JP7212982B1 - Cell library and its production method - Google Patents

Abstract

A method for producing a library containing genetically modified cells is provided.
Kind Code: A1 Disclosed is a cell library containing a plurality of modified cells having a wide variety of types of nucleotide sequences at a specific site of one allele in cells having a plurality of alleles. A cell library can be a combination of aqueous compositions containing one type of modified cell. This library is useful in evaluating the functionality of specific regions of the genome, introducing various types of mutations (eg, SNPs) into specific regions of the genome, and the like.
[Selection drawing] Fig. 2

Description

TECHNICAL FIELD The present invention relates to a cell library and its production method.

Since the CRISPR/Cas system was reported as a novel genome editing tool, CRI
Various studies using the SPR/Cas system are being conducted (for example, Patent Document 1).
In genome editing using the CRISPR/Cas system, the target region targeted by the guide RNA is double-strand cleaved by Cas9 nuclease. double-strand broken DNA
Homologous Directed Repair: HD
R) or Non-Homologous End-Joining
Repair: NHEJ) is known to be repaired. In HDR, any sequence can be incorporated into the target region by introducing donor DNA having sequences homologous to the regions surrounding the target region into cells with the CRISPR/Cas system.

Techniques for simultaneously and efficiently modifying two or more alleles by HDR have been developed using genome modification techniques (eg, Patent Document 2). In Patent Document 2, a large-scale deletion of several hundred kb is 2
It is disclosed that the above alleles could be modified simultaneously and efficiently.

WO2014/093661 WO2021/206054

The present disclosure provides cell libraries and methods for their production. More specifically, the present disclosure provides a cell library containing a plurality of modified cells having a rich variety of nucleotide sequences at a specific site of one allele in cells having multiple alleles. A cell library can be a combination of aqueous compositions containing one type of modified cell.

(1) a library of modified cells,
the library comprises combinations of a plurality of aqueous compositions;
each aqueous composition comprising one type of modified cell;
Each modified cell has a first allele and a second allele at the locus to be modified,
each of the modified cells has a cassette at the same position of the first allele and containing DNA fragments that differ from each other between the aqueous compositions;
Library.
(2) each modified cell has a second allele that is partially or wholly disrupted or deleted;
3. A library according to claim 1 or 2.
(3) The library according to (2) above, wherein the second allele is seamlessly deleted in part or in whole.
(4) The library according to any one of (1) to (3) above, wherein the sequences other than the cassette containing the DNA fragment of each modified cell are substantially the same before and after modification.
(5) Each of the sequences of the cassette consists of one or more modified portions (A) and one or more unmodified portions (B), and the modified portions (A) are sequence insertions, deletions, and sequences, respectively. having one or more modifications selected from the group consisting of substitutions, the modifications of the one or more modified portions differing between each aqueous composition with respect to the location or content of the modifications, and the one or more unmodified portions (B) is the same as the sequence of the corresponding site before modification, and the unmodified portion (B1) on the centromere side of the cassette is seamlessly linked to the adjacent sequence (C1) on the centromere side of the cassette, The unmodified portion (Bt) on the telomere side of the cassette is seamlessly linked to the flanking sequence (C2) on the telomere side of the cassette, and the region where the flanking sequence (C1) and the unmodified portion (B1) are linked , and the region where the unmodified portion (Bt) and the adjacent sequence (C2) are linked constitutes the same sequence as the sequence of the corresponding region before modification, (1) to
The library according to any one of (4).
(6) The modified cell does not contain the target sequence of the site-specific recombination enzyme, (1) to (5) above
).
(7) The number of types of the aqueous composition contained in the library is 50 or more, the above (1
) to (6).
(8) A method for producing a library of modified cells, comprising:
(α) in a cell having a genome containing a first allele and a second allele at the genetic locus to be modified, a cassette containing a selection marker gene and a target nucleic acid sequence for each of the first allele and the second allele; providing a population of cells having
Here, the selectable marker gene possessed by the first allele and the selectable marker gene possessed by the second allele are distinguishably different, the target nucleic acid sequence is the target of the genome modification system, and the genome modification system selects the first marker gene. each selectable marker gene is a negative selectable marker gene that can be used for negative selection, and is designed so that the allele and the second allele can be distinguished from each other;
(β) introducing the following (x) and (y) into the provided group of cells;
(x) a genome modification system comprising a sequence-specific nucleic acid cleaving molecule targeting the unique base sequence contained in the first allele, or a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule;
(y) a plurality of types of second recombination donor DNA {wherein the plurality of types of second recombination donor DNA are homologous to the base sequence adjacent to the upstream side of the target site in (x) above, respectively; and a downstream homology arm having a nucleotide sequence homologous to the nucleotide sequence adjacent to the target region downstream, and a modified base between the upstream homology arm and the downstream homology arm sequence, wherein the modified nucleotide sequence is different for each second recombination donor DNA and unique to each second recombination donor DNA},
(γ) selecting cells that do not express the selectable marker gene contained in the first allele after the step (β);
including
Thereby, a library of modified cells comprising a plurality of cells can be obtained, wherein in the plurality of cells obtained, the first allele has a modified nucleotide sequence unique to each cell, and the second allele has a common sequence between cells,
Method.
(9) The method according to (8) above, wherein, before step (α),
In a cell having a genome containing a first allele and a second allele at a genetic locus to be modified, a cassette containing a selection marker gene and a target nucleic acid sequence for the substituted sequences contained in the first allele and the second allele thereby removing the substituted sequence from the first allele and the second allele;
The method further comprising:
(10) The method according to (9) above,
Each modified nucleotide sequence of the first allele has one or more mutations selected from the group consisting of addition, insertion, substitution, deletion, and deletion of bases relative to the substituted sequence of the first allele,
Method.
(11) The method according to (9) or (10) above,
The sequence to be modified is a protein coding region,
The modified base sequence of the first allele has one or more mutations selected from the group consisting of base additions, insertions, substitutions, deletions, and deletions with respect to the substituted sequence of the first allele. (12) The modified base sequence has 80% or more sequence identity with the modified sequence (10)
Or the method according to (11).
(13) The method according to any one of (8) to (12) above, wherein step (α) and step (β)
)Between,
The method further comprising removing the cassette from the second allele.
(14) The method according to (13) above, wherein the cassette is seamlessly removed.
(15) A modified cell library containing a plurality of modified cells prepared by the method according to any one of (8) to (14) above.

FIG. 1 shows an outline of an example of the scheme for preparing the library of the present invention. FIG. 2 shows an example of a scheme for preparing the first modified cell library of the present invention. FIG. 3 is a diagram showing an example of the positional relationship between the modified portion and the unmodified portion when the modified base sequence contains the modified portion and the unmodified portion in the modified cell library. By doing so, a library containing various modified cells having various mutations at various sites in the modified base sequence can be constructed. FIG. 4 shows features (disadvantages) of genome modification methods using site-specific recombination enzymes.

[definition]
The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a nucleotide polymer in which the nucleotides are linked by phosphodiester bonds. "Polynucleotide" and "nucleic acid" may be DNA or RNA, and DNA and R
It may be configured from a combination with NA. "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). may be a polymer with modified nucleotides (eg, phosphorothioate backbone), or may be a polymer of non-natural nucleotides.

Base sequences of "polynucleotides" or "nucleic acids" are described in the generally accepted one-letter code unless otherwise specified. Unless otherwise specified, nucleotide sequences are 3
' is written towards the side. 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.

The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acids joined by amide bonds. "polypeptide", "
A "peptide" or "protein" may be a polymer of natural amino acids, a polymer of natural amino acids and non-natural amino acids (such as chemical analogues, modified derivatives of natural amino acids), or non-natural amino acids. It may be a polymer. Unless otherwise specified, amino acid sequences are written from the N-terminal side to the C-terminal side.

The term "allele" refers to a set of base sequences present at the same locus on the chromosomal genome. In some embodiments, diploid cells have 2 alleles at the same locus and triploid cells have 3 alleles at the same locus. Also, in some embodiments, additional alleles may be formed by aberrant copies of the chromosome or aberrant additional copies of the locus.

The terms "genome modification" or "genome editing" are used interchangeably and refer to the introduction of mutations at desired locations (target regions) on the genome. Genome modification is targeted region DNA
can include the use of sequence-specific nucleic acid cleaving molecules designed to cleave the In preferred embodiments, genomic modification may involve the use of nucleases engineered to cleave the DNA of the target region. In a preferred embodiment, genomic modification involves nucleases (e.g., TALENs) engineered to cleave target sequences with specific base sequences in the target region.
or ZFN). In a preferred embodiment, the genome modification involves the addition of 1 to the genome, such as a meganuclease, to cleave a target sequence having a specific base sequence in the target region.
Restriction enzymes with only one cleavage site (e.g., restriction enzymes with 16-base sequence specificity (theoretically, one in 4 ¹⁶ bases exists), restriction enzymes with 17-base sequence specificity (theoretically using sequence-specific endonucleases such as 1 in 4 ¹⁷ bases above) and restriction enzymes with 18-base sequence specificity (theoretically 1 in 4 ¹⁸ bases) sometimes it is possible. Typically, the use of site-specific nucleases induces double-strand breaks (DSBs) in the DNA of the target region, followed by homologous directed repair (HDR) and non-homologous end recombination (Non- Homologous End-Joining Repair: NH
The genome is repaired by endogenous processes of the cell, such as EJ). NHEJ is a repair method that joins the ends of double-strand breaks without using donor DNA, and insertions and/or deletions (indels) are frequently induced during repair. HDR is a repair mechanism using donor DNA, and it is also possible to introduce desired mutations into target regions. As a genome modification technology, for example, the CRISPR/Cas system is preferably exemplified. Examples of meganucleases include I-SceI, I-SceII, I-SceIII, I-SceIV
, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAII
P, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-Crepsb
IIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F
- SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-Am
aI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII,
I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-
DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-Lla
I, I-MsoI, I-NaaI, I-NanI, I-NclIP, I-NgrIP, I
- NitI, I - NjaI, I - Nsp236IP, I - PakI, I - PboIP, I
-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I
-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI
, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-Spom
IP, I-SpomIIP, I-SquiIP, I-Ssp68031, I-SthPhi
JP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I
-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPA
IP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-Mtul,
PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI
-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP
, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-Tli
A meganuclease and its cleavage site (or recognition site) selected from the group consisting of I, and PI-TliII, and functional derivative restriction enzymes thereof, preferably a restriction enzyme having a sequence specificity of 18 bases or more Certain meganucleases and their cleavage sites (or recognition sites) can be used, particularly meganucleases and their cleavage sites that do not cut one or more of the genome of the cell.

