JP7084021B2 - Targeting vector - Google Patents

Description

The present invention relates to a targeting vector. More specifically, the present invention relates to a highly versatile targeting vector capable of dramatically increasing the efficiency of homologous recombination in genetic recombination.

Genetically modified mice are an important tool for understanding gene function in embryology and disease science. In the conventional gene targeting method, a targeting vector designed for each target gene is prepared and introduced into embryonic stem (ES) cells, and mutation is introduced by homologous recombination (HR). The introduction of mutations by homologous recombination utilizes the repair mechanism of DNA double-strand break (DSB) inherent in cells, and the targeting vector introduced into the nucleus during the repair of DSB is used. It is done by referencing and repairing. The targeting vector is introduced into cells together with a drug resistance gene, and by screening cell colonies with drug resistance, targeted ES cells specifically inserted into the target genome are selected. Injecting targeted ES cells into wild-type blastocysts produces mice containing a modification of the target gene.

The target gene modification technique, that is, the genome editing technique, is premised on the introduction of DSB into the target DNA, and has been developed by the development and application of artificial restriction enzymes for introducing DSB. There are two types of artificial restriction enzymes: ZFN, which has a Zinc finger as a DNA binding domain, and TALEN, which has a binding domain derived from the transcription factor TALE protein derived from the plant non-pathogenic bacterium Xantomonas. When introduced into cells, artificial restriction enzymes bind to adjacent target sequences to form dimers and cleave double-stranded DNA.

In recent years, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated proteins) has been developed as a next-generation genome editing technology (see, for example, Patent Document 1, Non-Patent Documents 1 and 2). The CRISPR-Cas9 method co-expresses a Cas9 nuclease (DNA-cleaving enzyme) with a guide RNA vector expressed by a human U6 polymerase III promoter to cleave a specific target DNA sequence. When double-strand breaks are introduced by Cas9 nucleases, they are repaired as usual by the pathway of non-homologous end ligation or homologous recombination repair. Overexpression of CRISPR-Cas9 leads to repeated double-strand breaks, which induce repair errors. In the non-homologous end-linking pathway, errors in the repair process can introduce mutations such as deletions and insertions, which can cause frameshifts and disrupt genes if they are mutations within the gene. On the other hand, in the repair of the homologous recombination pathway, it is possible to insert a foreign gene into the cleavage site by co-introducing a donor vector to perform gene knock-in or replacement of SNPs. The CRISPR-Cas9 method is widely used as a third-generation genome editing tool following ZFN and TALEN as a gene modification technique capable of deleting, replacing, or inserting an arbitrary location in a genome sequence.

U.S. Pat. No. 8697359

Cell 153, 910-918 (2013) Cell 154, 1370-1379 (2013)

In genome editing using ZFNs and TALENs, most of the screenings for drug resistance genes are due to non-homologous end ligation, and there is a problem that the efficiency of obtaining homologous recombination is low. Although the CRISPR-Cas9 method has improved the efficiency of genome editing, the introduction of the modification itself is responsible for the repair mechanism for double-strand breaks in the original genomic DNA of the cell. There is no change. For example, in double-strand break repair via a homologous recombination pathway, a foreign gene can be inserted into the cut portion by co-introducing a donor vector, but such insertion is two of the genomic DNA inherent in the cell. It relies on a repair mechanism for chain breaks. Therefore, even with the CRISPR-Cas9 method, the drawback that the efficiency of homologous recombination is not high has not been solved yet.

In this way, homologous recombination itself is an intrinsic phenomenon carried out by the repair mechanism of the genomic DNA inherent in the cell for DSB, so that it is homologous between the gene inherent in the cell and the foreign gene introduced into the cell. It was the recognition among those skilled in the art that there was no room for improvement in the probability of recombination by artificial ingenuity. For this reason, no attention has been paid to improving the efficiency of endogenous homologous recombination.

Therefore, an object of the present invention is to provide a genome modification technique capable of increasing the efficiency of endogenous homologous recombination.

As a result of diligent studies, the present inventor has found that the shape of the terminal of the targeting vector has a great influence on the efficiency of homologous recombination. Furthermore, the present inventor has a dramatic 3'protruding shape at both ends of the targeting vectoron, and the length of the protruding end (overhang portion) is more than a predetermined value, which is not expected by those skilled in the art. It was found that homologous recombination occurs with high efficiency. The present invention has been completed by further studies based on this finding.

That is, the present invention provides the inventions of the following aspects.
Item 1. A targeting vector containing a foreign DNA fragment and a homologous DNA fragment having a homologous recombination sequence with a part of a target DNA site on both sides of the foreign DNA fragment.
A targeting vector having an overhang portion at both ends of the targeting vector having a protruding 3'end, the overhang portion having the homologous sequence, and a base length of 200 mer or more.
Item 2. Item 2. The targeting vector according to Item 1, wherein the base length of the overhang portion is 500 mer or less.
Item 3. Item 2. The targeting vector according to Item 1, wherein the foreign DNA fragment contains a marker gene.
Item 4. A targeting vector containing a foreign DNA fragment and a homologous DNA fragment having a homologous sequence capable of homologous recombination with a part of a target DNA site on both sides of the foreign DNA fragment; both ends of the targeting vector are 3'. An introduction step of introducing a targeting vector having an overhang portion having a protruding end, the overhang portion having the homologous sequence and a base length of 200 mer or more into a cell having the target DNA site on the chromosome. A method for producing a genetically modified cell, including.
Item 5. Item 6. The method for producing a gene-modified cell according to Item 4, wherein the introduction step is performed by a pronucleus injection method.
Item 6. Item 6. The method for producing a gene-modified cell according to Item 4 or 5, which does not include a double chain cleavage step of causing double chain cleavage at the target DNA site.
Item 7. Item 6. The method for producing a gene-modified cell according to Item 4 or 5, which comprises a double chain cleavage step of causing double chain cleavage at the target DNA site.
Item 8. Item 6. The method for producing a gene-modified cell according to Item 7, wherein the double-strand break step is performed by a zinc finger nuclease (ZFN), a transcriptional activator-like effector nuclease (TALEN), or a crisper-related (Cas9) nuclease.
Item 9. A linear DNA construct containing a foreign DNA fragment and a homologous DNA fragment having a homologous recombination sequence with a part of a target DNA site on both sides of the foreign DNA fragment, both ends of which are the homologous DNA fragments. The process of constructing a linear DNA construct composed of
Targeting comprising the step of allowing an exonuclease to act on the linear DNA construct to form an overhang portion at both ends of the linear DNA construct, the 3'end protruding with a base length of 200 mer or more. How to make a vector.
Item 10. Item 9. The method for producing a targeting vector according to Item 9 or 10, wherein a T7 exonuclease is used as the exonuclease in the step of generating the overhang portion, and the T7 exonuclease is allowed to act for 1 minute 20 seconds or longer.
Item 11. Item 9. A step of treating both ends of the DNA construct with a restriction enzyme that produces a 3'protruding end between the step of constructing the linear DNA construct and the step of causing the overhang portion. The method for producing a targeting vector according to.

