ＣＴＧ、ＴＴＧ、ＧＴＧ、または、ＡＴＡである、＜１＞に記載のポリヌクレオチド。
＜３＞
　上記組換え酵素は、Ｃｒｅ、ＦＬＰ、Ｄｒｅ、Ｔｒｅ、または、それらの改変体である、＜１＞または＜２＞に記載のポリヌクレオチド。
＜４＞
　＜１＞～＜３＞のいずれかに記載のポリヌクレオチドを含んでいる、ベクター。
＜５＞
　上記ポリヌクレオチドの５’末端側に、スプライシングアクセプター配列をさらに含んでいる、＜４＞に記載のベクター。
＜６＞
　＜１＞～＜３＞のいずれかに記載のポリヌクレオチド、または、＜４＞もしくは＜５＞に記載のベクターを備えている、遺伝子組換えキット。
＜７＞
　配列番号１０に記載のアミノ酸配列からなるＣｒｅ改変タンパク質、またはその融合タンパク質。
＜８＞
　＜１＞～＜３＞のいずれかに記載のポリヌクレオチド、または、＜４＞もしくは＜５＞に記載のベクターを、細胞または非ヒト被験体に導入する工程を含む、遺伝子組換え方法。 The present invention includes the following configurations.
<1>
The polynucleotide shown in any of the following (G1) to (G4):
(G1) (i) In a polynucleotide encoding a recombinant enzyme, a protein consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) a protein having recombinant activity. The polynucleotide encoding
(G2) (i) At least one translation initiation in a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme. A polynucleotide consisting of a polynucleotide whose codon has been replaced with an untranslated start codon and (ii) encoding a protein having recombinant activity;
(G3) (i) Consists of a polynucleotide in which at least one start codon is replaced with a non-translation start codon in a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme. And (ii) a polynucleotide encoding a protein having recombinant activity;
(G4) (i) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is untranslated. A polynucleotide consisting of a polynucleotide substituted with a codon and (ii) encoding a protein having recombinant activity.
<2>
The polynucleotide according to <1>, wherein the translation start codon is ATG, CTG, TTG, GTG, or ATA.
<3>
The polynucleotide according to <1> or <2>, wherein the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof.
<4>
A vector containing the polynucleotide according to any one of <1> to <3>.
<5>
The vector according to <4>, further comprising a splicing acceptor sequence on the 5'end side of the polynucleotide.
<6>
A genetic recombination kit comprising the polynucleotide according to any one of <1> to <3> or the vector according to <4> or <5>.
<7>
Cre modified protein consisting of the amino acid sequence of SEQ ID NO: 10, or a fusion protein thereof.
<8>
A genetic recombination method comprising the step of introducing the polynucleotide according to any one of <1> to <3> or the vector according to <4> or <5> into a cell or a non-human subject.
　本発明の一態様によれば、リークの発生を低減できるポリヌクレオチドが提供される。 According to one aspect of the present invention, a polynucleotide capable of reducing the occurrence of leaks is provided.
従来のドナーＤＮＡと、本発明の一実施形態におけるドナーＤＮＡとを比較した模式図である。It is a schematic diagram comparing the conventional donor DNA and the donor DNA in one embodiment of the present invention. 本発明の一実施形態に係るベクターおよびその使用方法を説明する模式図である。It is a schematic diagram explaining the vector which concerns on one Embodiment of this invention and the method of using it. 実施例１において使用したドナーＤＮＡの構成を表す模式図である。It is a schematic diagram which shows the composition of the donor DNA used in Example 1. FIG. 実施例１の結果を表す図である。It is a figure which shows the result of Example 1. FIG. 実施例２で作製した、Ｃｒｅ１～Ｃｒｅ６（野生型Ｃｒｅおよびその部分ペプチド）を表す模式図である。It is a schematic diagram showing Cre1 to Cre6 (wild type Cre and a partial peptide thereof) produced in Example 2. 実施例２の結果を表す図である。It is a figure which shows the result of Example 2. FIG. 実施例３の結果を表す図である。It is a figure which shows the result of Example 3. FIG. 実施例４で使用したドナーＤＮＡの構成と、当該ドナーＤＮＡのノックインサイトとを表す模式図である。It is a schematic diagram which shows the composition of the donor DNA used in Example 4 and knockin sight of the donor DNA. 実施例４の結果を表す図である。It is a figure which shows the result of Example 4. FIG. 実施例５で使用したドナーＤＮＡの構成と、当該ドナーＤＮＡのノックインサイトとを表す模式図である。It is a schematic diagram which shows the composition of the donor DNA used in Example 5 and knockin sight of the donor DNA. 実施例５の結果を表す図である。It is a figure which shows the result of Example 5. 実施例６で使用したドナーＤＮＡの構成と、当該ドナーＤＮＡのノックインサイトと表す模式図である。It is a schematic diagram which shows the composition of the donor DNA used in Example 6 and knockin sight of the donor DNA. 実施例６の結果を表す図である。It is a figure which shows the result of Example 6. 実施例７の結果であって、ネガティブコントロールのＥＧＦＰ発現率を表す図である。It is the result of Example 7, and is the figure which shows the EGFP expression rate of a negative control. 実施例７の結果であって、ポジティブコントロールのＥＧＦＰ発現率を表す図である。It is the result of Example 7, and is the figure which shows the EGFP expression rate of a positive control. 実施例７の結果であって、野生型Ｃｒｅを使用した群におけるＥＧＦＰ発現率を表す図である。It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used the wild type Cre. 実施例７の結果であって、Ｏｐｔｉ　Ｃｒｅ２を使用した群におけるＥＧＦＰ発現率を表す図である。It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used Opti Cre2. 実施例７の結果を表す図である。It is a figure which shows the result of Example 7. 実施例８の結果を表す図である。It is a figure which shows the result of Example 8.
　本発明の一実施形態について以下に説明するが、本発明は以下に説明する各構成に限定されるものではなく、特許請求の範囲に示した範囲で種々の変更が可能である。異なる実施形態や実施例にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態や実施例も、本発明の技術的範囲に含まれる。本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考文献として援用される。本明細書において特記しない限り、数値範囲を表す「Ａ～Ｂ」は、「Ａ以上、Ｂ以下」を意図する。 An embodiment of the present invention will be described below, but the present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims. The technical scope of the present invention also includes embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples. All academic and patent documents described herein are incorporated herein by reference. Unless otherwise specified in the present specification, "AB" representing a numerical range is intended to be "A or more and B or less".
　本明細書では、ポリヌクレオチドがＤＮＡである場合に基づいて説明する。なお、ポリヌクレオチドがＲＮＡである場合には、以下の説明において「Ｔ（チミン）」を「Ｕ（ウラシル）」に読み替えればよい。 In the present specification, the case where the polynucleotide is DNA will be described. When the polynucleotide is RNA, "T (thymine)" may be read as "U (uracil)" in the following description.
　〔１．ポリヌクレオチド〕
　［本発明の概要］
　以下、図１を参照しながら、本発明の概要を説明する。図１の上パネルは、従来技術におけるドナーＤＮＡ１０を表している。ドナーＤＮＡ１０には、組換え酵素をコードしているポリヌクレオチド１が含まれている。組換え酵素をコードしているポリヌクレオチド１には、翻訳開始コドン５（ＡＴＧなど）が含まれている。このような構成のドナーＤＮＡ１０を細胞内に導入した場合、たとえドナーＤＮＡ１０のノックインが生じていない状態であっても、ドナーＤＮＡ１０（組換え酵素をコードしているポリヌクレオチド１）から組換え酵素が発現してしまう。この現象を、本明細書では「リーク」と称する。 [1. Polynucleotide]
[Outline of the present invention]
Hereinafter, the outline of the present invention will be described with reference to FIG. The upper panel of FIG. 1 represents the donor DNA 10 in the prior art. The donor DNA 10 contains a polynucleotide 1 encoding a recombinant enzyme. The polynucleotide 1 encoding the recombinant enzyme contains a translation initiation codon 5 (such as ATG). When the donor DNA 10 having such a structure is introduced into the cell, the recombinant enzyme is released from the donor DNA 10 (polynucleotide 1 encoding the recombinant enzyme) even when the donor DNA 10 is not knocked in. It will be expressed. This phenomenon is referred to herein as a "leak".
　このリーク現象の原因は、以下のように推測される（もっとも、このメカニズムは本発明の理解を助けるための説明であり、本発明を限定するものではない）。通常、細胞内のＤＮＡは、エピジェネティックな修飾（クロマチン化など）によって、発現が制御されている。ところが、生命工学的な手法により、修飾されていないＤＮＡを大量に細胞内に導入した場合、当該ＤＮＡに含まれる遺伝子の発現を制御するプロモーターが機能しない状態であっても、ある程度は、遺伝子の転写・翻訳が生じてしまう（リーク）。このようにして発生した翻訳産物は、実験系を成立させなくなるなどの不利益をもたらす場合がある。 The cause of this leak phenomenon is presumed as follows (although this mechanism is an explanation for assisting the understanding of the present invention and does not limit the present invention). Expression of intracellular DNA is usually regulated by epigenetic modifications (such as chromatinization). However, when a large amount of unmodified DNA is introduced into a cell by a biotechnology method, even if the promoter that controls the expression of the gene contained in the DNA does not function, the gene can be introduced to some extent. Transcription / translation occurs (leak). The translation product generated in this way may bring disadvantages such as not establishing an experimental system.
　一方、図１の下パネルは、本発明の一実施形態におけるドナーＤＮＡ１０ａを表している。ドナーＤＮＡ１０ａには、改変されたポリヌクレオチド１ａが含まれている。具体的には、改変されたポリヌクレオチド１ａは、組換え酵素をコードしているポリヌクレオチド１において、少なくとも１つの翻訳開始コドン５が、非翻訳開始コドン５ａに置換されている。この構成によれば、転写レベルではなく翻訳レベルにおいて、リークを低減することができる（翻訳レベルでリークを低減する戦略の有効性については、実施例１、３を参照）。 On the other hand, the lower panel of FIG. 1 represents the donor DNA 10a in one embodiment of the present invention. The donor DNA 10a contains the modified polynucleotide 1a. Specifically, in the modified polynucleotide 1a, at least one translation initiation codon 5 is replaced with the untranslation initiation codon 5a in the polynucleotide 1 encoding the recombinant enzyme. According to this configuration, leaks can be reduced at the translation level rather than at the transcription level (see Examples 1 and 3 for the effectiveness of the leak reduction strategy at the translation level).
　本明細書において、「リークを低減する」とは、例えば、従来技術のドナーＤＮＡ１０よりも、本発明の一実施形態におけるドナーＤＮＡ１０ａの方が、ノックインされていないドナーＤＮＡに由来する組換え酵素の発現量が低減している状態を表す。一実施形態において、ノックインされていない状態におけるドナーＤＮＡ１０ａに由来する組換え酵素の発現量は、ノックインされていない状態における従来技術のドナーＤＮＡ１０に由来する組換え酵素の発現量の、５０％以下、４０％以下、３０％以下、２０％以下、１０％以下、５％以下、３％以下、１％以下、または０％である。 In the present specification, "reducing leakage" means, for example, that the donor DNA 10a in one embodiment of the present invention is a recombinant enzyme derived from a donor DNA that has not been knocked in, rather than the donor DNA 10 of the prior art. It represents a state in which the expression level is reduced. In one embodiment, the expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state is 50% or less of the expression level of the recombinant enzyme derived from the donor DNA 10 of the prior art in the non-knocked state. 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 1% or less, or 0%.
　ノックインされていない状態におけるドナーＤＮＡ１０ａに由来する組換え酵素の発現量、および、ノックインされていない状態におけるドナーＤＮＡ１０に由来する組換え酵素の発現量は、例えば、実施例で行った方法により測定され得る。具体的には、Ｃａｓ９（またはその発現ベクター）を伴わずにドナーＤＮＡ１０またはドナーＤＮＡ１０ａを細胞内に導入し、組換え酵素の発現量を測定することによって、測定され得る。組換え酵素の発現量を定量化し比較する方法は、本技術分野において周知の技術である。 The expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state and the expression level of the recombinant enzyme derived from the donor DNA 10 in the non-knocked state were measured by, for example, the method used in the examples. obtain. Specifically, it can be measured by introducing donor DNA10 or donor DNA10a into the cell without Cas9 (or its expression vector) and measuring the expression level of the recombinant enzyme. Methods for quantifying and comparing the expression levels of recombinant enzymes are well known in the art.