The term "target region" refers to the genomic region targeted for genomic modification. A "deletion" is
Including deletions of one or more bases relative to the reference genome, and deletions of one or more genes. Deletion is 100 bp or more deletion, 200 bp or more deletion, 300 bp or more deletion, 400 bp
≥ 500 bp deletion ≥ 600 bp deletion ≥ 700 bp deletion 8
00 bp or more deletion, 900 bp or more deletion, 1 kbp or more deletion, 10 kbp or more deletion, 50 kbp or more deletion, 100 kbp or more deletion, 200 kbp or more deletion, 300
kbp or greater deletion, 400 kbp or greater deletion, 500 kbp or greater deletion, or 1 Mbp
There can be more or less deletions. Deletions can be deletions of 1 Mbp or less.
Deletions can be deletions of 700 kbp or less. Deletions can be deletions of 600 kbp or less. Deletions can be deletions of 500 kbp or less. Deletion ≥ 10 kbp and 600 kbp
The deletion can be: Deletions can be deletions of 100 kbp or more and 600 kbp or less.
Deletions can be deletions of 100 kbp or more and 500 kbp or less.

The term "donor DNA" refers to DNA used to repair double-strand breaks in DNA that is capable of homologous recombination with the DNA surrounding the target region. The donor DNA contains a base sequence upstream of the target region and a base sequence downstream of the target region (eg, a base sequence flanking the target region) as homology arms. In the present specification, the nucleotide sequence upstream of the target region (e.g.,
A homology arm consisting of a nucleotide sequence adjacent to the upstream side is described as an "upstream homology arm", and a homology arm consisting of a nucleotide sequence downstream of the target sequence (for example, a nucleotide sequence adjacent to the downstream side) is described as a "downstream homology arm". sometimes. The donor DNA can contain the desired nucleotide sequence between the upstream homology arm and the downstream homology arm. The length of each homology arm is preferably 300 bp or more, and usually about 500 to 3000 bp. The lengths of the upstream homology arm and the downstream homology arm may be the same or different. When the target region successfully induces homologous recombination with the donor DNA after sequence-dependent cleavage, the sequence between the upstream and downstream sequences of the target region is the upstream sequence of the donor DNA. and the sequence flanked by the downstream nucleotide sequence.

“Upstream” of the target region means the DNA region located on the 5′ side of the reference nucleotide strand in the double-stranded DNA of the target region. "Downstream" of the target region means DNA located 3' to the reference nucleotide strand. It is arbitrary which strand of the double strand is used as the reference nucleotide strand. Conveniently, however, when the target region comprises a protein-coding sequence, the reference nucleotide strand is usually the sense strand. Generally, the promoter is positioned upstream from the protein-coding sequence. A terminator is located downstream of the protein-coding sequence.

The term "sequence-specific nucleic acid cleaving molecule" refers to a molecule capable of recognizing a specific nucleic acid sequence and cleaving nucleic acid at said specific nucleic acid sequence. A sequence-specific nucleic acid cleaving molecule is a molecule having a sequence-specific nucleic acid cleaving activity (sequence-specific nucleic acid cleaving activity).

The term "target sequence" refers to a DNA sequence in the genome that is targeted for cleavage by a sequence-specific nucleolytic molecule. When the sequence-specific nucleic acid cleaving molecule is a Cas protein, the target sequence refers to the DNA sequence in the genome to be cleaved by the Cas protein. When using the Cas9 protein as the Cas protein, the target sequence should be the sequence flanking the 5' side of the protospacer adjacent motif (PAM). The target sequence is usually 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 bases) immediately preceding the 5′ side of PAM.
A sequence of ~22 bases, more preferably 20 bases) is selected. For the design of target sequences, C
A known design tool such as RISPR DESIGN (crispr.mit.edu/) can be used.

The term "Cas protein" refers to the CRISPR-associated
ated) refers to proteins. In a preferred embodiment, the Cas protein is a guide RN
It forms a complex with A and exhibits endonuclease activity or nickase activity. Examples of Cas proteins include, but are not limited to, Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein.
the wild-type Cas protein and its homologs (paralogs and orthologs), as long as the Cas protein exhibits endonuclease activity or nickase activity in cooperation with the guide RNA;
and variants thereof.
In preferred embodiments, the Cas protein is involved in a class 2 CRISPR/Cas system, more preferably a type II CRISPR/Cas system. A preferred example of the Cas protein is the Cas9 protein. Cas
A preferred example of the protein is Cas3 protein.

The term "Cas9 protein" refers to the C
refers to the as protein. The Cas9 protein forms a complex with the guide RNA and exhibits the activity of cleaving the DNA of the target region in cooperation with the guide RNA. Cas9 proteins include wild-type Cas9 proteins and their homologues (paralogs and orthologs), and variants thereof, as long as they have the above activity. The wild-type Cas9 protein has the RuvC and HNH domains as nuclease domains, whereas the Ca
The s9 protein may have either one of the RuvC domain and the HNH domain inactivated. Cas9 with either one of the RuvC domain and HNH domain inactivated introduces a single-strand break (nick) into the double-stranded DNA. for that reason,
When one of the RuvC domain and HNH domain is inactivated Cas9 is used for cleaving double-stranded DNA, the target sequence of Cas9 is set for each of the sense strand and the antisense strand, and the sense strand and Modified systems can be constructed such that the antisense differential nicks occur in sufficiently close positions to induce a double-strand break.
The biological species from which the Cas9 protein is derived is not particularly limited, but Streptococcus (S
treptococcus) genus, Staphylococcus
Bacteria belonging to the genus Neisseria or Treponema are preferably exemplified. More specifically, S. pyogenes, S.; t
hermophilus, S. aureus, N. meningitidis, or T.
Cas9 protein derived from denticola and the like is preferably exemplified. In preferred embodiments, the Cas9 protein is derived from S. cerevisiae. pyogenes Cas9 protein.

Amino acid sequences of various Cas proteins and information on their coding sequences are available at GenBank
, UniProt, and Addgene. For example, S. The amino acid sequence of the Cas9 protein of pyogenes is plasmid number 422
30 registered with Addgene can be used. S. pyogen
An example of the amino acid sequence of the Cas9 protein of es is shown in SEQ ID NO:1.

The terms "guide RNA" and "gRNA" are used interchangeably and refer to an RNA capable of forming a complex with a Cas protein and directing the Cas protein to a target region.
point to In a preferred embodiment, the guide RNA is CRISPR RNA (crRNA)
and transactivating CRISPR RNA (tracrRNA). crRNA is involved in binding to target regions on the genome, and tracrRNA is involved in binding to Cas proteins. In preferred embodiments, the crRNA comprises a spacer sequence and a repeat sequence, and the spacer sequence binds to the complementary strand of the target sequence in the target region. In preferred embodiments, the tracrRNA comprises an anti-repeat sequence and a 3' tail sequence. The anti-repeat sequence has a sequence complementary to the repeat sequence of crRNA, forms a base pair with the repeat sequence, and the 3' tail sequence usually forms three stem loops.
The guide RNA may be a single guide RNA (sgRNA) in which the 5' end of tracrRNA is ligated to the 3' end of crRNA, wherein crRNA and tracrRNA are separate RNA molecules, and the repeat and anti-repeat sequences are base-paired. may be formed. In preferred embodiments, the guide RNA is an sgRNA.

The repeat sequence of crRNA and the sequence of tracrRNA can be appropriately selected according to the type of Cas protein, and those derived from the same bacterial species as the Cas protein can be used.
For example, S. When using a Cas9 protein derived from pyogenes, the length of the sgRNA can be about 50 to 220 nucleotides (nt), preferably about 60 to 180 nt, more preferably about 80 to 120 nt. The length of the crRNA can be about 25-70 bases including the spacer sequence, preferably about 25-50 nt. t
The length of the racrRNA can be about 10-130 nt, preferably about 30-80 nt.
The repeat sequence of the crRNA may be the same as in the bacterial species from which the Cas protein is derived, or may be partially truncated at the 3' end. tracrRNA is
It may have the same sequence as the mature tracrRNA in the bacterial species from which the Cas protein is derived, or may be a truncated form obtained by truncating the 5'-end and/or 3'-end of the mature tracrRNA. For example, the tracrRNA can be truncated by removing as many as 1-40 nucleotide residues from the 3' end of the mature tracrRNA. Also, trac
The rRNA can be truncated by removing as many as 1-80 nucleotide residues from the 5' end of the mature tracrRNA. In addition, tracrRNA is, for example, 1 to
It may be a truncated form with as many as 20 nucleotide residues removed and as many as 1-40 nucleotide residues removed from the 3' end.
Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNAs based on known techniques (for example, Jinek et al. (2012) Science, 337, 816
-21; Mali et al. (2013) Science, 339: 61
21, 823-6; Cong et al. (2013) Science, 3
39: 6121, 819-23; Hwang et al. (2013) Na
t. Biotechnol. 31: 3, 227-9; Jinek et al.
. (2013) eLife, 2, e00471).

The terms "protospacer adjacent motif" and "PAM" are used interchangeably and refer to sequences recognized by Cas proteins upon DNA cleavage by the Cas proteins. The sequence and position of PAM differ depending on the type of Cas protein. For example, Ca
For s9 proteins, the PAM should immediately flank the 3' side of the target sequence. Cas9
The PAM sequence corresponding to the protein varies depending on the bacterial species from which the Cas9 protein is derived. For example, S. The PAM corresponding to the Cas9 protein of pyogenes is "
NGG", and S. PAM corresponding to thermophilus Cas9 protein
is "NNAGAA"; The PAM corresponding to the Cas9 protein of N. aureus is "NNGRRT" or "NNGRR(N)"; C for meningitidis
The PAM corresponding to the as9 protein is "NNNNGATT"; dentico
"NAAAC" corresponding to the Cas9 protein of Cas9 ("R" is A or G; "N
' is A, T, G or C).

The terms "spacer sequence" and "guide sequence" are used interchangeably and refer to a sequence contained in a guide RNA that is capable of binding a complementary strand of a target sequence. The spacer sequence is usually the same sequence as the target sequence (provided that T in the target sequence becomes U in the spacer sequence). In an embodiment of the invention, the spacer sequence is 1
It can contain mismatches of a base or multiple bases. If it contains multiple base mismatches,
There may be mismatches at adjacent positions or mismatches at distant positions. In preferred embodiments, the spacer sequence may contain 1-5 base mismatches to the target sequence. In particularly preferred embodiments, the spacer sequence may contain a single base mismatch to the target sequence.
In the guide RNA, the spacer sequence is placed 5' to the crRNA.

The term "operably linked" as used in reference to polynucleotides means that a first base sequence is positioned sufficiently close to a second base sequence such that the first base sequence is either the second base sequence or the second base sequence. means that it can affect areas under the control of For example, that a polynucleotide is operably linked to a promoter means that the polynucleotide is linked so that it is expressed under the control of the promoter.

The term "expressible state" refers to a state in which a polynucleotide can be transcribed in a cell into which the polynucleotide has been introduced.
The term "expression vector" refers to a vector that contains a polynucleotide of interest and is equipped with a system that renders the polynucleotide of interest expressible in a cell into which the vector is introduced. For example, "expression vector for Cas protein" means a vector capable of expressing Cas protein in cells introduced with the vector. Also, for example,
“Guide RNA expression vector” means a vector capable of expressing a guide RNA in a cell into which the vector has been introduced.

