JP6246258B2 - Polypeptide comprising a DNA binding domain - Google Patents

Description

The present invention relates to a polypeptide comprising a DNA binding domain and a functional domain. The present invention also relates to a vector comprising a polynucleotide encoding the polypeptide.
Furthermore, the present invention relates to a vector library for producing the vector. The present invention also relates to a vector set for preparing the vector library.

Polypeptides containing a plurality of nuclease subunits consisting of a DNA binding domain and a functional domain include TALEN (TALE Nuclease) and ZFN (Zinc Fi).
nger Nuclease) (Patent Documents 1 to 4, Non-Patent Documents 1 to 5)
). For example, an artificial nuclease using a DNA cleavage domain as a functional domain is known. These artificial nucleases have multiple Ds at the binding site of the DNA binding domain.
Double-strand breaks in DNA are caused by the close proximity of NA cleavage domains to form multimers. The DNA-binding domain includes a plurality of DNA-binding modules repeatedly, and each DNA-binding module recognizes a specific base pair in the DNA strand. Specific cleavage can be performed. If you use errors and recombination that occur during repair of sequence-specific cleavage, genomic DNA
The above genes can be deleted, inserted, and mutated, and these nucleases can be applied to various gene modifications such as genome editing (see Non-Patent Document 6).

Non-Patent Document 1 discloses a polypeptide in which a DNA binding domain and a functional domain are connected by a 47 amino acid linker. Non-Patent Document 1 describes a nuclease in which one amino acid at a specific site other than an amino acid at a DNA recognition site in a DNA binding module is periodically changed for every four DNA binding modules used. However, in Non-Patent Document 1, there is no description that the used polypeptide has both a high level of desired function by the functional domain and a high sequence recognition specificity of the DNA binding domain,
In addition, there is no description on periodically changing a plurality of amino acids in the DNA binding module, and there is no description on the significance and purpose of the periodic change.

Non-Patent Document 2 discloses a polypeptide in which a DNA binding domain and a functional domain are connected by a 63 amino acid linker. Non-Patent Document 2 describes a polypeptide in which amino acids other than the amino acid at the DNA recognition site in the DNA binding module are periodically changed for each of the four DNA binding modules used. However, Non-Patent Document 2 does not describe that the polypeptide used has both the desired high function by the functional domain and the high sequence recognition specificity of the DNA binding domain. There is no mention of the purpose or significance of the periodic change for each DNA binding module.

Non-Patent Document 3 discloses a polypeptide in which a DNA binding domain and a functional domain are connected by a 47 amino acid linker. Non-Patent Document 3 describes a polypeptide in which amino acids other than the amino acid at the DNA recognition site in the DNA binding module are randomly changed for each DNA binding module used. However, Non-Patent Document 3 does not mention that the polypeptide used has both the desired high function by the functional domain and the high sequence recognition specificity of the DNA binding domain. There is no description about the purpose and significance of the function, and there is no description of changing periodically for each DNA binding module.

Non-Patent Document 4 discloses a polypeptide in which a DNA binding domain and a functional domain are connected by a 63 amino acid linker. Non-Patent Document 5 discloses a polypeptide in which a DNA binding domain and a functional domain are connected by a 47 amino acid linker. However, in both Non-Patent Document 4 and Non-Patent Document 5, it is described that the polypeptide used has both the desired high function by the functional domain and the high sequence recognition specificity of the DNA binding domain. No mention is made of having a periodic change for each DNA binding module.

International Publication No. 2011/072246 International Publication No. 2011/154393 International Publication No. 2011/159369 International Publication No. 2012/093833

Nucleic Acids Res. 2011 Nov; 39 (21): 9283-93. Nat Biotechnol. 2011 Aug 5; 29 (8): 697-8. Nat Biotechnol. 2011 Feb; 29 (2): 143-8. Nature. 2012 Nov 1; 491 (7422): 114-8. Genes Cells. 2013 Apr; 18 (4): 315-26. Cell. 2011 Jul 22; 146 (2): 318-31.

If a polypeptide containing a DNA binding domain that shows a high function by a functional domain is used, it is expected that the efficiency of obtaining a desired result is improved. For example, in the case of an artificial nuclease containing a DNA-binding domain and a DNA-cleaving domain, the use of the one having high DNA-cleaving activity improves the probability of DNA-cleaving and obtains cells with the desired genetic modification It is expected to improve the efficiency that can be achieved. However, the conventional D
In a polypeptide containing an NA-binding domain, those exhibiting high activity due to a functional domain exhibit high functions even in portions other than the target base sequence due to the DNA-binding domain,
There was a tendency that it was not enough from the viewpoint of safety. Thus, it was difficult to achieve both high recognition specificity for DNA sequences and high function by functional domains. Also,
Preparation of a polypeptide containing a DNA binding domain requires complicated operations such as introduction of a DNA binding module repeat corresponding to the target sequence into a vector, and a polypeptide that can be rapidly produced by a simpler operation. There is a need for peptides.

Therefore, the present invention achieves both a high function by a functional domain and a high recognition specificity for a DNA sequence, can exhibit a desired function with high probability, and can be produced by a simple operation. It is an object to provide a polypeptide capable of

The present inventors have intensively studied and found that the DNA binding domain and the functional domain are 35-5.
A polypeptide that is connected by a polypeptide consisting of 5 amino acids and in which the amino acids at two specific positions in the DNA binding module contained in the DNA binding domain have different repeating patterns for each of the four DNA binding modules is a functional domain It has been found that both the height of the function and the recognition specificity for DNA sequences can be compatible. A vector for expressing the polypeptide could be easily prepared by using a vector set having specific characteristics and a vector library having specific characteristics.