　ここで、改変されたポリヌクレオチド１ａは、組換え活性を有しているタンパク質をコードしている。つまり、改変されたポリヌクレオチド１ａの翻訳産物は、組換え活性を有している。一実施形態において、改変されたポリヌクレオチド１ａの翻訳産物の組換え活性は、組換え酵素をコードしているポリヌクレオチド１の翻訳産物の組換え活性の、５０％以上、６０％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、または９５％以上である。なお、改変されたポリヌクレオチド１ａの翻訳産物の組換え活性が、組換え酵素をコードしているポリヌクレオチド１の翻訳産物の組換え活性の１００％以上であってもよい。 Here, the modified polynucleotide 1a encodes a protein having recombinant activity. That is, the translated product of the modified polynucleotide 1a has recombinant activity. In one embodiment, the recombinant activity of the translated product of the modified polynucleotide 1a is 50% or more, 60% or more, 70% of the recombinant activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme. Above, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The recombination activity of the translation product of the modified polynucleotide 1a may be 100% or more of the recombination activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme.
　改変されたポリヌクレオチド１ａの翻訳産物が組換え活性を有しているか否かは、本技術分野で一般的なアッセイにより確認することができる（具体的な例としては、実施例２を参照）。あるいは、立体構造解析によって、改変されたポリヌクレオチド１ａの翻訳産物が組換え活性を有しているか否かを推定することもできる。また、組換え活性を定量化し比較する方法は、本技術分野において周知の技術である（具体的な例としては、実施例７、８を参照）。 Whether or not the translated product of the modified polynucleotide 1a has recombinant activity can be confirmed by an assay common in the art (see Example 2 for a specific example). .. Alternatively, it is possible to estimate whether or not the translated product of the modified polynucleotide 1a has recombination activity by three-dimensional structural analysis. Further, a method for quantifying and comparing recombinant activity is a technique well known in the art (see Examples 7 and 8 for specific examples).
　なお、本明細書における「ポリヌクレオチド」には、ＲＮＡおよびＤＮＡが含まれる。ＲＮＡの形態であるポリヌクレオチドの例としては、ｍＲＮＡが挙げられる。ＤＮＡの形態であるポリヌクレオチドの例としては、種々のＤＮＡ断片、ｃＤＮＡ、ゲノムＤＮＡが挙げられる。ＲＮＡおよびＤＮＡは、任意の構造を取りうる（二本鎖または一本鎖など）。 Note that the "polynucleotide" in the present specification includes RNA and DNA. Examples of polynucleotides in the form of RNA include mRNA. Examples of polynucleotides in the form of DNA include various DNA fragments, cDNAs, genomic DNAs. RNA and DNA can have any structure (such as double-stranded or single-stranded).
　［本発明の一実施形態に係るポリヌクレオチド］
　本発明の一実施形態に係るポリヌクレオチドは、下記の（Ｇ１）～（Ｇ４）のいずれかに示されるポリヌクレオチドである。
（Ｇ１）
　（i）組換え酵素をコードしているポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。
（Ｇ２）
　（i）組換え酵素をコードしているポリヌクレオチドの、１または数個の塩基が置換、欠失、挿入および／または付加された塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド。
（Ｇ３）
　（i）組換え酵素をコードしているポリヌクレオチドと配列同一性が９０％以上のポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。
（Ｇ４）
　（i）組換え酵素をコードしているポリヌクレオチドと相補的なＤＮＡ配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。 [Polynucleotide according to one embodiment of the present invention]
The polynucleotide according to one embodiment of the present invention is the polynucleotide shown in any of the following (G1) to (G4).
(G1)
(I) In a polynucleotide encoding a recombinant enzyme, a polynucleotide consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) encoding a protein having recombinant activity. Polynucleotide that is.
(G2)
(I) In a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme, at least one translation start codon is not present. A polynucleotide consisting of a polynucleotide substituted with a translation initiation codon and (ii) encoding a protein having recombinant activity.
(G3)
(I) In a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is substituted with a non-translation initiation codon, and (i) ii) A polynucleotide encoding a protein having recombinant activity.
(G4)
(I) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is replaced with a non-translation initiation codon. (Ii) A polynucleotide encoding a protein having recombinant activity.
　（Ｇ１）は、「野生型の組換え酵素をコードしているポリヌクレオチドから、当該ポリヌクレオチドに含まれている１つ以上の翻訳開始コドンを非翻訳開始コドンに置換したポリヌクレオチド」を意図している。一方、（Ｇ２）～（Ｇ４）は、「野生型の組換え酵素をコードしているポリヌクレオチドのバリアントまたは変異ポリヌクレオチドなどから、当該ポリヌクレオチドに含まれている１つ以上の翻訳開始コドンを非翻訳開始コドンに置換したポリヌクレオチド」を意図している。 (G1) is intended as "a polynucleotide in which one or more translation initiation codons contained in the polynucleotide are replaced with non-translation initiation codons from a polynucleotide encoding a wild-type recombinant enzyme". ing. On the other hand, (G2) to (G4) refer to "one or more start codons contained in the polynucleotide from a variant or mutant polynucleotide of the polynucleotide encoding the wild-type recombinant enzyme". It is intended as a "polynucleotide substituted with an untranslated start codon".
　（Ｇ１）～（Ｇ４）に関して、組換え活性を有するタンパク質をコードしているポリヌクレオチドであるか否かは、当該ポリヌクレオチドを所望の発現ベクターに挿入した後、当該発現ベクターを所望の宿主に導入し、当該宿主にて遺伝子の組換え（例えば、相同組換え、非相同組換え）が生じるか否かを検出することによって確認することができる。宿主にて遺伝子の組換えが生じていれば、上記ポリヌクレオチドを、「組換え活性を有するタンパク質をコードしているポリヌクレオチドである」と判定することができる。なお、遺伝子の組換えの検出は、公知の方法にしたがえばよい（例えば、実施例を参照）。 Regarding (G1) to (G4), whether or not the polynucleotide encodes a protein having recombinant activity is determined by inserting the polynucleotide into a desired expression vector and then using the expression vector as a desired host. It can be confirmed by introducing and detecting whether or not gene recombination (for example, homologous recombination, non-homologous recombination) occurs in the host. If the gene has been recombined in the host, the polynucleotide can be determined to be "a polynucleotide encoding a protein having recombination activity". The detection of gene recombination may be carried out according to a known method (see, for example, Examples).
　（Ｇ２）に関して、「数個の塩基」とは、例えば、５０個、４５個、４０個、３５個、３０個、２５個、２０個、１９個、１８個、１７個、１６個、１５個、１４個、１３個、１２個、１１個、１０個、９個、８個、７個、６個、５個、４個、３個、２個の塩基である。 With respect to (G2), "several bases" are, for example, 50, 45, 40, 35, 30, 25, 20, 19, 19, 18, 17, 16, 15 There are 14, 14, 12, 11, 10, 10, 9, 8, 7, 6, 5, 4, 3, and 2 bases.
　（Ｇ３）に関して、配列同一性は、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上、であってもよい。本明細書において、「配列同一性」とは、同一の塩基数の割合を意図する。 Regarding (G3), even if the sequence identity is 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. Good. As used herein, "sequence identity" is intended to be the proportion of the same number of bases.
　塩基配列の配列同一性は、例えば、BLASTNを利用して決定することができる（Altschul SF (1990) "Basic local alignment search tool", Journal of Molecular Biology, Vol.215 (Issue 3), pp.403-410）。このプログラムは、KarlinおよびAltschulによるBLASTアルゴリズムに基づいている（Karlin S and Altschul SF (1990) "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes", Proceedings of the National Academy of Sciences of the United States of America, Vol.87 (No.6), pp.2264-2268; Karlin S and Altschul SF (1993) "Applications and statistics for multiple high-scoring segments in molecular sequences", Proceedings of the National Academy of Sciences of the United States of America, Vol.90 (No.12), pp.5873-5877）。BLASTNによって塩基配列を解析する場合のパラメーターとしては、例えば、score＝１００、wordlength＝１２とすることができる。これらの解析方法の具体的な手法は周知である。比較対象となる塩基配列を最適な状態にアラインメントするために、付加または欠失（ギャップなど）を許容してもよい。上述した配列同一性は、好ましくは９１％以上、より好ましくは９２％以上、より好ましくは９３％以上、より好ましくは９４％以上、より好ましくは９５％以上、より好ましくは９６％以上、より好ましくは９７％以上、より好ましくは９８％以上、最も好ましくは９９％以上である。 The sequence identity of the base sequence can be determined using, for example, BLASTN (Altschul SF (1990) "Basic local alignment search tool", Journal of Molecular Biology, Vol.215 (Issue 3), pp.403). -410). This program is based on the BLAST algorithm by Karlin and Altschul (Karlin S and Altschul SF (1990) "Methods for assessing the statistical signature of molecular sequence features by using general scoring schemes", Proceedings of the National United States of America, Vol.87 (No.6), pp.2264-2268; Karlin S and Altschul SF (1993) "Applications and statistics for multiple high-scoring segments in molecular sequences", Proceedings of the National Academy of the United States of America, Vol.90 (No.12), pp.5873-5877). As parameters when analyzing the base sequence by BLASTN, for example, score = 100 and wordlength = 12 can be set. Specific methods of these analysis methods are well known. Additions or deletions (such as gaps) may be allowed to optimally align the base sequences to be compared. The above-mentioned sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably. Is 97% or more, more preferably 98% or more, and most preferably 99% or more.
　（Ｇ４）に関して、「ストリンジェントな条件でハイブリダイズする」とは、６×ＳＳＣの塩濃度のハイブリダイゼーション溶液中、５０～６０℃にて１６時間ハイブリダイゼーションを行い、０．１×ＳＳＣの塩濃度の溶液中で洗浄した後に、ハイブリダイズする条件を言う。ここで、１×ＳＳＣの組成は、塩化ナトリウム：１５０ｍＭ、クエン酸ナトリウム：１５ｍＭである。 Regarding (G4), "hybridizing under stringent conditions" means that hybridization is performed at 50 to 60 ° C. for 16 hours in a hybridization solution having a salt concentration of 6 × SSC to obtain a salt of 0.1 × SSC. The condition for hybridization after washing in a solution of a concentration. Here, the composition of 1 × SSC is sodium chloride: 150 mM and sodium citrate: 15 mM.
　［翻訳開始コドン］
　本発明の一実施形態に係るポリヌクレオチドにおいて、非翻訳開始コドンに置換される翻訳開始コドンは、特に限定されない。翻訳開始コドンの例としては、ＡＴＧ、ＣＴＧ、ＴＴＧ、ＧＴＧ、ＡＴＡなどが挙げられる。このうち、ＡＴＧは、広い生物種において共通する、典型的な翻訳開始コドンである。一方、ＣＴＧ、ＴＴＧ、ＧＴＧおよびＡＴＡは、翻訳開始コドンとして機能しうることが報告されている、非典型的な翻訳開始コドンである。したがって、本発明の一実施形態に係るポリヌクレオチドにおいては、ＡＴＧの１つ以上が非翻訳開始コドンに置換されていることが好ましい。さらに、本発明の一実施形態に係るポリヌクレオチドにおいては、ＡＴＧの１つ以上が非翻訳開始コドンに置換されている上に、更に、非典型的な翻訳開始コドンの１つ以上が非翻訳開始コドンに置換されていることがより好ましい。 [Translation start codon]
In the polynucleotide according to one embodiment of the present invention, the translation initiation codon substituted with the untranslation initiation codon is not particularly limited. Examples of translation start codons include ATG, CTG, TTG, GTG, ATA and the like. Of these, ATG is a typical translation initiation codon that is common to a wide range of species. On the other hand, CTG, TTG, GTG and ATA are atypical translation initiation codons that have been reported to function as translation initiation codons. Therefore, in the polynucleotide according to one embodiment of the present invention, it is preferable that one or more of the ATGs are replaced with non-translation initiation codons. Furthermore, in the polynucleotide according to one embodiment of the present invention, one or more of the ATGs are replaced with untranslated initiation codons, and one or more of the atypical translation initiation codons are untranslated initiation codons. It is more preferable that it is replaced with a codon.