According to the present invention, since a genome modification technique capable of improving the efficiency of endogenous homologous recombination is provided, accurate gene modification using the mechanism of endogenous homologous recombination is possible, and it can be applied to many biological systems. It can be applied.

An example of the manufacturing method (manufacturing method A) of the targeting vector of the present invention is schematically shown. An example of the manufacturing method (manufacturing method B) of the targeting vector of the present invention is schematically shown. Another example of the manufacturing method (manufacturing method B) of the targeting vector of the present invention is schematically shown. Still another example of the manufacturing method (manufacturing method B) of the targeting vector of this invention is schematically shown. The test result (a) of ExoIII activity and the test result (b) of T7 activity obtained in Test Example 1 are shown. The design of the substitution vector used for the Hprt gene targeting vector tested in Test Example 2 is shown. A schematic diagram of the shape of the Hprt gene targeting vector used in Test Example 2 and a photograph of a colony of cells subjected to gene targeting with the vector are shown. The design of the substitution vector used for the NudCD2 gene targeting vector tested in Test Example 3 is shown. The photograph (a) of the cell colony obtained by gene targeting with the NudCD2 gene targeting vector obtained in Test Example 3 and the result of Southern blotting analysis (b) are shown. The design of the substitution vector used for the Rosa26 gene targeting vector tested in Test Example 4 is shown. The results of Southern blotting analysis of clones obtained by gene targeting in Test Example 4 are shown. The design of the substitution vector used for the NudCD2 gene targeting vector tested in Test Example 5 is shown. The results of Southern blotting analysis of clones obtained by gene targeting in Test Example 5 are shown.

[1. Targeting vector]
The targeting vector of the present invention contains a foreign DNA fragment and homologous DNA fragments at both ends of the foreign DNA fragment. The homologous DNA fragment has a homologous sequence that can be homologously recombined with a part of the target DNA site. Foreign DNA fragments and homologous DNA fragments can be adjacent directly or indirectly via other sequences. Both ends of the targeting vector of the present invention each have an overhang portion with a protruding 3'end. That is, the overhang portion is single-stranded DNA. This overhang has the homologous sequence described above. Further, the overhang portion has a base length of 200 mer or more. Such a targeting vector of the present invention can dramatically improve the efficiency of homologous recombination.

[1-1. Overhang (homologous DNA fragment)]
The overhang portion composed of single-stranded DNA is composed of homologous DNA fragments, and the base length thereof is 200 mer. If the base length of the overhang portion is less than 200 mer, the desired effect of improving the homologous recombination efficiency cannot be obtained. From the viewpoint of better obtaining the effect of improving the homologous recombination efficiency, the base length of the overhang portion may be preferably 250 mer or more, more preferably 300 mer or more, and further preferably 400 mer or more. The upper limit of the base length of the overhang portion is not particularly limited, but from the viewpoint of satisfactorily obtaining the effect of improving the homologous recombination efficiency, for example, 500 mer or less, preferably 450 mer or less can be mentioned. The range of the base length of the homologous DNA fragment can be appropriately combined with the above upper limit value and lower limit value.

The homologous DNA fragment constituting the overhang portion has a homologous sequence capable of homologous recombination with a part of the target DNA site. The homology between the homologous DNA fragment and the target DNA site may be similar to the extent that homologous recombination occurs, for example, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more. , 98% or more, 99% or more, 99.9% or more, most preferably 100%. Homology can be appropriately determined by analysis using a DNA sequencer or the like.

The target vector of the present invention has a structural feature of being 3'protruding and having an overhang portion of a predetermined base length as described above, and when introduced into the cell, the DNA double-strand break (DSB) is introduced into the cell. ) Is introduced, the DNA double-strand break repair mechanism originally possessed by the cell can be made to function, and homologous recombination can be caused. As described above, the targeting vector of the present invention draws out the mechanism of endogenous homologous recombination to cause homologous recombination, so that the efficiency of homologous recombination can be dramatically improved. Since the mechanism of endogenous homologous recombination can be utilized in this way, the target vector of the present invention has an extremely wide range of versatility that is not bound by the target DNA site, foreign sequence, etc., and accurate homologous recombination is possible. Therefore, the off-target problem can also be reduced.