That is, the present invention in the first aspect,
A polypeptide comprising a DNA binding domain and a functional domain comprising:
Here, the DNA binding domain and the functional domain are connected by a polypeptide consisting of 35 to 55 amino acids,
The DNA binding domain includes a plurality of DNA binding modules continuously from the N-terminal side,
The combination of the x-th amino acid and the y-th amino acid in the 4n-3rd DNA binding module from the N-terminus is the same for any n,
The combination of the x-th amino acid and the y-th amino acid in the 4n-2nd DNA binding module from the N-terminus is the same for any n;
The combination of the x-th amino acid and the y-th amino acid in the 4n-1st DNA binding module from the N-terminus is the same for any n.
The combination of the xth and yth amino acids in the 4nth DNA binding module from the N-terminus is the same for any n
A combination of the x-th amino acid and the y-th amino acid in the 4n-3rd DNA binding module from the N-terminus, a combination of the x-th amino acid and the y-th amino acid in the 4n-2th DNA-binding module from the N-terminus, the N-terminus 4n-1 combination of the xth and yth amino acids in the 4n-1st DNA binding module, and the 4nth DN from the N-terminus
The combination of the xth amino acid and the yth amino acid in the A binding module is different from each other,
Here, n is a natural number of 1 to 10, x is a natural number of 1 to 40, and y is 1 to 1
40 natural numbers, and x and y are different natural numbers.
A polypeptide is provided.

In the second aspect, the present invention provides the polypeptide according to the first aspect, wherein the functional domain is a DNA cleavage domain.

In the third aspect, the present invention also provides a vector comprising a polynucleotide encoding the polypeptide according to the first aspect or the second aspect.

Furthermore, the present invention provides the fourth aspect,
A vector library for producing the vector according to the third aspect,
Here, the vector library is composed of a plurality of vectors having a first restriction enzyme cleavage site, a polynucleotide encoding four DNA binding modules, and a second restriction enzyme cleavage site in order from the 5 ′ end,
Here, the combination of the first restriction enzyme cleavage site and the second restriction enzyme cleavage site is a combination of an A type restriction enzyme cleavage site and a B type restriction enzyme cleavage site, an A type restriction enzyme cleavage site and a C type. A combination of restriction enzyme cleavage sites of type A, a combination of restriction enzyme cleavage sites of type A and a restriction enzyme cleavage site of type D, a combination of restriction enzyme cleavage sites of type A and a restriction enzyme cleavage site of type E, and a restriction enzyme cleavage of type B Any combination of a site and a C-type restriction enzyme cleavage site, a combination of a C-type restriction enzyme cleavage site and a D-type restriction enzyme cleavage site, and a combination of a D-type restriction enzyme cleavage site and an E-type restriction enzyme cleavage site Here, each of the A-type restriction enzyme cleavage site to the E-type restriction enzyme cleavage site causes cleavage ends different from each other by cleavage with the same restriction enzyme,
Of the four DNA binding modules,
The combination of the xth and yth amino acids in the first DNA binding module from the 5 ′ end is the same for any vector;
The combination of the xth and yth amino acids in the second DNA binding module from the 5 ′ end is the same for any vector;
The combination of the xth and yth amino acids in the third DNA binding module from the 5 ′ end is the same for any vector;
The combination of the xth and yth amino acids in the fourth DNA binding module from the 5 ′ end is the same for any vector;
Combination of the xth and yth amino acids in the first DNA binding module from the 5 ′ end. Combination of the xth and yth amino acids in the second DNA binding module from the 5 ′ end. The combination of the xth and yth amino acids in the third DNA binding module and the combination of the xth and yth amino acids in the fourth DNA binding module from the 5 ′ end are different from each other,
Here, x is a natural number of 1 to 40, y is a natural number of 1 to 40, and x and y are
A different natural number,
A vector library is provided.

Further, the present invention provides the fifth aspect,
A vector set for preparing the vector library according to the fourth aspect,
Here, the vector set includes a plurality of vectors including a first restriction enzyme cleavage site, a DNA binding module, and a second restriction enzyme cleavage site in order from the 5 ′ end,
The first restriction enzyme cleavage site and the second restriction enzyme cleavage site generate different cleavage ends by cleavage with the same restriction enzyme,
The combination of the xth amino acid and the yth amino acid in the DNA binding module is one of four different combinations,
Here, x is a natural number of 1 to 40, y is a natural number of 1 to 40, and x and y are
A different natural number,
A vector set is provided.

The polypeptide of the present invention achieves a high function by a functional domain and at the same time realizes a high recognition specificity for a DNA sequence, by introducing a vector containing a polynucleotide encoding the polypeptide of the present invention into a cell, A desired result can be realized safely and with high probability. In addition, by using the vector library of the present invention, a polypeptide expression vector that achieves both a high function by a functional domain and a high recognition specificity for a DNA sequence can be prepared easily and rapidly. Furthermore, if the vector set of the present invention is used, the vector library of the present invention can be easily prepared.

FIG. 1 is a schematic diagram showing the structural features of a vector set, vector library, and vector of the present invention, and a production method. FIG. 2 is a diagram showing an example of an amino acid sequence and a base sequence of a DNA binding module included in the vector set, vector library, and vector of the present invention. FIG. 3 is a diagram showing an example of an amino acid sequence and a base sequence contained in the vector of the present invention. FIG. 4 shows the structural features of vectors prepared by the methods of Example 3 and Comparative Examples 1 to 3 (FIGS. 4A and 4B), and shows high sequence specificity of nucleases expressed by various vector combinations. These are compared (FIG. 4C). FIG. 5 shows the design of vectors produced by the methods of Example 3 and Comparative Examples 1 to 3 (FIG. 5A), and compares the level of DNA cleavage activity of nucleases expressed by these vectors (FIG. 5B). ) FIG. 6 shows the design of vectors prepared by the methods of Example 3 and Comparative Examples 1 to 3 (FIG. 6A), and compares the sequence specificity of the DNA cleavage activity of nucleases expressed by these vectors (FIG. 6). 6B).

In a first aspect, the present invention provides a polypeptide comprising a DNA binding domain and a functional domain. The origin of the DNA binding domain is the plant pathogen Xanthomomon
as TALE (Transcribation Activator-Like Eff
e.g., zinc fingers) and the like.

Examples of functional domains include domains encoding enzymes, transcriptional regulatory factors, reporter proteins, and the like. Examples of the enzyme include DNA modifying enzymes such as recombinase, nuclease, ligase, kinase and phosphatase; and other enzymes such as lactamase. In the present specification, a domain encoding nuclease is referred to as a DNA cleavage domain. Examples of transcriptional regulatory factors include activators and repressors.
Reporter proteins include green fluorescent protein (GFP), humanized Renilla green fluorescent protein (hrGFP), enhanced green fluorescent protein (eGFP), enhanced blue fluorescent protein (eBFP), enhanced cyan fluorescent protein (eCFP), enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP or DsRed), fluorescent protein such as mCherry; bioluminescent protein such as firefly luciferase, Renilla luciferase; chemiluminescent such as alkaline phosphatase, peroxidase, chloramphenicol acetyltransferase, β-galactosidase Examples include enzymes that convert substrates. The DNA cleavage domain is preferably one that is in close proximity to other DNA cleavage domains to form multimers and obtain improved nuclease activity. Examples of such a DNA cleavage domain include those derived from FokI.