　翻訳開始コドンが置換される非翻訳開始コドンは、特に限定されない。ＡＴＧによってコードされているメチオニンは、ＡＴＧ以外のコドンによってコードされていない。それゆえ、ＡＴＧを非翻訳開始コドンに置換する場合は、必然的に、メチオニンをコードしているＡＴＧを、メチオニン以外のアミノ酸をコードしているコドンへと置換することになる。この場合、ＡＴＧは、ロイシンをコードしているコドン（ＴＴＡ、ＴＴＧ、ＣＴＴ、ＣＴＣ、ＣＴＡまたはＣＴＧ）に置換することが好ましい。これは、メチオニンとロイシンとでは、アミノ酸としての性質（親水性／疎水性、等電点、立体構造など）が類似しているためである。なお、ＡＴＧを、ロイシンをコードしているどのコドンに置換するかは、例えば、ポリヌクレオチドを導入する生物のコドン使用頻度に応じて、適宜決定できる。 The non-translation start codon in which the translation start codon is replaced is not particularly limited. The methionine encoded by ATG is not encoded by codons other than ATG. Therefore, when ATG is replaced with an untranslated start codon, the ATG encoding methionine is inevitably replaced with a codon encoding an amino acid other than methionine. In this case, the ATG is preferably replaced with a codon encoding leucine (TTA, TTG, CTT, CTC, CTA or CTG). This is because methionine and leucine have similar properties as amino acids (hydrophilic / hydrophobic, isoelectric point, three-dimensional structure, etc.). Which codon encoding leucine is replaced with ATG can be appropriately determined depending on, for example, the codon usage frequency of the organism into which the polynucleotide is introduced.
　一方、ＣＴＧ、ＴＴＧ、ＧＴＧおよびＡＴＡは、コードされているアミノ酸を変化させることなく、非翻訳開始コドンに置換することができる。一実施形態において、ＣＴＧは、ＣＴＣに置換される。一実施形態において、ＴＴＧは、ＴＴＡに置換される。一実施形態において、ＧＴＧは、ＧＴＡに置換される。一実施形態において、ＡＴＡは、ＡＴＴに置換される。 On the other hand, CTG, TTG, GTG and ATA can be replaced with non-translation start codons without changing the encoded amino acids. In one embodiment, CTG is replaced with CTC. In one embodiment, TTG is replaced with TTA. In one embodiment, GTG is replaced by GTA. In one embodiment, ATA is replaced with ATT.
　非翻訳開始コドンに置換される翻訳開始コドンの位置は、所望の翻訳産物が組換え活性を有している限り、特に限定されない。リークを低減しつつ、組換え活性を維持するという観点から、非翻訳開始コドンに置換される翻訳開始コドンの位置は、例えば、組換え酵素のアミノ末端、組換え酵素のアミノ末端側の領域、組換え酵素の中央のアミノ酸よりもアミノ末端側の領域、組換え酵素の触媒ドメインよりもアミノ末端側の領域、に対応する位置であってよい。 The position of the translation initiation codon to be replaced with the non-translation initiation codon is not particularly limited as long as the desired translation product has recombination activity. From the viewpoint of maintaining the recombinant activity while reducing the leak, the position of the translation initiation codon substituted with the non-translation initiation codon is, for example, the amino terminal of the recombinant enzyme, the region on the amino terminal side of the recombinant enzyme, and the like. The position may correspond to the region on the amino-terminal side of the central amino acid of the recombinant enzyme and the region on the amino-terminal side of the catalytic domain of the recombinant enzyme.
　非翻訳開始コドンに置換される翻訳開始コドンの数は、所望の翻訳産物が組換え活性を有している限り、特に限定されない。例えば、１個以上、２個以上、３個以上、４個以上、５個以上、６個以上、７個以上、８個以上、９個以上、１０個以上、１５個以上、２０個以上、２５個以上、３０個以上、３５個以上、４０個以上、４５個以上、または５０個以上の翻訳開始コドンが、非翻訳開始コドンに置換され得る。非翻訳開始コドンに置換される翻訳開始コドンの数が増えるほど、リークをより低減できる。非翻訳開始コドンに置換される翻訳開始コドンの数の上限値は、特に限定されず、１０個、２０個、３０個、４０個、５０個、または１００個であってもよい。 The number of translation initiation codons to be replaced with non-translation initiation codons is not particularly limited as long as the desired translation product has recombinant activity. For example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the number of translation initiation codons replaced by untranslation initiation codons increases. The upper limit of the number of translation initiation codons to be replaced with the untranslation initiation codon is not particularly limited, and may be 10, 20, 30, 40, 50, or 100.
　非翻訳開始コドンに置換される翻訳開始コドンの割合は、翻訳産物が組換え活性を有している限り、特に限定されない。例えば、５％以上、１０％以上、１５％以上、２０％以上、２５％以上、３０％以上、３５％以上、４０％以上、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、９５％以上、または１００％の翻訳開始コドンが、非翻訳開始コドンに置換され得る。非翻訳開始コドンに置換される翻訳開始コドンの割合が増えるほど、リークをより低減できる。非翻訳開始コドンに置換される翻訳開始コドンの割合の上限値は、特に限定されず、１０％以下、２０％以下、３０％以下、４０％以下、５０％以下、６０％以下、７０％以下、８０％以下、９０％以下、または１００％以下であってもよい。 The ratio of translation initiation codons replaced with non-translation initiation codons is not particularly limited as long as the translation product has recombinant activity. For example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the proportion of translation initiation codons replaced by untranslation initiation codons increases. The upper limit of the ratio of translation initiation codons substituted with untranslated initiation codons is not particularly limited, and is 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less. , 80% or less, 90% or less, or 100% or less.
　［組換え酵素］
　一実施形態において、組換え酵素は、Ｃｒｅ、ＦＬＰ、Ｄｒｅ、Ｔｒｅ、または、それらの改変体（例えば、野生型の塩基配列に対してコドン最適化を施したコドン改変体）である。好ましくは、組換え酵素は、Ｃｒｅ、ＦＬＰ、または、Ｄｒｅである。より好ましくは、組換え酵素は、Ｃｒｅである。 [Recombinant enzyme]
In one embodiment, the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof (eg, a codon variant obtained by codon-optimizing a wild-type nucleotide sequence). Preferably, the recombinant enzyme is Cre, FLP, or Dre. More preferably, the recombinant enzyme is Cre.
　これらの組換え酵素は公知の組換え酵素であり、これらをコードする塩基配列も公知である。例えば、上記の組換え酵素をコードしている塩基配列は、以下の通りである。
Ｃｒｅ：（配列番号１）
ＦＬＰ：（配列番号２）
Ｄｒｅ：（配列番号３）
Ｔｒｅ：（配列番号４）。 These recombinant enzymes are known recombinant enzymes, and the base sequences encoding them are also known. For example, the base sequence encoding the above recombinant enzyme is as follows.
Cre: (SEQ ID NO: 1)
FLP: (SEQ ID NO: 2)
Dre: (SEQ ID NO: 3)
Tre: (SEQ ID NO: 4).
　上述した組換え酵素の改変体としては、例えば、ＶＣｒｅ、ＳＣｒｅ、ＦＬＰＯ、ＦＬＰＥ、ＤｒｅＥなどが挙げられる。これらの改変体は公知の改変体であり、これらをコードする塩基配列も公知である。具体的な塩基配列は、以下の通りである：配列番号２５（ＶＣｒｅ）、配列番号２６（ＳＣｒｅ）、配列番号２７（ＦＬＰＯ）、配列番号２８（ＦＬＰＥ）。 Examples of the above-mentioned modified enzyme of the recombinant enzyme include VCre, SCre, FLPO, FLPE, DreE and the like. These variants are known variants, and the base sequences encoding them are also known. Specific nucleotide sequences are as follows: SEQ ID NO: 25 (VCre), SEQ ID NO: 26 (SCre), SEQ ID NO: 27 (FLPO), SEQ ID NO: 28 (FLPE).
　本発明の一実施形態に係るポリヌクレオチドは、以下の（Ｇ１’）～（Ｇ４’）からなる群より選択される１つ以上であってもよい。
（Ｇ１’）
　（i）配列番号１～４のいずれかに記載される塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド。
（Ｇ２’）
　（i）配列番号１～４のいずれかに記載される塩基配列の、１または数個の塩基が置換、欠失、挿入および／または付加された塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド。
（Ｇ３’）
　（i）配列番号１～４のいずれかに記載される塩基配列と９０％以上の配列同一性を有する塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド。
（Ｇ４’）
　（i）配列番号１～４のいずれかに記載される塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズするポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。 The polynucleotide according to one embodiment of the present invention may be one or more selected from the group consisting of the following (G1') to (G4').
(G1')
(I) In the polynucleotide consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 4, at least one translation initiation codon is substituted with a non-translation initiation codon, and (ii) set. A polynucleotide encoding a protein having a translating activity.
(G2')
(I) At least one translation of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 4 in a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added. A polynucleotide consisting of a polynucleotide in which the start codon is replaced with an untranslated start codon and (ii) encoding a protein having recombinant activity.
(G3')
(I) In a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 4, at least one translation initiation codon is replaced with a non-translation initiation codon. A polynucleotide consisting of a polynucleotide and encoding a protein having (ii) recombinant activity.
(G4')
(I) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence set forth in any of SEQ ID NOs: 1 to 4, at least one translation start codon initiates non-translation. A polynucleotide consisting of a polynucleotide substituted with a codon and (ii) encoding a protein having recombinant activity.
　なお、（Ｇ１’）～（Ｇ４’）に関する説明は、上述した（Ｇ１）～（Ｇ４）に関する説明にそれぞれ対応しているため、記載を省略する。 Since the explanations regarding (G1') to (G4') correspond to the above-mentioned explanations regarding (G1) to (G4), the description thereof will be omitted.
　〔２．ベクター〕
　本発明の一態様に係るベクターは、上述のポリヌクレオチドを含んでいる。このようなベクターを細胞内に導入することによって、当該ベクター（例えば、ドナーＤＮＡ）に由来するリークを低減することができる。 [2. vector〕
The vector according to one aspect of the present invention contains the above-mentioned polynucleotide. By introducing such a vector into cells, leakage derived from the vector (for example, donor DNA) can be reduced.
　ベクターの具体例としては、ファージベクター、プラスミドベクター、ウイルスベクター、レトロウイルスベクター、染色体ベクター、エピソームベクター、ウイルス由来ベクター（細菌プラスミド、バクテリオファージ、酵母エピソームなど）、酵母染色体エレメント、ウイルス（バキュロウイルス、パポバウイルス、ワクシニアウイルス、アデノウイルス、アデノ随伴ウイルス、トリポックスウイルス、仮性狂犬病ウイルス、ヘルペスウイルス、レンチウイルス、レトロウイルスなど）、これらの組み合わせに由来するベクター（コスミド、ファージミドなど）などが挙げられる。上述した中では、プラスミドベクターが、汎用性が高く好ましい。また、細胞内に大量のドナーＤＮＡを導入しうる点においても、プラスミドベクターが好ましい。 Specific examples of vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, virus-derived vectors (bacterial plasmids, bacteriophage, yeast episomes, etc.), yeast chromosomal elements, viruses (vaculovirus, etc.). Examples include papovavirus, vaccinia virus, adenovirus, adeno-associated virus, tripoxvirus, pseudomad dog disease virus, herpesvirus, lentivirus, retrovirus, etc.), and vectors derived from these combinations (cosmid, phagemid, etc.). Among the above, the plasmid vector is preferable because of its high versatility. In addition, a plasmid vector is preferable in that a large amount of donor DNA can be introduced into the cell.
　ベクターの細胞内への導入方法は、本技術分野において周知であるため、説明を省略する。 Since the method for introducing the vector into cells is well known in the present technical field, the description thereof will be omitted.
　一実施形態において、ベクターは、翻訳開始コドンを非翻訳開始コドンに置換したポリヌクレオチド配列の５’末端側に、スプライシングアクセプター配列（ＳＡ配列）を含んでいる。一実施形態において、ベクターは、翻訳開始コドンを非翻訳開始コドンに置換したポリヌクレオチド配列の５’末端側に、切断サイト（ｇＲＮＡ認識配列など）、緩衝用のポリヌクレオチド、およびスプライシングアクセプター配列（ＳＡ配列）をこの順で含んでいる。 In one embodiment, the vector contains a splicing acceptor sequence (SA sequence) on the 5'end side of a polynucleotide sequence in which the translation initiation codon is replaced with a nontranslation initiation codon. In one embodiment, the vector has a cleavage site (such as a gRNA recognition sequence), a buffering polynucleotide, and a splicing acceptor sequence (such as a gRNA recognition sequence) on the 5'end of a polynucleotide sequence in which the translation initiation codon has been replaced with a non-translation initiation codon. SA sequence) is included in this order.