[1-2. Target DNA site]
The target DNA site is not particularly limited as described above. For example, the target DNA site includes loci of all genes such as Hprt gene, Nud gene, and Rosa gene. In addition, the target DNA site may contain a plurality of loci. Since the Hprt gene is present on the X chromosome and is semi-conjugated in XY cells, targeted clones can be easily identified by negative selection using 6-TG or the like by inactivating the Hprt gene. The Nud gene is involved in nuclear transport, and members of the gene family include the NudC gene, NudE gene, NudF gene, NudG gene, etc., and the targeted clones inactivate LIS1 due to the inactivation of the Nud gene. It stabilizes and results in a phenotype similar to cytoplasmic dynein deficiency or LIS1 deficiency. The Rosa gene is widely expressed at all stages of growth and can be used for the purpose of producing transgenic mice derived from embryonic stem cells.

Other examples of the target DNA site include loci of antigen-presenting genes such as the HLA gene. Modification of the antigen-presenting gene can be performed for the purpose of replacing the antigen-presenting gene of the donor (human or non-human animal) with a gene compatible with the recipient (human) to prepare an organ for transplantation. In this case, it may be necessary to replace a relatively large region of the antigen presenting region in order to obtain good compatibility. Since the targeting vector of the present invention can perform accurate gene modification using the mechanism of endogenous homologous recombination as described above, it is also suitable for replacing such a wide region. The target DNA site is not limited to the above-mentioned example, and a wide range of the genome can be set. Extensive regions of the genome include those already cloned with yeast artificial chromosomes (YAC), human artificial chromosomes (HAC), and the like. For example, the entire region of various types of HLA in humans has been cloned and libraryized by HAC and can also be used in the present invention.

In the target DNA region, a foreign DNA fragment is incorporated into a region sandwiched between regions homologous to each of the two homologous DNA fragments of the targeting vector of the present invention by homologous recombination. In the present invention, the length of the target DNA region set is not limited. Since the targeting vector of the present invention is suitable for replacing not only a narrow region but also a wide region as described above, the target DNA region may be, for example, 1 kbp or more, 10 kbp or more, or 50 kbp or more. It may be 100 kbp or more, 200 kbp or more, or 300 kbp or more. The target DNA region is not particularly limited in its upper limit, and examples thereof include 500 kbp or less.

[1-3. Foreign DNA fragment]
The sequence contained in the foreign DNA fragment is not particularly limited, and examples thereof include foreign genes and marker genes. These foreign genes are used alone or in combination as appropriate.

The foreign gene is not particularly limited, and a person skilled in the art can appropriately select the gene according to the purpose of gene recombination, such as production of a target protein and production of a genetically modified organism. When producing a target protein, examples of the foreign gene include a gene encoding a protein useful as a drug or the like. When producing a genetically modified organism, examples of the foreign gene include a gene that the host does not have (human disease gene, a marker gene, etc.), a gene that overexpresses the gene possessed by the host, and the like. In the targeting vector of the present invention, one type of foreign gene may be integrated alone, or two or more types of foreign genes may be integrated in combination.

The foreign gene can be incorporated into the targeting vector as an expression unit appropriately combined with a sequence related to gene expression. Examples of the sequence related to the expression of a foreign gene include a promoter sequence, a sequence required for gene expression such as a transcription termination signal sequence, and a sequence that regulates gene expression such as a regulatory element. Examples of the promoter arrangement include CMV, SV40, RSV, EF1α, CAG, U6, pGK and the like. Examples of the transcription termination signal sequence include a BGH poly A signal sequence, an SV40 poly A signal sequence, and the like. Regulatory elements include enhancers, IRES (internal ribosome entry site) sequences, LuxP sequences, FRT sequences and the like.

The marker gene is not particularly limited as long as it is used to identify the host expressing the gene contained in the vector of the present invention, and is, for example, a drug resistance gene, a fluorescent protein gene, a reporter gene, or flanking. Examples include probe genes.

Examples of the drug resistance gene include a neomycin resistance gene, a hyglomycin resistance gene, a zeosin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicole resistance gene, and the like.

Examples of the fluorescent protein gene include genes encoding fluorescent proteins such as GFP, YFP, CFP, BFP, Venus, DsRed, HcRed, AsRed, ZsGreen, ZsYellow, AmCyan, AcGFP, and Kaede.

Examples of the reporter gene include a luciferase gene and β-galactosidase.

As the flanking probe gene, a person skilled in the art can appropriately determine an arbitrary sequence that can be used as a detection probe when homologous recombination is confirmed by Southern blotting after acquisition of the genetically modified cell.

The base length of the foreign DNA fragment is not particularly limited, and can be appropriately determined, for example, according to the length of the target DNA site and the like. Since the targeting vector of the present invention is suitable for replacing not only a narrow region but also a wide region as described above, the base length of the foreign DNA fragment may be, for example, 1 kbp or more or 10 kbp or more. However, it may be 50 kbp or more, 100 kbp or more, 200 kbp or more, or 300 kbp or more. The base length of the foreign DNA fragment is not particularly limited in its upper limit, and examples thereof include 500 kbp or less.

[1-4. Types of targeting vectors]
The type of the targeting vector of the present invention is not particularly limited, and for example, a plasmid vector, a cosmid vector, a phosmid vector, a virus vector (adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, a lentivirus vector), and the like. Artificial chromosome vector (bacterial artificial chromosome (BAC), P1 bacteriophage artificial chromosome (PAC), Yeast artificial chromosome (YAC), human artificial chromosome (HAC) Etc.) etc. These vectors are appropriately selected by those skilled in the art depending on the size of the foreign DNA fragment and the purpose of homologous recombination.

[2. Manufacturing method of targeting vector]
Methods for producing the targeting vector of the present invention include a method of constructing a linear DNA construct and then treating it with an exonuclease (manufacturing method A; see FIG. 1), and a method of constructing a circular DNA construct and then producing a nick. Further, a method of dissociating the double chain between nicks (manufacturing method B; see FIGS. 2 to 4) can be mentioned.

[2-1. Manufacturing method A]
The production method A includes a step a1 for constructing a linear DNA construct and a step a2 (FIG. 1) for forming an overhang portion. The production method A is suitable for producing a relatively short targeting vector.