In the polypeptide of the first aspect of the present invention, the DNA binding domain and the functional domain are:
35-55, preferably 40-50, more preferably 45-49, most preferably
It is connected by a polypeptide consisting of 47 amino acids. Examples of the polypeptide connecting the DNA binding domain and the functional domain include, for example, the 754th to 80th of SEQ ID NO: 34.
And a polypeptide having 85%, 90%, 95%, or 97% identity with the amino acid sequence of positions 754 to 801 of SEQ ID NO: 34. It is done.

In the polypeptide of the first aspect of the present invention, the DNA binding domain includes a plurality of DNA binding modules continuously from the N-terminal side. One DNA binding module specifically recognizes one base pair. The number of DNA binding modules contained in the DNA binding domain is preferably 8 to 40, more preferably 12 to 25, from the viewpoint of compatibility between the functional domain function and the DNA sequence recognition specificity. Even more preferably, it is 15-20.
As a DNA binding module, for example, a TAL effector repeat (effect
or repeat). As the length of one DNA binding module,
For example, 20-45, 30-38, 32-36, 34, etc. are mentioned. The length of the DNA binding module contained in the DNA binding domain is preferably the same for all DNA binding modules. Examples of DNA binding modules include SEQ ID NOs: 2, 4
, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, a polypeptide consisting of the first to 34th amino acid sequences. SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 3
12 in a polypeptide consisting of 0 or 32 amino acid sequence from 1 to 34
When the th amino acid and thirteenth amino acid are H and D in this order, the DNA binding domain recognizes C as a base, and when it is N and G in order, the DNA binding domain is a base When T is recognized as N and I in that order, the DNA binding domain recognizes A as a base, and when N and N in order, the DNA binding domain recognizes G as a base. To do. Examples of the DNA binding module include SEQ ID NOs: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32
A polypeptide having substantially 85%, 90%, 95%, or 97% identity with the first to 34th amino acid sequences and substantially retaining the function of recognizing one base pair.

In the polypeptide of the first aspect of the present invention, the combination of the x-th amino acid and the y-th amino acid in the 4n-3rd DNA binding module from the N-terminus is the same for any n. Further, the combination of the x-th amino acid and the y-th amino acid in the 4n-2nd DNA binding module from the N-terminus is the same for any n. 4 from the N-terminal
The combination of the xth and yth amino acids in the (n-1) th DNA binding module is the same for any n. Further, the combination of the x-th amino acid and the y-th amino acid in the 4n-th DNA binding module from the N-terminus is the same for any n. Here, n is a natural number of 1 to 10, preferably 1 to 7, more preferably 1
It is a natural number of ~ 5. n is preferably a natural number sufficient to indicate all of the DNA binding modules included in the DNA binding domain. Further, x is a natural number of 1 to 40, preferably 1 to 10, more preferably 2 to 6, even more preferably 3.
A natural number of ˜5, most preferably a natural number of 4. In addition, y is a natural number of 1 to 40, preferably a natural number of 25 to 40, more preferably a natural number of 30 to 36, still more preferably a natural number of 31 to 33, and most preferably a natural number 32. x and y are different natural numbers. The numerical values of x and y can vary depending on the length of the DNA binding module used. x is preferably a numerical value indicating a position corresponding to the second amino acid in a DNA binding module composed of 34 amino acids. y is preferably D consisting of 34 amino acids.
It is a numerical value indicating the position corresponding to the 32nd amino acid in the NA binding module.

In the polypeptide of the first aspect of the present invention, the combination of the x-th amino acid and the y-th amino acid in the 4n-3rd DNA binding module from the N-terminus, the xth in the 4n-2th DNA-binding module from the N-terminus A combination of the amino acid and the y-th amino acid, a combination of the x-th amino acid and the y-th amino acid in the 4n-1st DNA binding module from the N-terminus, and the x-th amino acid in the 4n-th DNA binding module from the N-terminus The combination of the yth amino acid is different from each other. Here, n is a natural number of 1 to 10, preferably a natural number of 1 to 7, and more preferably a natural number of 1 to 5. n is preferably a natural number sufficient to indicate all of the DNA binding modules included in the DNA binding domain. X is a natural number of 1 to 40, preferably a natural number of 1 to 10, more preferably a natural number of 2 to 6, even more preferably a natural number of 3 to 5, most preferably a natural number of 4. In addition, y is a natural number of 1 to 40, preferably a natural number of 25 to 40, more preferably a natural number of 30 to 36, still more preferably a natural number of 31 to 33, and most preferably a natural number 32. x and y are different natural numbers. Preferably, 4n from the N-terminus
A combination of the x-th amino acid and the y-th amino acid in the third DNA-binding module, a combination of the 4th-nth DNA-binding module from the N-terminal and the x-th amino acid and the y-th amino acid, and the 4n-th from the N-terminal The combination of the x-th amino acid and the y-th amino acid in the DNA binding module of FIG. 1 is from the combination of D and D in the order of x and y, the combination of E and A, the combination of D and A, and the combination of A and D, respectively. Chosen from the group of

In the second aspect, the present invention provides the polypeptide of the first aspect, wherein the functional domain is a DNA cleavage domain.

In the third aspect, the present invention provides a vector comprising a polynucleotide encoding the polypeptide of the first aspect or the second aspect. Examples of the vector include a plasmid vector, a cosmid vector, a virus vector, and an artificial chromosome vector. Artificial chromosome vectors include yeast artificial chromosome vector (YAC), bacterial artificial chromosome vector (BAC), P1 artificial chromosome vector (PAC), mouse artificial chromosome vector (MAC)
), Human artificial chromosome vector (HAC). In addition, as a component of the vector,
Examples thereof include nucleic acids such as DNA and RNA, and nucleic acid analogs such as GNA, LNA, BNA, PNA and TNA. The vector may be modified with components other than nucleic acids such as sugars.