　この点について、図２を参照しながら説明する。図２において、ポリヌクレオチド１ａがノックインされる遺伝子には、３つのエキソン（Ｅ１、Ｅ２、Ｅ３）と２つのイントロン（Ｉ１、Ｉ２）とが存在する。そしてベクターの構成は、改変されたポリヌクレオチド１ａの５’末端側に、切断サイト（ｇＲＮＡ認識配列など）、緩衝用のポリヌクレオチド、およびスプライシングアクセプター配列を配置したものである。 This point will be explained with reference to FIG. In FIG. 2, there are three exons (E1, E2, E3) and two introns (I1, I2) in the gene in which the polynucleotide 1a is knocked in. The composition of the vector is such that a cleavage site (gRNA recognition sequence, etc.), a polynucleotide for buffering, and a splicing acceptor sequence are arranged on the 5'terminal side of the modified polynucleotide 1a.
　このようなベクターをイントロンＩ２内にノックインすると、イントロンＩ２がスプライシングによって除去される際に、エキソンＥ２とイントロンＩ２との境界から、スプライシングアクセプター配列までが除去されることになる（ジーントラップ）。その結果、エキソンＥ１、エキソンＥ２、改変されたポリヌクレオチド１ａが連続した成熟ｍＲＮＡが生成される。 When such a vector is knocked into the intron I2, when the intron I2 is removed by splicing, the boundary between the exon E2 and the intron I2 to the splicing acceptor sequence is removed (gene trap). The result is a mature mRNA in which exon E1, exon E2, and the modified polynucleotide 1a are contiguous.
　スプライシングアクセプター配列を用いずに上述した成熟ｍＲＮＡを生成する場合、改変されたポリヌクレオチド１ａを、エキソンＥ２の直後にノックインする必要がある。一方、スプライシングアクセプター配列を用いれば、改変されたポリヌクレオチド１ａをエキソンＥ２の直後にノックインする必要がない。その代わりに、改変されたポリヌクレオチド１ａをイントロンＩ２の任意の箇所にノックインすればよい。したがって、スプライシングアクセプター配列を用いれば、改変されたポリヌクレオチド１ａのノックイン箇所の候補を増やせるという利点がある。この利点は、換言すれば、ｇＲＮＡの設計を容易にするという利点でもある。 When producing the above-mentioned mature mRNA without using the splicing acceptor sequence, it is necessary to knock in the modified polynucleotide 1a immediately after exon E2. On the other hand, if the splicing acceptor sequence is used, it is not necessary to knock in the modified polynucleotide 1a immediately after the exon E2. Instead, the modified polynucleotide 1a may be knocked in anywhere in the intron I2. Therefore, the use of the splicing acceptor sequence has an advantage that the number of knock-in sites of the modified polynucleotide 1a can be increased. This advantage is, in other words, the advantage of facilitating the design of gRNAs.
　また、上述したとおり、スプライシングアクセプター配列を用いずに、上述した成熟ｍＲＮＡを生成しようとすれば、改変されたポリヌクレオチド１ａをエキソンＥ２の直後にノックインする必要がある。この場合、ポリヌクレオチド１ａとエキソンＥ２との境界領域にてindelが生じる可能性がある。ポリヌクレオチド１ａとエキソンＥ２との境界領域にてindelが生じると、エキソンＥ２にコードされるアミノ酸配列が変化したり、ポリヌクレオチド１ａの翻訳時にフレームシフトが生じたりする虞がある。一方、スプライシングアクセプター配列および緩衝用のポリヌクレオチドを用いれば、ノックインの際にindelが生じたとしても、緩衝用のポリヌクレオチド内、または、イントロンＩ２内にてindelが生じる。そのため、エキソンＥ２にコードされるアミノ酸配列が変化したり、ポリヌクレオチド１ａの翻訳時にフレームシフトが生じたりすることを防ぐことができるという利点がある。なお、ノックインの標的となる塩基配列がコードしている内在性タンパク質の変質を可能な限り防止するためには、挿入サイトはイントロンＩ１（１番目のエキソンと２番目のエキソンの間のイントロン）に設定することが好ましい。 Further, as described above, if the above-mentioned mature mRNA is to be produced without using the splicing acceptor sequence, it is necessary to knock in the modified polynucleotide 1a immediately after the exon E2. In this case, indel may occur at the boundary region between the polynucleotide 1a and the exon E2. If indel occurs at the boundary region between polynucleotide 1a and exon E2, the amino acid sequence encoded by exon E2 may change, or a frame shift may occur during translation of polynucleotide 1a. On the other hand, if a splicing acceptor sequence and a buffering polynucleotide are used, even if indel occurs during knock-in, indel occurs in the buffering polynucleotide or intron I2. Therefore, there is an advantage that it is possible to prevent a change in the amino acid sequence encoded by the exon E2 and a frame shift during translation of the polynucleotide 1a. In order to prevent alteration of the endogenous protein encoded by the base sequence that is the target of knock-in as much as possible, the insertion site should be intron I1 (intron between the first exon and the second exon). It is preferable to set it.
　ＳＡ配列は、ノックイン箇所に固有の配列であってもよいし、より汎用性のある配列であってもよい（詳細は、実施例４、５を参照）。 The SA sequence may be a sequence unique to the knock-in site or a more versatile sequence (see Examples 4 and 5 for details).
　本発明の一実施形態に係るベクターは、改変されたポリヌクレオチド１ａの他に、任意の配列を含んでいてもよい。例えば、改変されたポリヌクレオチド１ａの３’末端側に、改変されたポリヌクレオチド１ａをノックインする遺伝子の３’ＵＴＲ配列を配置すると、改変されたポリヌクレオチド１ａが安定して発現するようになるので、好ましい（図８、１０を参照）。あるいは、他のタンパク質（Ｃａｓ９など）を発現させるポリヌクレオチド配列を含んでいてもよい。 The vector according to one embodiment of the present invention may contain an arbitrary sequence in addition to the modified polynucleotide 1a. For example, if the 3'UTR sequence of the gene that knocks in the modified polynucleotide 1a is placed on the 3'terminal side of the modified polynucleotide 1a, the modified polynucleotide 1a will be stably expressed. , Preferred (see FIGS. 8 and 10). Alternatively, it may contain a polynucleotide sequence that expresses another protein (such as Cas9).
　〔３．キット〕
　本発明の一態様は、上述したポリヌクレオチドまたは上述したベクターを備えている、遺伝子組み換えキットである。本明細書において、「キット」とは、任意の用途に用いられる、任意の試薬などの組み合わせを意味する。この用途は、医学用途であってもよいし、実験用途であってもよい。当該用途は、具体的に、ゲノム編集用途であってもよい。 [3. kit〕
One aspect of the present invention is a genetically modified kit comprising the above-mentioned polynucleotide or the above-mentioned vector. As used herein, the term "kit" means any combination of reagents and the like used for any purpose. This use may be for medical use or for experimental use. Specifically, the application may be a genome editing application.
　一実施形態において、キットには、遺伝子組換えに用いられる試薬および／または補助的な物質が備えられている。このような試薬および／または補助的な物質の例としては、ヌクレアーゼ（Ｃａｓなど）、ｇＲＮＡ、ＴＡＬＥＮ、ＺＦＮが挙げられる。 In one embodiment, the kit comprises reagents and / or ancillary substances used for genetic recombination. Examples of such reagents and / or ancillary substances include nucleases (such as Cas), gRNAs, TALENs, ZFNs.
　キットは、試薬および／または補助的な物質を格納する、１つ以上の格納容器（ボックス、ボトル、ディッシュなど）を備えていてもよい。キットは、２つ以上の容器を備えており、各容器が当該キットの構成要素の一部を格納している態様であってもよい。このとき、ユーザは、各容器をまとめて受け取ってもよいし、個別に受け取ってもよい。 The kit may include one or more containment vessels (boxes, bottles, dishes, etc.) for storing reagents and / or ancillary substances. The kit may include two or more containers, each container containing a portion of the components of the kit. At this time, the user may receive each container collectively or individually.
　〔４．タンパク質（改変された組換え酵素）〕
　本発明の一態様は、上述したポリヌクレオチド（改変されたポリヌクレオチド１ａ）の翻訳産物である、タンパク質（改変された組換え酵素）である。翻訳開始コドン「ＡＴＧ」が非翻訳開始コドンに置換されている場合、これらのタンパク質は、本来はメチオニンである箇所が、他のアミノ酸に置換されていることになる。したがって、これらのタンパク質は、天然物からは区別される。 [4. Protein (modified recombinant enzyme)]
One aspect of the present invention is a protein (modified recombinant enzyme) which is a translation product of the above-mentioned polynucleotide (modified polynucleotide 1a). When the translation start codon "ATG" is replaced with the untranslated start codon, these proteins are replaced with other amino acids where they are originally methionine. Therefore, these proteins are distinguished from natural products.
　一実施形態において、上記タンパク質は、配列番号１０、１１または１２に記載のアミノ酸配列からなる組換え酵素である。これらの組換え酵素は、後述する実施例における、「Ｏｐｔｉ　Ｃｒｅ２」、「Ｏｐｔｉ　ＦＬＰ」および「Ｏｐｔｉ　Ｄｒｅ」の翻訳産物である。 In one embodiment, the protein is a recombinant enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 10, 11 or 12. These recombinant enzymes are translation products of "Opti Cre2", "Opti FLP" and "Opti Dre" in Examples described later.
　一実施形態において、上記タンパク質は、他のタンパク質との融合タンパク質であってもよい。融合タンパク質の作製方法は、本技術分野において周知であるため、説明を省略する。 In one embodiment, the protein may be a fusion protein with another protein. Since the method for producing the fusion protein is well known in the art, the description thereof will be omitted.
　〔５．遺伝子導入方法〕
　本発明の一態様に係る遺伝子組換え方法は、上述のポリヌクレオチドまたは上述のベクターを、細胞または被験体（例えば、ヒト被検体、非ヒト被検体）に導入する工程を含む。ここで、「細胞に導入する」とは、例えば、ポリヌクレオチドまたはベクターを、in vitroで細胞内に導入することを意図する。また、「被験体に導入する」とは、例えば、ポリヌクレオチドまたはベクターを、in vivoで生体を構成する細胞内に導入することを意図する。 [5. Gene transfer method]
The genetic recombination method according to one aspect of the present invention includes the step of introducing the above-mentioned polynucleotide or the above-mentioned vector into a cell or a subject (for example, a human subject or a non-human subject). Here, "introducing into a cell" is intended to introduce, for example, a polynucleotide or vector into a cell in vitro. In addition, "introducing into a subject" is intended to introduce, for example, a polynucleotide or vector into cells constituting a living body in vivo.
　一実施形態において、被験体はヒトではない。一実施形態において、被験体は、非ヒト哺乳動物である。非ヒト哺乳動物の例としては、偶蹄類（ウシ、イノシシ、ブタ、ヒツジ、ヤギなど）、奇蹄類（ウマなど）、齧歯類（マウス、ラット、ハムスター、リスなど）、ウサギ目（ウサギなど）、食肉類（イヌ、ネコ、フェレットなど）などが挙げられる。上述した非ヒト哺乳動物には、家畜またはコンパニオンアニマル（愛玩動物）に加えて、野生動物も包含される。 In one embodiment, the subject is not human. In one embodiment, the subject is a non-human mammal. Examples of non-human mammals include cloven-hoofed animals (boars, wild boars, pigs, sheep, goats, etc.), cloven-hoofed animals (horses, etc.), carnivores (mouse, rats, hamsters, squirrels, etc.) Etc.), meats (dogs, cats, ferrets, etc.). The non-human mammals described above include wild animals in addition to livestock or companion animals (pets).