As used herein, sequence identity (or homology) between base sequences or between amino acid sequences refers to insertions and deletions of two base sequences or amino acid sequences such that the corresponding bases or amino acids match the most. The ratio of matching bases or amino acids to the entire base sequence or the entire amino acid sequence excluding the gaps in the resulting alignment is calculated. Sequence identity between nucleotide sequences or amino acid sequences can be determined using various homology search software known in the art. The sequence identity value (Identity value) of the base sequence is not particularly limited, but for example, BL installed in the known homology search software UCSC Genome Browser
Can be obtained by AT search.

For convenience herein, locations in the hg38 genome sequence are used as reference genomes when referring to locations in the human genome. hg38 is a reference genome released in December 2013 by the University of California, Santa Cruz (UCSC). A reference genome is a reference genome created by combining various genomes, and it does not mean that a human having this genome exists. However, the fragmentary sequence information decoded from the genomic DNA of the human individual is linked to the reference genome, and the fragmentary sequence information decoded is linked to construct a series of sequences on a computer. The sequence of an individual's genomic DNA can be deduced. In this way, it is common practice to decode the genomic DNA of an individual such as a human individual by associating the sequence of the genomic DNA of the human individual with the reference genome. Further, the position or region corresponding to the specific position or specific region of the hg38 genome sequence means the position or region linked to the specific position or specific region in separate genomes with different specific sequences. Specifically, a position or region having a sequence characteristic of that position or region based on sequence identity is a position or region corresponding to a specific position or region of the hg38 genome sequence. Corresponding positions can be determined by alignment of the partial sequences of the two genomic DNAs. Even if there are differences in the specific sequences, the two genomes D can be aligned by having an orthologous relationship or having sequence identity.
NA correspondences can be determined. In regions rich in paralogs generated by gene duplication, determining sequence correspondences based on simple individual sequences may not be sufficient to determine true correspondences between two genomes. This increases the difficulty of sequence decoding in regions where similar sequences accumulate. When determining the corresponding sequences, the correspondence between the two genomes can be clarified by seeking high sequence identity. Synteny can also be considered when the specified region is a large region containing multiple genes. Synteny means preservation of the physical positional relationship of orthologs on the genome. Can have synteny between individuals and between organisms. Therefore, the specific region can be determined in consideration of synteny.

[Method for preparing a library]
A method of making a library can include providing a cell. The cells to be prepared are cells before the modification of the present disclosure, and can be used as a reference for comparison with cells after modification, so they are sometimes referred to as “reference cells”. Cells may preferably be cloned, established or immortalized cells. In some preferred embodiments, cells may comprise a single type. The library of the present disclosure provides a library of cells in which only specific regions of specific loci have been replaced with sequences of interest. From the viewpoint that the technical significance of substituting the target sequence can be clarified by unifying the sequences other than the target sequence, the cells are preferably composed of a single type of cell. A single type of cell is a cloned cell.

A schematic overview of the first fabrication mode is shown in FIG.
A method of making a library may comprise using the genome modification method described below on a cell to obtain a first intermediate cell. The first intermediate cell is a cell having a genome containing a first allele and a second allele at the genetic locus to be modified, wherein each of the first allele and the second allele contains a selectable marker gene and a target nucleic acid. A cell with a cassette containing the sequence. In the first intermediate cell, the selectable marker gene possessed by the first allele and the selectable marker gene possessed by the second allele are distinguishably different, the target nucleic acid sequence is a target of a genome modification system, and genome modification is performed. Each selection marker gene is a negative selection marker gene that can be used for negative selection, and is designed so that the system can distinguishably cleave the first allele and the second allele. A marker gene for negative selection may be one that can also be used for positive selection (eg, a visualization marker gene, etc.).
A method of making a library can include obtaining a library of modified cells (sometimes referred to as a "first modified cell library" or simply a "first library") from a first intermediate cell. . The modified cells contained in the first library are sometimes referred to as "first modified cells".
The first intermediate cell can be appropriately obtained by those skilled in the art. Although not particularly limited, it can be easily produced by using the genome modification method described below. Obtaining a modified cell from an intermediate cell can be achieved by cleaving within or near the first cassette in the presence of the donor DNA for introducing the modified nucleotide sequence.

A schematic overview of the second fabrication mode is shown in FIG.
A method of making a library may comprise using the genome modification method described below on a cell to obtain a first intermediate cell. A method of making a library can include obtaining a second intermediate cell from a first intermediate cell. A method of making a library can include obtaining a library from a second intermediate cell. Here, the first intermediate cell is as described above. The second intermediate cell is a cell having a genome containing a first allele and a second allele at the genetic locus to be modified, and has a cassette containing a selection marker gene and a target nucleic acid sequence at the first allele. However, the second allele is a cell that does not contain the cassette. A second intermediate cell can be made by removing the cassette from the second allele of the first intermediate cell. Removal of the cassette can be appropriately carried out by those skilled in the art by a genome modification method. Specifically, in the presence of a donor DNA having an upstream homology arm capable of homologous recombination upstream of the cassette and a downstream homology arm capable of homologous recombination downstream of the cassette at the time of cleavage, included in the second allele A second intermediate cell can be obtained from the first intermediate cell by applying a genome modification system capable of cleaving the target sequence to the first intermediate cell. A method of making a library can include obtaining a library of modified cells (sometimes referred to as a "second modified cell library" or simply a "second library") from a second intermediate cell. . The modified cells contained in the second library are sometimes referred to as "second modified cells". Obtaining a second intermediate cell from a first intermediate cell can be accomplished by cleaving sequences in or near the second cassette in the presence of cassette removal donor DNA. Obtaining the modified cell from the second intermediate cell can be achieved by cleaving the first cassette in the presence of the donor DNA for introducing the modified nucleotide sequence.

Variations of Second Modification of Construction In a second modification of construction , a second intermediate cell was generated by removing the cassette from the second allele of the first intermediate cell. In a modification of the second production mode, the cassette in the second allele of the first intermediate cell is removed, and the second allele is returned to the sequence before modification, and the first allele is selected. A third intermediate cell is obtained having a cassette containing a marker gene and a target nucleic acid sequence, the second allele having the sequence prior to modification. In a variation of the second production mode, then donor DNA for library production against the first allele of the third intermediate cell
is applied to obtain a library of modified cells. In this embodiment, the modified cells contained in the library of modified cells contain the modified base sequence in the first allele and the second allele has the sequence before modification. The operation of returning the second allele of the first intermediate cell to the sequence before modification includes an upstream homology arm capable of homologous recombination upstream of the second cassette and a downstream homology arm capable of homologous recombination downstream of the cassette can be achieved by cleaving within or near the second cassette in the presence of donor DNA consisting of:

The present disclosure provides a first intermediate cell, a second intermediate cell, a composition comprising these intermediate cells, a first modified cell, a first library, a second modified cell, and a second A library is provided.

Generally, in genome editing, for a genome having a target region, an upstream homology arm (for example, having a complementary sequence) capable of homologous recombination upstream of the target region and a homologous recombination downstream of the target region In the presence of donor DNA that has a possible downstream homology arm (e.g., has a complementary sequence), the target region is isolated from the upstream homology arm in the donor DNA, typically by causing cleavage within the target region. It replaces the sequence flanked by the downstream homology arms. If the sequence flanked by the upstream homology arm and the downstream homology arm is absent, the target region is deleted (or seamlessly deleted). In genome editing, multiple cuts may be generated within the target region. Typically, it is beneficial to have the cleavage occur within the target region adjacent to the upstream homology arm and within the target region adjacent to the downstream homology arm, respectively.

In one preferred embodiment, in the first intermediate cell, the first allele and the second allele are each replaced by a cassette of the target region, and the first allele and the second allele are It may have deletions. Also, in certain preferred embodiments, the first allele and the second allele have an insertion of the cassette into the target region, and the first allele and the second allele have no deletion of the target region. good too. In certain preferred embodiments, the insertion of the target region cassette is into a non-functional target region, and thus is not accompanied by loss of function or loss associated with disruption of the target region.

Cells used in the genome modification method of the present embodiment are not particularly limited as long as they have a haploid, diploid or higher chromosomal genome. The cells may be diploid, triploid, tetraploid or higher. Cells include, but are not limited to, eukaryotic cells. The cells may be plant cells, animal cells, or fungal cells. Animal cells include, but are not limited to, humans, non-human mammals (
non-human primates such as monkeys, non-human mammals such as dogs, cats, cows, horses, sheep, goats, llamas, rodents), birds, reptiles, amphibians, fish, and other vertebrate cells may be

Examples of the cells include pluripotent cells (e.g., pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)), hematopoietic stem cells, hematopoietic progenitor cells, bone marrow cells, and spleen. cells, common myeloid progenitor cells, immune cells (e.g., T cells, B cells, NK cells, N
KT cells, macrophages, monocytes, neutrophils, eosinophils, basophils), erythrocytes, megakaryocytes, cardiac cells, cardiomyocytes, cardiac fibroblasts, pancreatic β cells, corneal cells (e.g., corneal epithelial cells, and corneal endothelial cells), epidermal cells, dermal cells, adipocytes, chondrocytes, osteocytes, osteoclasts, osteoblasts, mesenchymal stem cells (e.g., adipose-derived, bone marrow-derived, placental- and umbilical cord-derived), dental pulp cells, tendon cells, ligament cells, nerve cells (eg, cone cells, astrocytes, and granule cells), glial cells, Purkinje cells, retinal ganglion cells, retinal cells, optic nerve cells, and neural stem cells. In some preferred embodiments, the cells can be primary cells. In some preferred embodiments, the cells can be immortalized cells, or cell lines. In one preferred embodiment, the cells are human cells.

In some aspects, the cells can be isolated cells, cloned cells, or cell lines. The cells may be immortalized cells. In one preferred aspect, the cell is a cloned cell. In one preferred aspect, the cell is a cell line. In one preferred embodiment, the cells are immortalized cells. In one aspect, the cell is a primary somatic cell. Those skilled in the art will understand that cells are appropriately selected according to their intended use.

In some embodiments, cells (or libraries) can be frozen in a cell cryoprotectant. In some embodiments, the cell cryoprotectant containing said cells may be provided in an unfrozen or preferably frozen state. A cell cryoprotectant (also referred to as a "freeze stock") containing the cells in a frozen state can be used as a research cell bank (RCB), master cell bank (MCB), or working cell bank (WCB). Therefore, in the present invention, a research cell bank (RCB), a master cell bank (MCB) containing the freeze stock
, or a working cell bank (WCB) is provided.

[Genome modification method]
Methods described below (e.g. UKiS; see WO2021/206054)
can be preferably used for producing the above cells. This method is preferably used when producing a first intermediate cell from the cell, particularly in step S1 shown in FIG.