The polypeptide of the first aspect or the second aspect of the present invention can be prepared by introducing and expressing the vector of the third aspect of the present invention into a cell or the like. In addition, by introducing the vector of the third aspect of the present invention into a cell or the like and expressing it, DNA modification such as DNA recombination or DNA cleavage; expression of other enzyme activities such as transcriptional regulation in the cell A desired function corresponding to a functional domain such as labeling of a DNA region with a reporter protein can be exhibited. When the functional domain is a DNA cleavage domain, a plurality of, preferably two, vectors of the third aspect of the present invention are introduced into a cell or the like,
By expressing it, double-strand breaks specific to the base sequence can be generated on the genomic DNA of the cell into which the vector has been introduced, and mutations can be introduced into the genome of the cell. Examples of the origin of the cell into which the vector of the third aspect of the present invention is introduced include Drosophila,
Examples include animals such as zebrafish and mammals such as mice, plants such as Arabidopsis thaliana, and cultured cells such as ES cells and iPS cells.

In the fourth aspect, the present invention provides a vector library for producing the vector of the third aspect. The vector library according to the fourth aspect of the present invention is composed of a plurality of vectors. The vector library preferably contains exhaustively the vectors useful for producing the vector of the third aspect with respect to the four base combinations recognized by the combination of the four DNA binding modules. As long as the vector of
The included vectors need not be exhaustive. The vector library according to the fourth aspect of the present invention includes, for example, 6 to 9, 10 to 13, 14 to 17, or 18 to 21 DNA binding modules. A vector for exhaustively constructing the polypeptide of the two aspects, preferably the polypeptide of the first or second aspect of the present invention, preferably comprising 14 to 21 DNA binding modules Contains vectors for construction. When the polypeptide of the first aspect or the second aspect of the present invention includes 14 to 21 DNA binding modules, the compatibility of high recognition specificity and high function by the functional domain is particularly excellent. Therefore, such a vector library is excellent because it can easily produce the highly effective polypeptide of the first aspect or the second aspect of the present invention even though the number of vectors is small.

All vectors constituting the vector library of the fourth aspect of the present invention are, in order from the 5 ′ end, a first restriction enzyme cleavage site, a polynucleotide encoding four DNA binding modules, and a second restriction enzyme. Has a cleavage site.

The combination of the first restriction enzyme cleavage site and the second restriction enzyme cleavage site contained in the vector constituting the vector library of the fourth aspect of the present invention is a type A restriction enzyme cleavage site and a type B restriction enzyme. Combinations of cleavage sites, combinations of type A restriction enzyme cleavage sites and type C restriction enzyme cleavage sites, combinations of type A restriction enzyme cleavage sites and type D restriction enzyme cleavage sites, type A restriction enzyme cleavage sites and E Type restriction enzyme cleavage site combination, type B restriction enzyme cleavage site and type C restriction enzyme cleavage site combination, type C restriction enzyme cleavage site and type D restriction enzyme cleavage site combination, and type D restriction One of the combination of an enzyme cleavage site and an E-type restriction enzyme cleavage site. Type A to type E for restriction enzyme cleavage sites are notation set for convenience in this specification in order to show the difference in properties of restriction enzyme cleavage sites. If the type is different,
When the properties of the restriction enzyme cleavage sites are different from each other and the type is the same, it indicates that the properties of the restriction enzyme cleavage sites are common. In the vector library according to the fourth aspect of the present invention, the A-type restriction enzyme cleavage site to the E-type restriction enzyme cleavage site are cleaved by the same restriction enzyme. In addition, the A-type restriction enzyme cleavage site to the E-type restriction enzyme cleavage site all generate different cleavage ends by cleavage with the same restriction enzyme. Examples of such a restriction enzyme cleavage site include a cleavage site by a restriction enzyme (for example, BsaI, BbsI, BsmBI, etc.) that cleaves an arbitrary site adjacent to a recognition site by a restriction enzyme.

As shown in STEP 2 of FIG. 1, when it is intended to produce the vector of the third aspect of the present invention containing 18 to 21 DNA binding modules, the vector live of the fourth aspect of the present invention is used. The rally is a vector in which a combination of a first restriction enzyme cleavage site and a second restriction enzyme cleavage site is a combination of a restriction enzyme cleavage site of type A and a restriction enzyme cleavage site of type B, first
A combination of the restriction enzyme cleavage site and the second restriction enzyme cleavage site of B is a combination of a B-type restriction enzyme cleavage site and a C-type restriction enzyme cleavage site, a first restriction enzyme cleavage site and a second restriction enzyme A vector in which the combination of cleavage sites is a combination of a C-type restriction enzyme cleavage site and a D-type restriction enzyme cleavage site, and a combination of a first restriction enzyme cleavage site and a second restriction enzyme cleavage site is a D-type restriction enzyme A vector that is a combination of a cleavage site and an E-type restriction enzyme cleavage site is preferably comprehensively included for recognition bases by the DNA binding module.

In addition, as shown in STEP 2 of FIG. 1, when it is intended to produce the vector of the third aspect of the present invention containing 14 to 17 DNA binding modules, the fourth aspect of the present invention The vector library is a vector in which a combination of a first restriction enzyme cleavage site and a second restriction enzyme cleavage site is a combination of an A type restriction enzyme cleavage site and a C type restriction enzyme cleavage site, a first restriction enzyme cleavage site A combination of a site and a second restriction enzyme cleavage site is a combination of a C-type restriction enzyme cleavage site and a D-type restriction enzyme cleavage site, and a first restriction enzyme cleavage site and a second restriction enzyme cleavage site A vector whose combination is a combination of a D-type restriction enzyme cleavage site and an E-type restriction enzyme cleavage site is preferably comprehensively included with respect to recognition bases by the DNA binding module.

Furthermore, as shown in STEP2 of FIG. 1, when it is intended to produce the vector of the third aspect of the present invention containing 10 to 13 DNA binding modules, the fourth aspect of the present invention The vector library includes a vector in which a combination of a first restriction enzyme cleavage site and a second restriction enzyme cleavage site is a combination of a type A restriction enzyme cleavage site and a type D restriction enzyme cleavage site, and the first restriction enzyme A vector wherein the combination of the cleavage site and the second restriction enzyme cleavage site is a combination of a D-type restriction enzyme cleavage site and an E-type restriction enzyme cleavage site,
Comprehensive information on recognition bases by DNA binding modules.

