WO2022249775A1 - ゲノムを編集する方法 - Google Patents
ゲノムを編集する方法 Download PDFInfo
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- WO2022249775A1 WO2022249775A1 PCT/JP2022/017353 JP2022017353W WO2022249775A1 WO 2022249775 A1 WO2022249775 A1 WO 2022249775A1 JP 2022017353 W JP2022017353 W JP 2022017353W WO 2022249775 A1 WO2022249775 A1 WO 2022249775A1
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- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
Definitions
- the present invention relates to a method for editing genomes.
- genome editing tools such as zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas systems have been developed, and genome editing using these tools has been actively carried out around the world.
- ZFNs and TALENs have the disadvantage that it takes time and effort to design sequence-specific nucleases
- the CRISPR/Cas system is especially adopted as a genome editing tool around the world because it is extremely easy to target.
- the drawbacks of the CRISPR/Cas system include off-target issues and the difficulty of delivering large nucleases to cells.
- endonucleases which are enzymes that cleave DNA, are used to edit the genome, so the size and design of the nucleases are necessary.
- Patent Documents 1 and 2 enable genome editing using only single-stranded polynucleotides without introducing components other than single-stranded polynucleotides into cells.
- genome editing is possible not only in prokaryotic cells but also in eukaryotic cells.
- the present invention provides a method for editing genomes.
- the present invention provides methods for editing genomes in cells, particularly eukaryotic cells.
- the present inventors have studied various formulations for increasing the editing efficiency of single-stranded polynucleotides, and surprisingly found that the editing efficiency is improved in the presence of a specific single-stranded antisense polynucleotide. , led to the present invention. That is, the present inventors synthesized a single-stranded antisense polynucleotide (also referred to as an "editing-enhancing antisense polynucleotide”) and, in the presence of the editing-enhancing antisense polynucleotide, formed a single-stranded form for editing.
- a single-stranded antisense polynucleotide also referred to as an "editing-enhancing antisense polynucleotide
- the editing-enhancing antisense polynucleotide has a sequence complementary to the terminal portion of the editing sense polynucleotide or is complementary to It was found that the efficiency of genome editing is enhanced when the gene is designed to have a sequence having a specific property (that is, when it is designed to bind to the terminus of the sense polynucleotide for editing).
- a genomic region transcribed into RNA is to be edited, binding to genomic DNA occurs between the transcribed RNA present around the genomic DNA and the editing sense polynucleotide.
- a single-stranded form of an editing-enhancing polynucleotide for use in combination with a single-stranded form of an editing polynucleotide capable of modifying a target site of double-stranded genomic DNA The target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site,
- the editing polynucleotide is, in order from the 5' end side, a) a portion hybridizable with the region adjacent to the 3' end of the primary editing site x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) the 5' end of the primary editing site consisting of portions hybridizable with laterally adjacent regions, or portion a) and portion b) consists of
- the editing-enhancing polynucleotide is hybridizable with a nucleic acid sequence that is complementary to
- a portion consisting of a base sequence complementary to x) can be inserted into the secondary editing site, and A primary editing site when the editing polynucleotide consists of part a), part x) and part b), and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand can be replaced with a portion consisting of a base sequence complementary to the portion x), and the secondary editing site can be replaced with the portion x), Deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand.
- a single-stranded form of an editing-enhancing polynucleotide for use in combination with a single-stranded form of an editing polynucleotide capable of modifying a target site of double-stranded genomic DNA The target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site,
- the editing polynucleotide is, in order from the 5' end side, a) A portion consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site x) Any portion consisting of a nucleotide sequence not identical to the nucleotide sequence of the secondary editing site and b) a portion consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 3'
- a portion consisting of a base sequence complementary to x) can be inserted into the secondary editing site, and A primary editing site when the editing polynucleotide consists of part a), part x) and part b), and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand can be replaced with a portion consisting of a base sequence complementary to the portion x), and the secondary editing site can be replaced with the portion x), Deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand.
- a single-stranded form of an editing-enhancing polynucleotide for use in combination with a single-stranded form of an editing polynucleotide capable of modifying a target site of double-stranded genomic DNA The target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site,
- the editing polynucleotide is, in order from the 5' end side, a) a portion consisting of a base sequence identical to the base sequence of the region adjacent to the 5′ end of the secondary editing site x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) two Consisting of a portion consisting of the same base sequence as the base sequence of the region adjacent to the 3' end of the next editing site, or part a) and part b) consists of The editing-promoting
- region A When the region at the position corresponding to a) is defined as region A and the region at the position corresponding to part b) of the editing polynucleotide is defined as region B, does the 5′ end overlap with region A? , or overlaps region B at its 3′ end,
- the length of overlap with region A at the 5' end of the predetermined region is the same as or shorter than region A, and the length of overlap with region B at the 3' end of the predetermined region is equal to or shorter than B
- a portion at the primary editing site when the editing polynucleotide consists of portion a), portion x) and portion b) and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand.
- a portion consisting of a base sequence complementary to x) can be inserted into the secondary editing site, and A primary editing site when the editing polynucleotide consists of part a), part x) and part b), and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand can be replaced with a portion consisting of a base sequence complementary to the portion x), and the secondary editing site can be replaced with the portion x), Deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple contiguous nucleotides on one genomic DNA strand.
- [Section 3A] The editing-enhancing polynucleotide of item 1A, item 2A, or item 2B, its expression vector, or a conjugate of said editing-enhancing polynucleotide or its expression vector with another substance, and an editing polynucleotide, its expression vector , or a genome editing kit comprising a conjugate of the editing polynucleotide or its expression vector and other substances,
- the editing polynucleotide is, in order from the 5' end side, a) a portion hybridizable with the region adjacent to the 3' end of the primary editing site x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) the 5' end of the primary editing site consisting of portions hybridizable with laterally adjacent regions, or portion a) and portion b)
- the genome editing kit comprising: [Section 3B] The editing-enhancing polynucleotide of Item 1B, Item 2A or
- genome editing can be performed efficiently.
- FIG. 1 shows polynucleotides (1) and (2) that are editing polynucleotides (sense polynucleotides) used in the experiment of this example, and polynucleotides that are editing-promoting polynucleotides (antisense polynucleotides) ( 3)-(16) and (22)-(27) designs are shown.
- FIG. 2 shows intron-targeted genome editing by editing polynucleotides (sense polynucleotides) in the presence of some of the editing-enhancing polynucleotides (antisense polynucleotides), polynucleotides (3)-(16). Shows change in efficiency.
- FIG. 1 shows polynucleotides (1) and (2) that are editing polynucleotides (sense polynucleotides) used in the experiment of this example, and polynucleotides that are editing-promoting polynucleotides (antisense polynucleotides) ( 3)
- FIG. 3 shows editing polynucleotides (sense polynucleotides) in the presence of several of polynucleotides (11)-(16) and (22)-(27) that are editing-enhancing polynucleotides (antisense polynucleotides). shows changes in intron-targeted genome editing efficiency by .
- FIG. 4 shows polynucleotides (18), (17) and (1), which are editing polynucleotides (sense polynucleotides) used in the experiment of this example, and editing-promoting polynucleotides (antisense polynucleotides). Designs of certain polynucleotides (3) and (4) are shown.
- FIG. 5 shows the case of using a polynucleotide (18) that is an editing polynucleotide (sense polynucleotide) having a base deletion in the sequence and an editing polynucleotide (sense polynucleotide) having a base insertion in the sequence. It shows that the genome editing efficiency is improved in the presence of an editing-promoting polynucleotide (antisense polynucleotide) in any case of using the polynucleotide (17), which is a nucleotide).
- FIG. 6 shows a polynucleotide (19) that is an exon-targeted editing polynucleotide (sense polynucleotide) and a polynucleotide that is an editing-promoting polynucleotide (antisense polynucleotide) used in the experiment of this example. Designs of (20) and (21) are shown.
- FIG. 7 shows that even in the presence of one type of editing-promoting polynucleotide (antisense polynucleotide), the efficiency of genome editing using an editing sense polynucleotide is greatly improved.
- FIG. 8A shows the mechanism of conventional genome editing (replacement) by editing polynucleotides.
- FIG. 8B shows the mechanism of genome editing (replacement) by an editing polynucleotide and two types of editing-enhancing polynucleotides.
- FIG. 9A shows the mechanism of conventional genome editing by editing sense polynucleotides.
- FIG. 9B shows the mechanism of genome editing by an editing sense polynucleotide in the presence of an editing-promoting antisense polynucleotide.
- Genome editing or similar terms are a type of technique for modifying the genome. Genome editing involves targeting specific regions on the genome and selectively modifying those regions.
- genome editing tools such as zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas systems have been developed, and genome editing using these tools is actively carried out around the world. While ZFNs and TALENs have the disadvantage that it takes time and effort to design sequence-specific nucleases, the CRISPR/Cas system is especially adopted as a genome editing tool around the world because it is extremely easy to target.
- the drawbacks of the CRISPR/Cas system include off-target issues and the difficulty of delivering large nucleases to cells.
- cell is the structural and functional basic unit of an organism.
- a cell refers to a basic unit of life having at least genomic DNA, cytoplasm, and a membrane structure enveloping them.
- Cells can be eukaryotic and prokaryotic.
- Eukaryotic cells have a nucleus surrounded by a nuclear membrane and a cytoplasm.
- Prokaryotic cells do not have a nucleus.
- Genomic DNA includes DNA endogenous to the cell, but is not necessarily composed exclusively of factors endogenous to the cell.
- Eukaryotic cells may be human cells.
- Cells can be purified cells or isolated cells.
- the cells may be cultured cells.
- the cell may be an established cell line.
- a cell can be, for example, a somatic cell.
- a cell can be, for example, a pluripotent cell or a pluripotent stem cell.
- a cell can be, for example, a tissue stem cell.
- a cell can be, for example, a tissue progenitor cell.
- a cell can be, for example, a germ cell.
- Eukaryotes include animals, plants, and fungi.
- Animals include mammals (humans and non-human mammals), especially domestic animals such as cattle, pigs, goats, sheep, horses, llamas, and birds such as camels and chickens, and agricultural animals such as fish and shellfish.
- Animals, companion animals such as dogs, cats, rabbits, guinea pigs, hamsters and mice; vertebrates such as birds, fish, amphibians and reptiles; insects; crustaceans such as crabs and shrimps; Mollusks of.
- Plants include agricultural crops such as food crops such as rice, corn, potatoes, beans, wheat such as barley and wheat, horticultural crops (vegetables, fruit trees, and flowers) such as leafy vegetables (cabbage, asparagus, etc.). ), fruit vegetables (eggplants, tomatoes, cucumbers, etc.), root vegetables (radish, carrots, etc.), other vegetables, fruits stone fruits, shell fruits such as almonds, walnuts and chestnuts; citrus fruits such as mandarin oranges and lemons; tropical fruit trees such as tropical fruits); be done.
- food crops such as rice, corn, potatoes, beans, wheat such as barley and wheat
- horticultural crops vegetable (vegetables, fruit trees, and flowers)
- leafy vegetables cabbage, asparagus, etc.).
- fruit vegetables eggplants, tomatoes, cucumbers, etc.
- root vegetables radish, carrots, etc.
- other vegetables fruits stone fruits, shell fruits such as almonds, walnuts and chestnuts
- citrus fruits such as mandarin oranges and lemon
- eukaryotes are fungi such as yeasts, molds and basidiomycetes, examples of which include yeast, neurospora, matsutake, shiitake, enokitake, shimeji, nameko, king oyster mushroom, and the like.
- Eukaryotes can also be microalgae.
- Eukaryotes can be human or non-human.
- prokaryotes examples include Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Serratia, Sphingomonas, Brucella, Neisseria gonorrhoeae, Burkholderia, Shigella, Salmonella, Acinetobacter, Vibrio cholerae, Klebsiella pneumoniae, Legionella, Helicobacter pylori). , Campylobacter, etc.), Gram-positive bacteria (Tuberculosis, Mycoplasma, Staphylococcus aureus, Actinomycetes, Bacillus subtilis, Bacillus anthracis, etc.).
- Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Serratia, Sphingomonas, Brucella, Neisseria gonorrhoeae, Burkholderia, Shigella, Salmonella, Acinetobacter, Vibrio cholera
- the prokaryote may preferably be a Gram-negative bacterium, more preferably Pseudomonas aeruginosa, Escherichia coli, Serratia, Sphingomonas, more preferably Pseudomonas aeruginosa, Escherichia coli.
- a cell contains the genomic DNA of the cell, but may also contain the genomic DNA of a foreign invader (eg, pathogen).
- genomic DNA is referred to as the cell's own genomic DNA (ie, host genomic DNA) unless otherwise specified.
- the invader's genomic DNA may exist within the cell separately from the host's genomic DNA, but may also be integrated into the host's genomic DNA.
- Host genomic DNA may contain foreign elements (eg, insertion of all or part of genomic DNA, such as a virus).
- Host genomic DNA integrated with intruder genomic DNA and host genomic DNA containing exogenous factors are also included in the term genomic DNA as used herein.