In step a1, a linear DNA construct containing a foreign DNA fragment and a homologous DNA fragment having a homologous recombine homologous sequence with a part of a target DNA site is constructed on both sides of the foreign DNA fragment. In this linear DNA construct, homologous DNA fragments are configured to be located at both ends. The foreign DNA fragment and the target DNA are as described in detail in "1. Targeting vector" described above. The homologous DNA fragment is as described in detail in "1. Targeting vector" described above, except that it is double-stranded DNA.

A method for constructing a linear DNA construct can be appropriately selected by those skilled in the art according to a conventionally known method. For example, a linear DNA construct is constructed by inserting a fragment containing a foreign DNA fragment sequence cleaved with an appropriate restriction enzyme or the like into the restriction enzyme site of the base vector or a multicloning site by a ligation reaction. be able to.

The portion of the linear DNA construct except for both ends is intact with respect to the reaction system that causes an overhang portion in the production method A. Specifically, the linear DNA construct does not have a structure or nick in which the bond between adjacent bases of one of the DNA duplexes is broken. This makes it possible to prevent the exonuclease from acting at an undesired position other than the terminal of the linear DNA construct in step a2 described later. The production method A is suitable for producing a relatively short targeting vector from the viewpoint of preparing a linear DNA construct having no nick. Specifically, the production method A is preferably used when producing a targeting vector having a foreign DNA fragment having a length of, for example, 20 kbp or less, preferably 10 kbp or less. As the lower limit of the length of the foreign DNA fragment in the production method A, for example, 1 kbp or more can be mentioned.

In step a2, an exonuclease is allowed to act on the linear DNA construct for a predetermined time to form an overhang portion. FIG. 1 schematically shows step a2. As the exonuclease, a 5'-3'exonuclease that sequentially releases mononucleotides by hydrolysis from the 5'end of DNA is used. The 5'-3'exonuclease can be appropriately selected by those skilled in the art. For example, examples of the exonuclease include T7 exonuclease (shown as T7 in FIG. 1), T5 exonuclease, lambdaxonuclease, and the like.

The action of exonucleases yields 3'protruding single-stranded DNA from double-stranded homologous DNA fragments at the ends of linear DNA constructs. By allowing the exonuclease to act for a time until the length of the single-stranded DNA becomes 200 mer or more, an overhang portion having a length that improves the homologous recombination efficiency of the single-stranded DNA can be obtained. The length of the overhang portion can be adjusted by adjusting the treatment time with an exonuclease. The treatment time with an exonuclease can be determined depending on the type of exonuclease used. For example, the inventor has confirmed that T7 exonuclease releases about 150 bases per minute. Therefore, when T7 exonuclease is used as an exonuclease, it is effective to allow it to act for 1 minute and 20 seconds or more in order to obtain an overhang portion having a length of 200 mer or more.

The production method A may further include a step of treating both ends of the DNA construct with a restriction enzyme that produces a 3'protruding end between the steps a1 and a2. This step is performed as a pretreatment for step a2. By generating a 3'end having a short base length in advance by this step, the reaction of the exonuclease in step a2 can be started more easily. The restriction enzyme used in this step is not particularly limited as long as it produces a 3'protruding end, and examples thereof include SfiI, KpnI, Sse8387I, and SacI.

[2-2. Manufacturing method B]
The production method B includes a step b1 for constructing a DNA construct, a step b2 for generating a nick, and a step b3 (FIGS. 2 to 4) for dissociating double chains between the nicks. The production method B is suitable for producing a long targeting vector.

In step b1, a DNA construct containing a foreign DNA fragment and a homologous DNA fragment having a homologous sequence capable of homologous recombination with a part of a target DNA site is constructed. The foreign DNA fragment and the target DNA are as described in detail in "1. Targeting vector" described above. The homologous DNA fragment is as described in detail in "1. Targeting vector" described above, except that it is double-stranded DNA. The DNA construct may be either a circular DNA construct or a linear DNA construct.

A method for constructing a circular DNA construct or a linear DNA construct can be appropriately selected by those skilled in the art according to a conventionally known method. For example, by inserting a fragment containing a foreign DNA fragment sequence cleaved with an appropriate restriction enzyme or the like into a restriction enzyme site or a multicloning site of a base vector by a ligation reaction, a cyclic DNA construct or linear DNA can be used. You can build a construct. Examples of the vector on which the cyclic DNA construct is based include an Escherichia coli artificial chromosome (BAC), a P1 bacteriophage artificial chromosome (PAC), and the like. Examples of the vector on which the linear DNA construct is based include a yeast artificial chromosome (YAC), a human artificial chromosome (HAC), and the like.

In step b2, the DNA construct is nicked. In the circular DNA construct, as shown on the left of FIG. 2, a pair generated at one site each on one strand and the other strand separated by a length corresponding to the overhang portion of the targeting vector. Nicks (indicated by N in the figure) can be formed. In a linear DNA construct, as shown on the left in FIG. 3, a pair of nicks generated on one strand and the other strand separated by a length corresponding to the overhang portion of the targeting vector. (Indicated by N in the figure) can be formed at two locations.

As a method for generating a pair of nicks by separating them by a predetermined distance as described above, a double nicking method using a nickase-modified Cas9 of the CRISPR-Cas9 method can be applied. Specifically, in a circular DNA construct, two guide RNAs (gRNAs) targeting two sites according to the predetermined distance are designed, and these two gRNAs are docked at the target site of the circular DNA construct. The gRNA berthed at the target site recruits the nickase-modified Cas9, and the nickase-modified Cas9 undergoes single-strand breaks at each of the target sites.