　ポリヌクレオチドまたはベクターを、細胞または被験体に導入する方法は、特に限定されない。ベクターを細胞または被験体に導入する方法は、当該ベクターに応じた適切な方法が、本技術分野において広く知られている。ＤＮＡ分子（またはＲＮＡ分子）のまま、ポリヌクレオチドを細胞または被験体に導入する方法としては、エレクトロポレーション、マイクロインジェクション、ソノポレーション、レーザ照射、カチオン性物質（カチオン性ポリマー、カチオン性脂質、リン酸カルシウムなど）との複合化を利用したトランスフェクションなどが挙げられる。 The method for introducing a polynucleotide or vector into a cell or a subject is not particularly limited. As a method for introducing a vector into a cell or a subject, an appropriate method depending on the vector is widely known in the art. Methods for introducing a polynucleotide as a DNA molecule (or RNA molecule) into a cell or subject include electroporation, microinjection, sonoporation, laser irradiation, and cationic substances (cationic polymers, cationic lipids, etc.). Transfection using complexation with calcium phosphate, etc.) can be mentioned.
　［本発明の概要］で説明したように、リークの原因の１つとして、大量の修飾されていないＤＮＡの細胞内への導入があると考えられる。そのため、本発明の一実施形態に係る遺伝子組換え方法は、大量の修飾されていないＤＮＡを細胞内へ導入する導入形態と組み合わせることが好ましい。このような導入形態の例としては、エレクトロポレーション、ウイルス、マイクロインジェクション、脂質複合化トランスフェクションなどが挙げられる。 As explained in [Summary of the present invention], it is considered that one of the causes of the leak is the introduction of a large amount of unmodified DNA into cells. Therefore, the gene recombination method according to one embodiment of the present invention is preferably combined with an introduction form that introduces a large amount of unmodified DNA into a cell. Examples of such introduction forms include electroporation, virus, microinjection, lipid complex transfection and the like.
　〔６．Ｃｒｅに関する好ましい態様〕
　一実施形態において、改変されたポリヌクレオチド１ａがコードしているタンパク質は、Ｃｒｅ改変タンパク質である。 [6. Preferred embodiment regarding Cre]
In one embodiment, the protein encoded by the modified polynucleotide 1a is a Cre modified protein.
　好ましくは、改変されたポリヌクレオチド１ａは、野生型Ｃｒｅの翻訳開始コドンＡＴＧの１つ以上が、非翻訳開始コドン（好ましくは、ロイシンをコードしているコドン）に置換されているものである。より好ましくは、改変されたポリヌクレオチド１ａは、野生型Ｃｒｅの１番目、２８番目、３０番目、５８番目および７７番目のメチオニン残基に対応する翻訳開始コドン（ＡＴＧ）のうちの１つ以上が、非翻訳開始コドン（好ましくは、ロイシンをコードしているコドン）に置換されているものである。さらに好ましくは、改変されたポリヌクレオチド１ａは、野生型Ｃｒｅの１番目、２８番目、３０番目、５８番目および７７番目のメチオニン残基に対応する翻訳開始コドン（ＡＴＧ）が、非翻訳開始コドン（好ましくは、ロイシンをコードしているコドン）に置換されているものである。このような改変されたポリヌクレオチド１ａの塩基配列の例としては、配列番号５に記載の塩基配列が挙げられる。この塩基配列は、実施例における「Ｏｐｔｉ　Ｃｒｅ」に該当する。 Preferably, the modified polynucleotide 1a has one or more of the wild-type Cre translation initiation codons ATG replaced with non-translation initiation codons (preferably leucine-encoding codons). More preferably, the modified polynucleotide 1a contains one or more of the translation initiation codons (ATGs) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild Cre. , Is replaced with an untranslated start codon (preferably a codon encoding leucine). More preferably, the modified polynucleotide 1a has a translation initiation codon (ATG) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild-type Cre, which is a non-translation initiation codon (ATG). Preferably, it is substituted with a codon encoding leucine). An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 5. This base sequence corresponds to "Opti Cre" in the examples.
　より好ましくは、改変されたポリヌクレオチド１ａは、上述した翻訳開始コドンＡＴＧの置換に加えて、野生型Ｃｒｅの非典型翻訳開始コドンの１つ以上が、非翻訳開始コドンに置換されているものである。一実施形態において、非典型翻訳開始コドンから非翻訳開始コドンへの置換は、（i）ＣＴＧからＣＴＣへの置換、（ii）ＴＴＧからＴＴＡへの置換、（iii）ＧＴＧからＧＴＡへの置換、または、（iv）ＡＴＡからＡＴＴへの置換、である。一実施形態において、非翻訳開始コドンに置換される非典型翻訳開始コドンは、野生型Ｃｒｅをコードするポリヌクレオチドの５’末端から、３０ｂｐ以内、６０ｂｐ以内、９０ｂｐ以内、１２０ｂｐ以内、１８０ｂｐ以内、２１０ｂｐ以内、２４０ｂｐ以内、２７０ｂｐ以内、３００ｂｐ以内、３３０ｂｐ以内、３６０ｂｐ以内、３９０ｂｐ以内、４２０ｂｐ以内、４８０ｂｐ以内、５１０ｂｐ以内、５４０ｂｐ以内、５７０ｂｐ以内、または、６００ｂｐ以内の領域に存在する。このような改変されたポリヌクレオチド１ａの塩基配列の例としては、配列番号６に記載の塩基配列が挙げられる。この塩基配列は、実施例における「Ｏｐｔｉ　Ｃｒｅ２」に該当する。 More preferably, the modified polynucleotide 1a is one in which one or more of the atypical translation initiation codons of wild Cre is substituted with the untranslation initiation codon in addition to the substitution of the translation initiation codon ATG described above. is there. In one embodiment, the atypical translation start codon to non-translation start codon substitutions are (i) CTG to CTC substitution, (ii) TTG to TTA substitution, (iii) GTG to GTA substitution, Or (iv) substitution from ATA to ATT. In one embodiment, the atypical translation initiation codon substituted for the untranslation initiation codon is within 30 bp, within 60 bp, within 90 bp, within 120 bp, within 180 bp, 210 bp from the 5'end of the polynucleotide encoding the wild-type Cre. Within, within 240 bp, within 270 bp, within 300 bp, within 330 bp, within 360 bp, within 390 bp, within 420 bp, within 480 bp, within 510 bp, within 540 bp, within 570 bp, or within 600 bp. An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 6. This base sequence corresponds to "Opti Cre2" in the examples.
　本項目に記載した態様の改変されたポリヌクレオチド１ａの翻訳産物である、Ｃｒｅ改変タンパク質もまた、本発明の好ましい実施態様である。Ｃｒｅ改変タンパク質のアミノ酸配列の例としては、配列番号１０に記載のアミノ酸配列が挙げられる。このアミノ酸配列は、実施例における「Ｏｐｔｉ　Ｃｒｅ」または「Ｏｐｔｉ　Ｃｒｅ２」の翻訳産物である。この翻訳産物は、野生型Ｃｒｅに見られる毒性が低減されている可能性が示唆されている（詳細には、実施例７を参照）。 Cre-modified protein, which is a translation product of the modified polynucleotide 1a of the embodiment described in this item, is also a preferred embodiment of the present invention. Examples of the amino acid sequence of the Cre modified protein include the amino acid sequence set forth in SEQ ID NO: 10. This amino acid sequence is a translation of "Opti Cre" or "Opti Cre 2" in the Examples. It has been suggested that this translation product may have reduced toxicity found in wild-type Cre (see Example 7 for details).
　〔材料および方法〕
　特に記載がない限り、実験に使用した材料および方法は以下の通りである。 〔Materials and methods〕
Unless otherwise stated, the materials and methods used in the experiment are as follows.
　［実験動物］
・マウス：ＩＣＲマウス（日本ＳＬＣより購入）
・フェレット：セーブルフェレット（Marshal bioResourcesより購入）。 [Experimental animals]
・ Mouse: ICR mouse (purchased from Japan SLC)
-Ferret: Sable ferret (purchased from Marshal bioResources).
　［プラスミド］
　プラスミドのボディとして、pLeaklessIII（2016 developmentで開発したベクター）を使用した。プラスミドへの導入配列の組み込みには、infusion（TAKARA製）を使用した。 [Plasid]
As the body of the plasmid, pLeaklessIII (vector developed in 2016 development) was used. Infusion (manufactured by TAKARA) was used for integration of the introduction sequence into the plasmid.
　［組換え酵素（Ｃｒｅ）］
　野生型Ｃｒｅ、翻訳開始コドン（ＡＴＧ）を非翻訳開始コドンに置換したＣｒｅ（Ｏｐｔｉ　Ｃｒｅ）、および、非典型翻訳開始コドンも非翻訳開始コドンに置換したＣｒｅ（Ｏｐｔｉ　Ｃｒｅ２）を使用した。Ｏｐｔｉ　Ｃｒｅをコードしている塩基配列では、野生型Ｃｒｅの１番目、２８番目、３０番目、５８番目および７７番目のメチオニン残基に対応するコドン（ＡＴＧ）を、ロイシン残基をコードしているコドン（ＴＴＡ、ＴＴＧ、ＣＴＴ、ＣＴＣ、ＣＴＡまたはＣＴＧ）に置換した。さらに、Ｏｐｔｉ　Ｃｒｅ２をコードしている塩基配列では、Ｏｐｔｉ　Ｃｒｅに含まれている非典型翻訳開始コドンも非翻訳開始コドンに置換した。具体的には、５’末端から６００ｂｐまでに存在する、ＣＴＧをＣＴＣに置換し、ＴＴＧをＴＴＡに置換し、ＧＴＧをＧＴＡに置換し、ＡＴＡをＡＴＴに置換した。なお、Ｏｐｔｉ　Ｃｒｅ２において、置換前のコドンがコードしているアミノ酸と、置換後のコドンがコードしているアミノ酸とは、同じアミノ酸である。 [Recombinant enzyme (Cre)]
Wild-type Cre, Cre (Opti Cre) in which the translation start codon (ATG) was replaced with the untranslated start codon, and Cre (Opti Cre2) in which the atypical translation start codon was also replaced with the untranslated start codon were used. In the nucleotide sequence encoding Opti Cre, the codon (ATG) corresponding to the 1st, 28th, 30th, 58th, and 77th methionine residues of wild-type Cre is encoded by the leucine residue. Substituted with codons (TTA, TTG, CTT, CTC, CTA or CTG). Furthermore, in the base sequence encoding Opti Cre2, the atypical translation initiation codon contained in Opti Cre was also replaced with the non-translation initiation codon. Specifically, CTG, which exists from the 5'end to 600 bp, was replaced with CTC, TTG was replaced with TTA, GTG was replaced with GTA, and ATA was replaced with ATT. In Opti Cre2, the amino acid encoded by the codon before substitution and the amino acid encoded by the codon after substitution are the same amino acid.
　野生型Ｃｒｅをコードしている塩基配列は配列番号１、野生型Ｃｒｅのアミノ酸配列は配列番号９である。Ｏｐｔｉ　Ｃｒｅをコードしている塩基配列は配列番号５、Ｏｐｔｉ　Ｃｒｅのアミノ酸配列は配列番号１０である。Ｏｐｔｉ　Ｃｒｅ２をコードしている塩基配列は配列番号６、Ｏｐｔｉ　Ｃｒｅ２のアミノ酸配列はＯｐｔｉ　Ｃｒｅのアミノ酸配列と同じである。 The nucleotide sequence encoding the wild-type Cre is SEQ ID NO: 1, and the amino acid sequence of the wild-type Cre is SEQ ID NO: 9. The nucleotide sequence encoding Opti Cre is SEQ ID NO: 5, and the amino acid sequence of Opti Cre is SEQ ID NO: 10. The nucleotide sequence encoding Opti Cre2 is the same as SEQ ID NO: 6, and the amino acid sequence of Opti Cre2 is the same as the amino acid sequence of Opti Cre.
　［組換え酵素以外のタンパク質］
　組換え酵素の導入には、ヒト化Ｃａｓ９（ｈＣａｓ９）を使用した。ｈＣａｓ９をコードしている塩基配列は、配列番号１３である。 [Proteins other than recombinant enzymes]
Humanized Cas9 (hCas9) was used for the introduction of the recombinant enzyme. The nucleotide sequence encoding hCas9 is SEQ ID NO: 13.
　レポーターとしては、ｍＣｈｅｒｒｙ（赤色蛍光タンパク質）またはｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列（Ｃｒｅの発現によってＥＧＦＰが発現するように構築された配列）を使用した。ｍＣｈｅｒｙをコードしている塩基配列は、配列番号１４である。ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列をコードしている塩基配列は、配列番号１５である。 As a reporter, mCherry (red fluorescent protein) or flox-STOP-EGFP sequence (a sequence constructed so that EGFP is expressed by the expression of Cre) was used. The base sequence encoding mChery is SEQ ID NO: 14. The nucleotide sequence encoding the flox-STOP-EGFP sequence is SEQ ID NO: 15.