- Isolating means separating a cell of interest from at least one other component. Isolation can be performed, for example, by separating the cells in their natural state of existence from other components with which they are present in their natural state of existence. Isolation can be performed, for example, by separating and removing some cells from a multicellular organism. Techniques dealing with isolated cells are referred to in this disclosure as in vitro techniques.
- purification means further separation of the isolated cells of interest from other components with which they are present. Purification can be performed, for example, by separating the cells of interest from other constituents based on morphology or surface markers. Purification may be performed by limiting dilution and/or cloning of cells. Purification can be performed by establishing a cell line of interest. Purification can be performed based on the expression of the marker gene when the target cell has a marker gene such as a drug resistance gene or a gene encoding a fluorescent protein.
- polynucleotide and “nucleic acid” are used interchangeably and refer to a nucleotide polymer in which nucleotides are linked by phosphodiester bonds.
- Polynucleotides” and “nucleic acids” may be DNA, RNA, or may be composed of combinations of DNA and RNA (eg, gapmers).
- Polynucleotides” and “nucleic acids” may also be polymers of natural nucleotides, including natural nucleotides and non-natural nucleotides (analogs of natural nucleotides, base moieties, sugar moieties and phosphate moieties).
- a polynucleotide may be a peptide nucleic acid.
- a polynucleotide may be a morpholino oligo.
- base sequences of "polynucleotide” or “nucleic acid” are described in generally accepted one-letter code unless otherwise specified. Nucleotide sequences are written from the 5' side to the 3' side unless otherwise specified. Nucleotide residues constituting a “polynucleotide” or “nucleic acid” may simply be described as adenine, thymine, cytosine, guanine, uracil, etc., or their one-letter codes.
- the term "gene” refers to a polynucleotide containing at least one open reading frame that encodes a particular protein.
- a gene may contain both exons and introns.
- a "region in which RNA is transcribed” includes regions in which RNA is transcribed, such as regions in which genes occur and regions in which non-coding RNA is transcribed.
- a region from which RNA is transcribed may be a region that exhibits transcriptional activity of RNA. From the RNA-transcribed region, RNA is transcribed using the minus strand as a template. The minus strand is called the antisense strand, and the complementary strand of the minus strand is called the sense strand.
- operably linked when used in reference to polynucleotides means that a regulatory sequence, such as a promoter, is placed sufficiently close to a gene sequence to affect expression of the gene sequence.
- a polynucleotide being operably linked to a promoter means that the polynucleotide is linked so that it is expressed under the control of the promoter.
- expressible state refers to a state in which the polynucleotide can be transcribed in a cell into which the polynucleotide has been introduced.
- expression vector refers to a vector containing a polynucleotide of interest and having a mechanism for expressing the polynucleotide of interest in a cell into which the vector has been introduced.
- a "polynucleotide expression vector” means a vector capable of expressing a polynucleotide in a cell into which the vector has been introduced.
- the polynucleotide is, for example, operably linked to regulatory sequences.
- sequence identity (or homology) between base sequences is obtained by aligning two base sequences so that the corresponding bases match the most with gaps inserted and deleted. It is calculated as the percentage of matched bases relative to the entire base sequence, excluding gaps in the generated alignment.
- Sequence identity between nucleotide sequences can be determined using various homology search software known in the art. For example, the sequence identity value of base sequences can be obtained by calculation based on alignments obtained by the known homology search software BLASTN.
- the nucleotide sequences to be aligned contain uracil
- the identity is calculated using the nucleotide sequence after replacing uracil with thymine.
- identity calculations replace the modified nucleotides or nucleotide analogues with the original natural deoxyribonucleotides, i.e. adenine, thymine, guanine, or cytosine. It is carried out using the later nucleotide sequence.
- portion and region regarding polynucleotides are used interchangeably, and both mean one nucleotide or multiple consecutive nucleotides contained in a polynucleotide.
- portion and region with respect to double-stranded genomic DNA are used interchangeably and both refer to a single nucleotide pair or multiple contiguous nucleotide pairs contained in the double-stranded genomic DNA.
- a portion or region of a polynucleotide and a portion or region “at a corresponding position” refer to a portion or region of the polynucleotide on a different polynucleotide from the polynucleotide, or a portion or region of the polynucleotide. point to the area.
- a polynucleotide or portion or region thereof is “hybridizable” with another polynucleotide or portion or region thereof means that a polynucleotide or portion or region thereof is capable of or have the ability to bind to a region through hydrogen bonding between complementary bases.
- Ability to hybridize can be confirmed by maintaining the hybridized state under stringent conditions. Stringent conditions are the melting temperature (Tm) of the nucleic acid that binds the probe, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA).
- Two polynucleotides, or portions or regions thereof, are hybridizable to each other if the hybridized state between them is maintained by washing under stringent conditions, such as 1 ⁇ SSC, 0.1% SDS, at 37° C. .
- Stringent conditions may be washing in 0.5 x SSC, 0.1% SDS at 42°C or washing in 0.1 x SSC, 0.1% SDS at 65°C.
- editing polynucleotides are polynucleotides in single-stranded form that are capable of modifying a target site in double-stranded genomic DNA.
- the editing polynucleotide is, in order from the 5' end side, a) a portion hybridizable with the region adjacent to the 3' end of the primary editing site x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) the 5' end of the primary editing site consisting of portions hybridizable with laterally adjacent regions, or portion a) and portion b) consists of Details of the editing polynucleotide are described below (see also FIG. 8A).
- the target site refers to a position between one nucleotide pair, a plurality of consecutive nucleotide pairs, or two consecutive nucleotide pairs that are desired to be modified, existing in double-stranded genomic DNA.
- the target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site located on the other genomic DNA strand at a position corresponding to the primary editing site.
- the primary editing site may be replaced or deleted on one genomic DNA strand. It is set to one nucleotide or multiple consecutive nucleotides desired to be deleted.
- the primary editing site may be a single nucleotide or multiple nucleotide pairs on one genomic DNA strand. It is set between two consecutive nucleotides where it is desired to insert consecutive nucleotides.
- the primary editing site can be set between any one nucleotide or multiple consecutive nucleotides, or between any two consecutive nucleotides present in one genomic DNA strand, and a specific single nucleotide or multiple nucleotides It is not limited to contiguous nucleotides or between any particular two contiguous nucleotides.
- the region adjacent to the 5' end of the primary editing site and the region adjacent to the 3' end of the primary editing site are genome They are separated by the length of the primary editing site on the DNA.
- the primary editing site is set at a position between two consecutive nucleotides, the region adjacent to the 5' end of the primary editing site and the region adjacent to the 3' end of the primary editing site are genome It becomes a region adjacent to each other on the DNA.
- the number of consecutive nucleotides is not particularly limited. may be more than or equal to 6 nucleotides, more than or equal to 7 nucleotides, or more than or equal to 8 nucleotides, and also for example 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides 40 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, 15 nucleotides or less, 12 nucleotides or less, 10 nucleotides or less, or 9 nucleotides or less,
- the primary editing site may be set on either strand of the double strands that make up the genomic DNA.
- a secondary editing site is set on the genomic DNA strand on which the primary editing site is not set.
- the primary editing site may be set on the sense strand or the antisense strand.
- Part a) of the editing polynucleotide can hybridize with the region adjacent to the 3' end of the primary editing site.
- Part a) may also consist of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site.
- the identity is preferably 95% or more, more preferably 97% or more, still more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.
- part a) has a nucleotide sequence complementary to the nucleotide sequence of the region adjacent to the 3′ end of the primary editing site, that is, the nucleotide sequence of the region adjacent to the 5′ end of the secondary editing site. It consists of the same base sequence.
- Part b) of the editing polynucleotide can hybridize with the region adjacent to the 5' end of the primary editing site.
- Part b) may also be a part consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site.
- the identity is preferably 95% or more, more preferably 97% or more, still more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.
- part b) has a nucleotide sequence complementary to the nucleotide sequence of the region adjacent to the 5' end of the primary editing site, that is, the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site. It consists of the same base sequence.
- the lengths of part a) and part b) are not particularly limited, and can be set independently.
- the length of part a) and part b) is, for example, 10 nucleotides or more, 20 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 45 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, or 70 nucleotides or more.
- nucleotides or less 80 nucleotides or more, 90 nucleotides or more, or 100 nucleotides or more; , 150 nucleotides or less, 140 nucleotides or less, 130 nucleotides or less, 120 nucleotides or less, 110 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, or 50 nucleotides or less.
- part a) and part b) are, respectively, It can be 70 nucleotides, or 35-50 nucleotides.
- Part x) is an arbitrary part consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site, and is determined depending on how the target site is desired to be modified. For example, if the desired modification is to replace a single nucleotide pair or multiple consecutive nucleotide pairs within the double-stranded genomic DNA, portion x) is the single nucleotide or A plurality of consecutive nucleotides. Also, portion x) is not included in the editing polynucleotide if the desired modification is to delete a single nucleotide pair or multiple consecutive nucleotide pairs within the double-stranded genomic DNA.
- portion x is the single nucleotide desired to be inserted into the secondary editing site. or multiple consecutive nucleotides.
- Part x) consists of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site.
- Part x) may consist of any base sequence as long as it does not completely match the base sequence of the secondary editing site, and for example, bases having less than 90% identity with the base sequence of the secondary editing site. It may be an array.
- the identity is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, even more preferably 10% or less, and particularly preferably 5% or less.
- the length of portion x) is not particularly limited, but is, for example, 2000 nucleotides or less, 1000 nucleotides or less, 900 nucleotides or less, 800 nucleotides or less, 700 nucleotides or less, 600 nucleotides or less, 500 nucleotides or less, 400 nucleotides or less, 300 nucleotides or less.
- nucleotides or less 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides or less, 40 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, 10 nucleotides or less, 9 nucleotides or less, 8 It can be no more than 7 nucleotides, no more than 6 nucleotides, no more than 5 nucleotides, no more than 4 nucleotides, no more than 3 nucleotides, no more than 2 nucleotides, or 1 nucleotide.
- the editing polynucleotide consists of part a), part x) and part b), or consists of part a) and part b) in order from the 5' end. Therefore, the length of the editing polynucleotide can be obtained by summing the lengths of the parts constituting the editing polynucleotide, that is, the lengths of part a), part x) and part b), or part a) and part b). It can be determined by summing the lengths of parts b).
- the editing polynucleotide may contain one or more DNA high-affinity nucleotide analogues in one or both of part a) and part b).
- the DNA high affinity nucleotide analogue is replaced with a DNA high affinity nucleotide analogue that is identical to the original base, eg, adenine or an adenine analogue when the original base is adenine.
- the DNA high-affinity nucleotide analogues may be contained adjacently or intermittently so as not to be adjacent to each other. Also, when included in both part a) and part b), the DNA high-affinity nucleotide analogues can be included at positions selected independently of each other, and the number of inclusions need not be the same.
- DNA high-affinity nucleotide analogues are artificial nucleic acid building blocks with high binding affinity to DNA that can form polymeric structures similar to those of natural nucleic acids, e.g. Nucleotides bridged between the oxygen atom and the 4' carbon atom, peptide nucleic acids (PNAs) with deoxyribose or N-(2-aminoethyl)glycine in place of ribose, and deoxyribose or ribose in place of A morpholino nucleic acid having a morpholine ring and the like can be mentioned.
- PNAs peptide nucleic acids
- the DNA high affinity nucleotide analog is a nucleotide bridged between the 2' oxygen atom and the 4' carbon atom of the ribose ring, examples of which include the 2' oxygen LNA (Locked Nucleic Acid) in which the atom and the 4'-position carbon atom are bridged through methylene, ENA bridged through ethylene, BNA (Bridged Nucleic Acid) bridged through -CH2OCH2- COC , BNA NC bridged through -NR- CH2- (R is a methyl or hydrogen atom), cMOE bridged through -CH2 ( OCH3 )-, -CH2 ( CH3 ) Examples include cEt bridged through -, AmNA bridged through amide, and scpBNA bridged through methylene to form cyclopropane at the 6' position.
- LNA Locked Nucleic Acid
- BNA Bridged Nucleic Acid
- BNA NC bridged through -NR- CH2- (
- the editing polynucleotide may further contain chemically modified nucleotides as its constituent units in addition to the DNA high affinity nucleotide analogues.
- chemically modified nucleotides include nucleotides in which the phosphate group (phosphate) has been replaced with a chemically modified phosphate group such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc., at the 2' position of the sugar (ribose) moiety.
- PS phosphorothioate
- methylphosphonate methylphosphonate
- phosphorodithionate etc.
- the hydroxyl group is -OR (R is, for example, CH3 ( 2'-O-Me), CH2CH2OCH3 ( 2'-MOE), CH2CH2NHC (NH) NH2 , CH2CONHCH3 , CH 2 CH 2 CN, etc.), nucleotides in which a methyl group or a cationic functional group is introduced at the 5-position of the pyrimidine base, nucleotides in which the carbonyl group at the 2-position of the pyrimidine base is substituted with a thiocarbonyl group etc. can be mentioned. Furthermore, nucleotides whose phosphate moieties and hydroxyl moieties have been modified with, for example, biotin, amino groups, lower alkylamine groups, acetyl groups, etc., but are not limited to these.