The number of nicks generated in step b2 may be at least one pair in the circular DNA construct and at least two pairs in the linear DNA construct. In order to facilitate the dissociation of DNA between nicks in step b3 described later, one and / or the other of the pair of nicks may be a plurality of nicks (that is, a plurality of nicks on one strand and / or a plurality of nicks). Multiple nicks may be generated on the other single chain in small steps). Each of the plurality of nicks is preferably 20 to 30 bases apart from each other. In order to introduce a plurality of nicks, a plurality of sets of corresponding nickase-modified Cas9 and guide RNA may be prepared.

In step b3, the double-stranded DNA between the nicks is dissociated. The method for dissociating the double-stranded DNA is not particularly limited and can be appropriately determined by those skilled in the art. For example, a method of denaturing double-stranded DNA by applying heat or making the pH alkaline can be mentioned. In the alkaline method, the pH can be returned to neutral after dissociation with an appropriate buffer. When a plurality of nicks are introduced as described above, the dissociation may be performed in small steps (for example, 20 to 30 bases at a time), so that the possibility of rear kneeling is lower and the dissociation can be performed more easily. The dissociation of the double strand between the nicks results in a 3'protruding overhang portion of a predetermined length consisting of single-stranded DNA, and the targeting vector of the present invention can be obtained.

Also in the production method B, the cyclic DNA construct is intact with respect to the reaction system that causes the overhang portion. In the production method B, since an exonuclease is not used to generate the overhang portion, the cyclic DNA construct has a nick on other sites other than the site that causes the nick in step b2 as long as the gRNA is not compatible. You may have it. Therefore, the production method B is suitable for producing a relatively long targeting vector. Specifically, the production method B is preferably used when producing a targeting vector having a foreign DNA fragment having a length of, for example, 20 kbp or more, preferably 200 kbp or more, and more preferably 300 kbp or more. The upper limit of the length of the foreign DNA fragment in the production method B is, for example, 500 kbp or less.

[3. Manufacturing method of genetically modified cells]
By introducing the above-mentioned targeting vector of the present invention into cells, it is possible to efficiently obtain genetically modified cells in which a foreign DNA fragment is incorporated into a target DNA site by homologous recombination. The method for producing a gene-modified cell of the present invention comprises an introduction step of introducing the above-mentioned targeting vector of the present invention into a cell having a target DNA site on a chromosome.

[3-1. Introduction process]
When the targeting vector having the characteristics of the present invention is introduced into the cell, the cell recognizes that the DNA double-strand break (DSB) has been introduced, so that the DNA double-strand break repair mechanism originally possessed by the cell functions. Then, homologous recombination occurs. That is, the method for producing a gene-modified cell of the present invention utilizes an endogenous homologous recombination mechanism. The mechanism of homologous recombination functions as a DNA double-strand break repair mechanism in eukaryotes, and its basic mechanism is highly conserved. Therefore, the method for producing a gene-modified cell of the present invention can be applied to cells derived from any eukaryote. Examples include cells derived from mammals (eg, mice, rats, pigs, and cattle, as well as humans, primates such as common marmosets), Xenopus laevis, zebrafish, gypsum flies, nematodes, plants, and the like.

The type of cell is not particularly limited, and examples thereof include embryonic stem cells, induced pluripotent stem cells, and fertilized eggs. Due to the high efficiency of homologous recombination of the targeting vector of the present invention, homologous recombination can also be caused by pronuclear injection into a fertilized egg, which has been used only for the production of genetically modified animals by gene insertion. Therefore, in the present invention, homologous recombinant cells can be more easily produced by introducing a pronucleus into a fertilized egg for the introduction of a targeting vector.

The method for introducing the targeting vector is not particularly limited, and a conventionally known method can be used. For example, as a viral vector method, a method of infecting a virus vector such as a retrovirus, a lentivirus, an adeno-associated virus, etc. can be mentioned, and as a non-viral vector method, a lipofection method, an electroporation method, a microinjection method, etc. Examples include physicochemical methods.

As described above, the method for producing a genetically modified cell of the present invention causes homologous recombination by recognizing that DNA double-strand break (DSB) has been introduced into the cell by introducing the target vector, and therefore, the target DNA. The double-strand break step that causes double-strand breaks at the site may not be performed.

On the other hand, the method for producing a gene-modified cell of the present invention does not exclude performing a double-strand break step that causes double-strand breaks at a target DNA site. When performing a double chain cleavage step, the targeting vector of the present invention can be used as a donor vector for repairing the double chain cleavage that has occurred. Enzymes such as zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), or crisper-related (Cas9) nucleases can be utilized for double-strand breaks. In this case, nucleic acids expressing these enzymes can be introduced into cells.

In addition, depending on the type of vector and foreign gene, a site-specific introduction system using a recombinant enzyme such as Cre / LuxP system or Flp / FRT system may be appropriately applied in consideration of acquisition efficiency of gene-modified cells. You can also do it.

[3-2. Other processes]
After the introduction step of the targeting vector, the step of selecting the genetically modified cells can be performed. The selection step can be performed by positive selection when the drug resistance gene is incorporated into the targeting vector, and by negative selection based on the inactivation of the Hprt gene when the Hprt locus is set as the target DNA site. Can be done. In addition, cell selection may be performed with high accuracy by combining these positive selection and negative selection. In addition, a promoter trap method, a poly A trap method, or the like can be used in combination as appropriate.

For the gene-modified cells obtained by the selection step, genomic DNA can be extracted as needed, and homologous recombination can be confirmed by PCR method, Southern blotting method, or the like. The genetically modified cells are then subjected to a step according to the purpose of the genetic recombination. For example, when the purpose is to produce the target protein, it can be cultured by an appropriate culture method (for example, batch culture method, fed-batch culture method, reflux culture method, etc.). In addition, when the purpose is to produce genetically modified organisms, after aggregating genetically modified cells with early embryos, transplanting them into pseudopregnant individuals to produce chimeric individuals, and mating them with wild-type individuals to obtain hetero-F1 generations. , F1 generations can be mated to obtain a homo F2 generation.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Test Example 1: Estimating the enzyme activity of exonuclease III (ExoIII) and T7 exonuclease (T7)]
The vector was intercalated with a fluorescent reporter SYBR Green I (Invitrogen) and the enzymatic activity of ExoIII or T7 was examined by detecting dsDNA using real-time quantitative PCR while digesting with ExoIII or T7.