In one embodiment, in the present invention, the following (a) and (b) are used to prepare the first intermediate cell
can be used.
(a) introducing the following (i) and (ii) into a cell containing two or more alleles to introduce a selectable marker gene into each of the two or more alleles;
(i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of said chromosomal genome, or a polynucleotide encoding said sequence-specific nucleic acid cleaving molecule; genome modification system, including
(ii) two or more selection marker donor DNAs, each of which has an upstream homology arm having a base sequence capable of homologous recombination with the base sequence upstream of the target region; and bases downstream of the target region. a sequence and a downstream homology arm having a base sequence capable of homologous recombination, and comprising a base sequence of a selectable marker gene between the upstream homology arm and the downstream homology arm, wherein the two or more types of selectable marker donors Each of the DNAs has a selectable marker gene that is distinguishably different from each other, the selectable marker gene is unique for each type of selectable marker donor DNA, and the number of types of the selectable marker donor DNA is determined by genome modification. Two or more selectable marker donors D, which are the same or more than the number of target alleles
NA,
(b) after the step (a), by homologous recombination of different types of donor DNAs for selection markers with respect to the two or more alleles, unique selection markers that are distinguishably different from each other for the two or more alleles; selecting cells into which the gene has been introduced and expressing all of the introduced distinctly different selectable marker genes (a step for positive selection);
can be a method comprising:

(Step (a))
In step (a), the above (i) and (ii) are introduced into cells containing chromosomes.

<(i) genome modification system>
By "genomic modification system" is meant a molecular mechanism capable of modifying a desired target region. The genome modification system comprises a sequence-specific nucleic acid cleaving molecule targeted to a target region of the chromosomal genome, or a polynucleotide encoding said sequence-specific nucleic acid cleaving molecule. More specifically, the genome modification system can cut at least one, preferably two, in or near the target region.

A target region for genome modification can be any region on the genome having one or more alleles. The size of the target region is not particularly limited. The genome modification method of this embodiment can modify a region of a larger size than conventionally. The target region may be, for example,
It may be 10 kbp or more. The target region may be, for example, 100 bp or more, 200 bp or more, 400 bp or more, 800 bp or more, 1 kbp or more, 2 kbp or more, 3 kbp or more, 4 kbp or more.
bp or more, 5 kbp or more, 8 kbp or more, 10 kbp or more, 20 kbp or more, 40 kbp
80kbp or more, 100kbp or more, 200kbp or more, 300kbp, 400kbp
bp or more, 500 kbp or more, 600 kbp or more, 700 kbp or more, 800 kbp or more, 900 kbp or more, or 1 Mbp or more, or any of the above numbers or less. In one aspect, the target region is deleted in the modified cell.

The sequence-specific nucleic acid cleaving molecule is not particularly limited as long as it is a molecule having sequence-specific nucleic acid cleaving activity, and may be a synthetic organic compound or a biopolymer such as a protein. Proteins having sequence-specific site-cleaving activity include, for example, sequence-specific endonucleases.

A sequence-specific endonuclease is an enzyme capable of cleaving nucleic acids at a given sequence. A sequence-specific endonuclease can cleave double-stranded DNA at a given sequence. Examples of sequence-specific endonucleases include, but are not limited to, zinc finger nuclease (ZFN), TA
LEN (Transcription activator-like effect)
r nuclease), Cas protein and the like, but are not limited to these.

ZFNs are artificial nucleases containing a nucleic acid cleaving domain conjugated to a binding domain containing a zinc finger array. As a cleavage domain, the type II restriction enzyme Fok
The cleavage domain of I. A zinc finger nuclease capable of cleaving a target sequence can be designed by a known method.

TALENs are artificial nucleases that contain transcriptional activator-like (TAL) effector DNA-binding domains in addition to a DNA-cleaving domain (eg, a FokI domain). A TALE construct capable of cleaving a target sequence can be designed by a known method (e.g.,
Zhang, Feng et. al. (2011) Nature Biotec
hnology 29 (2)).

When using a Cas protein as the sequence-specific nucleic acid cleaving molecule, the genome modification system includes the CRISPR/Cas system. That is, the genome modification system preferably contains a Cas protein and a guide RNA having a nucleotide sequence homologous to the nucleotide sequence in the target region. The guide RNA may contain, as a spacer sequence, a sequence homologous to the sequence in the target region (target sequence). The guide RNA only needs to be able to bind to DNA within the target region, and does not need to have a completely identical sequence to the target sequence. This bond may be formed under physiological conditions within the cell nucleus. The guide RNA can contain, eg, 0-3 base mismatches, eg, to the target sequence. The number of mismatches is preferably 0 to 2 bases, more preferably 0 to 1 bases, and more preferably no mismatches. Guide RNA can be designed based on known methods. The genome modification system is CR
It is preferably an ISPR/Cas system, preferably comprising a Cas protein and a guide RNA. Preferably, the Cas protein is the Cas9 protein.

A sequence-specific endonuclease may be introduced into the cell as a protein or as a polynucleotide encoding the sequence-specific endonuclease. For example, a sequence-specific endonuclease mRNA may be introduced, or a sequence-specific endonuclease expression vector may be introduced. In expression vectors, a sequence-specific endonuclease coding sequence (sequence-specific endonuclease gene) is operably linked to a promoter. The promoter is not particularly limited, and for example, various pol II promoters can be used. Examples of pol II promoters include, but are not limited to, CMV promoter, EF1 promoter (EF1α promoter)
, SV40 promoter, MSCV promoter, hTERT promoter, β actin promoter, CAG promoter, CBh promoter and the like.

The promoter may be an inducible promoter. An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to it only in the presence of an inducer driving the promoter. Inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters. Inducible promoters also include promoters in which the inducer driving the promoter is a drug. Such drug-inducible promoters include, for example, Cumate operator sequences, λ operator sequences (eg, 12×λOp), tetracycline system-inducible promoters, and the like. Tetracycline-based inducible promoters include, for example, promoters that drive gene expression in the presence of tetracycline or its derivatives (eg, doxycycline), or reverse tetracycline-regulated transactivator (rtTA). Examples of tetracycline-inducible promoters include the TRE3G promoter.

Any known expression vector can be used without particular limitation. Examples of expression vectors include plasmid vectors and virus vectors. When the sequence-specific endonuclease is a Cas protein, the expression vector may contain, in addition to the Cas protein coding sequence (Cas protein gene), a guide RNA coding sequence (guide RNA gene). In this case, the guide RNA coding sequence (guide RNA gene) is preferably functionalized to a pol III-based promoter. pol I
II system promoters include, for example, mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, human valine-tRNA promoters and the like.

<(ii) Donor DNA for selection marker>
Selectable marker donor DNA is donor DNA for knocking in a selectable marker into a target region. The donor DNA for selection marker has an upstream homology arm having a nucleotide sequence homologous to the nucleotide sequence adjacent to the upstream side of the target region, and a downstream homology arm having a nucleotide sequence homologous to the nucleotide sequence adjacent to the downstream side of the target region. contains the base sequences of one or more selectable marker genes.

The donor DNA for the selection marker is not particularly limited, but is, for example, 1 kb or more, 2 kb or more, 3 kb or more, 4 kb or more, 5 kb or more, 6 kb or more, 7 kb or more, 8 kb or more, 9 kb or more.
b or longer, 9.5 kb or longer, or 10 kb or longer. The donor DNA for the selection marker is not particularly limited, but is, for example, 50 kb or less, 45 kb or less, 40 kb or less,
35 kb or less, 30 kb or less, 25 kb or less, 20 kb or less, 15 kb or less, 14 kb or less, 13 kb or less, 12 kb or less, 11 kb or less, 10 kb or less, 9 kb or less, 8 kb or less, 7 kb or less, 6 kb or less, 5 kb or less, or 4 kb or less in length can have

By "selectable marker" is meant a protein that allows cells to be selected based on the presence or absence of its expression. A selectable marker gene is a gene that encodes a selectable marker. In a cell population in which selectable marker-expressing cells and non-expressing cells coexist, when selectable marker-expressing cells are selected, the selectable marker is referred to as a "positive selectable marker" or "selective marker for positive selection". In a cell population in which selectable marker-expressing cells and non-expressing cells coexist, when selectable marker-non-expressing cells are selected, the selectable marker is referred to as a "negative selectable marker" or "selective marker for negative selection". The selectable markers differ from each other means that they are distinguishable from each other (e.g., distinguishably different), and may have physiological or other properties such as drug resistance properties that confer on cells into which the selectable markers have been introduced. It means at least distinguishable from each other in physicochemical properties. i.e.
The selection markers being different from each other means that multiple selection markers that are different can be detected in a distinguishable manner from other selection markers, or that a drug can be selected in a manner distinguishable from other selection markers. In addition, the expression that the selectable marker gene is unique to each type of selectable marker donor DNA means that the selectable marker gene possessed by one type of selectable marker donor DNA is not included in the types of selectable marker donor DNAs other than the above. or multiple types of donor D
When it is contained in NA, it means that it is constructed so as not to be expressed from two or more donor DNAs at the same time. At this time, the two or more donor DNAs may be identical except for the selectable marker, or may differ in sequence and/or composition other than the selectable marker.

The positive selectable marker is not particularly limited as long as it can select cells expressing it. Positive selectable marker genes include, for example, drug resistance genes, fluorescent protein genes, luciferase genes, chromogenic enzyme genes and the like.

The negative selectable marker is not particularly limited as long as it can select cells that do not express it. Negative selectable marker genes include, for example, suicide genes (thymidine kinase, etc.), fluorescent protein genes, luciferase genes, chromogenic enzyme genes, and the like. When the negative selectable marker gene is a gene that negatively affects cell survival (eg, a suicide gene), the negative selectable marker gene can be operably linked to an inducible promoter. By being operably linked to an inducible promoter, the negative selectable marker gene can be expressed only when it is desired to eliminate cells bearing the negative selectable marker gene. Optically detectable marker genes (visible marker genes; visible mark
If the negative effect on cell survival is small, such as when the gene is an er gene), it may be expressed constitutively.

Examples of drug resistance genes include puromycin resistance gene, blasticidin resistance gene, geneticin resistance gene, neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene, zeocin resistance gene, hygromycin resistance gene, chloramphenicol resistance gene, and the like. include, but are not limited to:
Examples of fluorescent protein genes include, but are not limited to, green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, and the like.
Luciferase genes include, but are not limited to, luciferase genes.
Examples of the chromogenic enzyme gene include, but are not limited to, β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene and the like.
Suicide genes include, for example, herpes simplex virus thymidine kinase (HSV-T
K), inducible caspase 9, etc., but are not limited thereto.

The selectable marker gene possessed by the selectable marker donor DNA is preferably a positive selectable marker gene. That is, cells expressing the selectable marker can be selected as cells in which the selectable marker gene has been knocked in.