- An editing polynucleotide containing DNA high-affinity nucleotide analogues and optionally further chemically modified nucleotides can be produced by known synthetic methods.
- the editing polynucleotide in the present disclosure can modify genomic DNA as desired in cells in the same manner as the polynucleotide in single-stranded form disclosed in Patent Document 1 (Patent No. 6899564). It does not require any intracellular introduction of site-specific nucleases (ZFN proteins, TALEN proteins, Cas proteins, etc.), guide RNAs, or their expression vectors, unlike conventional genome editing techniques.
- site-specific nucleases ZFN proteins, TALEN proteins, Cas proteins, etc.
- guide RNAs or their expression vectors, unlike conventional genome editing techniques.
- the editing polynucleotide has a region in which part a) is adjacent to the 3′ end of the primary editing site set on one genomic DNA strand, and part b) is Hybridizes with the region flanking the 5' end of the primary editing site.
- part x) is on the genomic DNA when the editing polynucleotide consists of part a) and part b).
- the primary editing site sticks out like a boil without hybridizing with other sequences. Then, this protruding portion is recognized by the repair system, and the target site of the double-stranded genomic DNA can be modified.
- the primary editing site can be inserted on one genomic DNA strand. set between two consecutive nucleotides desired; set portion a) and portion b) as described above; portion x) the single nucleotide or series of sequences desired to be inserted into the secondary editing site set to the nucleotide that By performing genome editing using an editing polynucleotide composed of part a), part x) and part b) designed in this way, a part consisting of a base sequence complementary to part x) is added to the primary editing site. , the portion x) can be inserted at the secondary editing site.
- the primary editing site may be replaced on one genomic DNA strand by the single nucleotide pair desired to be replaced. set to a nucleotide or multiple consecutive nucleotides; set portion a) and portion b) as described above; set portion x) to a nucleotide or multiple consecutive nucleotides where it is desired to replace the secondary editing site set.
- part a) and part b) are set as described above.
- the primary editing site is deleted from one genomic DNA strand, and two genomic DNA strands are deleted from the other genomic DNA strand. Deletion of the next-editing site, ie deletion of the target site from the double-stranded genomic DNA, can be performed.
- the target site is within the genomic DNA region that is transcribed into RNA
- the primary editing site is When set on the genomic DNA sense strand, the editing polynucleotide is more Since it preferentially binds to a region having the same base sequence as the above two regions contained in surrounding RNA, the genome editing efficiency is considered to be poor. If the genome editing efficiency is emphasized, it is preferable to set the primary editing site on the antisense strand rather than on the genomic DNA sense strand.
- portion a) of the editing polynucleotide hybridizes with the region adjacent to the 3′ end of the primary editing site set on one genomic DNA strand, and the portion b) is By hybridizing with the region flanking the 5' end of the primary editing site, the target site of the double-stranded genomic DNA can be modified.
- portion a) of the editing polynucleotide hybridizes to the region 3' to the primary editing site, and portion b) of the editing polynucleotide is 5' to the primary editing site.
- the editing polynucleotide may have any base sequence at one or both ends thereof, i. may be added.
- the polynucleotides for editing in the present disclosure include those to which such arbitrary base sequences are added.
- the calculation of the identity with the region adjacent to the 5' end side and the region adjacent to the 3' end side of the secondary editing site is partially Between the nucleotide sequence of a) and the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site, or the nucleotide sequence of part b) and the nucleotides of the region adjacent to the 3' end of the secondary editing site Any base sequence added shall be considered absent in the calculation of identity.
- Editing-enhancing polynucleotides can be used in combination with editing-enhancing polynucleotides to improve genome editing efficiency.
- An editing-promoting polynucleotide is a polynucleotide that can hybridize with a nucleic acid sequence that is complementary to a predetermined region on a genomic DNA strand that has a primary editing site (also referred to as a complementary sequence of the predetermined region). Details of the editing-enhancing polynucleotide are described below (see also FIG. 8B).
- the predetermined region, on the genomic DNA strand on which the primary editing site is set, is defined as region A at a position corresponding to part a) of the editing polynucleotide, and at a position corresponding to part b) of the editing polynucleotide. Assuming that a certain region is region B, it is set so that it overlaps with region A at its 5' end, or overlaps with region B at its 3' end.
- the length of the overlap with the region A at the 5' end of the predetermined region is not limited as long as it is the same as or shorter than the region A.
- the length of overlap with region B at the 3' end of the predetermined region is not limited as long as it is the same as region B or shorter.
- the length of these overlaps can be set independently, for example, 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, 8 can be ⁇ 20, ⁇ 19, ⁇ 18, ⁇ 17, ⁇ 16, ⁇ 15, ⁇ 14, ⁇ 13, 12 It can be no more than a nucleotide, or 11 nucleotides. Also, the length of these overlaps can each independently be, for example, 1-20 nucleotides, 5-20 nucleotides, 5-15 nucleotides, 8-12 nucleotides, or 9-11 nucleotides.
- a complementary sequence of a given region is a region on another polynucleotide that is complementary to the given region on a genomic DNA strand.
- the complementary sequence of the given region is naturally contained in RNA transcribed from the genomic DNA strand in which the given region is typically defined.
- the editing-promoting polynucleotide can hybridize with the complementary sequence of the predetermined region on the genomic DNA strand on which the primary editing site is set.
- the editing-promoting polynucleotide may be a polynucleotide consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the predetermined region.
- the identity is preferably 95% or higher, more preferably 97% or higher, even more preferably 99% or higher, even more preferably 99.5% or higher, particularly preferably 99.9% or higher.
- the editing-promoting polynucleotide consists of the same base sequence as the base sequence of the predetermined region.
- the editing-promoting polynucleotide includes an editing-promoting polynucleotide in which a predetermined region overlaps region A at its 5' end, and a predetermined region overlaps region B at its 3' end.
- the length of the editing-promoting polynucleotide is equal to or longer than the overlap length with the region A when the predetermined region is set to overlap with the region A at the 5' end thereof, When the predetermined region is designed to overlap region B at its 3' end, the length of overlap with region B is the same or longer.
- the length of each of the editing-promoting polynucleotides does not need to be the same, and can be set independently.
- the length of the editing-promoting polynucleotide is, for example, 20 nucleotides or more, 30 nucleotides or more, 40 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70 nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, 100 nucleotides or more, can be 110 nucleotides or more, 120 nucleotides or more, or 130 nucleotides or more; It can be 160 nucleotides or less, 150 nucleotides or less, 140 nucleotides or less or 130 nucleotides or less.
- the length of the editing-promoting polynucleotide is, for example, 10 to 1000 nucleotides, 20 to 500 nucleotides, 30 to 300 nucleotides, 40 to 200 nucleotides, 50 to 200 nucleotides, 60 to 200 nucleotides, and 70 to 150 nucleotides. , or 70-130 nucleotides.
- the editing polynucleotide in an environment where there are many other polynucleotides having regions complementary to the primary editing site or regions in the vicinity thereof (the region A and the region B), the editing polynucleotide It is thought that the binding of the gene to the genomic DNA strand having the primary editing site is competitively inhibited by the other polynucleotide, and as a result, the efficiency of genome editing by the editing polynucleotide is reduced.
- the editing polynucleotide will be peripheral with respect to binding to the genomic DNA antisense strand. Since the binding of the editing polynucleotide and the genomic DNA antisense strand is suppressed, the genome editing efficiency is considered to be lower than when there is no surrounding RNA.
- the other polynucleotide for example, RNA
- RNA binds to the editing-promoting polynucleotide, resulting in the editing polynucleotide and Suppression of binding with a genomic DNA strand (for example, an antisense strand) having a primary editing site is eliminated, and the genome editing efficiency is considered to be improved (Fig. 8B).
- the editing-enhancing polynucleotide can have a high binding affinity for RNA.
- An editing-enhancing polynucleotide may comprise one or more RNA high affinity nucleotide analogues.
- the RNA high-affinity nucleotide analogue is replaced with an RNA high-affinity nucleotide analogue that is identical to the original base, eg, adenine or an adenine analogue containing an adenine analogue when the original base is adenine.
- RNA high-affinity nucleotide analogues may be contained adjacently or intermittently so as not to be adjacent to each other.
- each of the editing-promoting polynucleotides may contain RNA high-affinity nucleotide analogues at independently selected positions, and the number thereof should be the same. no.
- the target site is in the pre-mRNA.
- the target site is set within the region transcribed into the intron portion, there is a greater difference between the editing polynucleotide and the RNA than when the target site is set within the region transcribed into the exon portion of the pre-mRNA. It is thought that competition for binding with the genomic DNA antisense strand becomes more intense, and genome editing efficiency becomes lower. This is because a large amount of introns excised from the pre-mRNA by splicing exists around the genomic DNA.
- the editing-promoting polynucleotide is used when the target site is within the genomic DNA region transcribed into the intron portion of the pre-mRNA, when the primary editing site is set on the genomic DNA antisense strand, and when the target site is mRNA
- Genome editing efficiency can be improved in any case where the primary editing site is set on the genomic DNA antisense strand within the genomic DNA region transcribed into the exon portion of the precursor, but particularly when the target site is It is considered that the genome editing efficiency improvement effect can be strongly exhibited when the primary editing site is set on the genomic DNA antisense strand within the genomic DNA region that is transcribed into the intron portion of the pre-mRNA.
- the editing polynucleotide comprises an editing sense polynucleotide and an editing-promoting Polynucleotides for editing are also called antisense polynucleotides for editing.
- the present disclosure provides a genome editing kit comprising the above-described editing polynucleotide and the above-described editing-enhancing polynucleotide.
- the genome editing kit comprises an editing-promoting polynucleotide set such that the predetermined region overlaps region A at its 5' end, and the predetermined region overlaps region B at its 3' end. It preferably contains two types of editing-promoting polynucleotides designed to:
- the genome editing kit can contain an editing polynucleotide expression vector, or an editing polynucleotide or a conjugate of the editing polynucleotide expression vector and another substance.
- the genome editing kit contains an expression vector for an editing-enhancing polynucleotide, or a conjugate of an editing-enhancing polynucleotide or an editing-enhancing polynucleotide expression vector and another substance, instead of the editing-enhancing polynucleotide. can contain.
- Other substances that are conjugated to the editing polynucleotide are capable of forming a conjugate with the editing polynucleotide, so long as they do not interfere with the function of the editing polynucleotide, i.e., to modify the target site of genomic DNA. There are no restrictions.
- other substances that are conjugated to the editing-enhancing polynucleotide can form a conjugate with the editing-enhancing polynucleotide and enhance the function of the editing-enhancing polynucleotide, that is, the efficiency of genome editing by the editing polynucleotide. There are no restrictions as long as they do not interfere with the function to be enhanced.
- Examples of other substances conjugated to editing polynucleotides and editing-enhancing polynucleotides include functional peptides such as nuclear localization signals and membrane-permeable peptides; hydrophilic polymers such as polyethylene glycol and dextran; , fluorescein and its derivatives, rhodamine and its derivatives, fluorescent proteins, phycobiliproteins, biotin, and avidin.
- the genome editing kit can further contain reagents such as nucleic acid introduction reagents such as transfection reagents or buffers.
- Components contained in the genome editing kit for example, the above-mentioned editing polynucleotide, its expression vector or its conjugate, the above-mentioned editing-promoting polynucleotide, its expression vector or its conjugate, each reagent, etc. may be included in the kit in separate forms, or may be included in the kit in the form of a composition in which a plurality of components are combined.
- the genome editing kit may further include, for example, instruments used for genome editing, instructions on how to use, etc.
- the genome editing kit is a kit for treating or preventing a disease, and contains a therapeutically or prophylactically effective amount of the above-described editing polynucleotide, its expression vector, or its editing polynucleotide or its expression vector and other
- other pharmaceutically acceptable ingredients such as pharmaceutically acceptable excipients (buffers, stabilizers, preservatives, excipients, etc.), pharmaceutically acceptable medium (water, saline, phosphate-buffered saline (PBS), etc.
- a kit for treating or preventing a disease can be used to treat or prevent a disease caused by some abnormality in genomic DNA.
- Genome editing method and method for producing genome-edited cells or organisms includes a step of treating a cell or organism with the genome editing kit described above.
- a method for modifying a target site in genomic DNA (also referred to in this disclosure as a "genome editing method") is provided.
- the present disclosure also provides a method for producing a cell or organism in which the target site of the double-stranded genomic DNA is modified, comprising the step of treating the cell or organism using the genome editing kit described above (this Also referred to in the disclosure as "methods for producing genome-edited cells or organisms").
- Cells to be processed using the genome editing kit are cells whose genomic DNA is desired to be modified, and can originate from various organisms including the prokaryotes and eukaryotes mentioned above. If the cell is of a multicellular organism, the cell can be derived from various tissues of that organism.
- the cells may be somatic or germ cells.
- Cells may be differentiated cells or undifferentiated cells, and may be tissue stem cells, embryonic stem cells or induced pluripotent stem cells (iPS cells).
- Cells to be processed using the genome editing kit may be single cells or may be in the form of cell populations containing one or more types of cells.
- the organism to be processed using the genome editing kit is an organism whose genomic DNA is desired to be modified, and can be various organisms including the prokaryotes and eukaryotes mentioned above.