Furthermore, as shown in STEP 2 of FIG. 1, when the purpose is to produce a vector of the third aspect of the present invention containing 6 to 9 DNA binding modules, the fourth aspect of the present invention The vector library is a vector in which a combination of a first restriction enzyme cleavage site and a second restriction enzyme cleavage site is a combination of a restriction enzyme cleavage site of type A and a restriction enzyme cleavage site of type E, preferably DNA binding It contains comprehensively about the recognition base by the module.

The vector constituting the vector library of the fourth aspect of the present invention includes a DNA binding module. The length of one DNA binding module is, for example, 30 to 38, 32 to
36 or 34. The length of the DNA binding module contained in the DNA binding domain is preferably the same for all the vectors constituting the vector library. The length of the DNA binding module contained in the DNA binding domain is preferably the same for all four DNA binding modules contained in the vector constituting the vector library. Examples of the DNA binding module include SEQ ID NOs: 2, 4, 6
, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 3
2 is a polypeptide having the amino acid sequence from the 1st to the 34th amino acid. DN
As the A coupling module, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16
, 18, 20, 22, 24, 26, 28, 30, or 32 with 1% to 34th amino acid sequence, 85%, 90%, 95%, or 97% identity and one base A polypeptide that substantially retains the function of recognizing a pair can be mentioned.

Four DNs contained in the vector constituting the vector library of the fourth aspect of the present invention
Among the A binding modules, the combination of the x-th amino acid and the y-th amino acid in the first DNA binding module from the 5 ′ end is the same for any vector constituting the vector library. Similarly, x in the second DNA binding module from the 5 ′ end
The combination of the amino acid and the y-th amino acid is the same for any vector constituting the vector library. In addition, the combination of the x-th amino acid and the y-th amino acid in the third DNA binding module from the 5 ′ end is the same for any vector constituting the vector library. In addition, the combination of the x-th amino acid and the y-th amino acid in the fourth DNA binding module from the 5 ′ end is the same for any vector constituting the vector library. Where x is a natural number from 1 to 40, preferably
A natural number of 1 to 10, more preferably a natural number of 2 to 6, even more preferably a natural number of 3 to 5, most preferably a natural number of 4. Further, y is a natural number of 1 to 40, preferably a natural number of 25 to 40, more preferably a natural number of 30 to 36, still more preferably,
A natural number of 31 to 33, most preferably a natural number of 32. x and y are different natural numbers. The numerical values of x and y can vary depending on the length of the DNA binding module used. x is preferably a numerical value indicating a position corresponding to the second amino acid in a DNA binding module composed of 34 amino acids. y is preferably a numerical value indicating a position corresponding to the 32nd amino acid in a DNA binding module composed of 34 amino acids.

Four DNs contained in the vector constituting the vector library of the fourth aspect of the present invention
Among the A-binding modules, the combination of the x-th amino acid and the y-th amino acid in the first DNA-binding module from the 5 ′ end, The combination of the xth and yth amino acids in the third DNA binding module from the 5 ′ end, and 4 from the 5 ′ end
The combination of the x th amino acid and the y th amino acid in the th DNA binding module is
Different from each other. Here, x is a natural number of 1 to 40, preferably a natural number of 1 to 10, more preferably a natural number of 2 to 6, even more preferably a natural number of 3 to 5, most preferably a natural number of 4. . Y is a natural number of 1 to 40, preferably a natural number of 25 to 40,
More preferably, it is a natural number of 30 to 36, even more preferably, a natural number of 31 to 33, and most preferably a natural number of 32. x and y are different natural numbers. Examples of the numerical values of x and y include the same numerical values as described above. Preferably, the first D from the 5 ′ end
The combination of the xth amino acid and the yth amino acid in the NA binding module is D and D in the order of x and y, and the combination of the xth amino acid and the yth amino acid in the second DNA binding module from the 5 ′ end Are E and A in the order of x and y, and the third D from the 5 ′ end
The combination of the xth and yth amino acids in the NA binding module is D and A in the order of x and y, and the xth and yth amino acids in the fourth DNA binding module from the 5 ′ end The combination of x and y is A and D.

If the vector library according to the fourth aspect of the present invention is used, the vector according to the third aspect of the present invention can be easily prepared. Specifically, a vector corresponding to the sequence of the DNA binding module contained in the vector of the third aspect of the present invention is selected from the vector library of the fourth aspect of the present invention, and the selected vector is type A The vector according to the third aspect of the present invention can be prepared by digesting with a restriction enzyme that cleaves the restriction enzyme cleavage site to the restriction enzyme cleavage site of type E and connecting the vector fragments obtained by digestion. . All the vectors constituting the vector library of the fourth aspect of the present invention have two restriction enzyme cleavage sites that are cleaved by the same restriction enzyme, and the restriction enzyme cleavage sites are cleaved by the enzyme. Gives different cut ends. Therefore, in the production of the vector of the third aspect of the present invention, the digestion of the selected vector and the connection of the vector fragments can be carried out in the same reaction solution, respectively. Therefore, the vector of the third aspect of the present invention can be prepared very simply by using the vector library of the fourth aspect of the present invention.

In the fifth aspect, the present invention provides a vector set for preparing the vector library of the fourth aspect of the present invention.

The vector set according to the fifth aspect of the present invention includes a plurality of vectors. Vector set is
It is preferable to comprehensively include vectors useful for preparing the vector library of the fourth aspect, but the included vectors are not exhaustive as long as the vector library of the fourth aspect can be prepared. Also good.

All the vectors included in the vector set of the fifth aspect of the present invention include a first restriction enzyme cleavage site, a DNA binding module, and a second restriction enzyme cleavage site in order from the 5 ′ end. The first restriction enzyme cleavage site and the second restriction enzyme cleavage site are the fourth restriction site of the present invention.
It is preferable that it is a site | part which is not cut | disconnected by the restriction enzyme which cut | disconnects the 1st restriction enzyme cutting site and the 2nd restriction enzyme cutting site contained in the vector which comprises the vector library of aspect. In this case, the vector of the third aspect from the vector library of the fourth aspect,
It can be produced more easily.