The upstream homology arm has a base sequence capable of homologous recombination with the base sequence upstream of the target region in the genome to be modified, for example, has a base sequence homologous to the base sequence adjacent to the upstream side of the target sequence. . The downstream homology arm has a base sequence capable of homologous recombination with the base sequence downstream of the target region in the genome to be modified, for example, has a base sequence homologous to the base sequence adjacent to the downstream side of the target sequence. . The length and sequence of the upstream homology arm and the downstream homology arm are not particularly limited as long as they are capable of homologous recombination with the region surrounding the target region. The upstream homology arm and downstream homology arm do not necessarily have to completely match the upstream or downstream sequence of the target region as long as homologous recombination is possible. For example, the upstream homology arm can be a sequence having 90% or more sequence identity (homology) with the nucleotide sequence adjacent to the upstream side of the target region, such as 92% or more, 93% or more, 94% or more, Preferably, they have a sequence identity of 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. For example, the downstream homology arm includes the base sequence flanking the downstream side of the target region and 9
It can be a sequence having a sequence identity (homology) of 0% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% % or greater sequence identity is preferred. In addition, when at least one of the upstream homology arm and the downstream homology arm is closer to the cleavage site in the target region, the efficiency of allele modification can be increased. Here, "close" means that the distance between the two sequences is 100 bp or less, 50 bp
Hereafter can mean 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.

In the selectable marker donor DNA, the selectable marker gene is located between the upstream homology arm and the downstream homology arm. As a result, when donor DNA for a selection marker is introduced into cells together with the genome modification system of (i) above, a selection marker gene is introduced into the target region by HDR (when the gene is disrupted by this, gene knockout When a desired gene is introduced by this, it is possible to knock in another gene while knocking out a gene, called gene knock-in).

A selectable marker gene is preferably operably linked to a promoter so that it is expressed under the control of a suitable promoter. A promoter can be appropriately selected according to the type of cell into which the donor DNA is introduced. Examples of promoters include SRα
promoter, SV40 early promoter, retroviral LTR, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-T
K (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoter, heat shock promoter and the like. The selectable marker donor DNA may have any regulatory sequences such as enhancers, poly(A) addition signals, terminators, and the like.

The selectable marker donor DNA may have an insulator sequence. An "insulator" is a DN sandwiched between regions that blocks or mitigates the influence of the adjacent chromosomal environment.
A sequence that ensures or enhances the independence of A transcriptional regulation. Insulators have both an enhancer-blocking effect (the effect of blocking the effect of the enhancer on the promoter activity by inserting it between the enhancer and the promoter) and a position-effect suppressing effect (the effect of sandwiching the transgene with insulators on both sides). , the action of rendering the expression of the transgene unaffected by the position on the genome where it was inserted). The donor DNA for the selectable marker is
An insulator sequence may be present between the upstream arm and the selectable marker gene (or between the upstream arm and the promoter controlling the selectable marker gene). The selectable marker donor DNA may have an insulator sequence between the downstream arm and the selectable marker gene.

The selectable marker donor DNA may be linear or circular, but preferably circular. Preferably, the selectable marker donor DNA is a plasmid. Donor DNA for selectable markers can contain any sequence in addition to the above sequences. For example, all or part of each sequence between the upstream homology arm, the insulator, the selectable marker gene, and the downstream homology arm may contain a spacer sequence.

In step (a), the same or more types of donor DNAs for selection markers as the number of target alleles for genome modification are introduced into cells. Different types of selectable marker donor DNAs have mutually different (distinguishable) types of selectable marker genes. In certain aspects, the different types of selectable marker donor DNAs do not have the exact same selectable marker gene or set thereof. That is, the first type of selectable marker donor DNA has the first type of selectable marker gene, and the second type of selectable marker donor DNA has the second type of selectable marker gene. The third type of selectable marker donor DNA has the third type of selectable marker gene, and the same applies to subsequent types of selectable marker donor DNAs. When there are two target alleles for genome modification, there are two or more types of donor DNAs for selection markers. When there are three target alleles for genome modification, there are three or more types of selectable marker donor DNAs. In one aspect, donor D for one selectable marker
NA may have two or more mutually different (distinguishable) selection markers (even in this case, different types of donor DNA for selection markers are mutually different (distinguishable) must have a species (eg, unique) selectable marker gene). In some embodiments, the selectable marker donor DNA does not have recombination sequences for site-specific recombinase (eg, loxP sequences and variants thereof that are recombined by Cre recombinase).
Also, in certain aspects, the methods of the invention do not employ site-specific recombinases and recombination sequences thereof (eg, loxP sequences and variants thereof that are recombined by Cre recombinase). When a site-directed recombinase is used, usually one recombination sequence of the site-directed recombinase remains in the edited genome. In contrast, in one aspect, the modified genome of the cell obtained by the method of the invention does not contain the recombination sequence (which is foreign) of the site-specific recombinase.

The number of types of selectable marker donor DNAs may be equal to or greater than the number of target alleles for genome modification, and the upper limit is not particularly limited. Two or more alleles can be stably modified by using the same or more types of selective marker donor DNAs as the number of target alleles for genome modification. From the viewpoint of the selection operation in the step (b) described later, the number of types of donor DNA for selection markers is preferably the same as the number of alleles to be modified or about 1 to 2 more than the number of alleles to be modified. More preferably, the number is the same as the number of alleles.

The method for introducing the above (i) and (ii) into cells is not particularly limited, and known methods can be used without particular limitations. Methods for introducing (i) and (ii) into cells include, for example,
Examples include, but are not limited to, virus infection method, lipofection method, microinjection method, calcium phosphate method, DEAE-dextran method, electroporation method, particle gun method and the like. By introducing (i) and (ii) above into a cell, the DNA in the target region is cleaved by the sequence-specific nucleic acid cleaving molecule of (i) above,
HDR knocks in the selectable marker in the selectable marker donor DNA of (ii) into the target region. At this time, two or more selection marker donor DNAs can be randomly knocked into two or more alleles of the target region when their respective upstream homology arms and downstream homology arms are the same. However, donor DNA for two or more selection markers
can modify each of two or more alleles as long as they have base sequences of homology arms that allow homologous recombination with the upstream and downstream sequences of the target regions of each of the two or more alleles. need not have the nucleotide sequence of the homology arms of In one aspect, in two or more types of selectable marker donor DNAs, the nucleotide sequences of the upstream and downstream homology arms have nucleotide sequences that are more identical to the upstream and downstream sequences of the target regions of the respective alleles. (eg, optimized to do so).

In one aspect, the donor DNA for the selectable marker has an upstream homology arm and a downstream homology arm, has a selectable marker gene between the upstream homology arm and the downstream homology arm, and preferably has a meganuclease cleavage site or the like. It may further have a target sequence for an endonuclease (sequence-specific nucleic acid cleaving molecule). In this aspect, in one preferred aspect, the selectable marker comprises a selectable marker gene for positive selection and a marker gene for negative selection. In another preferred embodiment, the selectable marker includes a selectable marker for positive selection, but may not separately include a negative selectable marker gene. In certain preferred embodiments, selectable marker genes for positive selection can also be used for negative selection, and such marker genes include visualization marker genes.
The set of two or more selectable marker donor DNAs includes the above selectable marker donor DNAs
and each have selectable marker genes for positive selection that are distinguishable from each other. The set may further have a target sequence for an endonuclease (base sequence-specific nucleic acid cleaving molecule), such as a cleavage site for a meganuclease, and the target sequences may be different from each other, but are identical (or is cleavable with a base sequence-specific nucleic acid cleaving molecule). The length of the donor DNA for the selectable marker is as described above, but can be, for example, 5 kbp or longer, 8 kbp or longer, or 10 kbp or longer.

(Step (b))
After the step (a), the step (b) is performed. In step (b), cells into which two or more alleles have been introduced with distinguishably different selectable marker genes or a combination thereof are selected based on the expression of the distinguishably different selectable marker genes. More specifically, step (b
), by homologous recombination of different types of selectable marker donor DNAs with respect to the two or more alleles, unique selectable marker genes that are distinguishably different from each other are introduced into the two or more alleles. Cells are selected that express all of the distinguishably distinct selectable marker genes. In one aspect, in step (b), different donors for selection markers are selected based on the expression of all the selection marker genes that are included in the two or more types of donor DNAs for selection markers and that are integrated into the chromosomal genome. Cells in which each allele has been modified by introduction of the DNA are selected. In one embodiment, in step (b), cells are selected based on all the selectable marker genes possessed by the two or more selectable marker donor DNAs. In one aspect, in step (b), expression of all selectable marker genes (positive selection marker genes) integrated into the chromosomal genome that are selectable marker genes possessed by the two or more selectable marker donor DNAs Based on this, cells in which each allele has been modified by introduction of a distinguishable selection marker donor DNA are selected. In one aspect, the cells obtained in step (b) have a marker gene for positive selection with each allele being different. In one embodiment, the cells obtained in step (b) each allele carries a common marker gene for positive selection, and in one embodiment, in step (b), single cell cloning is not performed {however, performing single-cell cloning after selecting cells in which two or more alleles have been modified in step (b);
may not be included}. In one aspect, in step (b), the selection of cells is based on multiple expression of the introduced distinct positive selection marker gene at each allele. In some embodiments, step (b) is not performed in a manner that estimates the abundance of modified alleles based on the expression intensity of a single selectable marker gene (eg, fluorescent protein expression intensity or fluorescence intensity). When cells are selected by a method of estimating the number of modified alleles based on the intensity of expression of a single selectable marker gene, the gene expression level of each cell fluctuates, and two or more alleles are modified. This is because it becomes difficult to completely separate the cells from the cells modified in one allele, thus necessitating single cell cloning in step (b).

In step (b), cells may be appropriately selected according to the type of selectable marker gene used in step (a). At this time, cells are selected based on the expression of all of the selectable marker genes used in step (a).

For example, when the selectable marker gene is a positive selectable marker gene, cells expressing all selectable marker genes integrated (or integrated) into the chromosomal genome to be modified can be selected. Cells can be selected that express as many positive selectable markers as there are. When the positive selectable marker gene is a drug resistance gene, cells expressing the positive selectable marker can be selected by culturing cells in a medium containing the drug. When the positive selectable marker gene is a fluorescent protein gene, a luciferase gene, or a chromogenic enzyme gene, the positive selectable marker is selected by selecting cells exhibiting fluorescence, luminescence, or color development by the fluorescent protein, luciferase, or chromogenic enzyme. can be selected for cells that express In this process,
When the same number of selectable marker donor DNAs as the number of alleles to be modified are incorporated into the genome, the number of alleles is modified. In nploid cells, the number of alleles to be modified is n or less, and the donor DN for selection marker of the number or more and n or less types
When A is incorporated into the genome, at least the alleles to be modified (which are two or more alleles) have been modified. In one aspect, the number of alleles to be modified is n, the number of types of donor DNAs for selection markers are incorporated into the chromosomal genome, and all alleles are modified. In one aspect, in this step, the number of types of donor DNA for selection markers equal to or more than the number of alleles to be modified is used. means modified. From the viewpoint of enhancing the cell selection efficiency in step (b), the number of alleles to be modified is preferably the same as the number of types of selectable marker donor DNAs.