- the organism may be a mature individual or a developing embryo or fetus, and in the case of plants it may be a seed, seedling or bulb.
- cells do not include human gametes and fertilized eggs, and can be referred to as cells (but excluding human gametes and fertilized eggs).
- organisms do not include humans and can be referred to as non-human organisms. In another embodiment, organisms exclude humans and animals, and can be referred to as non-human, non-animal organisms.
- the cells do not include human gametes and fertilized eggs, and the organisms do not include humans. In yet another preferred embodiment, the cells do not include human gametes and fertilized eggs, and the organisms do not include humans and animals.
- the treatment of cells or organisms using a genome editing kit includes an editing polynucleotide, an expression vector thereof, or a conjugate of the editing polynucleotide or the expression vector and another substance and promoting editing It means coexistence of a polynucleotide for editing, its expression vector, or a conjugate of an editing-promoting polynucleotide or its expression vector with another substance in a cell, or administration thereof to an organism.
- an editing polynucleotide, an expression vector thereof, or a conjugate of the expression vector and an editing polynucleotide or a conjugate of the expression vector and another substance, and an editing-promoting polynucleotide, or the An expression vector or an editing-promoting polynucleotide or a conjugate of the expression vector and other substances can be introduced to edit the genomic DNA of the cell or organism.
- the treatment of cells or organisms using a genome editing kit can be carried out by various known methods appropriately selected by those skilled in the art in consideration of factors such as the type or properties of cells or organisms.
- Examples of cell or organism treatment include microinjection, electroporation, DEAE-dextran treatment, transfection using a transfection reagent (jetPEI (PolyPlus-transfection), lipofectamine, etc.), and lipid nanoparticles. Transfection, hydrodynamics methods can be mentioned.
- treatment of human cells is performed in vitro or ex vivo.
- genome editing increases or decreases the characteristics of cells or organisms, such as auxotrophy, sensitivity or resistance to chemical substances, or imparts characteristics not found in cells or organisms before editing, Alternatively, the characteristics of the cells or organisms before editing are lost.
- Cells or organisms in which the desired genome editing has been performed can be selected using such a change in properties as an index.
- cells treated with a genome editing kit are cultured using an appropriate medium containing the drug, or genome editing
- Genome-edited cells or organisms can be selected by breeding organisms treated with the kit using appropriate feed containing the drug.
- the above-described genome editing method and the method for producing a genome-edited cell or organism include, in addition to the step of processing the cell or organism using the genome editing kit, the characteristics of the cell or organism that are changed by the editing polynucleotide.
- the index can include a step of selecting cells or organisms whose genomic DNA has been edited.
- Table 1 shows an example of correspondence between terms used in the following invention and terms used in the above invention.
- a method for editing a genome in a eukaryotic cell comprising: (A) Hybridizable to sequences flanking the upstream and downstream of the target region of the genome (for example, the target region corresponding to the antisense strand of the genomic region where RNA is transcribed) in the cell allowing a single-stranded form of the editing polynucleotide to interact in a sequence-specific manner with the genome;
- the single-stranded form of an editing polynucleotide e.g., an editing sense polynucleotide
- the upstream homology arm has a sequence hybridizable with a sequence flanking the target region upstream
- the downstream homology arm has a sequence hybridizable with a sequence flanking the target region downstream
- the upstream capable of hybridizing at least upstream and downstream of a target region by means of a homology arm and said downstream homology arm, with any or no sequence between said upstream homo
- the step (A) is performed in the presence of at least two other hybridizable single-stranded polynucleotides, and at least one other single-stranded polynucleotide (first 1 other polynucleotide) has a sequence (e.g., a sequence having complementarity) that can hybridize to a region having a sequence corresponding to the 5' end portion of the editing polynucleotide, and at least one The other single-stranded form of the polynucleotide (second other polynucleotide) is a sequence (e.g., complementary The method according to any one of [1] to [8] above, wherein the sequence has a [10] A genome editing kit for use in the method according to any one of [1] to [8] above, wherein the single-stranded editing polynucleotide and the other single-stranded polynucleotide A genome editing kit comprising a combination with nucleotides.
- a genome editing kit for use in the method according to [9] above, wherein the editing polynucleotide in single-stranded form and the at least two other single-stranded polynucleotides A kit for genome editing, comprising a combination.
- kit for use in the method of [9] above, comprising a DNA encoding the editing polynucleotide in single-stranded form, and the at least two other single-stranded forms wherein the DNA encoding the single-stranded form of the editing polynucleotide is expressably contained in an expression vector, and the other single-stranded form of the polynucleotide is DNA.
- a method for editing a genome in a eukaryotic cell comprising: (A) for editing in a single-stranded form hybridizable to sequences flanking upstream and downstream of the target region corresponding to the antisense strand of the genomic region to which the RNA is transcribed in the cell; allowing the sense polynucleotide to interact with the genome in a sequence-specific manner;
- the single-stranded form of the editing sense polynucleotide has an upstream homology arm and a downstream homology arm,
- the upstream homology arm has a sequence hybridizable with a sequence flanking the target region upstream
- the downstream homology arm has a sequence hybridizable with a sequence flanking the target region downstream
- the upstream capable of hybridizing at least upstream and downstream of a target region by means of a homology arm and said downstream homology arm, with any or no sequence between said upstream homology arm and said downstream homology arm; may be
- the step (A) is performed in the
- [2A] The method of [1A] above, wherein the editing sense polynucleotide in single-stranded form is a polynucleotide selected from the group consisting of DNA, RNA, and combinations thereof.
- [3A] The method of [1A] or [2A] above, wherein the antisense polynucleotide in single-stranded form is DNA.
- [4A] The method according to any one of [1A] to [3A] above, wherein the terminal portion has a length of 5 bases or more.
- [5A] The method according to any one of [1A] to [4A] above, wherein the terminal portion has a length of 20 bases or less.
- [6A] The method according to any one of [1A] to [5A] above, wherein the single-stranded form of the editing sense polynucleotide has a length of 60 to 200 bases.
- [7A] The method of any one of [1A] to [6A] above, wherein the antisense polynucleotide in single-stranded form has a length of 50 to 200 bases.
- [8A] The method of any one of [1A] to [7A] above, wherein the target region is in an intron.
- the step (A) is performed in the presence of at least two types of hybridizable single-stranded antisense polynucleotides, and at least one type of antisense polynucleotide is transcribed from a genomic region.
- a partial region of RNA which has a sequence (for example, a sequence having complementarity) that can hybridize to a region having a sequence corresponding to the 5′ terminal portion of the editing sense strand polynucleotide, and at least one A class of antisense polynucleotides is a partial region of RNA transcribed from a genomic region, the sequence capable of hybridizing to a region having a sequence corresponding to the 3' terminal portion of the editing sense strand polynucleotide ( For example, the method according to any one of [1A] to [8A] above, which has complementary sequences).
- a genome editing kit for use in the method according to any one of [1A] to [8A] above comprising: the single-stranded form of the editing sense polynucleotide and the single-stranded form of the antisense
- a kit for genome editing comprising a combination with a polynucleotide.
- a genome editing kit for use in the method according to any one of [1A] to [8A] above comprising a DNA encoding the single-stranded form of an editing sense polynucleotide; and DNAs encoding antisense polynucleotides in chain form, which DNAs are expressably contained in one or more expression vectors.
- a DNA comprising an antisense polynucleotide in stranded form and encoding the editing sense polynucleotide in single-stranded form is expressably contained in an expression vector, wherein the antisense polynucleotide in single-stranded form is DNA.
- a genome editing kit for use in the method of [9A] above comprising: the single-stranded form of the editing sense polynucleotide; and the at least two types of single-stranded form of antisense polynucleotides;
- a kit for genome editing comprising a combination of [15A]
- a genome editing kit for use in the method described in [9A] above comprising a DNA encoding the editing sense polynucleotide in single-stranded form, and at least two types of single-stranded forms of and DNAs encoding antisense polynucleotides, wherein these DNAs are expressably contained in one or more expression vectors.
- corresponding sequence in the item "Further invention provided by the present disclosure” means the sequence (especially the base sequence) of the corresponding part of the sequence.
- DNA and RNA transcribed from that DNA have corresponding sequences.
- the correspondence between DNA and RNA sequences is known that A, T, G, and C in DNA are A, U, G, and C, respectively.
- the term “having complementarity” means that two polynucleotides can hybridize by Watson-Crick binding in an intracellular environment or an equivalent in vitro environment. Generally, A and T (or U) are complementary, and G and C are complementary.
- “Complementarity” in the term “having complementarity” can be 90% or greater, 95% or greater, or perfect complementarity (ie, 100% complementarity).
- target region refers to the region targeted by the genome modification system.
- a method of editing a genome is provided.
- methods of targeting a target region of the genome are provided.
- a method for obtaining a cell with an edited genome is provided.
- the cells can be eukaryotic cells. In all embodiments, the cells may preferably be cloned cells.
- the method of the invention may also be an in vitro method, preferably in all embodiments.
- isolated cells can be obtained.
- the isolated cells obtained by the present invention can be cells into which the base sequence to be inserted into the target sequence has been inserted.
- the cells obtained in the present invention may or may not be cloned thereafter.
- the cells obtained in the present invention are cloned and can be obtained as cloned cells. Making the genome uniform has the advantage of facilitating subsequent industrial applications, genetic manipulations, and the like.
- FIGS. 9A and 9B a method for editing genomes (or a method for producing cells having edited genomes) provided by the present invention will be described using FIGS. 9A and 9B.
- the method described in FIG. 9A is disclosed in detail in WO2018/030536 and JP2016-220639A.
- the genomic RNA transcription region (100) is composed of a sense strand (110) and an antisense strand (120).
- a method of editing a genome comprising: (A) hybridize to the sequence (122) adjacent upstream and the sequence (123) adjacent downstream of the target region (121) corresponding to the antisense strand of the genomic region to which the RNA is transcribed in the cell; A method is provided comprising allowing sequence-specific interaction of a possible single-stranded form of an editing sense polynucleotide (10) with a genome (100).
- “adjacent” means existing next to each other (that is, existing consecutively on the sequence).
- a method of genome editing in a cell comprising: (A) hybridize to the sequence (122) adjacent upstream and the sequence (123) adjacent downstream of the target region (121) corresponding to the antisense strand of the genomic region to which the RNA is transcribed in the cell; A method is provided comprising allowing a sequence-specific interaction of the editable sense polynucleotide (10) in possible single-stranded form with the genome.
- a method of targeting the genome in a cell comprising: (A) hybridize to the sequence (122) adjacent upstream and the sequence (123) adjacent downstream of the target region (121) corresponding to the antisense strand of the genomic region to which the RNA is transcribed in the cell; A method is provided comprising allowing sequence-specific interaction of a possible single-stranded form of an editing sense polynucleotide (10) with a genome (100).
- the single-stranded form of the editing sense polynucleotide (10) has an upstream homology arm (11) and a downstream homology arm (12).
- Said upstream homology arm (11) has a sequence hybridizable with a sequence (122) that flanks upstream of said target region
- said downstream homology arm (12) has a sequence that flanks downstream of said target region (121). (123), the sequence (122) flanking at least upstream and the flanking sequence (123) downstream of the target region by said upstream homology arm (11) and said downstream homology arm (12) ), respectively.
- the single-stranded form of the editing sense polynucleotide (10) can have an arbitrary sequence (13) between the upstream homology arm (11) and the downstream homology arm (12).
- Any sequence (13) replaces sequence (111) of the corresponding region of the complementary strand of the target region (121).
- the target region (121) replaces the sequence (23) complementary to any sequence (13) and the region (111) corresponding to the target sequence on the complementary strand replaces any sequence (13) is replaced by an array of Arbitrary sequence (13) is therefore determined by how one wishes to modify the region (111) corresponding to the target sequence on the complementary strand. That is, region (111) can be replaced with any sequence (13).
- part or all of the region (111) corresponding to the target sequence on the complementary strand can be replaced with a new sequence (ie, any sequence (13)). If the sequence of any sequence (13) has an insertion of a sequence into the region (111) corresponding to the target sequence on the complementary strand, then the new sequence can be inserted into the region (111). Also, if the sequence of the arbitrary sequence (13) has a deletion of part or all of the sequence with respect to the region (111) corresponding to the target sequence on the complementary strand, the sequence from the region (111) can be deleted.
- the editing sense polynucleotide (10) in single-stranded form may have no bases in any sequence (13) ⁇ i.e.
- the upstream homology arm (11) and the downstream homology arm (12) are seamlessly may be connected ⁇ . If any sequence (13) has no bases, the target region (121) and the region (111) corresponding to the target sequence on the complementary strand will be deleted in their entirety.
- This is a genome editing method using a single-stranded polynucleotide that applies the PODiR mechanism. Genome-edited cells can be obtained, for example, by cloning and analyzing the sequence of a region containing the target region of the genome.
- said step (A) removes either or both of the single-stranded form of the antisense polynucleotide (20) and the single-stranded form of the antisense polynucleotide (30).
- the antisense polynucleotide (20) is a partial region of the RNA (50) transcribed from the genomic region (120) and has a sequence corresponding to the 5' terminal portion of the editing sense strand polynucleotide. It has a sequence capable of hybridizing to the region (51) and its upstream sequence (53).