The length of the DNA binding module included in the vector included in the vector set of the fifth aspect of the present invention is preferably the same for all vectors included in the vector set. Examples of the DNA binding module include, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 1
Polypeptides consisting of amino acid sequences from the 1st to the 34th amino acids of 4, 16, 18, 20, 22, 24, 26, 28, 30, or 32 are mentioned. Examples of the DNA binding module include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, or 32 amino acid sequence 1 to 34, 85%,
Polypeptides that have 90%, 95%, or 97% identity and substantially retain the function of recognizing one base pair.

The first restriction enzyme cleavage site and the second restriction enzyme cleavage site contained in the vector included in the vector set of the fifth aspect of the present invention are cleaved by the same restriction enzyme. In addition, the first restriction enzyme cleavage site and the second restriction enzyme cleavage site cause different cleavage ends from being cleaved by the same restriction enzyme. Examples of such a restriction enzyme cleavage site include a cleavage site by a restriction enzyme (for example, BsaI, BbsI, BsmBI, etc.) that cleaves an arbitrary site adjacent to a recognition site by a restriction enzyme.

The combination of the xth amino acid and the yth amino acid in the DNA binding module included in the vector included in the vector set of the fifth aspect of the present invention is any one of four different combinations. The four different combinations of the xth amino acid and the yth amino acid include, for example, in the order of x and y, a combination of D and D, a combination of E and A, a combination of D and A, and A and D
The combination of these is mentioned. Here, x is a natural number of 1 to 40, preferably a natural number of 1 to 10, more preferably a natural number of 2 to 6, even more preferably a natural number of 3 to 5, most preferably a natural number of 4. . In addition, y is a natural number of 1 to 40, preferably a natural number of 25 to 40, more preferably a natural number of 30 to 36, still more preferably a natural number of 31 to 33, and most preferably a natural number 32. x and y are different natural numbers. x and y
The number of can vary depending on the length of the DNA binding module used. x is preferably
It is a numerical value indicating the position corresponding to the second amino acid in a DNA binding module consisting of 34 amino acids. y is preferably a numerical value indicating a position corresponding to the 32nd amino acid in a DNA binding module composed of 34 amino acids.

As a vector included in the vector set of the fifth aspect of the present invention, the combination of the first restriction enzyme cleavage site and the second restriction enzyme cleavage site is an α-type restriction enzyme cleavage site and β
A combination of restriction enzyme cleavage sites of the type, wherein the DNA binding module recognizes bases A, T, G or C; the combination of the first restriction enzyme cleavage site and the second restriction enzyme cleavage site is β Type restriction enzyme cleavage site and γ type restriction enzyme cleavage site, wherein the DNA binding module recognizes base A, T, G, or C; first restriction enzyme cleavage site and second restriction The combination of the enzyme cleavage site is a combination of a γ-type restriction enzyme cleavage site and a δ-type restriction enzyme cleavage site, and the DNA binding module recognizes the base A, T, G, or C; first restriction The combination of the enzyme cleavage site and the second restriction enzyme cleavage site is δ
Examples include a combination of a type restriction enzyme cleavage site and an ε type restriction enzyme cleavage site, wherein the DNA binding module recognizes bases A, T, G, or C. Here, α type to ε type for the restriction enzyme cleavage site are notation set for convenience in this specification in order to show the difference in the properties of the restriction enzyme cleavage sites. When the types are different, the properties of the restriction enzyme cleavage sites are different from each other, and when the types are the same, the properties of the restriction enzyme cleavage sites are common. The vector set of the fifth aspect of the present invention preferably includes all the vectors exemplified above. In this case, the vector library according to the fourth aspect of the present invention of any aspect can be prepared.

By using the vector set of the fifth aspect of the present invention, the vector library of the fourth aspect of the present invention can be easily prepared. Specifically, four vectors included in the vector set of the fifth aspect of the present invention are based on a combination of four DNA binding modules included in the vector constituting the vector library of the fourth aspect of the present invention. The selected vector is digested with a restriction enzyme that cleaves the first restriction enzyme cleavage site and the second restriction enzyme cleavage site, and the vector fragment obtained by digestion is ligated, thereby The vector library of the fourth aspect can be prepared. All the vectors included in the vector set of the fifth aspect of the present invention have two restriction enzyme cleavage sites that are cleaved by the same restriction enzyme, and the restriction enzyme cleavage sites are cleaved by the enzyme. Create different cut ends. Therefore, in the preparation of the vector library according to the fourth aspect of the present invention, the selected vector can be digested and the vector fragments can be connected in the same reaction solution. Therefore, if the vector set of the fifth aspect of the present invention is used, the vector library of the fourth aspect of the present invention can be prepared very simply.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the scope of the examples.

Example 1 Preparation of vector set:
A sequence in which a recognition site for the restriction enzyme BsaI is added to both ends of the base sequence shown in FIG. 2 is prepared by artificial gene synthesis, inserted into pBluescript SK vector, and used for STEP1 in FIG. 1 (p1HD-p4HD, p1NG-p4NG, p
1NI-p4NI, p1NN-p4NN).

Example 2 Preparation of a vector library:
Using the pFUS_B6 vector (Addgene) as a template, a pFUS2 vector shown in STEP 1 of FIG. 1 was prepared by In-Fusion cloning (Clontech). As shown in STEP 1 of FIG. 1, a Golden Gate reaction was performed using the prepared pFUS2 vector and the vector set prepared in Example 1 to prepare a vector library.