　［プロモーター配列］
　非特異的な遺伝子発現のためのプロモーター配列としては、ＣＡＧプロモーターを使用した。神経細胞において部位特異的に遺伝子を発現させるためのプロモーター配列としては、ＣＡＧプロモーターを改変したＣＡＸプロモーター配列を使用した。ＣＡＧプロモーターおよびＣＡＸプロモーターのプロモーター領域は同じであり、配列番号は２４である。 [Promoter sequence]
The CAG promoter was used as the promoter sequence for non-specific gene expression. As a promoter sequence for site-specific expression of a gene in a nerve cell, a CAX promoter sequence modified from the CAG promoter was used. The promoter regions of the CAG promoter and the CAX promoter are the same, and the SEQ ID NO: 24.
　［スプライシングアクセプター配列（ＳＡ配列）］
　マウスＴｕｂｂ３遺伝子第４エキソンのＳＡ配列（配列番号１６）、または、ヒト免疫グロブリンのＳＡ配列（chimeric intron；配列番号１７）を使用した。 [Splicing acceptor sequence (SA sequence)]
The SA sequence of mouse Tubb3 gene 4th exon (SEQ ID NO: 16) or the SA sequence of human immunoglobulin (chimeric intron; SEQ ID NO: 17) was used.
　［実験動物への遺伝子導入］
　マウス（またはフェレット）胎児の大脳皮質に、in uteroエレクトロポレーションによってプラスミドを導入した。 [Gene transfer into experimental animals]
The plasmid was introduced into the cerebral cortex of a mouse (or ferret) fetal by in utero electroporation.
　〔実施例１：野生型Ｃｒｅ遺伝子のリーク〕
　野生型Ｃｒｅ（翻訳開始コドンを置換していないＣｒｅ）を含んでいるドナーＤＮＡを用いて、ドナーＤＮＡからのリークの有無を検討した。具体的には、神経細胞に特異的に発現しているＴｕｂｂ３遺伝子座に、野生型Ｃｒｅをde novoノックインした。遺伝子の導入には、ＨＩＴＩ法を採用した。ＨＩＴＩ法はホモロジーアームなどの配列を必要としないため、Ｃｒｅ以外の配列がリークに及ぼす影響を最小限に抑制できる。 [Example 1: Leakage of wild-type Cre gene]
The presence or absence of leaks from the donor DNA was examined using donor DNA containing wild-type Cre (Cr that did not replace the translation initiation codon). Specifically, wild-type Cre was knocked in to the Tubb3 locus specifically expressed in nerve cells. The HITI method was adopted for gene transfer. Since the HITI method does not require a sequence such as a homology arm, the influence of sequences other than Cre on the leak can be minimized.
　野生型Ｃｒｅを含んでいるドナーＤＮＡの具体的な構成を、図３に示す。このドナーＤＮＡは、野生型Ｃｒｅをコードしている塩基配列の両端に、Ｔｕｂｂ３遺伝子の３’末端に存在するｇＲＮＡ認識配列（配列番号１８）を配置したものである。 The specific composition of the donor DNA containing the wild-type Cre is shown in FIG. In this donor DNA, a gRNA recognition sequence (SEQ ID NO: 18) existing at the 3'end of the Tubb3 gene is arranged at both ends of the base sequence encoding the wild-type Cre.
　胎生１４日目のマウス胎児の大脳皮質に、下記のベクターを導入した。また、ｈＣａｓ９の発現ベクター以外の発現ベクターを導入したマウスを、ネガティブコントロールとした。
・pCAX-hCas9（ｈＣａｓ９の発現ベクター）
・pCAG-mCherry-gRNA（ｍＣｈｅｒｒｙおよびｇＲＮＡの発現ベクター）
・pCAG-flox-STOP-EGFP（ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるレポーターベクター）
・ドナーＤＮＡ（Ｃｒｅを含んでいる）。 The following vector was introduced into the cerebral cortex of a mouse fetal on the 14th day of embryonic development. In addition, mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
-PCAX-hCas9 (expression vector of hCas9)
-PCAG-mCherry-gRNA (expression vector of mCherry and gRNA)
-PCAG-flox-STOP-EGFP (reporter vector containing the fluorx-STOP-EGFP sequence)
-Donner DNA (containing Cre).
　ベクターの導入から４８時間後に脳を摘出し、大脳皮質の切片を作製した。この切片を、ｍＣｈｅｒｒｙに対する抗体およびＥＧＦＰに対する抗体で抗体染色し、顕微鏡で観察した。その結果を図４に示す。 48 hours after the introduction of the vector, the brain was excised to prepare a section of the cerebral cortex. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
　図４より、ｈＣａｓ９を導入した群だけでなく、ｈＣａｓ９を導入していない群においても、ＥＧＦＰの発現が観察された。つまり、ノックインが生じていない細胞においても、ドナーＤＮＡからのリークによってＣｒｅが発現していることが判った。実施例１において用いたドナーＤＮＡは、Ｃｒｅをコードしている配列以外は、２３ｂｐしか有していなかったにもかかわらず、リークが生じていた。そのため、転写の制御によりリークを抑制することは困難であると考えられる。 From FIG. 4, the expression of EGFP was observed not only in the group into which hCas9 was introduced but also in the group in which hCas9 was not introduced. That is, it was found that Cre was expressed by the leak from the donor DNA even in the cells in which knock-in did not occur. The donor DNA used in Example 1 had a leak even though it had only 23 bp except for the sequence encoding Cre. Therefore, it is considered difficult to suppress the leak by controlling transcription.
　〔実施例２：野生型Ｃｒｅに含まれるメチオニンと組換え活性の関係〕
　野生型Ｃｒｅに含まれているメチオニンが、Ｃｒｅの組換え活性に寄与しているか否かを検討した。具体的には、（i）全長の野生型Ｃｒｅタンパク質、および、（ii）野生型Ｃｒｅタンパク質の２８番目、３０番目、５８番目、７７番目または１４５番目のメチオニンから翻訳を開始して得られるポリペプチド、の発現ベクターを作製した。これらをそれぞれ、Ｃｒｅ１～Ｃｒｅ６と称する（図５を参照）。 [Example 2: Relationship between methionine contained in wild-type Cre and recombinant activity]
It was examined whether or not methionine contained in wild-type Cre contributed to the recombination activity of Cre. Specifically, the poly obtained by initiating translation from (i) the full-length wild-type Cre protein and (ii) the 28th, 30th, 58th, 77th or 145th methionine of the wild-type Cre protein. An expression vector for the peptide was prepared. These are referred to as Cre1 to Cre6, respectively (see FIG. 5).
　胎生１４日目のマウス胎児の大脳皮質に、下記のベクターを導入した。
・pCAG-Cre-1-6（Ｃｒｅ１～Ｃｒｅ６のいずれかの発現ベクター）
・pCAG-mChery（ｍＣｈｅｒｒｙの発現ベクター）
・pCAG-flox-STOP-EGFP（ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるレポーターベクター）。 The following vector was introduced into the cerebral cortex of a mouse fetal on the 14th day of embryonic development.
-PCAG-Cre-1-6 (expression vector of any of Cre1 to Cre6)
-PCAG-mChery (expression vector of mCherry)
-PCAG-flox-STOP-EGFP (reporter vector containing the fluorx-STOP-EGFP sequence).
　ベクターの導入から４８時間後に脳を摘出し、大脳皮質の切片を作製した。この切片を、ｍＣｈｅｒｒｙに対する抗体およびＥＧＦＰに対する抗体で抗体染色し、顕微鏡で観察した。その結果を図６に示す。 48 hours after the introduction of the vector, the brain was excised to prepare a section of the cerebral cortex. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
　図６より、Ｃｒｅ５およびＣｒｅ６は、組換え活性を全く有していないことが判った。この結果から、野生型Ｃｒｅタンパク質の１～７７番目のアミノ酸が欠失していたとしても、翻訳産物であるポリペプチドは組換え活性を有していることが示唆される。そこで、本発明者らは、野生型Ｃｒｅタンパク質の１番目、２８番目、３０番目、５８番目および７７番目のメチオニン残基に対応するコドン（ＡＴＧ）を、ロイシン残基をコードしているコドンに置換した改変型Ｃｒｅ遺伝子（Ｏｐｔｉ　Ｃｒｅ）を作製し、Ｏｐｔｉ　Ｃｒｅの機能を検討することにした。 From FIG. 6, it was found that Cre5 and Cre6 had no recombinant activity. From this result, it is suggested that the polypeptide which is a translation product has recombinant activity even if the amino acids 1 to 77 of the wild-type Cre protein are deleted. Therefore, we have changed the codons (ATG) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of the wild-type Cre protein to the codons encoding the leucine residues. We decided to prepare a substituted modified Cre gene (Opti Cre) and examine the function of Opti Cre.
　〔実施例３：Ｏｐｔｉ　Ｃｒｅ遺伝子のリーク〕
　Ｏｐｔｉ　Ｃｒｅを含んでいるドナーＤＮＡを用いて、ドナーＤＮＡからのリークの有無を検討した。具体的には、野生型ＣｒｅをＯｐｔｉ　Ｃｒｅに変更して、実施例１と同じ条件で実験を行った。その結果を、図７に示す。 [Example 3: Leakage of Opti Cre gene]
Using donor DNA containing Opti Cre, the presence or absence of leaks from the donor DNA was examined. Specifically, the wild-type Cre was changed to Opti Cre, and the experiment was conducted under the same conditions as in Example 1. The result is shown in FIG.
　図７より、ｈＣａｓ９を導入した群ではＥＧＦＰの発現が観察されたのに対し、ｈＣａｓ９を導入していない群ではＥＧＦＰの発現が観察されなかった。つまり、ノックインが生じていない細胞においては、ドナーＤＮＡからのリークが抑制されていた。このように、Ｃｒｅ遺伝子に含まれている翻訳開始コドン（ＡＴＧ）を非翻訳開始コドンに置換することによって、ドナーＤＮＡからのリークを低減することができる。 From FIG. 7, EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. In this way, by substituting the translation start codon (ATG) contained in the Cre gene with the untranslated start codon, leakage from the donor DNA can be reduced.
　〔実施例４：ＳＡ配列を有するドナーＤＮＡ〕
　スプライシングアクセプター配列（ＳＡ配列）およびＯｐｔｉ　Ｃｒｅを含んでいるドナーＤＮＡを用いて、Ｏｐｔｉ　Ｃｒｅをイントロン中に導入して、発現させた。具体的には、５’末端側から順に、ＳＡ配列（マウスＴｕｂｂ３遺伝子第４エキソンのＳＡ配列）、Ｏｐｔｉ　Ｃｒｅをコードしている塩基配列、マウスＴｕｂｂ３遺伝子の３’ＵＴＲ領域を順に配置したドナーベクターを作製した（より詳細な構成は、図８の上パネル参照）。このドナーＤＮＡを、ＨＩＴＩ法によって、マウスＴｕｂｂ３遺伝子のイントロン領域に導入した（図８の下パネル参照）。使用したｇＲＮＡ認識配列は、配列番号１９である。 [Example 4: Donor DNA having SA sequence]
Opti Cre was introduced into the intron and expressed using donor DNA containing the splicing acceptor sequence (SA sequence) and Opti Cre. Specifically, a donor vector in which the SA sequence (SA sequence of the 4th exon of the mouse Tubb3 gene), the base sequence encoding Opti Cre, and the 3'UTR region of the mouse Tubb3 gene are arranged in order from the 5'end side. (See the upper panel in FIG. 8 for a more detailed configuration). This donor DNA was introduced into the intron region of the mouse Tubb3 gene by the HITI method (see the lower panel of FIG. 8). The gRNA recognition sequence used is SEQ ID NO: 19.