- the antisense polynucleotide (30) is a partial region of the RNA (50) transcribed from the genomic region (120) and has a sequence corresponding to the 3' terminal portion of the editing sense strand polynucleotide. It has a sequence capable of hybridizing to the region (52) and its downstream sequence (54).
- the length of region (51) and region (52) can each be 1-20 bases, such as 2-19 bases, 3-18 bases, 4-17 bases, and 4-17 bases.
- base can be 5-16 bases, can be 6-15 bases, can be 7-14 bases, can be 8-13 bases, can be 9-12 bases, 10 bases, or 11 bases It can be a base.
- the length of the region (21) of the single-stranded form of the antisense polynucleotide (20) and the region (31) of the single-stranded form of the antisense polynucleotide (30) binding to this region are also 1 to 20 bases, such as 2-19 bases, 3-18 bases, 4-17 bases, 5-16 bases, 6-15 bases, 7- It can be 14 bases, it can be 8-13 bases, it can be 9-12 bases, it can be 10 bases, or it can be 11 bases.
- the upstream homology arm and the downstream homology arm are each 10 nucleotides or longer, 20 nucleotides or longer, 30 nucleotides or longer, 40 nucleotides or longer, 50 nucleotides or longer, 60 nucleotides or longer, 70 nucleotides or longer, It can be 80 nucleotides or longer, 90 nucleotides or longer, or 100 nucleotides or longer.
- the upstream homology arm and the downstream homology arm are each 200 nucleotides or less, 190 nucleotides or less, 180 nucleotides or less, 170 nucleotides or less, 160 nucleotides or less, 150 nucleotides or less, 140 nucleotides or less, 130 nucleotides or less, or 130 nucleotides or less. 120 nucleotides or less, 110 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides or less, or 40 nucleotides or less could be.
- the upstream homology arm and the downstream homology arm can each be 10-200 nucleotides in length, 20-150 nucleotides in length, 30-100 nucleotides in length, 35-70 nucleotides in length, or 40-50 nucleotides in length.
- the upstream homology arm and the downstream homology arm may have different lengths or may have the same length.
- the shorter arm is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% of the length of the long arm. It can be longer than, 90% or longer, or 95% or longer.
- said antisense polynucleotide (20) and antisense polynucleotide (30) are independently greater than 10 bases, greater than 15 bases, greater than 20 bases, 25 bases more than 30 bases, more than 35 bases, or more than 40 bases.
- the antisense polynucleotide (20) and the antisense polynucleotide (30) independently have a length of 50 bases or more, a length of 60 bases or more, a length of 70 bases or more, or 80 bases. 90 bases or longer, 100 bases or longer, 110 bases or longer, 120 bases or longer, or 130 bases or longer.
- the antisense polynucleotide (20) and the antisense polynucleotide (30) are independently, for example, 200 bases or less in length, 190 bases in length or less, 180 bases in length or less, 170 bases in length or less length, 160 bases or less, 150 bases or less, 140 bases or less, or 130 bases or less. Said antisense polynucleotide (20) and antisense polynucleotide (30) may have the same length.
- the antisense polynucleotide (20) is within ⁇ 50%, within ⁇ 40%, within ⁇ 30%, within ⁇ 25%, within ⁇ 20%, within ⁇ 15%, within ⁇ It can be within 10%, or within ⁇ 5% of the length.
- the antisense polynucleotide (30) is within ⁇ 50%, within ⁇ 40%, within ⁇ 30%, within ⁇ 25%, within ⁇ 20%, within ⁇ 15% of the antisense polynucleotide (20) , within ⁇ 10%, or within ⁇ 5%.
- RNA (50) when present, hybridizes to the genomic antisense strand (120), It is possible to reduce the effect of the inability of the editing sense polynucleotide (10) to bind to the antisense strand (120). This facilitates binding of the editing sense polynucleotide (10) to the antisense strand (120).
- step (A) is performed in the presence of at least two hybridizable single-stranded forms of antisense polynucleotides, wherein at least one antisense polynucleotide comprises a genomic region at least one type of antisense polynucleotide having a sequence capable of hybridizing to a region having a sequence corresponding to the 5' terminal portion of the editing sense strand polynucleotide is a partial region of RNA transcribed from a genomic region, having a sequence capable of hybridizing to a region having a sequence corresponding to the 3' terminal portion of the editing sense strand polynucleotide.
- This makes it easier for the editing sense polynucleotide (10) to bind to the antisense strand (120) than when using one type of single-stranded form of the antisense polynucleotide.
- An editing sense polynucleotide comprises, as constituent units, for example, nucleotides selected from the group consisting of DNA and RNA and derivatives thereof.
- Derivatives can be modified nucleotides or modified nucleic acids.
- Modified nucleic acids include, for example, fluorescent dye-modified nucleic acids, biotinylated nucleic acids, and cholesteryl group-introduced nucleic acids.
- RNA may have 2′-O-methyl, 2′-fluoro or 2′-methoxyethyl (MOE) modifications to the bases to increase stability, and phosphodiester bonds in the nucleic acid backbone may be modified. It may be replaced by a phosphorothioate bond.
- MOE 2′-O-methyl, 2′-fluoro or 2′-methoxyethyl
- Examples of artificial nucleic acids include nucleic acids in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position are bridged.
- Examples of such artificial nucleic acids include locked nucleic acid (LNA), which is a crosslinked DNA in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position are crosslinked via methylene, the oxygen atom at the 2'-position and ENA in which the carbon atom at the 4'-position is bridged through ethylene, BNA COC in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position are bridged through -CH 2 OCH 2 -, oxygen at the 2'-position Bridged Nucleic Acids (BNA) such as BNA NC where the atom and the carbon atom at the 4′ position are bridged via —NR—CH 2 — (where R is a methyl or hydrogen atom), cMOE in which the oxygen atom and the carbon atom at the 4′ position are bridge
- the antisense polynucleotide (20) and the antisense polynucleotide (30) may use polynucleotides with strong binding affinity to RNA from the viewpoint of improving the affinity to RNA.
- the polynucleotide can be DNA, LNA, BNA, morpholino oligos, etc. or combinations thereof.
- the sense polynucleotide for editing can be a polynucleotide with strong binding affinity to DNA from the viewpoint of improving binding to DNA, for example, it is selected from the group consisting of RNA, LNA, BNA, and modified RNA. or a combination thereof.
- the method of the present invention allows genome editing in eukaryotic cells, in particular the target region (121) of the genome is linked to said upstream homology arm (11) and said downstream homology arm ( 12) can be replaced with the sequence of the complementary strand of any sequence (13) between, and the sequence of the region (111) corresponding to the region of the complementary strand of the target region (121) can be replaced with the arbitrary sequence (13) can be replaced. If any sequence (13) is absent, the target region of the genome (121) and the corresponding region of the complementary strand (111) will be deleted.
- the target region is present in a region where RNA is transcribed.
- a region in which RNA is transcribed may be a transcriptionally activated region.
- the target region can be a portion of an exon.
- the target region is an intron segment.
- the size of the target region is not particularly limited. 700bp or less, 600bp or less, 500bp or less, 400bp or less, 300bp or less, 200bp or less, 100bp or less, 90bp or less, 80bp or less, 70bp or less, 60bp or less, 50bp or less, 40bp or less, 30bp or less, 20bp or less, 10bp or less, 9bp or less .
- the methods of the present invention do not involve endonuclease cleavage of the target region of exogenous DNA. In one embodiment, the methods of the invention do not employ exogenous DNA endonucleases in editing and targeting the target region. In one aspect, the methods of the invention do not use ZFNs, TALENs and CRISPR/Cas systems in editing and targeting the target region. In some embodiments, other genome editing tools may be used in combination to edit regions other than the target regions of the methods of the invention.
- a method for targeting a target region of a genome comprising: (A) a sequence upstream and flanking the target region corresponding to the antisense strand of the genomic region to which RNA is transcribed in the cell; allowing sequence-specific interaction with the genome of an editing sense polynucleotide in single-stranded form hybridizable to the downstream flanking sequence;
- the single-stranded form of the editing sense polynucleotide has an upstream homology arm and a downstream homology arm,
- the upstream homology arm has a sequence hybridizable with a sequence flanking the target region upstream
- the downstream homology arm has a sequence hybridizable with a sequence flanking the target region downstream
- the upstream capable of hybridizing at least upstream and downstream of a target region by means of a homology arm and said downstream homology arm, with any or no sequence between said upstream homology arm and said downstream homology arm; may be
- the step (A) is performed in the presence of a single-
- An agent e.g., label or protein
- Labels can be labels such as fluorescent labels, dyes, RI labels, and tags.
- the editing sense polynucleotide (10) in single-stranded form is complementary to the target region as any sequence (13) between said upstream homology arm (11) and said downstream homology arm (12). It may have the same sequence as the sequence (111) of the region corresponding to the target region in the strand.
- a genome targeting kit or genome editing kit comprising a single-stranded form of an editing sense polynucleotide (10) and a single-stranded form of an antisense polynucleotide (20) and a single-stranded form
- a kit comprising at least one, preferably both, antisense polynucleotides (30) of The editing sense polynucleotide in single-stranded form (10), the antisense polynucleotide in single-stranded form (20) and the antisense polynucleotide in single-stranded form (30) are respectively as described above.
- a genome targeting kit or genome editing kit comprising a polynucleotide encoding an editing sense polynucleotide (10) in single-stranded form and an antisense polynucleotide (20) in single-stranded form and at least one, preferably both, antisense polynucleotides (30) in single-stranded form.
- a polynucleotide encoding an editing sense polynucleotide (10) in single-stranded form is operably linked to a control sequence. These polynucleotides can then be expressed.
- a genome targeting kit or genome editing kit comprising polynucleotides encoding an editing sense polynucleotide in single-stranded form (10) and an antisense polynucleotide in single-stranded form (20) and at least one, preferably both, of a polynucleotide encoding an antisense polynucleotide (30) in single-stranded form.
- the polynucleotide encoding the single-stranded form of the antisense polynucleotide (20) and the polynucleotide encoding the single-stranded form of the antisense polynucleotide (30) are each operably linked to a regulatory sequence. These polynucleotides can then be expressed.
- a genome targeting kit or genome editing kit comprising a polynucleotide encoding an editing sense polynucleotide (10) in single-stranded form and an antisense polynucleotide (20) in single-stranded form and at least one, preferably both, of a polynucleotide encoding an antisense polynucleotide (30) in single-stranded form.
- Each encoding polynucleotide is operably linked to a control sequence.
- the polynucleotide encoding the editing sense polynucleotide (10) in single-stranded form the polynucleotide encoding the antisense polynucleotide (20) in single-stranded form and the antisense polynucleotide in single-stranded form
- the polynucleotides encoding nucleotide (30) can each be operably linked to regulatory sequences and contained in one or more expression vectors.
- the genome targeting kit is preferably used in the genome targeting method of the present invention.
- the editing sense polynucleotide (10) in single-stranded form is RNA and can target the target region of the genome in the form of RNA.
- the genome editing kit is preferably used in the genome editing method of the present invention.
- the genome editing kit may further include an introduction reagent for introducing polynucleotides into cells.
- the single-stranded form of the editing sense polynucleotide (10), the single-stranded form of the antisense polynucleotide (20) and the single-stranded form of the antisense polynucleotide (30) are It may be provided in a chained state.
- the single-stranded form of the antisense polynucleotide (20), the single-stranded form of the editing sense polynucleotide (10), and the single-stranded form of the antisense polynucleotide (30) are A chimeric polynucleotide in single-stranded form comprising in sequence is provided.
- the present invention also provides an expression vector that expressably contains a DNA encoding the chimeric polynucleotide in single-stranded form.
- a cell having a genome modified by the genome editing method of the present invention and an organism containing the cell are provided.
- Example 1 Genome Editing by Sense Polynucleotides for Editing and Antisense Polynucleotides for Promoting Editing
- Single-stranded sense strand polynucleotides that directly contribute to genome editing bind to genomic DNA and alter the genomic DNA to the sequence of the editing sense polynucleotide.
- the first step in genome editing is to hybridize a sense polynucleotide for editing with DNA on the antisense side of genomic DNA.
- newly synthesized RNA such as mRNA or a fragment thereof (for example, a fragment removed by splicing) is hybridized on the DNA. It may inhibit the sense polynucleotide from hybridizing to the antisense side of the DNA.
- intron-targeted genome editing is less efficient than exon-targeted genome editing.
- intron it is possible that an RNA fragment from the processed and excised mRNA binds to the intron portion of the genomic DNA and inhibits genome editing.
- a detachment antisense polynucleotide was designed for the purpose of dissociating this newly synthesized RNA hybridized to the antisense strand of the genomic DNA from the genomic DNA.
- the stripping antisense polynucleotide was designed to bind to a DNA region that partially overlaps with the DNA region to which the editing sense polynucleotide hybridizes.
- the synthesized antisense polynucleotide for stripping was introduced into the system, and genome editing by the sense polynucleotide for editing was tested in the presence of the antisense polynucleotide for stripping.