Example 3 Preparation of TALEN expression vector:
Bsm incorporating the module after making by In-Fusion cloning using pTALEN_v2 and pcDNA-TAL-NC2 (both Addgene)
A sequence around the BI site was prepared by In-Fusion cloning, and a globin leader sequence was inserted upstream of the start codon by In-Fusion cloning to produce a ptCMV vector shown in STEP 2 of FIG. The prepared ptCMV vector, the vector library prepared in Example 2, and Golden Gat
e TALEN and TAL Effector Kit, Yamamoto La
b Using the vectors included in TALEN Accessory Pack (both Addgene), as shown in STEP 2 of FIG. 1, by the Golden Gate method,
A DNA binding domain was inserted to produce a TALEN expression vector. As a ptCMV vector, the region adjacent to the N-terminal side of the DNA binding domain (TALEN-N ′) has 153 amino acids, and the region between the C-terminal side of the DNA binding domain and the DNA cleavage domain (TALEN-C) The thing whose amino acid number of ') is 47 was used. An example of the base sequence and amino acid sequence of the prepared TALEN is shown in FIG.
As shown in FIG. 3, the 4th and 32nd amino acids of the DNA binding module (total 34 amino acids) in the TALEN expression vector of Example 3 are 4n-3, 4n-2, 4n-1 and The 4nth (n is a natural number) DNA binding module was different. Further, the 4th and 32nd amino acids in the 4n-3rd (n is a natural number) DNA binding module were common to the respective DNA binding modules. This means that the 4n-2nd, 4n-1th, and 4nth (n is a natural number) D
The same was true for the NA binding module. As described above, by using the vector set of Example 1 and the vector library of Example 2, a TALEN expression vector having a repetitive pattern for every four DNA binding modules could be produced.

Comparative Example 1 Preparation of TALEN expression vector:
Instead of the vector set prepared in Example 1, Golden Gate TA
PH contained in LEN and TAL Effector Kit (Addgene)
D1-6, pNG1-6, pNI1-6, pNN1-6 are used, and Yamamoto Lab TALEN Accessory is used as the pFUS vector used in the reaction.
Golden Gat is the same as in Example 2 except that Pack (Addgene) is used.
e Reaction was performed to prepare a vector library. Using the prepared vector library, a Golden Gate reaction was performed in the same manner as in Example 3 to prepare a TALEN expression vector.

Comparative Example 2 Preparation of TALEN expression vector:
As a ptCMV vector, a region adjacent to the N-terminal side of the DNA binding domain (TALE
N—N ′) has 136 amino acids, and the C-terminal side of the DNA binding domain and the region between the DNA cleavage domains (TALEN-C ′) have 63 amino acids,
The same method as in Example 3 was performed to prepare a TALEN expression vector.

Comparative Example 3 Preparation of TALEN expression vector:
As a ptCMV vector, a region adjacent to the N-terminal side of the DNA binding domain (TALE
N—N ′) has 136 amino acids, and the C-terminal side of the DNA binding domain and the region between the DNA cleavage domains (TALEN-C ′) have 63 amino acids,
A TALEN expression vector was prepared in the same manner as in Comparative Example 1.

Test Example 1 Evaluation of recognition specificity of TALEN:
TALEN expression vectors (L14 to L20 and R14 to R20) for recognizing sites as shown in FIG. 4B were prepared in the same manner as in Example 3 or Comparative Examples 1 to 3.
As shown in the table of FIG. 4C, 1 type from L14 to L20 and 1 type from R14 to R20 were combined into left and right TALEN expression vectors, and the sequence shown in FIG. 4B was used as the target sequence of TALEN. TALEN activity was evaluated by a single-strand annealing assay using HEK293T cells (see Non-Patent Document 5).
Specifically, the single strand annealing assay was performed as follows. First, a reporter vector in which a target sequence of the target TALEN was inserted into a reporter vector (pGL4-SSA; Addgene) in which a firefly luciferase gene was fragmented and ligated downstream of the CMV promoter was prepared. At this time, the target sequence of TALEN was prepared by annealing a synthetic oligo and the BsaI-treated pGL4-SSA vector was labeled with Li.
It inserted using gate-Convenience Kit (Nippon Gene).
Thereafter, the prepared reporter vector was combined with a TALEN expression vector and a pRL-CMV vector (Promega) which is an expression vector for Renilla luciferase.
After introduction into HEK293T cells by a lipofection method on a well plate and culturing for 24 hours, Dual-Glo Luciferase Assay System (
Reporter activity was measured using Promega). The amount of DNA introduced is determined by the left and right TALEN
Each expression vector is 200 ng, the reporter vector is 100 ng, and pRL-CM
The V vector is 20 ng. For the measurement of chemiluminescence, TriStar LB 941 pl
An ate reader was used.
FIG. 4C shows the relative value of the reporter activity in each of the combinations of the left and right TALEN expression vectors in Example 3 and Comparative Examples 1 to 3, compared to the combination of L20 and R17 in Comparative Example 1. Left and right TAL for each left and right TALEN combination
The length (number of bases) of the spacer region sandwiched between EN recognition sites is shown in the table of FIG. 4B.
As shown in FIG. 4C, in the case of Example 3 and Comparative Example 1, the spacer region was 12 to
It can be seen that, in a limited case such as 15, specific high activity is obtained, and specific cleavage to the limited spacer region is possible. On the other hand, in the case of Comparative Example 2 and Comparative Example 3, the height of the activity is not particularly related to the length of the spacer region, and it is possible to recognize and cleave sequences having different spacer sequence lengths. Is high. From this, it can be seen that by using the TALEN expression vector of Example 3, it is possible to carry out effective DNA cleavage with less non-specific cleavage for different sequences of the spacer region.

Test Example 2 Evaluation of TALEN activity:
A left and right pair of TALEN expression vectors (ATM (
L17 on the left, R17 on the right), APC (L17 on the left, R17 on the right), and eGFP (L20 on the left, R18 on the right)) were prepared in the same manner as in Example 3 or Comparative Examples 1-3. did. Left and right TA
Using the LEN expression vector, the sequence shown in FIG. 5A as the target sequence of TALEN, and using the same method as in Test Example 1, single strand annealing assay using HEK293T cells (Non-patent Document 5) And TALEN activity was evaluated.
The result is shown in FIG. 5B. In FIG. 5B, the numerical value on the vertical axis is the relative value of the reporter activity compared to the combination of L20 and R17 of Comparative Example 1 prepared in Test Example 1. As shown in FIG. 5B, in the case of Example 3, a higher TALEN DNA cleavage activity was observed in any of ATM, APC, and eGPF as compared with Comparative Example 1. From this, it was found that the DNA cleavage activity of TALEN can be improved by having the repeating structure of the present invention for the DNA binding module.