As described above, in the genome modification method of the present embodiment, by inducing HDR using n types of selection marker donor DNAs to modify n alleles in n-ploid cells, all the cells have Allele-modified cells can be obtained efficiently.
In addition, since cells in which all alleles have been modified can be reliably obtained, cells in which the target region has been modified can be efficiently obtained even if the target region has a large size (eg, 10 kbp or more). can. Therefore, large-scale genome modification becomes possible.

In a preferred embodiment, the selectable marker donor DNA may contain a negative selectable marker gene in addition to the positive selectable marker gene between the upstream homology arm and the downstream homology arm. In a preferred embodiment, in the selection marker donor DNA, the positive selection marker gene may be a marker gene that can also be used for negative selection (a marker gene that can be used for both positive selection and negative selection). In one preferred embodiment, the positive selectable marker gene can be a drug resistance gene. In a preferred embodiment, the positive selection marker gene can be a visualization marker gene or the like that can also be used for negative selection.

The selectable marker donor DNA contains a positive selectable marker gene and a negative selectable marker gene between the upstream homology arm and the downstream homology arm, but may further contain a target nucleic acid sequence. A target nucleic acid sequence is a sequence cleavable by the genome modification system described above. The target nucleic acid sequence is preferably an allele-specific sequence whereby the first of the first allele cassette (or first cassette) and the second allele cassette (second cassette) one allele or only the second allele. In this way, selective editing of only one allele is possible by allele-specific induction of cleavage. In one preferred embodiment, the selectable marker donor DNA comprises a single target nucleic acid sequence between the upstream homology arm and the downstream homology arm. In one preferred embodiment, the selectable marker donor DNA comprises a first target nucleic acid sequence and a second target nucleic acid sequence between an upstream homology arm and a downstream homology arm, wherein the first target nucleic acid sequence and the second target nucleic acid sequence are A selectable marker gene is included between the nucleic acid sequences. Another selectable marker donor DNA comprises a third target nucleic acid sequence and a fourth target nucleic acid sequence between the upstream homology arm and the downstream homology arm, wherein the third target nucleic acid sequence and the fourth target nucleic acid sequence are Including a selectable marker gene in between. The first target nucleic acid sequence and the second target nucleic acid sequence may be the same or different, and the third and fourth target nucleic acid sequences may be the same or different. For example, when cleaving a first target nucleic acid sequence and a second target nucleic acid sequence,
designed such that the third target nucleic acid sequence and the fourth target nucleic acid sequence are not cleaved and/or the first target nucleic acid sequence is cleaved when the third target nucleic acid sequence and the fourth target nucleic acid sequence are cleaved and the second target nucleic acid sequence is designed not to be cleaved, specifically cleaves only one of the first cassette and the second cassette, and selectively edits one of the cassettes It can be carried out. It will be appreciated that the first to fourth target nucleic acid sequences may be identical if the first and second cassettes are edited simultaneously.

In one aspect, in step (b), from the pool comprising the cells obtained by step (a),
Modified cells can be selected without cloning the cells. Omitting the cloning step can reduce the time required for the process.

A first intermediate cell can be obtained from the pre-modification cell by steps (a) and (b) above (see step S1 in FIG. 1). In step (a), the selectable marker donor DNA is a positive selectable marker gene and a negative selectable marker gene, or both positive and negative selectable marker genes between the upstream homology arm and the downstream homology arm for the target sequence. contains a marker gene that can be used for both. Donor DNAs for selectable markers preferably contain two target nucleic acid sequences between upstream and downstream homology arms to the target sequences. A positive selection marker gene and a negative selection marker gene, or a marker gene that can be used for both positive selection and negative selection, are preferably present between the two target nucleic acid sequences. By doing so, a first intermediate cell can be obtained by positive selection.

The first intermediate cell is a cell having a genome containing a first allele and a second allele at the genetic locus to be modified, wherein each of the first allele and the second allele contains a selectable marker gene and a target nucleic acid. A cell with a cassette containing the sequence. In a preferred embodiment, in the first intermediate cell, the selectable marker gene possessed by the first allele is distinguishably different from the selectable marker gene possessed by the second allele. In one preferred embodiment, the first intermediate cell comprises
The target nucleic acid sequence is a target of a genome modification system and is designed so that the genome modification system can cleave the first allele and the second allele in a distinguishable manner. In one preferred embodiment, in the first intermediate cell, each selectable marker gene is a negative selectable marker gene that can be used for negative selection. A positive selectable marker gene is useful when obtaining the first intermediate cell, but the positive selectable marker gene is not required in the steps after obtaining the first intermediate cell. Thus, positive selectable marker genes may be removed. Removal can be performed, for example, by what is referred to as genome editing technology. Thus, the first intermediate cell may not have a positive selection potential.

A second intermediate cell may be obtained from the first intermediate cell (see step S3 in FIG. 1).
A second intermediate cell can be made by removing the second cassette of the first intermediate cell. Cassette removal is preferably performed in the presence of a donor DNA (donor DNA for cassette removal) containing an upstream homology arm capable of homologous recombination with the upstream of the second cassette and a downstream homology arm capable of homologous recombination with the downstream of the cassette. by specifically cleaving the target nucleic acid sequence within the second cassette. By cleaving the target nucleic acid sequence in the presence of a donor DNA consisting of an upstream homology arm capable of homologous recombination with the upstream of the cassette and a downstream homology arm capable of homologous recombination with the downstream of the cassette, the cassette is removed when the cassette is removed. It is also possible to connect upstream and downstream seamlessly. The second intermediate cell thus obtained has, in the first allele, the cassette containing the selectable marker gene and the target nucleic acid sequence, but does not contain the second cassette.

A library of the present disclosure can be generated from a first intermediate cell and a second intermediate cell (see steps S2 and S4 in Figure 1). The first intermediate cell and the second intermediate cell (collectively, "intermediate cells") have at the first allele a cassette comprising a selectable marker gene and a target nucleic acid sequence. In the library preparation process, the first cassette can be removed and a modified nucleotide sequence can be introduced instead. That is, the first cassette can be replaced with a modified base sequence. This replacement can be performed by a genome modification system. A donor D for introducing a modified base sequence comprising an upstream homology arm capable of homologous recombination with the upstream of the first cassette and a downstream homology arm capable of homologous recombination with the downstream of the cassette
It can be performed by specifically cleaving the target nucleic acid sequence inside the first cassette in the presence of NA (or donor DNA for library construction). The donor DNA for library construction has a modified base sequence between the upstream homology arm and the downstream homology arm. Basically, the region where the upstream homology arm on the genome undergoes homologous recombination and the region where the downstream homology arm homologously recombines into the sequence sandwiched between the upstream homology arm and the downstream homology arm of the donor DNA for library construction. Since the region sandwiched between the two is replaced, a donor DNA for library construction having the sequence after replacement between the upstream homology arm and the downstream homology arm can be preferably used. Therefore, the cassette can be replaced with the modified nucleotide sequence by the above operation. The donor DNA for library construction can be a group of DNAs containing various modified base sequences. By doing so, the cassette of each intermediate cell can be replaced with various modified base sequences by the above operation. When the cassette is replaced, the negative selectable marker gene in the cassette is removed, so that non-expression of the negative selectable marker gene can be used as an index to obtain cells in which the cassette has been replaced with the modified base sequence. . In one aspect, the donor DNA for library construction is linear (not circular). By doing so, the donor DNA for library preparation
(see, for example, drawback 2 in FIG. 4). Replacement of the modified nucleotide sequence of the cassette can be confirmed by a person skilled in the art by well-known and commonly used techniques, for example, the presence or absence of restriction enzyme cleavage, the presence or absence of PCR amplification (e.g., junction PCR), and sequencing. . The modified base sequence may have a length of 0 bases, that is, may be absent, but preferably has a length of 1 base or more. Modified base sequences are not particularly limited, but may be, for example, 1 to 1,000,000 bases long, 1 to 500,000 bases long, 10,000 to 100,000 bases long, 10,000 to 20,000 bases long,
It can be 10,000 to 15,000 bases long, or 10,000 to 10,000 bases long. The modified base sequence is not particularly limited, but may be, for example, 3 bases or longer, 10 bases or longer, 30 bases or longer, 50 bases or longer, 10 bases or longer,
It can have a length of 0 bases or longer. The modified nucleotide sequences of the donor DNAs for library construction may be the same or different. The modified base sequence of each library-construction donor DNA can independently have any of the above lengths.

If the intermediate cell has three or more alleles in the target region, at least the first allele needs to have a distinct negative marker gene. By doing so, the cassette other than the first allele can be removed, or an operation is performed to replace the cassette of the first allele with the modified base sequence while maintaining the cassette other than the first allele. be able to. Therefore, as the intermediate cells, only the first allele is a cell having a unique negative marker gene in which at least the first allele can be distinguished, and such cells can be selected and used as the intermediate cells. .

Although the method of the present disclosure may not solve any of the drawbacks 1-3 shown in FIG. 4, it may preferably solve any one or more of the drawbacks 1-3 shown in FIG. Specifically, the method of the present disclosure has a higher efficiency of recombination of a foreign gene into the target genome than the GatewayTM method and the LoxP/Cre method. In a preferred embodiment of the present disclosure, the donor DNA for introducing modified nucleotide sequences is linear and not circular. In preferred aspects of the present disclosure, the modified cell is
It does not have a site-specific recombination enzyme recognition site. In certain preferred aspects of the methods of the present disclosure, G
Compared to the atweyTM method and the LoxP/Cre method, the recombination efficiency of the foreign gene into the target genome is high, the donor DNA for introducing the modified base sequence is linear, and the modified cell is a site-specific recombination enzyme does not have a recognition site for The feature of not having a recognition site for a site-specific recombination enzyme is useful, for example, when seamlessly ligating an introduction cassette and a genome.

The library of the present disclosure is
comprising a combination of multiple aqueous compositions;
each aqueous composition comprising one type of modified cell;
Each modified cell has a first allele and a second allele at the locus to be modified (target locus or target locus),
Each modified cell has a cassette containing DNA fragments at the same position of the first allele that differ from each other among the aqueous compositions. Such libraries can be obtained as follows. For example, the target nucleic acid sequence of the first cassette of the intermediate cell is cleaved in the presence of library construction donor DNA containing various modified base sequences. Then the D in the cell
The NA damage repair mechanism replaces the first cassette of the intermediate cell with the modified nucleotide sequence.
Cells with altered nucleotide sequences can be subjected to single cell cloning. Single-cell cloning can involve forming multiple droplets or aqueous compositions containing a single cell and subjecting them to culture to generate cell clones derived from a single cell in the droplets or aqueous compositions. . In this way, a plurality (or a large number) of aqueous compositions containing cell clones are obtained. A combination of multiple (or multiple) such aqueous compositions can be used as a library of modified cells.

In some embodiments of intermediate cells or modified cells, the first allele lacks part or all of the target region (originally genomic). In certain aspects of intermediate or modified cells, the second allele lacks part or all of the target region. In certain preferred embodiments of intermediate cells or modified cells, the first allele and the second allele lack part or all, more preferably all, of the target region.