The eGFP empty vector (Clontech) was straightened with EcoRI (leaving a 5'protruding end), SmaI (leaving a blunt end), or KpnI (leaving a 3'protruding end). The linearized eGFP empty vector was mixed with SYBR Green I and then treated with ExoIII or T7. In each well, 10 μg eGFP empty vector and 20 units of enzyme / well were contained in 20 μl of the reaction mixture. Vectors were treated with ExoIII and T7 at 37 ° C. and 30 ° C. for 50-60 minutes, respectively. The digestive activity of ExoIII or T7 was estimated from the decrease in fluorescence intensity every minute by the Applied Biosystems® 7500 Real-Time PCR System (Thermo Fisher Scientific). The experiment was performed 5 times and the obtained data were averaged.

FIG. 5 shows the test results (a) of ExoIII activity and the test results (b) of T7 activity using SYBR Green. In the figure, the X-axis shows the elapsed time (minutes), and the Y-axis shows the relative intensity of SYBR Green (standardized with a starting point of 1.0). The eGFP empty vector linearized with KpnI to have a 3'protruding end tended to be relatively resistant to ExoIII digestion, whereas T7 was very resistant to any restriction site. The activity was high. The eGFP empty vector has a length of 4.7 kbp. Considering the decrease in fluorescence intensity, the digestion rate of ExoIII was derived as 100 bases / minute, and the digestion rate of T7 was derived as 150 bases / minute.

[Test Example 2: Hprt gene targeting: Examination of terminal shape and overhang base length of targeting vector]
Using the Hprt gene on the X chromosome, targeting vectors having different terminal shapes and / or base lengths at the overhangs of the protruding ends were prepared, and the efficiency of homologous recombination by these targeting vectors was examined.

The substitution vector shown in FIG. 6 was designed for homologous targeting of the mouse Hprt gene. That is, the neomycin resistance cassette (V. L. Tybulewicz, C. E. Crawford, P. K. Jackson, R. T. Bronson, R. C. Mulligan, Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. A targeting vector containing 65, 1153-1163 (1991)) was constructed. This vector was constructed from Hprt genomic sequences isolated from isogenic 129 / Sv mouse genomic DNA by PCR amplification. As shown in FIG. 6, the targeting vector has a neomycin resistance cassette PGK-neo in which exon 3 of the Hprt gene and an adjacent intron are substituted, and has 3.4 kbp homologous regions of the Hprt gene on both sides.

C. Deng, A. Wynshaw-Boris, F. Zhou, A. Kuo, P. Leder, Fibroblast growth factor receptor 3 is a negative regulator of bone growth. 25 μg of the vector was enzymatically treated according to the conditions shown in Tables 1 and 2. Comparative Example 1 was a control (without vector), Comparative Example 2 was a cyclic vector before enzymatic treatment, Comparative Example 3 was a vector linearized with NotI (blunt end), and Comparative Example 4-1 was linearized with NotI. Later, a vector treated with ExoIII for 1 minute (5'overhang on one side, base length of the overhang portion is 100 mer), and Comparative Example 4-2 is a vector linearized with NotI and then treated with ExoIII for 3 minutes (5'overhang on one side). , The base length of the overhang portion is 300 mer), Comparative Example 4-3 is a vector treated with ExoIII for 5 minutes after linearizing with NotI (5'overhang on one side, base length of the overhang portion is 500 mer), Comparative Example 5 -1 is a vector linearized with NotI and then treated with T7 for 1 minute (3'overhang on one side, the base length of the overhang portion is 150 mer), and Comparative Example 5-2 is linearized with NotI and then treated with T7 for 3 minutes. Vector (3'overhang on one side, base length of overhang part is 450 mer), Comparative Example 5-3 is a vector treated with T7 for 5 minutes after straightening with NotI (3'overhang on one side, base of overhang part). The length is 750 mer), Comparative Example 6 is a vector (blunt end) subjected to double digestion of EcoRI and NotI (the vector skeleton is also removed), and Comparative Example 7-1 is after double digestion of EcoRI and NotI. A vector treated with ExoIII for 1 minute (5'overhang on both sides, base length of the overhang portion is 100 mer), and Comparative Example 7-2 is a vector treated with ExoIII for 3 minutes after double digestion with EcoRI and NotI. Both sides 5'overhang, base length of overhang part is 300 mer), Comparative Example 8 is a vector treated with T7 for 1 minute after double digestion with EcoRI and NotI (both sides 3'overhang, overhang part). The base length is 150 mer), and Example 1 is a vector treated with T7 for 3 minutes after performing double digestion with EcoRI and NotI (both sides 3'overhang, base length of overhang portion is 450 mer).

Vectors treated with these enzymes were transfected into TC1 embryonic stem (ES) cells. After electroporation, ES cells were sown on mitomycin-treated MEF cells to a concentration of ¹⁰⁶ ES cells per 100 mm diameter Petri dish. The cells were fed fresh medium every other day.

After 24 hours of incubation at 37 ° C., G418 (250 μg / ml, Roche) was added to the medium. The cells were fed fresh medium every other day and maintained for 7 days under G418 selection, followed by 7 days under selection with G418 plus 6-thioguanine (6-TG: 1 μg / ml, Sigma-Aldrich). Maintained. At the end of these periods, the number of surviving colonies was determined.