- an editing sense polynucleotide (101 base length) represented by (1) was prepared.
- antisense for exfoliation represented by (3), (4), (7), (8), (11) to (16), and (22) to (27)
- a polynucleotide was provided.
- the detachment antisense polynucleotides represented by (3), (4), (7), (8), (11) to (16), and (22) to (27) are represented by (1) It is designed to have a sequence capable of hybridizing to the terminal 10 bases (sometimes referred to as "glue") of the editing sense polynucleotide.
- (3) and (4) are 130 bases long, (7) and (8) are 10 bases long, (11) and (12) are 100 bases long, (13) and ( 14) is 70 bases long, (15) and (16) are 40 bases long, (22) and (23) are 80 bases long, (24) and (25) are 60 bases long. (26) and (27) are 50 bases long.
- an editing sense polynucleotide (81 base length) represented by (2) was prepared.
- a 130-base-long antisense polynucleotide for detachment represented by (9) and (10) designed to have a sequence capable of hybridizing to bases was prepared.
- the sense polynucleotides for editing represented by (17) and (18) were prepared.
- a sense polynucleotide for editing represented by (19) and an antisense polynucleotide for detachment represented by (20) and (21) were prepared.
- the above polynucleotides (1) to (18) and (22) to (27) are set within the first intron of the region encoding ARFGEF3 (BIG3) of human genomic DNA.
- the sense polynucleotides for editing (1) and (2) have base sequences in which one nucleotide is substituted in the partial sequence on the sense strand side of the intron, and the sense polynucleotide for editing (17) is on the sense strand side of the intron.
- the sense polynucleotide for editing (18) has a nucleotide sequence in which 2 nucleotides are deleted in the partial sequence on the sense strand side of the intron.
- the antisense polynucleotides (3) to (18) for detachment have a partial sequence on the antisense strand side in the partial sequence on the antisense strand side of the intron.
- the 5′ terminal 10 nucleotides of the editing sense polynucleotides (1), (17) and (18) are replaced with the stripping antisense polynucleotides (3), (11), (13), (15), (22). ), (24) and (26), and to the 5′-terminal 10 nucleotides of the stripping antisense polynucleotide (7).
- the 3′-terminal 10 nucleotides of the editing sense polynucleotides (1), (17) and (18) are the stripping antisense polynucleotides (4), (12), (14), (16), (23). ), (25) and (27), and to the 3′-terminal 10 nucleotides, and to the stripping antisense polynucleotide (8).
- the 5'-terminal 10 nucleotides of the editing sense polynucleotide (2) are complementary to the 5'-terminal 10 nucleotides of the stripping antisense polynucleotide (9), and the 3'-terminal 10 nucleotides are It is complementary to the 3' terminal 10 nucleotides of the detachment antisense polynucleotide (10).
- the sense polynucleotide for editing (1) and the antisense polynucleotides for stripping (5) and (6) do not have complementary sequences, and the sense polynucleotide for editing (2) and the antisense polynucleotide for stripping (3) and (4) have no complementary sequence.
- the sense polynucleotide for editing (19) is set at a position containing the entire first exon of the region encoding ARFGEF3 (BIG3) of human genomic DNA, and one nucleotide is substituted in the sequence on the sense strand side of the position. base sequence.
- the antisense polynucleotide for stripping (20) is located at a position that includes a portion of the 5′-terminal untranslated region and a portion of the first exon of the region encoding ARFGEF3, and the antisense polynucleotide for stripping (21) is ARFGEF3. are set within the first intron of the coding region, and each has a partial sequence on the antisense strand side.
- the 5'-terminal 10 nucleotides of the editing sense polynucleotide (19) are complementary to the 5'-terminal 10 nucleotides of the stripping antisense polynucleotide (20), and the 3'-terminal 10 nucleotides are It is complementary to the 10 nucleotides at the 3' end of the detachment antisense polynucleotide (21).
- test was carried out as follows. [1] First, Dulbecco's Modified Eagle's Medium-high containing 10% (v/v) Fetal Bovine Serum (Sigma, F7524), 1% (v/v) Antibiotic-Antimycotic (Gibco, 15240-062) HEK293T cells were cultured in a 10 cm dish in a glucose; DMEM (Sigma, D5796) medium to confluent cells.
- Genome DNA was extracted from the cell pellet (2.0 ⁇ 10 6 cells) remaining in the 1.5 mL tube using the Macherey-Nagel genome extraction kit (NucleoSpin Tissue Kit), and the next-generation sequencer was used. The percentage of genomes that were edited as designed was calculated using this method, and the genome editing efficiency was calculated as the number of edited reads relative to the total number of reads.
- the genome editing efficiency with the editing sense polynucleotide represented by (2) was 0.0056%, whereas the detachment anti-stripping of (9) and (10) was 0.0056%. In the presence of the sense polynucleotide, the genome editing efficiency was 12.1425%.
- Genome editing was performed using the editing sense polynucleotide of (1) in the presence of the antisense polynucleotides for detachment of ) to (27).
- genome editing efficiency with the editing sense polynucleotides of (1) was improved in the presence of any combination of stripping antisense polynucleotides.
- the genome editing efficiency was significantly improved.
- the efficiency of genome editing was confirmed using eukaryotic cells.
- the efficiency of genome editing by the editing sense polynucleotide was improved in the presence of the stripping antisense polynucleotide.
- the antisense polynucleotide for exfoliation removed the factor that prevented genome editing by the sense polynucleotide for editing.
- Stripping antisense polynucleotides were originally designed to inhibit RNA transcribed from genomic DNA from binding to DNA. It was suggested that the transcribed RNA was probably linked on the genomic DNA by DNA-RNA interaction, which inhibited the hybridization of the editing sense polynucleotide to the region. Since the DNA-RNA interaction is stronger than the DNA-DNA interaction, the inhibitory effect is considered to be particularly large when DNA is used as the editing sense polynucleotide.
- an editing sense polynucleotide (18) having a base deletion and an editing sense polynucleotide having a base insertion was performed using (17). The results were as shown in FIG. As shown in FIG. 5, both when using the editing sense polynucleotide (18) having a base deletion and when using the editing sense polynucleotide (17) having a base insertion, the antisense polynucleotide The editing efficiency was greatly improved in the presence of nucleotides.
- an antisense polynucleotide designed to bind to a DNA region that partially overlaps with a DNA region to which a sense polynucleotide for editing hybridizes can be used as an antisense polynucleotide for promoting editing. shown.
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Abstract
Description
即ち、本発明者は、一本鎖アンチセンスポリヌクレオチド(「編集促進用アンチセンスポリヌクレオチド」ともいう)を合成し、編集促進用アンチセンスポリヌクレオチド存在下で、編集のための一本鎖形態のポリヌクレオチド(「編集用センスポリヌクレオチド」ともいう)によるゲノム編集効率を試験したところ、編集促進用アンチセンスポリヌクレオチドを、編集用センスポリヌクレオチドの末端部分と相補する配列を有するようにもしくは相補性を有する配列を有するように設計した場合(すなわち、編集用センスポリヌクレオチドの末端と結合するように設計した場合)に、ゲノム編集の効率が高まることを見出した。理論に拘束されるものではないが、RNAに転写されるゲノム領域が編集対象である場合、ゲノムDNAの周辺に存在する転写されたRNAと編集用センスポリヌクレオチドとの間でゲノムDNAへの結合に関して競合が生じ得ることから、上記のゲノム編集効率の向上は、編集促進用アンチセンスポリヌクレオチドがRNAと結合した結果として前記競合が抑制されるためと考えられる。ゲノム編集技術において、このようなゲノムDNAへの結合に関する競合を抑制することで編集効率を高めるという技術思想はこれまで全くなかったものであり、本発明はこの技術思想に立脚して行われたものである。
[項1A]
二本鎖ゲノムDNAの標的部位を改変することができる一本鎖形態の編集用ポリヌクレオチドと組み合わせて用いるための、一本鎖形態の編集促進用ポリヌクレオチドであって、
標的部位は、一方のゲノムDNA鎖上に設定される一次編集部位と、他方のゲノムDNA鎖上の、一次編集部位と対応する位置にある二次編集部位とからなり、
編集用ポリヌクレオチドは、5'末端側から順に、
a)一次編集部位の3’末端側に隣接する領域とハイブリダイズ可能な部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)一次編集部位の5’末端側に隣接する領域とハイブリダイズ可能な部分
からなるか、又は
部分a)及び部分b)
からなり、