In some embodiments of the first intermediate cell, the first allele has the entire target region replaced by the first cassette. In some embodiments of the first intermediate cell, the second allele has the entire target region replaced by the second cassette. In one preferred embodiment of the first intermediate cell, the first and second alleles are replaced by the first and second cassettes respectively over the entire target region.

In the second intermediate cell the second cassette has been removed. In a preferred embodiment, the second intermediate cell is seamlessly linked upstream and downstream of the target region of the second allele (originally on the genome). "Seamlessly linked" means that upstream and downstream are linked without the addition of new bases. In seamless connection of upstream and downstream, it is preferable that upstream and downstream are connected without additional omission of bases.

In some embodiments of the modified cell, the first allele comprises a modified sequence, and the modified sequence comprises (
It has one or more mutations selected from the group consisting of base additions, insertions, substitutions, deletions, and deletions in the target region (also referred to as the substituted sequence) originally on the genome. Modified cells can be advantageously used to compare with the original cells (reference cells or reference cells) and to assess the effects of mutations. Also, by including a variety of cells that differ in their mutations, it is advantageous to assess the function of each mutation site in the target region through cell-to-cell comparisons.

In some embodiments of the first modified cell, the second allele comprises a cassette containing a selectable marker. Selectable markers can include positive selectable marker genes. In some embodiments of the second modified cell, the second allele is seamlessly linked upstream and downstream of the substituted sequence. In certain preferred embodiments of the modified cell, the substituted sequence of the first allele and the substituted sequence of the second allele have corresponding sequences. Having corresponding sequences means that the start and end of the sequences are located at the same position on the genome. Corresponding sequences typically have high identity (
for example, 80% or more, 90% or more, or 95% or more) and have the same length.

In some embodiments, each sequence of the cassette in the modified cell consists of one or more modified portions (A) and one or more unmodified portions (B) (see, eg, Figure 3). Each of the modified portions (A) has one or more modifications selected from the group consisting of sequence insertions, deletions, and substitutions, and the modifications (A) of the one or more modified portions are modified positions or Content differs between each aqueous composition. Each of the one or more unmodified portions (B) is identical to the sequence of the corresponding site before modification. Here, when the sequence before modification to be replaced by the insertion cassette and the sequence in the insertion cassette are aligned and aligned at the same position, the two nucleic acid sequences are sequences at corresponding sites. The unmodified portion (B1) on the centromere side of the cassette is seamlessly linked to the adjacent sequence (C1) on the centromere side of the cassette, and the unmodified portion (Bt) on the telomere side of the cassette is the cassette. flanking sequences on the telomeric side of (
C2), and the region where the adjacent sequence (C1) and the unmodified portion (B1) are linked, and the region where the unmodified portion (Bt) and the adjacent sequence (C2) are linked,
It may constitute the same sequence as the sequence of the corresponding region before modification. In order to incorporate such a cassette into DNA, an intermediate cell may be modified using a donor DNA for library construction having the structure of the cassette between the upstream homology arm and the downstream homology arm. In one preferred embodiment, the total length of the modified portion (A) is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less of the length of the insertion cassette. , 20%
or less, 10% or less, or 5% or less. A library containing modified cells of this embodiment can be a library having different mutations at different positions within the same region, for example, to investigate which mutation at which position changes cell activity, or to determine the desired property. can be preferably used for screening of cells having The donor DNA for introducing a modified nucleotide sequence may have the above cassette structure between the upstream homology arm and the downstream homology arm. The modified nucleotide sequence-introducing donor DNA is a modified nucleotide sequence-introducing donor D having a different cassette structure.
It can be included in a library of NA.

In some embodiments, the modified base sequence in the modified cell consists entirely of mutations.

In some embodiments, there are no recombination sequences (recognition sites) for the site-specific recombination enzyme inside or outside (near) the insertion cassette. In some embodiments, the cell is unmodified or has the same base sequence as the cell before modification, except for the insertion cassette. By doing so, it is possible to obtain a modified cell that does not contain modifications other than the desired modification, and it is possible to eliminate unexpected effects of modifications other than the desired modification (for example, see defect 3 in FIG. 4) ).

The library is not particularly limited, but is preferably 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more. , 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40
types or more, 45 types or more, 50 types or more, 60 types or more, 70 types or more, 80 types or more,
90 types or more, 100 types or more, 150 types or more, 200 types or more, 300 types or more, 4
00 types or more, 500 types or more, 600 types or more, 700 types or more, 800 types or more, 9
00 types or more, 1000 types or more, 2000 types or more, 3000 types or more, 4000 types or more, 5000 types or more, 6000 types or more, 7000 types or more, 8000 types or more, 9
000 types or more, or 10000 or more types of modified cells (or aqueous compositions containing modified cells) may be included.

A library typically contains a combination of a plurality of aqueous compositions. Each aqueous composition contains 1
Contains different types of cells. In some embodiments, the library separately comprises a plurality of combinations of aqueous compositions. In some embodiments, the library may contain combinations of multiple aqueous compositions as a mixture for some purposes. For example, when screening cells with high proliferative ability or viability of cells, the library may contain a combination of a plurality of aqueous compositions as a mixture. By analyzing the cells, cells with the highest proliferative capacity and viability are enriched, and cells with high proliferative capacity and viability can be obtained.

In one aspect, in one library, genomic sequences other than the modified nucleotide sequence (or DNA fragment) of each modified cell contained in the library are designed to be identical. In one aspect, in one library, the genomic sequences other than the modified base sequence (or DNA fragment) of each modified cell contained in the library are substantially identical. Substantially identical allows for the existence of mutational differences that can occur between cells after only 10 passages of the cloned cells under conditions suitable for cell culture (under normal conditions). .

Since they are common except for the modified base sequence, they are suitable for evaluating the influence of the modified base sequence, for example. Elimination of the cassette in the second allele minimizes the potential impact of the cassette. Seamless removal of the cassette in the second allele minimizes the potential impact of introducing additional new bases into the second allele.

In another aspect, the cassette in the second allele in the intermediate cell (second cassette) may also be replaced by the second modified nucleotide sequence. The second modified sequence may be a sequence common to the modified cells (ie, the same sequence across the modified cells) or may be a sequence that differs from aqueous composition to aqueous composition. In some cases, the second modified sequence may differ from cell to cell even within the same aqueous composition. Also, the second modified sequence may be the same as or different from the modified base sequence (first modified base sequence) in the first allele. The second modified base sequence can be designed in the same manner as the modified base sequence in the first allele and introduced in the same manner as the modified base sequence in the first allele. Substitution of the second modified base sequence of the second cassette in the presence of the second library preparation donor DNA,
The vicinity of the second cassette (preferably, the target nucleic acid sequence within the second cassette) may be specifically cleaved. In the present specification, the content of the second modified base sequence and the method of introducing it are the same as those described for the first modified base sequence, so this description will be referred to and the description will be omitted here.

Application example 1
Application 1 is an application to analysis of a specific region of the genome. According to the present disclosure, by introducing various mutations into the base sequence of a specific region of the genome and observing the gain or loss of function of the specific region due to mutation, important bases in the specific region of the genome are identified. It is possible to Examples of specific regions include regions of unknown function, promoter regions, enhancer regions, regions corresponding to introns, regions corresponding to 5′ untranslated regions (UTR), and regions corresponding to 3′ untranslated regions (UTR). , regions encoding non-coding RNAs, and the like.

Application example 2
Application 2 is an application to regulation of protein or RNA expression levels. In this application, the transcriptional control region of a protein or RNA (including a region suspected of being involved in transcriptional control) and the translational control region of said protein (including a region suspected of being involved in translational control) A region involved or suspected of being involved in regulation of the expression level of said protein or RNA is modified to regulate the expression level of said protein. Thus, in Application Example 2, a modified cell in which the expression level of protein or RNA is regulated can be obtained. Modulation can be an increase or decrease in expression levels. RNA is mRNA, tRNA, r
RNA, other non-coding RNA (eg, microRNA).

Application example 3
Application Example 3 is an application example to a protein or RNA coding region. According to the present disclosure, by introducing various mutations into a region encoding a protein or RNA, respectively, and observing functional alteration (e.g., gain or loss of function) of the protein or RNA due to mutation, the protein or RNA It is possible to identify key amino acids or key sequences in the function of . Also, for example, by observing gain or loss of function of protein or RNA due to mutation, obtaining mutant protein or RNA having improved or reduced function or new function and modified cells expressing the mutant protein or RNA can be done. When various modifications are made to part or all of a protein or RNA, it is desirable to seamlessly ligate the part or all containing the modified site in-frame to the region encoding the protein or RNA. RNA is mRNA
A, tRNA, rRNA, other non-coding RNAs (e.g., microRNAs)
can be According to Application Example 3, in combination with Application Example 2, it is possible to obtain a modified cell that expresses the mutant protein or RNA and whose expression level is regulated.

Application example 4
Application Example 5 is an application to screening for cells with high proliferative ability or viability. According to the present disclosure, libraries can be obtained that contain diverse modified cells with different mutations in genomic regions that may be involved in cell proliferation or viability. Such a library may contain separate aqueous compositions containing each type of cell, but may also be a mixture of aqueous compositions containing each type of cell. Preferably, the mixture contains equal amounts of each modified cell. By culturing the mixture in an environment suitable for culturing cells or in the presence of selective pressure on the cells, the relative concentration of cells with high proliferation or viability is increased, and the relative concentration of cells with low proliferation or viability is reduced. lower. Therefore, after culturing, cells with high proliferation or viability are concentrated, and it is advantageous to be able to obtain these cells. Screening can also be performed under conditions that impose a certain selective pressure. In this way, cells with high proliferative or viable viability can be screened against the particular selective pressure.
Examples of selective pressure include, but are not limited to, poor nutrition, high salt concentration, low salt concentration, high temperature, low temperature, hypoxia, and the presence of drugs (eg, bioactive substances such as toxic substances and antibiotics).
An existing gene on the genome can be modified by replacing the gene with a modified gene. Alternatively, safe harbor regions (e.g., AAVS1 locus, ROSA26
It is also possible to simply insert the modified nucleotide sequence into the gene locus, CLBYL locus, CXCR4 locus, CCR5 locus, etc.).

In one embodiment, the present invention provides a cell in which two or more alleles of the chromosomal genome are modified, the cell having a mutually different (distinguishable) selectable marker gene in each of the two or more alleles. offer. In some aspects, the cell can be a cell of a unicellular organism. In some aspects, the cells can be isolated cells. In some embodiments, the cells can be cells selected from the group consisting of pluripotent cells and pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells. In some aspects, the cells can be tissue stem cells. In some aspects, the cell can be a somatic cell. In some aspects, the cells can be germline cells (eg, germ cells). In some aspects, the cell can be a cell line. In some aspects, the cells can be immortalized cells. In some embodiments, the cells can be cancer cells. In some embodiments, the cells can be non-cancer cells. In some aspects, the cells can be cells of a diseased patient. In some embodiments, the cells can be cells from healthy individuals. In some aspects, the cells are animal cells (e.g., human cells), e.g., insect cells (e.g., silkworm cells), HEK293 cells, HEK293T cells, Expi293F™ cells, FreeStyle™ 293F cells, Chinese hamster ovary cells. (CHO cells), CHO-S cells, CHO-K1 cells, and E
xpiCHO cells, as well as cells derived from these cells. In a preferred embodiment, in the cell, all alleles of the target region of the chromosomal genome are modified, and the modified regions have mutually different (distinguishable) selectable marker genes.