As a result of Hprt gene targeting, a schematic diagram of the shape of each vector and a photograph of the colony are shown in FIG. In the photographs of the colonies in FIG. 7, selection by G418 only for the first 7 days (left colony photograph) and selection by G418 and 6-TG for the next 7 days (right colony photograph) are shown. Table 1 shows an outline of the shape of each vector, and Table 2 shows the results of summarizing the number of colonies. Table 2 shows the number of colonies selected by G418 alone for the first 7 days, and the ratio of the number of colonies selected by G418 and 6-TG to the number of 6-TG resistant colonies.

Cells into which the neomycin resistance gene has been introduced by homologous recombination have resistance to G418, and cells lacking the Hprt gene by homologous recombination have resistance to 6-TG. In the absence of transfection of the targeting vector (Comparative Example 1), only a few colonies were present after selection of G418 and 6-TG. As shown in the number of colonies selected by G418 and 6-TG in Table 2, the homologous recombination efficiency was hardly improved in Comparative Example 3 and Comparative Example 6 using the linear vector having no overhang portion. .. Further, when Comparative Example 3 using a linear vector having no overhang portion and Comparative Example 4-1 to Comparative Example 4-3 using a targeting vector having a 5'overhang on one side are compared, 5'over on one side. In Comparative Example 4-1 to Comparative Example 4-3 using the targeting vector of the hang, the homologous recombination efficiency was improved only about 2 times, about 4 times, and about 5 times, respectively. Similarly, even when Comparative Example 6 using a linear vector having no overhang portion and Comparative Example 7-1 to Comparative Example 7-2 using a targeting vector having 5'overhangs on both sides are compared, both sides are compared. In Comparative Example 7-1 to Comparative Example 7-2 using the 5'overhang targeting vector, the homologous recombination efficiency was improved only about 2 times and about 4 times, respectively.

Further, even when Comparative Example 3 using a linear vector having no overhang portion and Comparative Examples 5-1 to 5-3 using a targeting vector having a 3'overhang on one side are compared, 3'over on one side is achieved. In Comparative Examples 5-1 to 5-3 using the hang targeting vector, the homologous recombination efficiency was not improved or improved at most about 3 times, respectively. On the other hand, in Comparative Example 8 using a targeting vector having 3'overhangs on both sides and a base length of the 3'overhang portion of 150, a linear vector having no overhang portion is used for homologous recombination efficiency. It was improved 25 times as compared with Comparative Example 6 which had been used.

Further, in Example 1 using a targeting vector having 3'overhangs on both sides and a base length of the 3'overhang portion of 450 mer, a linear vector having no overhang portion was used for the homologous recombination efficiency. Compared with Comparative Example 6, the improvement effect was well over 40 times, and compared with Comparative Example 3, the improvement effect was well over 400 times. The demonstrated high homologous recombination efficiency was astonishingly unpredictable to those of skill in the art. Thus, the use of the targeting vector of the present invention could dramatically improve the efficiency of homologous recombination.

[Test Example 3: NudCD2 gene targeting]
In order to verify that the target vector of the present invention can be applied to other loci of ES cells, the mouse NutCD2 gene was targeted.

The substitution vector shown in FIG. 8 was designed for homologous targeting of the mouse NutCD2 gene. As shown in FIG. 8, the targeting vector contains a neomycin cassette PGK-neo inserted before the first exon of the NudCD2 gene. As shown in FIG. 8, a neomycin cassette having a first and second loxP sequence pair at both ends was inserted into the beginning of exon 1, and a third loxP sequence was inserted into intron 1 of NudCD2. .. Substitutions based on homologous recombination introduce new KpnI sites. Therefore, homologous recombination events can be detected by performing Southern blot analysis with a flanking probe after digesting the DNA of the obtained clone with KpnI.

25 μg of the above vector was enzymatically treated according to the conditions described in Modification in Table 3. Comparative Example 9 is a vector linearized by double digestion of EcoRI and NotI (the vector skeleton is also removed), and Example 2 is linearized with EcoRI and NotI and then treated with T7 for 3 minutes. Vector (3'overhang on both sides, base length of overhang is 450 bases).

Vectors treated with these enzymes were transfected into TC1 embryonic stem (ES) cells. After electroporation, ES cells were sown on mitomycin-treated MEF cells to a concentration of ¹⁰⁶ ES cells per 100 mm diameter Petri dish. The cells were fed fresh medium every other day.

After 24 hours of incubation at 37 ° C., G418 (250 μg / ml, Roche) was added to the medium. The cells were fed fresh medium every other day and maintained for 7 days under G418 selection. Following that, Southern blotting analysis using a flanking probe was performed. Homologous recombination clones were determined by restricted polymorphisms, which are newly introduced KpnI sites into the targeted alleles.

FIG. 9 shows the results of Southern blotting analysis (b) of ES cell colonies (a) after selection by G418 for 7 days and G418 resistant clones after KpnI digestion using a flanking probe. The band represented by WT represents the wild-type allele (3.1 kbp) and the band represented by HR represents the introduced target gene (2.5 kbp). Table 3 also shows the results of genotyping of G418 resistant clones. In Comparative Example 9, only 5 clones out of all 168 G418 resistant colonies were homologous recombinants, whereas in Example 2, as many as 237 clones out of all 252 G418 resistant colonies were homologous. It was a recombinant. As these results show, the number of homologous recombination clones could be dramatically increased by using the target vector of the present invention. Moreover, in Example 2, it was even confirmed that 22 of the 237 homologous recombinations had undergone homologous recombination in both alleles.

[Test Example 4: Rosa26 gene targeting of fertilized mouse eggs by pronuclear injection]
Using the targeting vector of the present invention, direct gene targeting of the Rosa locus was performed by pronuclear injection into fertilized mouse eggs (Example 3).