Test Example 3 Evaluation of recognition specificity of TALEN:
A left and right pair (L19 as left and R18 as right) of a TALEN expression vector that recognizes the site as shown in FIG. 6A was prepared in the same manner as in Example 3 or Comparative Examples 1 to 3. Using the left and right pairs produced by each method as the left and right TALEN expression vectors, the sequences shown in FIG. 6A (no mismatches, left 1 mismatch, right 0 mismatch (L: 1 mismatch / R: 0 mismatch), left 1 Mismatch Right 1 mismatch (L: 1 missmatch / R: 1 missmatch), Left 2 mismatch Right 2 mismatch (L: 2 missmatches / R: 2 missmatch)
ches)) as the target sequence of TALEN, and using the same method as in Test Example 1,
Single-strand annealing assay using EK293T cells (Non-patent Document 5)
) And TALEN activity was evaluated. When each sequence in FIG. 6A is used as the target sequence of TALEN, a mismatch occurs in the lower case part in FIG. 6A. Therefore, by comparing the results of the TALEN activity evaluation for the target sequence of TALEN, It is possible to compare the recognition specificity of TALENs.
The result is shown in FIG. 6B. The numerical value on the vertical axis in FIG. 6B shows the relative activity obtained by dividing the measured value of firefly luciferase activity by the measured value of Renilla luciferase activity. As shown in FIG. 6B,
When the TALEN expression vector of Comparative Example 2 was used, high activity was observed even when the left 2 mismatch right 2 mismatch sequence was used, and the recognition specificity of the TALEN expression vector of Comparative Example 2 was It was low. In contrast, when the TALEN expression vector of Example 3 was used, almost complete loss of activity was observed when the left 2 mismatch right 2 mismatch sequence was used. Therefore, the TALEN expression vector of Example 3 can perform highly specific DNA cleavage while retaining high cleavage activity as shown in Test Example 2, and the TALEN expression vector of Example 3 It was found that the target DNA can be safely cleaved with high probability by using.

Claims (6)

A polypeptide comprising a DNA binding domain and a functional domain comprising:
Here, the DNA binding domain and the functional domain are connected by a polypeptide consisting of 40 to 50 amino acids,
The DNA binding domain comprises 16-20 DNA binding modules consisting of 34 amino acids continuously from the N-terminal side,
The combination of the 4th and 32nd amino acids in the 4n-3rd DNA binding module from the N-terminus is the same for any n;
The combination of the 4th and 32nd amino acids in the 4n-2nd DNA binding module from the N-terminus is the same for any n;
The combination of the 4th and 32nd amino acids in the 4n-1st DNA binding module from the N-terminus is the same for any n;
The combination of the 4th and 32nd amino acids in the 4nth DNA binding module from the N-terminus is the same for any n
Combination of the 4th amino acid and the 32nd amino acid in the 4n-3rd DNA binding module from the N-terminal, combination of the 4th amino acid and the 32nd amino acid in the 4n-2nd DNA binding module from the N-terminal, the N-terminal The combination of the 4th amino acid and the 32nd amino acid in the 4n-1st DNA binding module to 4n-1 and the combination of the 4th amino acid and the 32nd amino acid in the 4nth DNA binding module from the N-terminus are different from each other,
Here, n is a natural number of 1 to 5,
A polypeptide whose origin of the DNA binding domain is TALE.   A vector comprising a polynucleotide encoding the polypeptide of claim 1. A vector library for producing the vector according to claim 2,
Here, the vector library is composed of a plurality of vectors having a first restriction enzyme cleavage site, a polynucleotide encoding four DNA binding modules, and a second restriction enzyme cleavage site in order from the 5 ′ end,
Here, the combination of the first restriction enzyme cleavage site and the second restriction enzyme cleavage site is a combination of an A type restriction enzyme cleavage site and a B type restriction enzyme cleavage site, an A type restriction enzyme cleavage site and a C type. A combination of restriction enzyme cleavage sites of type A, a combination of restriction enzyme cleavage sites of type A and a restriction enzyme cleavage site of type D, a combination of restriction enzyme cleavage sites of type A and a restriction enzyme cleavage site of type E, and a restriction enzyme cleavage of type B Any combination of a site and a C-type restriction enzyme cleavage site, a combination of a C-type restriction enzyme cleavage site and a D-type restriction enzyme cleavage site, and a combination of a D-type restriction enzyme cleavage site and an E-type restriction enzyme cleavage site Here, each of the A-type restriction enzyme cleavage site to the E-type restriction enzyme cleavage site causes cleavage ends different from each other by cleavage with the same restriction enzyme,
Among the four DNA binding modules, the combination of the fourth amino acid and the 32nd amino acid in the first DNA binding module from the 5 ′ end is the same for any vector, and the second DNA binding module from the 5 ′ end. The combination of the 4th and 32nd amino acids in is the same for any vector, and the combination of the 4th and 32nd amino acids in the 3rd DNA binding module from the 5 ′ end is the same for any vector. The combination of amino acids 4 and 32 in the fourth DNA binding module from the 5 ′ end is the same for any vector,
Combination of the 4th and 32nd amino acids in the first DNA binding module from the 5 'end Combination of the 4th and 32nd amino acids in the 2nd DNA binding module from the 5' end From the 5 'end The combination of the 4th amino acid and the 32nd amino acid in the 3rd DNA binding module and the combination of the 4th amino acid and the 32nd amino acid in the 4th DNA binding module from the 5 ′ end are different from each other.
Vector library. A vector set for preparing the vector library according to claim 3,
Here, the vector set includes a plurality of vectors including a first restriction enzyme cleavage site, a DNA binding module, and a second restriction enzyme cleavage site in order from the 5 ′ end,
The first restriction enzyme cleavage site and the second restriction enzyme cleavage site generate different cleavage ends by cleavage with the same restriction enzyme,
Here, the first restriction enzyme cleavage site and the second restriction enzyme cleavage site are the first restriction enzyme cleavage site and the second restriction enzyme contained in the vector constituting the vector library according to claim 3. It is a site that is not cleaved by a restriction enzyme that cleaves the cleavage site,
The vector set, wherein the combination of the fourth amino acid and the 32nd amino acid in the DNA binding module is one of four different combinations.   A method for producing a modified cell, comprising introducing the vector according to claim 2 into a cell (excluding a cell in a human individual) and expressing the same. A method for producing a plant or non-human animal containing a modified cell, comprising the step of introducing the vector according to claim 2 into a cell (excluding cells in a human individual) and expressing the vector .