編集促進用ポリヌクレオチドは、前記一方のゲノムDNA鎖上の所定領域に対して相補的である核酸配列とハイブリダイズ可能であって、ここで前記所定領域は、前記一方のゲノムDNA鎖上の、編集用ポリヌクレオチドの部分a)と対応する位置にある領域を領域Aとし、編集用ポリヌクレオチドの部分b)と対応する位置にある領域を領域Bとしたときに、その5'末端部において領域Aとオーバーラップするか、又はその3'末端部において領域Bとオーバーラップし、
前記所定領域の5'末端部における領域Aとのオーバーラップの長さは、領域Aと同一又はそれより短く、前記所定領域の3'末端部における領域Bとのオーバーラップの長さは、領域Bと同一又はそれより短く、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の2つの連続するヌクレオチドの間に設定された場合に、一次編集部位に部分x)と相補的な塩基配列からなる部分を、二次編集部位に部分x)を挿入することができ、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、一次編集部位を部分x)と相補的な塩基配列からなる部分に、二次編集部位を部分x)に置換することができ、
編集用ポリヌクレオチドが部分a)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、標的部位を欠失させることができる、前記編集促進用ポリヌクレオチド。
[項1B]
二本鎖ゲノムDNAの標的部位を改変することができる一本鎖形態の編集用ポリヌクレオチドと組み合わせて用いるための、一本鎖形態の編集促進用ポリヌクレオチドであって、
標的部位は、一方のゲノムDNA鎖上に設定される一次編集部位と、他方のゲノムDNA鎖上の、一次編集部位と対応する位置にある二次編集部位とからなり、
編集用ポリヌクレオチドは、5'末端側から順に、
a)二次編集部位の5’末端側に隣接する領域の塩基配列と90%以上の同一性を有する塩基配列からなる部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)二次編集部位の3’末端側に隣接する領域の塩基配列と90%以上の同一性を有する塩基配列からなる部分
からなるか、又は
部分a)及び部分b)
からなり、
編集促進用ポリヌクレオチドは、前記一方のゲノムDNA鎖上の所定領域の塩基配列と90%以上の同一性を有する塩基配列からなり、ここで前記所定領域は、前記一方のゲノムDNA鎖上の、編集用ポリヌクレオチドの部分a)と対応する位置にある領域を領域Aとし、編集用ポリヌクレオチドの部分b)と対応する位置にある領域を領域Bとしたときに、その5'末端部において領域Aとオーバーラップするか、又はその3'末端部において領域Bとオーバーラップし、
前記所定領域の5'末端部における領域Aとのオーバーラップの長さは、領域Aと同一又はそれより短く、前記所定領域の3'末端部における領域Bとのオーバーラップの長さは、領域Bと同一又はそれより短く、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の2つの連続するヌクレオチドの間に設定された場合に、一次編集部位に部分x)と相補的な塩基配列からなる部分を、二次編集部位に部分x)を挿入することができ、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、一次編集部位を部分x)と相補的な塩基配列からなる部分に、二次編集部位を部分x)に置換することができ、
編集用ポリヌクレオチドが部分a)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、標的部位を欠失させることができる、前記編集促進用ポリヌクレオチド。
[項1C]
二本鎖ゲノムDNAの標的部位を改変することができる一本鎖形態の編集用ポリヌクレオチドと組み合わせて用いるための、一本鎖形態の編集促進用ポリヌクレオチドであって、
標的部位は、一方のゲノムDNA鎖上に設定される一次編集部位と、他方のゲノムDNA鎖上の、一次編集部位と対応する位置にある二次編集部位とからなり、
編集用ポリヌクレオチドは、5'末端側から順に、
a)二次編集部位の5’末端側に隣接する領域の塩基配列と同一の塩基配列からなる部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)二次編集部位の3’末端側に隣接する領域の塩基配列と同一の塩基配列からなる部分
からなるか、又は
部分a)及び部分b)
からなり、
編集促進用ポリヌクレオチドは、前記一方のゲノムDNA鎖上の所定領域の塩基配列と同一の塩基配列からなり、ここで前記所定領域は、前記一方のゲノムDNA鎖上の、編集用ポリヌクレオチドの部分a)と対応する位置にある領域を領域Aとし、編集用ポリヌクレオチドの部分b)と対応する位置にある領域を領域Bとしたときに、その5'末端部において領域Aとオーバーラップするか、又はその3'末端部において領域Bとオーバーラップし、
前記所定領域の5'末端部における領域Aとのオーバーラップの長さは、領域Aと同一又はそれより短く、前記所定領域の3'末端部における領域Bとのオーバーラップの長さは、領域Bと同一又はそれより短く、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の2つの連続するヌクレオチドの間に設定された場合に、一次編集部位に部分x)と相補的な塩基配列からなる部分を、二次編集部位に部分x)を挿入することができ、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、一次編集部位を部分x)と相補的な塩基配列からなる部分に、二次編集部位を部分x)に置換することができ、
編集用ポリヌクレオチドが部分a)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、標的部位を欠失させることができる、前記編集促進用ポリヌクレオチド。
[項2A]
編集促進用ポリヌクレオチドの長さが、50ヌクレオチド以上である、項1A、項1B又は項1Cに記載の編集促進用ポリヌクレオチド。
[項2B]
前記オーバーラップの長さが、5ヌクレオチド以上である、項1A、項1B、項1C又は項2Aに記載の編集促進用ポリヌクレオチド。
[項3A]
項1A、項2A又は項2Bに記載の編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
編集用ポリヌクレオチド、その発現ベクター、又は前記編集用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、ゲノム編集用キットであって、
編集用ポリヌクレオチドが、5'末端側から順に、
a)一次編集部位の3’末端側に隣接する領域とハイブリダイズ可能な部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)一次編集部位の5’末端側に隣接する領域とハイブリダイズ可能な部分
からなるか、又は
部分a)及び部分b)
からなる、前記ゲノム編集用キット。
[項3B]
項1B、項2A又は項2Bに記載の編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
編集用ポリヌクレオチド、その発現ベクター、又は前記編集用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、ゲノム編集用キットであって、
編集用ポリヌクレオチドが、5'末端側から順に、
a)二次編集部位の5’末端側に隣接する領域の塩基配列と90%以上の同一性を有する塩基配列からなる部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)二次編集部位の3’末端側に隣接する領域の塩基配列と90%以上の同一性を有する塩基配列からなる部分
からなるか、又は
部分a)及び部分b)
からなる、前記ゲノム編集用キット。
[項3C]
項1C、項2A又は項2Bに記載の編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
編集用ポリヌクレオチド、その発現ベクター、又は前記編集用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、ゲノム編集用キットであって、
編集用ポリヌクレオチドが、5'末端側から順に、
a)二次編集部位の5’末端側に隣接する領域の塩基配列と同一の塩基配列からなる部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)二次編集部位の3’末端側に隣接する領域の塩基配列と同一の塩基配列からなる部分
からなるか、又は
部分a)及び部分b)
からなる、前記ゲノム編集用キット。
[項4]
前記所定領域がその5'末端部において領域Aとオーバーラップするように設定された編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
前記所定領域がその3'末端部において領域Bとオーバーラップするように設定された編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、項3A、項3B又は項3Cに記載のゲノム編集用キット。
[項5]
編集促進用ポリヌクレオチドに関する前記オーバーラップの長さが、5ヌクレオチド以上である、項3A、項3B、項3C又は項4に記載のゲノム編集用キット。
[項6]
細胞又は生物を、項3A、項3B、項3C、項4又は項5に記載のゲノム編集用キットを用いて処理する工程を含む、細胞又は生物の二本鎖ゲノムDNAの標的部位を改変する方法。
[項7]
細胞又は生物を、項3A、項3B、項3C、項4又は項5に記載のゲノム編集用キットを用いて処理する工程を含む、二本鎖ゲノムDNAの標的部位が改変された細胞又は生物を製造する方法。
本開示において、編集用ポリヌクレオチドは、二本鎖ゲノムDNAの標的部位を改変することができる、一本鎖形態のポリヌクレオチドである。編集用ポリヌクレオチドは、5'末端側から順に、
a)一次編集部位の3’末端側に隣接する領域とハイブリダイズ可能な部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)一次編集部位の5’末端側に隣接する領域とハイブリダイズ可能な部分
からなるか、又は
部分a)及び部分b)
からなる。以下、編集用ポリヌクレオチドの詳細を説明する(図8Aも参照されたい)。
編集用ポリヌクレオチドは、ゲノム編集効率を向上させるため、編集促進用ポリヌクレオチドと組み合わせて用いることができる。編集促進用ポリヌクレオチドは、一次編集部位が設定されたゲノムDNA鎖上の所定領域に対して相補的である核酸配列(所定領域の相補配列とも呼ぶ)とハイブリダイズ可能なポリヌクレオチドである。以下、編集促進用ポリヌクレオチドの詳細を説明する(図8Bも参照されたい)。
本開示は、一態様として、上述の編集用ポリヌクレオチドと、上述の編集促進用ポリヌクレオチドとを含むゲノム編集用キットを提供する。ゲノム編集用キットは、前記所定領域がその5'末端部において領域Aとオーバーラップするように設定された編集促進用ポリヌクレオチド、及び前記所定領域がその3'末端部において領域Bとオーバーラップするように設定された編集促進用ポリヌクレオチドの2種類を含むことが好ましい。
本開示は、一態様において、細胞又は生物を、上述のゲノム編集用キットを用いて処理する工程を含む、細胞又は生物の二本鎖ゲノムDNAの標的部位を改変する方法(本開示において「ゲノム編集方法」とも呼ぶ)を提供する。また本開示は、一態様において、細胞又は生物を、上述のゲノム編集用キットを用いて処理する工程を含む、二本鎖ゲノムDNAの標的部位が改変された細胞又は生物を製造する方法(本開示において「ゲノム編集された細胞又は生物の製造方法」とも呼ぶ)を提供する。
(A)当該細胞内において、ゲノムの標的領域(例えば、RNAが転写されるゲノム領域のアンチセンス鎖に対応する標的領域)の上流に隣接する配列と下流に隣接する配列に対してハイブリダイズ可能な一本鎖形態の編集用ポリヌクレオチドをゲノムに配列特異的に相互作用させる工程を含み;
前記一本鎖形態の編集用ポリヌクレオチド(例えば、編集用センスポリヌクレオチド)は、上流ホモロジーアームと下流ホモロジーアームを有し、
前記上流ホモロジーアームは前記標的領域の上流に隣接する配列とハイブリダイズ可能な配列を有し、前記下流ホモロジーアームは前記標的領域の下流に隣接する配列とハイブリダイズ可能な配列を有し、前記上流ホモロジーアームと前記下流のホモロジーアームにより標的領域の少なくとも上流および下流にハイブリダイズすることができ、前記上流ホモロジーアームと前記下流のホモロジーアームの間に任意の配列を有するか、または配列を有しなくてもよく、
前記工程(A)が、他の一本鎖形態のポリヌクレオチド(例えば、一本鎖形態のアンチセンスポリヌクレオチド)の存在下で行われ、前記他のポリヌクレオチド(例えば、一本鎖形態のアンチセンスポリヌクレオチド)は、当該編集用ポリヌクレオチドの末端部分と対応する配列を有する領域(例えば、ゲノム領域から転写されるRNAの部分領域であって、当該編集用センスポリヌクレオチドの末端部分に対応するヌクレオチド配列を有する領域)にハイブリダイズすることができる配列(例えば、当該部分領域と相補性を有する配列)を有し、
前記他のポリヌクレオチド(例えば、一本鎖形態のアンチセンスポリヌクレオチド)は、40塩基以上の長さを有し、
前記工程により、前記標的領域が、前記一本鎖形態の編集用ポリヌクレオチド(例えば、一本鎖形態の編集用センスポリヌクレオチド)の前記上流ホモロジーアームと前記下流のホモロジーアームの間の塩基配列に置き換わる、但し、前記上流ホモロジーアームと前記下流のホモロジーアームの間に塩基配列が存在しない場合には前記標的領域が欠失する、
方法。
[2]前記一本鎖形態の編集用ポリヌクレオチドが、DNAおよびRNA並びにその組合せからなる群から選択されるポリヌクレオチドである、上記[1]に記載の方法。
[3]前記他の一本鎖形態のポリヌクレオチドが、DNAである、上記[1]または[2]に記載の方法。
[4]前記末端部分と対応する配列を有する領域が、5塩基以上の長さを有する、上記[1]~[3]のいずれかに記載の方法。
[5]前記末端部分と対応する配列を有する領域が、20塩基以下の長さを有する、上記[1]~[4]のいずれかに記載の方法。
[6]前記一本鎖形態の編集用ポリヌクレオチドが、60~200塩基の長さを有する、上記[1]~[5]のいずれかに記載の方法。
[7]前記他の一本鎖形態のポリヌクレオチドが、50~200塩基の長さを有する、上記[1]~[6]のいずれかに記載の方法。
[8]標的領域が、イントロンに存在する、上記[1]~[7]のいずれかに記載の方法。
[9]前記工程(A)が、少なくとも2種類のハイブリダイズ可能な前記他の一本鎖形態のポリヌクレオチドの存在下で行われ、少なくとも1種類の他の一本鎖形態のポリヌクレオチド(第1の他のポリヌクレオチド)は、当該編集用ポリヌクレオチドの5’末端部分と対応する配列を有する領域にハイブリダイズすることができる配列(例えば、相補性を有する配列)を有し、少なくとも1種類の他の一本鎖形態のポリヌクレオチド(第2の他のポリヌクレオチド)は、当該編集用ポリヌクレオチドの3’末端部分と対応する配列を有する領域にハイブリダイズすることができる配列(例えば、相補性を有する配列)を有する、上記[1]~[8]のいずれかに記載の方法。
[10]上記[1]~[8]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドと前記他の一本鎖形態のポリヌクレオチドとの組合せを含む、ゲノム編集用キット。
[11]上記[1]~[8]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAと、前記他の一本鎖形態のポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[12]上記[1]~[8]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAと、前記他の一本鎖形態のポリヌクレオチドを含み、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAは、発現ベクター中に発現可能に含まれ、前記他の一本鎖形態のポリヌクレオチドはDNAである、ゲノム編集用キット。
[13]上記[1]~[8]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするポリヌクレオチドと、前記他の一本鎖形態のポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[14]上記[9]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドと前記少なくとも2種類の他の一本鎖形態のポリヌクレオチドとの組合せを含む、ゲノム編集用キット。
[15]上記[9]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAと、前記少なくとも2種類の他の一本鎖形態のポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[16]上記[9]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAと、前記少なくとも2種類の他の一本鎖形態のポリヌクレオチドを含み、前記一本鎖形態の編集用ポリヌクレオチドをコードするDNAは、発現ベクター中に発現可能に含まれ、前記他の一本鎖形態のポリヌクレオチドはDNAである、ゲノム編集用キット。
[17]上記[9]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用ポリヌクレオチドをコードするポリヌクレオチドと、前記少なくとも2種類の他の一本鎖形態のポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
(A)当該細胞内において、RNAが転写されるゲノム領域のアンチセンス鎖に対応する標的領域の上流に隣接する配列と下流に隣接する配列に対してハイブリダイズ可能な一本鎖形態の編集用センスポリヌクレオチドをゲノムに配列特異的に相互作用させる工程を含み;