A substitution vector (Rosa26-H2B-eGFP) shown in FIG. 10 was designed to directly target the Rosa26 gene by pronuclear injection. Rosa26-H2B-eGFP is T. Abe, H. Kiyonari, G. Shioi, K. Inoue, K. Nakao, S. Aizawa, T. Fujimori, Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging. Genesis. It was made according to 49, 579-590 (2011). Substitutions based on homologous recombination introduce new EcoRI sites for Southern blotting analysis.

The designed vector Rosa26-H2B-eGFP was treated with the restriction enzyme SfiI to remove the vector skeleton and treated with T7 for 3 minutes to obtain a targeting vector.

The obtained targeting vector was injected into fertilized eggs of C57BL / 6 mice. A total of 459 fertilized eggs were injected with pronucleus to obtain 110 pups. DNA was extracted from the tail of the obtained pups and subjected to Southern blotting analysis using a flanking probe of the Rosa26 gene. Homologous recombination events were determined by restricted polymorphisms at the newly introduced EcoRI site into the targeted allele. That is, the polymorphism after EcoRI digestion was detected by the flanking probe shown in FIG. In addition, HindIII digested product of λDNA was used as a size marker.

The results of Southern blotting analysis are shown in FIG. The band represented by WT represents the wild-type allele (15.5 kbp) and the band represented by HR represents the introduced target gene (10.5 kbp). According to this Example 3, two homologous recombinants were found. The detection intensity of the band derived from the introduced target gene was lower than that of the wild type. This result indicates that the produced Rosa targeting mice are mosaic. This means that the introduction of foreign DNA occurred after the first round of chromosomal DNA replication.

[Test Example 5: NudCD2 gene targeting of fertilized mouse eggs by pronuclear injection]
In order to verify that direct gene targeting of fertilized mouse eggs by pronuclear injection is applicable to other loci, direct gene targeting of the NudCD2 locus was performed by pronuclear injection into fertilized mouse eggs ( Example 4).

In order to directly target the NudCD2 gene by pronuclear injection, a substitution vector without the neomycin cassette shown in FIG. 12 was designed. As shown in FIG. 12, in the vector, the first exon was flanked by two loxP sites. Substitutions based on homologous recombination introduce new ApaI sites for Southern blotting analysis.

The designed vector was treated with the restriction enzyme SfiI to remove the vector skeleton and treated with T7 for 3 minutes to obtain a targeting vector.

The obtained targeting vector was injected into fertilized eggs of FVB mice by pronuclear injection. A total of 213 fertilized eggs were injected with pronucleus to obtain 41 pups. DNA was extracted from the tails of the obtained pups and subjected to Southern blotting analysis using a flanking probe for the NudCD2 gene (HindIII digested product of λDNA was used as a size marker). Homologous recombination events were determined by restricted polymorphisms at the newly introduced ApaI site into the targeted allele. That is, the polymorphism after ApaI digestion was detected by the flanking probe shown in FIG. In addition, HindIII digested product of λDNA was used as a size marker.

The results of Southern blotting analysis are shown in FIG. The band represented by WT represents the wild-type allele (15.5 kbp) and the band represented by HR represents the introduced target gene (11 kbp). According to this Example 4, one homologous recombination was found. The detection intensity of the band derived from the introduced target gene was similar to that of the wild type. This means that the introduction of foreign DNA occurred after the first round of chromosomal DNA replication.

Claims (11)

A targeting vector containing a foreign DNA fragment and a homologous DNA fragment having a homologous recombination sequence with a part of a target DNA site on both sides of the foreign DNA fragment.
A targeting vector having an overhang portion at both ends of the targeting vector having a protruding 3'end, the overhang portion having the homologous sequence, and a base length of 200 mer or more. The targeting vector according to claim 1, wherein the base length of the overhang portion is 500 mer or less. The targeting vector according to claim 1, wherein the foreign DNA fragment contains a marker gene. A targeting vector containing a foreign DNA fragment and a homologous DNA fragment having a homologous sequence capable of homologous recombination with a part of a target DNA site on both sides of the foreign DNA fragment; both ends of the targeting vector are 3'. An introduction step of introducing a targeting vector having an overhang portion having a protruding end, the overhang portion having the homologous sequence and a base length of 200 mer or more, into a cell having the target DNA site on the chromosome. Including
A method for producing a gene-modified cell , wherein the cell is a eukaryotic cell or a human cell, and the human cell is an induced pluripotent stem cell (except when producing a human) . The method for producing a gene-modified cell according to claim 4, wherein the introduction step is performed by a pronucleus injection method. The method for producing a gene-modified cell according to claim 4 or 5, which does not include a double-strand break step that causes double-strand break at the target DNA site. The method for producing a genetically modified cell according to claim 4 or 5, which comprises a double chain cleavage step of causing double chain cleavage at the target DNA site. The method for producing a gene-modified cell according to claim 7, wherein the double-strand break step is performed by a zinc finger nuclease (ZFN), a transcriptional activator-like effector nuclease (TALEN), or a crisper-related (Cas9) nuclease. A linear DNA construct containing a foreign DNA fragment and a homologous DNA fragment having a homologous recombination sequence with a part of a target DNA site on both sides of the foreign DNA fragment, both ends of which are the homologous DNA fragments. The process of constructing a linear DNA construct composed of
Targeting comprising the step of allowing an exonuclease to act on the linear DNA construct to form an overhang portion at both ends of the linear DNA construct, the 3'end protruding with a base length of 200 mer or more. How to make a vector. The method for producing a targeting vector according to claim 9, wherein a T7 exonuclease is used as the exonuclease in the step of generating the overhang portion, and the T7 exonuclease is allowed to act for 1 minute and 20 seconds or longer. A claim comprising treating both ends of the DNA construct with a restriction enzyme that produces a 3'protruding end between the step of constructing the linear DNA construct and the step of creating the overhang. The method for producing a targeting vector according to 9 or 10.

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