　胎生１３日目のマウス胎児の大脳皮質に、下記のベクターを導入した。また、ｈＣａｓ９の発現ベクター以外の発現ベクターを導入したマウスを、ネガティブコントロールとした。
・pCAX-hCas9（ｈＣａｓ９の発現ベクター）
・pgRNA（ｇＲＮＡの発現ベクター）
・pCAG-mCherry（ｍＣｈｅｒｒｙの発現ベクター）
・pCAG-flox-STOP-EGFP（ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるレポーターベクター）
・pCAG hyper piggybase（ｈｙｐｅｒ　ｐｉｇｇｙｂａｓｅの発現ベクター）
・ドナーＤＮＡ（ＳＡ配列およびＯｐｔｉ　Ｃｒｅを含んでいる）。 The following vector was introduced into the cerebral cortex of a mouse fetal on the 13th day of embryonic development. In addition, mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
-PCAX-hCas9 (expression vector of hCas9)
・ PgRNA (expression vector of gRNA)
-PCAG-mCherry (expression vector of mCherry)
-PCAG-flox-STOP-EGFP (reporter vector containing the fluorx-STOP-EGFP sequence)
-PCAG hyper piggybase (expression vector of hyper piggybase)
-Donner DNA (containing SA sequence and Opti Cre).
　ベクターの導入から７２時間後に脳を摘出し、大脳皮質の切片を作製した。この切片を、ｍＣｈｅｒｒｙに対する抗体およびＥＧＦＰに対する抗体で抗体染色し、顕微鏡で観察した。その結果を図９に示す。 72 hours after the introduction of the vector, the brain was excised to prepare a section of the cerebral cortex. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
　図９より、ｈＣａｓ９を導入した群ではＥＧＦＰの発現が観察されたのに対し、ｈＣａｓ９を導入していない群ではＥＧＦＰの発現が観察されなかった。つまり、ノックインが生じていない細胞においては、ドナーＤＮＡからのリークが抑制されていた。このことから、Ｏｐｔｉ　Ｃｒｅの５’末端側にＳＡ配列を配置したドナーＤＮＡを用いると、gene trap法によって、Ｏｐｔｉ　Ｃｒｅを部位特異的に発現させられることが示唆された。 From FIG. 9, EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. From this, it was suggested that Opti Cre can be expressed site-specifically by the gene trap method when a donor DNA in which the SA sequence is arranged on the 5'terminal side of Opti Cre is used.
　〔実施例５：ＳＡ配列を有するドナーＤＮＡ（その２）〕
　実施例４では、遺伝子導入の標的箇所に固有の内在性ＳＡ配列を使用した。これに対して、実施例５では、より汎用性のある内在性ＳＡ配列を使用してＯｐｔｉ　Ｃｒｅを使用した。具体的には、ヒト免疫グロブリンのＳＡ配列（chimeric intron）をＳＡ配列として用いた（具体的なドナーＤＮＡの構成は、図１０を参照）。ＳＡ配列を変更した以外は実施例４と同様にして、実験を行った。結果を図１１に示す。 [Example 5: Donor DNA having SA sequence (No. 2)]
In Example 4, an endogenous SA sequence unique to the target site for gene transfer was used. In contrast, in Example 5, Opti Cre was used with a more versatile endogenous SA sequence. Specifically, the SA sequence (chimeric intron) of human immunoglobulin was used as the SA sequence (see FIG. 10 for the specific composition of the donor DNA). The experiment was carried out in the same manner as in Example 4 except that the SA sequence was changed. The results are shown in FIG.
　図１１より、ｈＣａｓ９を導入した群ではＥＧＦＰの発現が観察されたのに対し、ｈＣａｓ９を導入していない群ではＥＧＦＰの発現が観察されなかった。つまり、ノックインが生じていない細胞においては、ドナーＤＮＡからのリークが抑制されていた。このことから、内在性ではないＳＡ配列を用いた場合にも、gene trap法によって、Ｏｐｔｉ　Ｃｒｅを部位特異的に発現させられることが示唆された。 From FIG. 11, EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. This suggests that Opti Cre can be expressed site-specifically by the gene trap method even when a non-endogenous SA sequence is used.
　〔実施例６：非マウスモデルおよびＨＤＲを使用するモデルへの応用〕
　上記の実施例で検討した遺伝子組換え方法が、非マウスモデルおよびＨＤＲを使用するモデルにも応用できるかどうかを検討した。具体的には、モデル動物としてフェレットを用いた。相同組換え修復（Homology Directed Repair；ＨＤＲ）を利用して、ドナーＤＮＡをゲノムに導入した。ＨＤＲを利用する場合、ドナーＤＮＡには、５００～１０００ｂｐのホモロジーアームを持たせる必要がある。そのため、ＨＤＲを利用する実験系でもリークを低減できるならば、本発明の一実施形態に係る遺伝子組換え方法は、リークを低減する有力な方法と言うことができよう。実施例６で用いた具体的なドナーＤＮＡの構成は、図１２を参照。 [Example 6: Application to a non-mouse model and a model using HDR]
It was examined whether the gene recombination method examined in the above example could be applied to a non-mouse model and a model using HDR. Specifically, a ferret was used as a model animal. Donor DNA was introduced into the genome using Homology Directed Repair (HDR). When using HDR, the donor DNA needs to have a homology arm of 500 to 1000 bp. Therefore, if the leak can be reduced even in an experimental system using HDR, the gene recombination method according to the embodiment of the present invention can be said to be a promising method for reducing the leak. See FIG. 12 for the specific composition of the donor DNA used in Example 6.
　なお、実施例６では、ＡＴＧに加えて非典型翻訳開始コドンも置換した改変型Ｃｒｅ（Ｏｐｔｉ　Ｃｒｅ２）を用いた。また、遺伝子導入の標的は、未分化細胞に特異的に発現しているＰａｘ６遺伝子とした（使用したｇＲＮＡ認識配列は、配列番号２０）。さらに、（i）ｍＣｈｅｒｒｙおよびｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるベクターにＬＲ配列（配列番号２１および２２）を含ませ、（ii）トランスポゾンの一種であるｈｙｐｅｒ　ｐｉｇｇｙｂａｓｅもゲノム内に導入した。これにより、ｍＣｈｅｒｒｙおよびｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列が、ランダムにゲノムに組込まれるようになる。それゆえ、ｍＣｈｅｒｒｙおよびｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列が組込まれた細胞の割合が、細胞分裂によって低下することを防げる。 In Example 6, a modified Cre (Opti Cre2) in which the atypical translation start codon was substituted in addition to ATG was used. The target of gene transfer was the Pax6 gene specifically expressed in undifferentiated cells (the gRNA recognition sequence used was SEQ ID NO: 20). Furthermore, (i) a vector containing mCherry and flox-STOP-EGFP sequences contained LR sequences (SEQ ID NOs: 21 and 22), and (ii) hyper piggybase, which is a type of transposon, was also introduced into the genome. This allows the mCherry and flox-STOP-EGFP sequences to be randomly integrated into the genome. Therefore, it is possible to prevent the proportion of cells incorporating the mCherry and flox-STOP-EGFP sequences from being reduced by cell division.
　胎生３０日目のフェレット胎児の大脳皮質に、下記のベクターを導入した。また、ｈＣａｓ９の発現ベクター以外の発現ベクターを導入したフェレットを、ネガティブコントロールとした。
・pCAX-hCas9（ｈＣａｓ９の発現ベクター）
・pLR5 CAG-mCherry（ｍＣｈｅｒｒｙの発現ベクター；ＬＲ配列を有している）
・Pax6 gRNA 発現ベクター（ｇＲＮＡの発現ベクター）
・pLR5 CAG-flox-STOP-EGFP（ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるレポーターベクター；ＬＲ配列を有している）
・HDR Cre Donor（Ｏｐｔｉ　Ｃｒｅ２のドナーＤＮＡ）
・pCAX hyper piggybase（ｈｙｐｅｒ　ｐｉｇｇｙｂａｓｅの発現ベクター）。 The following vector was introduced into the cerebral cortex of a ferret fetal on the 30th day of embryonic development. Further, a ferret into which an expression vector other than the expression vector of hCas9 was introduced was used as a negative control.
-PCAX-hCas9 (expression vector of hCas9)
-PLR5 CAG-mCherry (mCherry expression vector; has LR sequence)
-Pax6 gRNA expression vector (gRNA expression vector)
-PLR5 CAG-flox-STOP-EGFP (reporter vector containing fluorx-STOP-EGFP sequence; having LR sequence)
・ HDR Cre Donor (Donator DNA of Opti Cre2)
-PCAX hyper piggybase (expression vector of hyper piggybase).
　ベクターの導入から４日間後に脳を摘出し、大脳皮質の切片を作製した。この切片を、ｍＣｈｅｒｒｙに対する抗体およびＥＧＦＰに対する抗体で抗体染色し、顕微鏡で観察した。その結果を図１３に示す。 Four days after the introduction of the vector, the brain was excised and a section of the cerebral cortex was prepared. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
　図１３より、ｈＣａｓ９を導入した群ではＥＧＦＰの発現が観察されたのに対し、ｈＣａｓ９を導入していない群ではＥＧＦＰの発現が観察されなかった。つまり、ノックインが生じていない細胞においては、ドナーＤＮＡからのリークが抑制されていた。このように、Ｏｐｔｉ　Ｃｒｅ２を用いれば、非マウスモデルおよびＨＤＲを使用するモデルにおいても、リークを低減できることが判った。 From FIG. 13, EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. As described above, it was found that the leakage can be reduced even in the non-mouse model and the model using HDR by using Opti Cre2.
　〔実施例７：ＯｐｔｉＣｒｅ２と野生型Ｃｒｅとの活性の比較〕
　培養細胞を利用したアッセイによって、ＯｐｔｉＣｒｅ２の組換え活性と、野生型Ｃｒｅの組換え活性とを比較した。 [Example 7: Comparison of activity between OptiCre2 and wild-type Cre]
The recombinant activity of OptiCre2 was compared with the recombinant activity of wild-type Cre by an assay using cultured cells.
　２９３Ｔ細胞を６ウェルプレートに播種し、lipofectamine 2000を用いて下記のベクターを導入した。
・pCAG-CreまたはpCAG optiCre2（それぞれ、ＣｒｅまたはＯｐｔｉ　Ｃｒｅ２の発現ベクター）
・CAG-flox-STOP-EGFP（ｆｌｏｘ－ＳＴＯＰ－ＥＧＦＰ配列を含んでいるレポーターベクター）
　なお、pCAG optiCre2においては、Ｏｐｔｉ　Ｃｒｅ２をコードしている塩基配列の５’末端側にＡＴＧを配置し、Ｏｐｔｉ　Ｃｒｅ２が通常に翻訳されるようにした。また、pCAG-CreまたはpCAG optiCre2は、稀釈系列を作製し、段階的な濃度でウェルに注入した（１μｇ／ウェル、１００ｎｇ／ウェル、１０ｎｇ／ウェル、１ｎｇ／ウェル、および１０ｐｇ／ウェル）。 293T cells were seeded on a 6-well plate and the following vector was introduced using lipofectamine 2000.
-PCAG-Cre or pCAG optiCre2 (Expression vector of Cre or OptiCre2, respectively)
-CAG-flox-STOP-EGFP (reporter vector containing the fluorx-STOP-EGFP sequence)
In pCAG optiCre2, ATG was placed on the 5'end side of the base sequence encoding OptiCre2 so that OptiCre2 could be translated normally. Also, pCAG-Cre or pCAG optiCre2 was made into a dilution series and injected into wells at gradual concentrations (1 μg / well, 100 ng / well, 10 ng / well, 1 ng / well, and 10 pg / well).
　遺伝子を導入してから２日後、ＦＡＣＳによって、ＥＧＦＰを発現している細胞が全細胞に占める割合を測定した。この測定値を、ポジティブコントロールにおいてＥＧＦＰを発現している細胞が全細胞に占める割合で除して、標準化した。ここで、ポジティブコントロールとしては、pCAG-EGFP（ＥＧＦＰの導入ベクター）を導入した細胞を用いた（１μｇ／ウェル）。ネガティブコントロールとしては、トランスフェクトしていない細胞を用いた。ＦＡＣＳによる測定結果を図１４～１７に、標準化後の結果を図１８に、それぞれ示す。 Two days after the gene was introduced, the ratio of EGFP-expressing cells to all cells was measured by FACS. This measurement was standardized by dividing by the percentage of all cells expressing EGFP in positive control. Here, as a positive control, cells into which pCAG-EGFP (EGFP introduction vector) was introduced were used (1 μg / well). As a negative control, untransfected cells were used. The measurement results by FACS are shown in FIGS. 14 to 17, and the results after standardization are shown in FIGS.