In one embodiment, a method for culturing cells is provided in which two or more alleles of the chromosomal genome are modified, and the two or more alleles have mutually different (distinguishable) selectable marker genes. be done. When the selectable marker gene is a drug resistance marker gene, the culture can be grown in the presence of drugs directed against the respective drug resistance marker gene. Culturing may be performed under conditions suitable for cell maintenance or growth.

In one embodiment, the present invention provides a non-human organism having a chromosomal genome in which two or more alleles are modified, wherein the two or more alleles each have a different (distinguishable) selectable marker gene. , a non-human organism is provided. In some aspects, the cell can be a cell of a unicellular organism. In certain aspects, the non-human organism is a yeast (e.g., fission yeast or budding yeast, e.g., Saccharomyces cerevisiae).
evisiae), Saccharomyces carlsbergensis
scarlsbergensis), Saccharomyces fragilis (Saccharo
myces fragilis), Saccharomyces rouxii (Saccharomyces)
ces rouxii), Candida utilis (Can
dida utilis, Candida tropicalis
calis), Pichia, Kluyveromyces (K
luyveromyces, Yarrowia, Hansenula (Ha
nsenula), Endomyces, etc., selected from the group consisting of yeast. In some aspects, the non-human organism is a filamentous fungus (e.g., Aspe
rgillus, Trichoderma, Humicola, Acremonium,
Fusarium, and Penicillium species). In some aspects, a non-human organism can be a multicellular organism. In some aspects, the non-human organism can be a non-human animal. In some aspects, the non-human organism can be a plant. In a preferred embodiment, in the non-human organism, all alleles of the target region of the chromosomal genome are modified, and the modified regions each have mutually different (distinguishable) selectable marker genes.

In such cells, one or more desired genes, such as genes required for cell survival or proliferation, may be contained or clustered in other regions of the chromosomal genome. Other regions can be, for example, safe harbor regions such as the AAVS1 locus, ROSA26 locus, CLBYL locus, CXCR4 locus, and CCR5 locus. The other region can be, for example, the region of (ii) above that has a deletion.

As shown in Figure 1, a first intermediate cell is obtained from a pre-modified cell (referred to as a reference cell). The step of obtaining the first intermediate cell from the pre-modification cell is called step S1. A library of first modified cells (hereinafter simply referred to as the "first library") can be generated from the first intermediate cells. The step of obtaining the first library from the first intermediate cell is called step S2. A second intermediate cell can be obtained from the first intermediate cell. The step of obtaining the second intermediate cell from the first intermediate cell is called step S3. A second library of modified cells (hereinafter simply referred to as the "second library") can be generated from the second intermediate cells. The step of obtaining the second library from the second intermediate cells is called step S4.

Prepare reference cells. A reference cell is a cell to be made into a library. Reference cells are eukaryotic cells, for example, and can be used to generate the libraries of the present disclosure. A reference cell may be a naturally occurring cell, but may also be a cell with modifications. Reference cells are typically diploid, but may be triploid or higher.

For example, a first intermediate cell can be made as shown in FIG. Specifically, a target region on the genome of a reference cell is replaced with a cassette containing a selectable marker gene. In FIG. 2, two donor DNAs and a target region are subjected to homologous recombination. Donor D
NAs each contain a distinguishably different drug selectable marker gene (for positive selection) and a distinguishably different visualization marker gene (for negative selection), each flanked by a CRI
It has a target sequence (gRNA1-4) by SPR/Cas9. After homologous recombination, drug selection is performed with two drugs in order to select cells in which fragments derived from the two donor DNAs have replaced the target sequences on the paternal and maternal chromosomes, respectively (Fig. 2). Step (1)
reference). Cells that survive the selection are cells (first intermediate cells) that have distinguishably different drug resistance genes in the two alleles of the target region. A first intermediate cell can be subjected to single cell cloning. It is possible to confirm whether a cassette containing a selection marker gene has been inserted at the intended position.

Either the paternal or maternal cassette is then removed. For example, as shown in FIG. 2, target sequences (gRNA
1 and gRNA 2) were subjected to CRISPR/Ca in the presence of donor DNA for cassette removal.
It can be cut by the s9 system. The donor DNA for cassette removal consists of an upstream homology arm capable of homologous recombination with the upstream of the target region on the paternal allele and a downstream homology arm capable of recombination with the downstream of the target region. Then, by sorting cells in which the GFP signal has disappeared, a second intermediate having a genome in which the upstream and downstream of the target region are seamlessly linked in the paternal allele (that is, the entire target region has been dropped) is selected by using a cell sorter. Somatic cells can be obtained.

A second library of modified cells (a second library) can be generated from the second intermediate cells. The target sequences (gRN
A3 and gRNA4) can be cleaved by the CRISPR/Cas9 system in the presence of donor DNA for introducing modified nucleotide sequences (donor DNA for library preparation).
The donor DNA for introducing the modified base sequence has an upstream homology arm capable of homologous recombination with the upstream of the target region on the maternal allele and a downstream homology arm capable of recombination with the downstream of the target region, and the upstream homology arm and the downstream A modified base sequence is included between the homology arms. In this way, a modified cell is obtained that has a genome containing a modified base sequence between the upstream and downstream of the target sequence in the maternal allele. By preparing modified nucleotide sequence-introducing donor DNAs having different modified nucleotide sequences, modified cells having different modified nucleotide sequences can be obtained, thereby obtaining a second library.

The donor DNA for introducing the modified nucleotide sequence consists of one or more modified portions (A) and one or more unmodified portions (B) as described above. It can have the same sequence as the sequence of the region.

Claims (20)

A library of modified cells,
the library comprises combinations of a plurality of aqueous compositions;
each aqueous composition comprising one type of modified cell;
Each modified cell has a first allele and a second allele at the locus to be modified,
Each of the modified cells has at the same position of the first allele a cassette containing DNA fragments that differ from each other among the aqueous compositions , said locus of the modified cell being the recognition site for the site-specific recombination enzyme or the recombination sequence. does not contain
Library. 2. The library of claim 1 , wherein each modified cell has a disruption or deletion of part or all of the target region in the second allele. 3. The library of claim 2, wherein the second allele seamlessly lacks said part or all. 2. The library of claim 1, wherein the sequences other than the cassette containing the DNA fragment of each modified cell are substantially the same before and after modification. Each of the sequences of said cassette consists of one or more modified portions (A) and one or more unmodified portions (B), each of said modified portions (A) consisting of sequence insertions, deletions and substitutions. having one or more modifications selected from the group, the modifications of the one or more modified portions differing between each aqueous composition with respect to the location or content of the modifications, and the one or more unmodified portions (B) each It is identical to the sequence of the corresponding site before modification, and the unmodified portion (B1) on the centromere side of the cassette is seamlessly linked to the adjacent sequence (C1) on the centromere side of the cassette, and the cassette The unmodified portion (Bt) on the telomere side is seamlessly linked to the flanking sequence (C2) on the telomere side of the cassette, and the region where the flanking sequence (C1) and the unmodified portion (B1) are linked, and the non- The library according to claim 1, wherein the region where the modified portion (Bt) and the flanking sequence (C2) are linked constitutes the same sequence as the sequence of the corresponding region before modification. 2. The library according to claim 1, wherein the library contains 50 or more aqueous compositions . A method of producing a library of modified cells, comprising:
(α) in a cell having a genome containing a first allele and a second allele at the genetic locus to be modified, a cassette containing a selection marker gene and a target nucleic acid sequence for each of the first allele and the second allele; providing a population of cells having
Here, the selectable marker gene possessed by the first allele and the selectable marker gene possessed by the second allele are distinguishably different, the target nucleic acid sequence is the target of the genome modification system, and the genome modification system selects the first marker gene. each selectable marker gene is a negative selectable marker gene that can be used for negative selection, and is designed so that the allele and the second allele can be distinguished from each other;
(β) introducing the following (x) and (y) into the provided group of cells;
(x) a genome modification system comprising a sequence-specific nucleic acid cleaving molecule targeting the unique base sequence contained in the first allele, or a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule;
(y) a plurality of types of second recombination donor DNA {wherein the plurality of types of second recombination donor DNA are homologous to the base sequence adjacent to the upstream side of the target site in (x) above, respectively; and a downstream homology arm having a nucleotide sequence homologous to the nucleotide sequence adjacent to the target region downstream, and a modified base between the upstream homology arm and the downstream homology arm sequence, wherein the modified nucleotide sequence is different for each second recombination donor DNA and unique to each second recombination donor DNA},
(γ) selecting cells that do not express the selectable marker gene contained in the first allele after the step (β);
including
Thereby, a library of modified cells comprising a plurality of cells can be obtained, wherein in the plurality of cells obtained, the first allele has a modified nucleotide sequence unique to each cell, and the second allele has a common sequence between cells,
Method. 8. The method of claim 7 , wherein prior to step (a),
In a cell having a genome containing a first allele and a second allele at a genetic locus to be modified, a cassette containing a selection marker gene and a target nucleic acid sequence for the substituted sequences contained in the first allele and the second allele thereby removing the substituted sequence from the first allele and the second allele;
The method further comprising: 9. The method of claim 8 , wherein
Each modified nucleotide sequence of the first allele has one or more mutations selected from the group consisting of addition, insertion, substitution, deletion, and deletion of bases relative to the substituted sequence of the first allele,
Method. 9. The method of claim 8 , wherein
The sequence to be modified is a protein coding region,
The modified base sequence of the first allele has one or more mutations selected from the group consisting of base additions, insertions, substitutions, deletions, and deletions with respect to the substituted sequence of the first allele. . 11. The method according to claim 9 or 10 , wherein the modified base sequence has 80% or more sequence identity with the modified sequence. 8. The method of claim 7 , wherein between steps (α) and (β),
The method further comprising removing the cassette from the second allele. 13. The method of claim 12 , wherein the cassette is seamlessly removed. 8. A modified cell library comprising a plurality of modified cells produced by the method of claim 7 . 9. A modified cell library comprising a plurality of modified cells produced by the method of claim 8. 10. A modified cell library comprising a plurality of modified cells produced by the method of claim 9. 11. A modified cell library comprising a plurality of modified cells produced by the method of claim 10. 12. A modified cell library comprising a plurality of modified cells produced by the method of claim 11. 13. A modified cell library comprising a plurality of modified cells produced by the method of claim 12. 14. A modified cell library comprising a plurality of modified cells produced by the method of claim 13.

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