前記一本鎖形態の編集用センスポリヌクレオチドは、上流ホモロジーアームと下流ホモロジーアームを有し、
前記上流ホモロジーアームは前記標的領域の上流に隣接する配列とハイブリダイズ可能な配列を有し、前記下流ホモロジーアームは前記標的領域の下流に隣接する配列とハイブリダイズ可能な配列を有し、前記上流ホモロジーアームと前記下流のホモロジーアームにより標的領域の少なくとも上流および下流にハイブリダイズすることができ、前記上流ホモロジーアームと前記下流のホモロジーアームの間に任意の配列を有するか、または配列を有しなくてもよく、
前記工程(A)が、一本鎖形態のアンチセンスポリヌクレオチドの存在下で行われ、前記アンチセンスポリヌクレオチドは、ゲノム領域から転写されるRNAの部分領域であって、当該編集用センス鎖ポリヌクレオチドの末端部分と対応する配列を有する領域にハイブリダイズすることができる配列(例えば、当該部分領域と相補性を有する配列)を有し、
前記アンチセンスポリヌクレオチドは、40塩基以上の長さを有し、
前記工程により、前記標的領域が、前記一本鎖形態の編集用センスポリヌクレオチドの前記上流ホモロジーアームと前記下流のホモロジーアームの間の塩基配列に置き換わる、但し、前記上流ホモロジーアームと前記下流のホモロジーアームの間に塩基配列が存在しない場合には前記標的領域が欠失する、
方法。
[2A]前記一本鎖形態の編集用センスポリヌクレオチドが、DNAおよびRNA並びにその組合せからなる群から選択されるポリヌクレオチドである、上記[1A]に記載の方法。
[3A]前記一本鎖形態のアンチセンスポリヌクレオチドが、DNAである、上記[1A]または[2A]に記載の方法。
[4A]前記末端部分が、5塩基以上の長さを有する、上記[1A]~[3A]のいずれかに記載の方法。
[5A]前記末端部分が、20塩基以下の長さを有する、上記[1A]~[4A]のいずれかに記載の方法。
[6A]前記一本鎖形態の編集用センスポリヌクレオチドが、60~200塩基の長さを有する、上記[1A]~[5A]のいずれかに記載の方法。
[7A]前記一本鎖形態のアンチセンスポリヌクレオチドが、50~200塩基の長さを有する、上記[1A]~[6A]のいずれかに記載の方法。
[8A]標的領域が、イントロンに存在する、上記[1A]~[7A]のいずれかに記載の方法。
[9A]前記工程(A)が、少なくとも2種類のハイブリダイズ可能な一本鎖形態のアンチセンスポリヌクレオチドの存在下で行われ、少なくとも1種類のアンチセンスポリヌクレオチドは、ゲノム領域から転写されるRNAの部分領域であって、当該編集用センス鎖ポリヌクレオチドの5’末端部分と対応する配列を有する領域にハイブリダイズすることができる配列(例えば、相補性を有する配列)を有し、少なくとも1種類のアンチセンスポリヌクレオチドは、ゲノム領域から転写されるRNAの部分領域であって、当該編集用センス鎖ポリヌクレオチドの3’末端部分と対応する配列を有する領域にハイブリダイズすることができる配列(例えば、相補性を有する配列)を有する、上記[1A]~[8A]のいずれかに記載の方法。
[10A]上記[1A]~[8A]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドと前記一本鎖形態のアンチセンスポリヌクレオチドとの組合せを含む、ゲノム編集用キット。
[11A]上記[1A]~[8A]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAと、前記一本鎖形態のアンチセンスポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[12A]上記[1A]~[8A]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAと、前記一本鎖形態のアンチセンスポリヌクレオチドを含み、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAは、発現ベクター中に発現可能に含まれ、前記一本鎖形態のアンチセンスポリヌクレオチドはDNAである、ゲノム編集用キット。
[13A]上記[1A]~[8A]のいずれかに記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするポリヌクレオチドと、前記一本鎖形態のアンチセンスポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[14A]上記[9A]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドと前記少なくとも2種類の一本鎖形態のアンチセンスポリヌクレオチドとの組合せを含む、ゲノム編集用キット。
[15A]上記[9A]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAと、前記少なくとも2種類の一本鎖形態のアンチセンスポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
[16A]上記[9A]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAと、前記少なくとも2種類の一本鎖形態のアンチセンスポリヌクレオチドを含み、前記一本鎖形態の編集用センスポリヌクレオチドをコードするDNAは、発現ベクター中に発現可能に含まれ、前記一本鎖形態のアンチセンスポリヌクレオチドはDNAである、ゲノム編集用キット。
[17A]上記[9A]に記載の方法において用いるためのゲノム編集用キットであって、前記一本鎖形態の編集用センスポリヌクレオチドをコードするポリヌクレオチドと、前記少なくとも2種類の一本鎖形態のアンチセンスポリヌクレオチドをコードするDNAとを含み、これらのDNAは、1以上の発現ベクター中に発現可能に含まれる、ゲノム編集用キット。
本発明によれば、ゲノムを編集する方法が提供される。本発明によれば、ゲノムの標的領域を標的化する方法が提供される。本発明によれば、編集されたゲノムを有する細胞を得る方法が提供される。
(A)当該細胞内において、RNAが転写されるゲノム領域のアンチセンス鎖に対応する標的領域(121)の上流に隣接する配列(122)と下流に隣接する配列(123)に対してハイブリダイズ可能な一本鎖形態の編集用センスポリヌクレオチド(10)をゲノム(100)に配列特異的に相互作用させる工程を含む、方法が提供される。ここで、「隣接する」とは、隣りに存在する(つまり、配列上では連続して存在する)ことを意味する。
(A)当該細胞内において、RNAが転写されるゲノム領域のアンチセンス鎖に対応する標的領域(121)の上流に隣接する配列(122)と下流に隣接する配列(123)に対してハイブリダイズ可能な一本鎖形態の編集用センスポリヌクレオチド(10)をゲノムに配列特異的に相互作用させる工程を含む、方法が提供される。
(A)当該細胞内において、RNAが転写されるゲノム領域のアンチセンス鎖に対応する標的領域(121)の上流に隣接する配列(122)と下流に隣接する配列(123)に対してハイブリダイズ可能な一本鎖形態の編集用センスポリヌクレオチド(10)をゲノム(100)に配列特異的に相互作用させる工程を含む、方法が提供される。
前記一本鎖形態の編集用センスポリヌクレオチドは、上流ホモロジーアームと下流ホモロジーアームを有し、
前記上流ホモロジーアームは前記標的領域の上流に隣接する配列とハイブリダイズ可能な配列を有し、前記下流ホモロジーアームは前記標的領域の下流に隣接する配列とハイブリダイズ可能な配列を有し、前記上流ホモロジーアームと前記下流のホモロジーアームにより標的領域の少なくとも上流および下流にハイブリダイズすることができ、前記上流ホモロジーアームと前記下流のホモロジーアームの間に任意の配列を有するか、または配列を有しなくてもよく、
前記工程(A)が、一本鎖形態のアンチセンスポリヌクレオチドの存在下で行われ、前記アンチセンスポリヌクレオチドは、ゲノム領域から転写されるRNAの部分領域であって、当該編集用センス鎖ポリヌクレオチドの末端部分と対応する配列を有する領域にハイブリダイズすることができる配列を有し、
前記アンチセンスポリヌクレオチドは、40塩基以上の長さを有する、
方法が提供される。
[1]まず、10% (v/v)Fetal Bovine Serum (Sigma, F7524)、1% (v/v)Antibiotic-Antimycotic (Gibco、15240-062)を含むDulbecco’s Modified Eagle’s Medium-high glucose; DMEM (Sigma、D5796)培地で、10cm dishでconfluent cellsまでHEK293T細胞を培養した。
[2]次に、上清をアスピレーターで除き、そこに1mLのPBS(-) (富士フイルム和光純薬、166-23555)および1mL 0.05% Trypsin-EDTA (Gibco、25300-062)を加え、37℃、3min処理した。
[3]次に、3.6mLのDMEM(Sigma、D5796)と0.36mLのFetal Bovine Serum (Biowest、S1760)及び0.04mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、ピペットバックにより細胞をプレートから剥がし、15mLの遠沈管に回収後、1000rpm 5min遠心し、細胞を遠沈管の底に集め、上清をアスピレーターで除いた。
[4]そこへ、4.5mLのDMEM(Sigma、D5796)と0.45mLのFetal Bovine Serum (Sigma, F7524)及び0.05mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、再懸濁した。
[5]懸濁液中の細胞数を測定し、2.5×105 cells/wellになるように、6wellのマルチウェルプレート(Corning、3516)に分注した。
[6]CO2インキュベータで37℃、2days培養し、60-80% confluent cellsまで培養した。
[7]そこへ、各ポリヌクレオチドを5μgずつ含むDNA mixture 液150 μl(Opti-MEM (Gibco、11058-021)+ Fugene HD液)を添加し、CO2 インキュベータで37℃、一日培養した。
[8]HEK293T細胞の上清をアスピレーターで除き、そこに0.3mLのPBS(-) (富士フイルム和光純薬、166-23555)および0.3mL 0.05% Trypsin-EDTA (Gibco、25300-062)を加え、37℃、3minで処理した。
[9]次に、2.7mLのDMEM(Sigma、D5796)と0.27mLのFetal Bovine Serum (Biowest、S1760)及び0.03mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、ピペットバックにより細胞をプレートから剥がし、15mLの遠沈管に回収し、1000 rpm, 5minで細胞を遠沈管の底に集め、上清をアスピレーターで除いた。
[10]そこへ、9mLのDMEM(Sigma、D5796)と0.9mLのFetal Bovine Serum (Sigma, F7524)及び0.1mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、再懸濁し、10cm dishに播種した。
[11]CO2 インキュベータで37℃, 4days培養し、細胞を十分に増殖させた。
[12]HEK293T細胞の上清をアスピレーターで除き、そこに1mLのPBS(-)(富士フイルム和光純薬、166-23555)および1mL 0.05% Trypsin-EDTA(Gibco、25300-062)を加え、37℃、3min処理した。
[13]次に、3.6mLのDMEM(Sigma、D5796)と0.36mLのFetal Bovine Serum (Biowest、S1760)及び0.04mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、ピペットバックにより細胞をプレートから剥がし、15mLの遠沈管に回収し、1000 rpm, 5minで細胞を遠沈管底に集め、上清をアスピレーターで除いた。
[14]そこへ、4.5mLのDMEM(Sigma、D5796)と0.45mLのFetal Bovine Serum (Sigma, F7524)及び0.05mLのAntibiotic-Antimycotic (Gibco、15240-062)を加え、再懸濁した。
[15]懸濁液中の細胞数を測定し、2.0×106 cells分の細胞懸濁液を1.5mLのtubeに移し、1000 rpm、 5minで細胞をtube底に集め、上清をアスピレーターで除いた。
[16]1.5mL tubeに残っている細胞ペレット(2.0×106 cells分)からMacherey-Nagel社製genome抽出キット(NucleoSpin Tissue Kit)を用いてgenome DNAを抽出し、次世代sequencerを用いてデザイン通りに編集されているゲノムの割合を求め、全リード数に対する編集リード数としてゲノム編集効率を算出した。
Claims (7)
- 二本鎖ゲノムDNAの標的部位を改変することができる一本鎖形態の編集用ポリヌクレオチドと組み合わせて用いるための、一本鎖形態の編集促進用ポリヌクレオチドであって、
標的部位は、一方のゲノムDNA鎖上に設定される一次編集部位と、他方のゲノムDNA鎖上の、一次編集部位と対応する位置にある二次編集部位とからなり、
編集用ポリヌクレオチドは、5'末端側から順に、
a)一次編集部位の3’末端側に隣接する領域とハイブリダイズ可能な部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)一次編集部位の5’末端側に隣接する領域とハイブリダイズ可能な部分
からなるか、又は
部分a)及び部分b)
からなり、
部分a)及び部分b)の長さは、それぞれ独立して10~100ヌクレオチドであり、
編集促進用ポリヌクレオチドは、前記一方のゲノムDNA鎖上の所定領域に対して相補的である核酸配列とハイブリダイズ可能であって、ここで前記所定領域は、前記一方のゲノムDNA鎖上の、編集用ポリヌクレオチドの部分a)と対応する位置にある領域を領域Aとし、編集用ポリヌクレオチドの部分b)と対応する位置にある領域を領域Bとしたときに、その5'末端部において領域Aとオーバーラップするか、又はその3'末端部において領域Bとオーバーラップし、
前記所定領域の5'末端部における領域Aとのオーバーラップの長さは、領域Aと同一又はそれより短く、前記所定領域の3'末端部における領域Bとのオーバーラップの長さは、領域Bと同一又はそれより短く、
編集促進用ポリヌクレオチドの長さは、50ヌクレオチド以上であり、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の2つの連続するヌクレオチドの間に設定された場合に、一次編集部位に部分x)と相補的な塩基配列からなる部分を、二次編集部位に部分x)を挿入することができ、
編集用ポリヌクレオチドが部分a)、部分x)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、一次編集部位を部分x)と相補的な塩基配列からなる部分に、二次編集部位を部分x)に置換することができ、
編集用ポリヌクレオチドが部分a)及び部分b)からなり、かつ一次編集部位が一方のゲノムDNA鎖上の1つのヌクレオチド又は複数の連続するヌクレオチドに設定された場合に、標的部位を欠失させることができる、前記編集促進用ポリヌクレオチド。 - 前記オーバーラップの長さが、5ヌクレオチド以上である、請求項1に記載の編集促進用ポリヌクレオチド。
- 請求項1又は2に記載の編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
編集用ポリヌクレオチド、その発現ベクター、又は前記編集用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、ゲノム編集用キットであって、
編集用ポリヌクレオチドが、5'末端側から順に、
a)一次編集部位の3’末端側に隣接する領域とハイブリダイズ可能な部分
x)二次編集部位の塩基配列と同一でない塩基配列からなる任意の部分、及び
b)一次編集部位の5’末端側に隣接する領域とハイブリダイズ可能な部分
からなるか、又は
部分a)及び部分b)
からなる、前記ゲノム編集用キット。 - 前記所定領域がその5'末端部において領域Aとオーバーラップするように設定された編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート、及び
前記所定領域がその3'末端部において領域Bとオーバーラップするように設定された編集促進用ポリヌクレオチド、その発現ベクター、又は前記編集促進用ポリヌクレオチド若しくはその発現ベクターと他の物質とのコンジュゲート
を含む、請求項3に記載のゲノム編集用キット。 - 編集促進用ポリヌクレオチドに関する前記オーバーラップの長さが、5ヌクレオチド以上である、請求項3又は4に記載のゲノム編集用キット。
- 細胞(ただし、ヒトの配偶子及び受精卵を除く)又は非ヒト生物を、請求項3~5のいずれか一項に記載のゲノム編集用キットを用いて処理する工程を含み、ただし細胞がヒトの配偶子及び受精卵を除くヒト細胞の場合には前記工程がインビトロで行われる、細胞(ただし、ヒトの配偶子及び受精卵を除く)又は非ヒト生物の二本鎖ゲノムDNAの標的部位を改変する方法。
- 細胞(ただし、ヒトの配偶子及び受精卵を除く)又は非ヒト生物を、請求項3~5のいずれか一項に記載のゲノム編集用キットを用いて処理する工程を含み、ただし細胞がヒトの配偶子及び受精卵を除くヒト細胞の場合には前記工程がインビトロで行われる、二本鎖ゲノムDNAの標的部位が改変された細胞(ただし、ヒトの配偶子及び受精卵を除く)又は非ヒト生物を製造する方法。
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