　図１８より、ＯｐｔｉＣｒｅ２は、野生型Ｃｒｅと同等の組換え活性を有していることが判る。 From FIG. 18, it can be seen that OptiCre2 has a recombination activity equivalent to that of wild-type Cre.
　また、１μｇ／ウェルの注入量においては、野生型ＣｒｅよりもＯｐｔｉ　Ｃｒｅ２の方がＥＧＦＰを発現している細胞の割合が多かった。従前、野生型Ｃｒｅには毒性が知られていたところ、この結果によると、Ｏｐｔｉ　Ｃｒｅ２は野生型Ｃｒｅの毒性を低減している可能性がある。つまり、Ｏｐｔｉ　Ｃｒｅ２は、ドナーＤＮＡからのリークを低減するだけでなく、野生型Ｃｒｅの毒性も低減した改変体であることが示唆された。 In addition, at the injection amount of 1 μg / well, the proportion of cells expressing EGFP was higher in Opti Cre2 than in wild-type Cre. Previously, the toxicity of wild-type Cre was known, and according to this result, Opti Cre2 may reduce the toxicity of wild-type Cre. That is, it was suggested that Opti Cre2 is a variant that not only reduces the leakage from the donor DNA but also reduces the toxicity of the wild-type Cre.
　〔実施例８：ＦＬＰおよびＤｒｅの改変型の活性〕
　実施例８では、Ｃｒｅ以外で使用頻度の高い酵素である、ＦＬＰおよびＤｒｅの改変体を作製し、活性を検討した。具体的には、ＯｐｔｉＣｒｅ２と同様に、野生型ＦＬＰおよび野生型Ｄｒｅに含まれている翻訳開始コドンを非翻訳開始コドンに置換した改変体を作製した。この改変体を、それぞれ、Ｏｐｔｉ　ＦＬＰおよびＯｐｔｉ　Ｄｒｅと称する。具体的な配列は、Ｏｐｔｉ　ＦＬＰの塩基配列が配列番号７、Ｏｐｔｉ　ＦＬＰのアミノ酸配列が配列番号１１、Ｏｐｔｉ　Ｄｒｅの塩基配列が配列番号８、Ｏｐｔｉ　Ｄｒｅのアミノ酸配列が配列番号１２である。 [Example 8: Modified activity of FLP and Dre]
In Example 8, variants of FLP and Dre, which are enzymes frequently used other than Cre, were prepared and their activities were examined. Specifically, similarly to OptiCre2, a variant in which the translation initiation codon contained in the wild-type FLP and the wild-type Dre was replaced with the non-translation initiation codon was prepared. These variants are referred to as Opti FLP and Opti Dre, respectively. The specific sequence is SEQ ID NO: 7 for the base sequence of Opti FLP, SEQ ID NO: 11 for the amino acid sequence of Opti FLP, SEQ ID NO: 8 for the base sequence of Opti Dre, and SEQ ID NO: 12 for the amino acid sequence of Opti Dre.
　これらの改変体について、実施例７と同様のアッセイで、組換え活性を検討した。その結果を図１９に示す。同図より、Ｏｐｔｉ　ＦＬＰおよびＯｐｔｉ　Ｄｒｅのいずれもが、野生型Ｃｒｅと同等の組換え活性を有していることが示された。このことから、Ｏｐｔｉ　ＦＬＰおよびＯｐｔｉ　Ｄｒｅは、組換え活性を有していることが示唆される。 The recombinant activity of these variants was examined by the same assay as in Example 7. The result is shown in FIG. From the figure, it was shown that both Opti FLP and Opti Dre have the same recombination activity as wild-type Cre. This suggests that Opti FLP and Opti Dre have recombinant activity.
　本発明は、例えば、生命工学分野（例えば、ゲノム編集）に利用することができる。 The present invention can be used, for example, in the field of biotechnology (for example, genome editing).
　１　：組換え酵素をコードしているポリヌクレオチド
　１ａ：改変されたポリヌクレオチド（本発明の一実施形態に係るポリヌクレオチド）
　５　：翻訳開始コドン
　５ａ：非翻訳開始コドン 1: Polynucleotide encoding a recombinant enzyme 1a: Modified polynucleotide (polynucleotide according to one embodiment of the present invention)
5: Translation start codon 5a: Non-translation start codon

Claims (8)

  1. 　以下の（Ｇ１）～（Ｇ４）の何れかに示される、ポリヌクレオチド：
    　（Ｇ１）（i）組換え酵素をコードしているポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド；
    　（Ｇ２）（i）組換え酵素をコードしているポリヌクレオチドの、１または数個の塩基が置換、欠失、挿入および／または付加された塩基配列からなるポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしている、ポリヌクレオチド；
    　（Ｇ３）（i）組換え酵素をコードしているポリヌクレオチドと配列同一性が９０％以上のポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド；
    　（Ｇ４）（i）組換え酵素をコードしているポリヌクレオチドと相補的なＤＮＡ配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドにおいて、少なくとも１つの翻訳開始コドンが非翻訳開始コドンに置換されているポリヌクレオチドからなり、かつ、（ii）組換え活性を有するタンパク質をコードしているポリヌクレオチド。     The polynucleotide shown in any of the following (G1) to (G4):
    (G1) (i) In a polynucleotide encoding a recombinant enzyme, a protein consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) a protein having recombinant activity. The polynucleotide encoding
    (G2) (i) At least one translation initiation in a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme. A polynucleotide consisting of a polynucleotide whose codon has been replaced with an untranslated start codon and (ii) encoding a protein having recombinant activity;
    (G3) (i) Consists of a polynucleotide in which at least one start codon is replaced with a non-translation start codon in a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme. And (ii) a polynucleotide encoding a protein having recombinant activity;
    (G4) (i) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is untranslated. A polynucleotide consisting of a polynucleotide substituted with a codon and (ii) encoding a protein having recombinant activity.
  2. 　上記翻訳開始コドンは、ＡＴＧ、ＣＴＧ、ＴＴＧ、ＧＴＧ、または、ＡＴＡである、請求項１に記載のポリヌクレオチド。 The polynucleotide according to claim 1, wherein the translation start codon is ATG, CTG, TTG, GTG, or ATA.
  3. 　上記組換え酵素は、Ｃｒｅ、ＦＬＰ、Ｄｒｅ、Ｔｒｅ、または、それらの改変体であるである、請求項１または２に記載のポリヌクレオチド。 The polynucleotide according to claim 1 or 2, wherein the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof.
  4. 　請求項１～３のいずれか１項に記載のポリヌクレオチドを含んでいる、ベクター。 A vector containing the polynucleotide according to any one of claims 1 to 3.
  5. 　上記ポリヌクレオチドの５’末端側に、スプライシングアクセプター配列をさらに含んでいる、請求項４に記載のベクター。 The vector according to claim 4, further comprising a splicing acceptor sequence on the 5'terminal side of the polynucleotide.
  6. 　請求項１～３のいずれか１項に記載のポリヌクレオチド、または、請求項４もしくは５に記載のベクターを備えている、遺伝子組換えキット。 A genetic recombination kit comprising the polynucleotide according to any one of claims 1 to 3 or the vector according to claim 4 or 5.
  7. 　配列番号１０に記載のアミノ酸配列からなるＣｒｅ改変タンパク質、またはその融合タンパク質。 Cre modified protein consisting of the amino acid sequence shown in SEQ ID NO: 10, or a fusion protein thereof.
  8. 　請求項１～３のいずれか１項に記載のポリヌクレオチド、または、請求項４もしくは５に記載のベクターを、細胞または非ヒト被験体に導入する工程を含む、遺伝子組換え方法。 A gene recombination method comprising the step of introducing the polynucleotide according to any one of claims 1 to 3 or the vector according to claim 4 or 5 into a cell or a non-human subject.
PCT/JP2020/040738 2019-10-31 2020-10-30 Polynucleotide and use for same WO2021085580A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019199153 2019-10-31
JP2019-199153 2019-10-31

Publications (1)

Publication Number Publication Date
WO2021085580A1 true WO2021085580A1 (en) 2021-05-06

Family

ID=75714516

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/040738 WO2021085580A1 (en) 2019-10-31 2020-10-30 Polynucleotide and use for same

Country Status (1)

Country Link
WO (1) WO2021085580A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070931A2 (en) * 2002-02-21 2003-08-28 Vision 7 Gmbh Methods for conducting site-specific dna recombination
US20140283156A1 (en) * 2013-03-14 2014-09-18 Cold Spring Harbor Laboratory Trans-splicing ribozymes and silent recombinases
JP2017526341A (en) * 2014-09-02 2017-09-14 ハインリヒ−ペッテ−インスティトゥート・ライブニッツ−インスティトゥート・フューア・エクスペリメンテレ・ヴィロロギー − シュティフトゥング・ビュルガーリヒェン・レッヒツＨｅｉｎｒｉｃｈ−Ｐｅｔｔｅ−Ｉｎｓｔｉｔｕｔ Ｌｅｉｂｎｉｚ−Ｉｎｓｔｉｔｕｔ Ｆｕｒ Ｅｘｐｅｒｉｍｅｎｔｅｌｌｅ Ｖｉｒｏｌｏｇｉｅ − Ｓｔｉｆｔｕｎｇ Ｂｕｒｇｅｒｌｉｃｈｅｎ Ｒｅｃｈｔｓ A well-tolerated and highly specific tailor-made recombinase for recombination of asymmetric target sites in multiple retrovirus strains
JP2018500037A (en) * 2014-12-31 2018-01-11 シンセティック ジェノミクス インコーポレーテッド Compositions and methods for highly efficient in vivo genome editing
JP2018506293A (en) * 2015-02-24 2018-03-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Binding-induced transcription switch and method of using the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070931A2 (en) * 2002-02-21 2003-08-28 Vision 7 Gmbh Methods for conducting site-specific dna recombination
US20140283156A1 (en) * 2013-03-14 2014-09-18 Cold Spring Harbor Laboratory Trans-splicing ribozymes and silent recombinases
JP2017526341A (en) * 2014-09-02 2017-09-14 ハインリヒ−ペッテ−インスティトゥート・ライブニッツ−インスティトゥート・フューア・エクスペリメンテレ・ヴィロロギー − シュティフトゥング・ビュルガーリヒェン・レッヒツＨｅｉｎｒｉｃｈ−Ｐｅｔｔｅ−Ｉｎｓｔｉｔｕｔ Ｌｅｉｂｎｉｚ−Ｉｎｓｔｉｔｕｔ Ｆｕｒ Ｅｘｐｅｒｉｍｅｎｔｅｌｌｅ Ｖｉｒｏｌｏｇｉｅ − Ｓｔｉｆｔｕｎｇ Ｂｕｒｇｅｒｌｉｃｈｅｎ Ｒｅｃｈｔｓ A well-tolerated and highly specific tailor-made recombinase for recombination of asymmetric target sites in multiple retrovirus strains
JP2018500037A (en) * 2014-12-31 2018-01-11 シンセティック ジェノミクス インコーポレーテッド Compositions and methods for highly efficient in vivo genome editing
JP2018506293A (en) * 2015-02-24 2018-03-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Binding-induced transcription switch and method of using the same

Similar Documents

Publication Publication Date Title
JP6486979B2 (en) Methods for modifying eukaryotic cells
US20160177336A1 (en) Expression Tools for Multiprotein Applications
Chiu et al. Transgenic mice that express Cre recombinase in osteoclasts
US20100129914A1 (en) Tol1 FACTOR TRANSPOSASE AND DNA INTRODUCTION SYSTEM USING THE SAME
KR100490188B1 (en) Sequence-specific DNA recombination in eukaryotic cells
EP1507865B1 (en) The frog prince, a transposon vector for gene transfer in vertebrates
US9161995B2 (en) Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
WO2018023014A1 (en) Mice comprising mutations resulting in expression of c-truncated fibrillin-1
US11622547B2 (en) Genetically modified mouse that expresses human albumin
JP4769796B2 (en) Hybrid recombinase for genome manipulation
WO2021085580A1 (en) Polynucleotide and use for same
US20080104723A1 (en) Development of Mammalian Genome Modification Technique Using Retrotransposon
US20210230637A1 (en) Method for producing homozygous cells
US20230180725A1 (en) Production of Human Cells, Tissues, and Organs in a Growth Factor Receptor-Deficient Animal Host

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20880975

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20880975

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP