JP2003502015A - Methods and materials for rapid and mass production of gene knockout libraries in organisms - Google Patents

Abstract

(57) Abstract: A method for promoting site-directed homologous recombination in an organism to produce mutants, the method comprising: 1) a large-insert vector comprising one or more large-insert vectors; Constructing a library, 2) constructing a second vector containing a transposable element, 3) combining the product of step 2) with step 1) in the presence of an inverted repeat-specific transposase on said plasmid vector. 4) amplifying the disturbed large insert vector produced as a result of step 3) 5) disturbing the disturbed large insert vector produced by step 4) in the target host cell And 6) selecting the successful homologous recombination obtained by step 5) with said second selectable marker.

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001]   (background)   The present invention provides a method for systematic and random insertion of genetic material into a biological genome.
Laws and materials. The present invention enables rapid mutagenesis of organisms,
Mutating essentially all genes of organisms, especially fungi, and mutagenesis
Reliable and efficient identification of genes knocked out in elephants
And enable. The present invention also relates, in particular, to the previously mentioned events, such as filamentous fungi.
Promotes highly efficient homologous recombination in species known to be infrequent
Proceed.       [0002]   Numerous methods for introducing exogenous genetic material into living cells have been around for a quarter century.
It has been normal since the first case in genetic engineering earlier. Exogenous inheritance
Introduction of the child material can be via a vector that can be replicated or the host cell genome.
This is made possible by the incorporation into it. With the introduction of the exogenous genetic material
In a species that does not normally produce a certain protein, expression of the protein is possible.
Noh. Also, the transcription and translation of the protein may be higher or higher.
By introducing modified control sequences that result in lower efficiency,
Control (overexpression and underexpression) became possible. Another use of genetic engineering is
Amino acid of the protein produced by modifying the coding region of the gene
Alter the biological activity of a structural protein or enzyme by altering the sequence
Was that. Modified amino acid sequences can alter conformations and surface charges.
And changes in the higher-order structure (tertiary and quaternary structure) of the protein
All of which will result in a change in biological activity.       [0003]   "Functional genomics" derived from the principles of genomics or gene sequencing
With the recent developments in the field of DNA, the manipulation of DNA in living organisms is another urgent task.
It is a screen. Functional genomics involves sequencing the genetic material of an organism.
In addition, we are seeking to identify the functions of genes in target organisms on an industrial scale. Functional Geno
Mixes contain the functions of most, if not all, genes and organisms.
By determining the products of those genes in
Accelerate the identification of quality targets and identify their genes and gene products
Identify compounds that may be modified to reduce disease and improve human and animal health
And improve the quality and quantity of food crops. To achieve this,
Systematically alters the expression of essentially any gene in and identifies the corresponding gene,
And rapid monitoring of the effect of genetic modification on the phenotype of said organism
, Mass operation techniques are needed.       [0004]   Automated processes in molecular genetics allow microorganisms such as yeasts and fungi
Systematic analysis by determining the DNA sequence of genomics derived from genus
. Attention has been focused on quickly assigning functions to newly discovered genes. Remains
In the area of genetics, gene function refers to organisms (variants) that contain defined genetic mutations.
It is widely accepted that the most appropriate distribution can be obtained by analysis.       [0005]   Traditional methods of introducing genetic material into eukaryotes involve mutating a single gene.
It is enough to do just that. The above methods include protoplast fusion, electro-
Transformation by poration, particle bombardment, cell envelope (membranes and walls)
Chemical disruption, phage and viral infection, DNA transduction into cells and
And physical insertion. Many of these methods involve placing the vector into cells.
Is limited to the introduction of DNA in the form of
Produces gene products. Hope for useful gene insertion method for functional genomics
The characteristics that can be identified include essentially all genes in the genome of the target organism,
Includes efficient and systematic insertion of DNA. DNA in the genome of an organism
In the method of incorporating the DNA, usually, the DNA is randomly inserted into the genome.
You. Methods for integrating DNA at specific locations in the genome of an organism are unreliable and often
Often, the efficiency is low.       [0006]   To randomly insert DNA into another piece of DNA containing genomic DNA, Cre
-Lox (Sauer (1996) Nucleic Acid Res. 24:
4608-4613) and Flp recombinase (Seibler and
Bode (1997) Biochemistry 36: 1740-1747).
And virus systems using recombinases such as In these systems, the host
The DNA is inserted into specific sites of the DNA in the nome DNA.
It was randomly engineered into the genome. Recently, transposer
Enzymes, known as enzymes, change from one position in DNA to another random position in DNA.
The ability to transfer DNA fragments to a location has been discovered (Devine et al., US Pat.
No. 67,170; Devine et al., US Pat. No. 5,728,551; Hack.
ett et al., WO 98/40510; Plasternak et al., WO 97/292.
02; Reznikoff et al., WO 98/10077; Craig, WO 98 /.
37205; Strathman et al., (1991) Proc. Natl. Aca
d. Sci. USA 88: 1247-1250; Phadnis et al., (1989)
) Proc. Nat. Acad. Sci. USA 86: 5908-5912; W
ay et al., (1984) Gene 32: 269-279; Kleckner et al.
(1991) Method. Enzymol. 204: 139-180; Lee
Et al., (1987) Proc. Natl. Acad. Sci. USA 84:78
76; Brown et al. (1987) Cell 49: 347-356; Eichi.
(1988) Cell 54: 955-966; Eichinger.
(1990) Genes Dev. 4: 324-330). Usually, tran
Posases are known as inverted repeats located adjacent to an internal piece of DNA.
Recognize short DNA sequences. The internal DNA sequence and its two adjacent internal repeats
DNA sequences, including sequences, are known as transposons or transposable elements.
is there. This transposase cuts out the transposon and converts it into another DNA.
It has the ability to insert it, which will come into contact with it. Position of the insertion
The positions are usually not all random, but occur preferentially at the target sequence position.
(So-called "hot spots") (Kleckner et al., (1991) Me
thod. Enzymol. 204: 139-180). Same as virus
, The insertion is site specific, but
The sites are randomly located in the genome and are site-directed (site dir).
ected) Does not allow insertion.       [0007]   Transposons are used to introduce desired genes into organisms.
However, in this case, the introduction can also be via a plasmid in the genome of the organism.
Are performed randomly (site-specific but not site-directed).
Another transposon application is that transposon sequences are known, especially at their boundaries.
And as a sequencing tool, a primer designed for this transposon.
Can be used to determine the sequence of the DNA into which the transposon is inserted
Become. If the insertion site lacks randomness, essentially all genes in the organism
Use or transposons as a tool for systematically determining the sequence of
Transcription as a means to systematically knock out essentially all genes in a product
The use of spozons will be reduced. Therefore, functional genomics
Will be limited in their use.       [0008]   Therefore, to use a transposon, place the transposon in the plasmid.
Engineering (eg, Reznikoff et al., WO 98/10077).
And the gene into which this plasmid was introduced into the target organism and transposed
Has previously been required (Devine et al.,
U.S. Patent No. 5,677,170; Devine et al., U.S. Patent No. 5,728,5.
No. 51). Separately, in the presence of mobile transposase inside cells,
Moving the transposon directly from the plasmid into the genome of the target organism
Thus, genetic material has been introduced into the genome of organisms (Hackett et al., WO
98/40510; Plasternak et al., WO 97/29202). This is
For this to occur, the transposable element on the plasmid is located inside the cell to be transposed.
The corresponding transposase must be included. As a result,
Of transposons that use transposons to insert DNA into the genome of
The only use is for transposable DNA in the presence of transposase.
Position, and not a site-directed position (Hackett
WO 98/40510; Plasternak et al., WO 97/29202.
). In addition, vectors containing transposon events are limited to plasmids
The use of transposition vectors is to express gene products of transposable genes.
Was that. In addition, transposon introduction is usually not random, but
It happens at one of the spots. Transfer DNA to filamentous fungi using transposons
Inter alia, directly or indirectly introducing DNA into the filamentous fungal genome
Has only recently been achieved (Migheli et al. (1999) Gene.
tics 15: 1005-1013).       [0009]   In order to achieve site-directed DNA insertion into the genome of an organism,
A method of homologous recombination wherein essentially all genes of said organism are mutated
is necessary. However, transforming filamentous fungal cells by homologous recombination
Usually involves difficulties. It was known that such recombination was inefficient.       [0010]   Genome-wide mutagenesis is particularly problematic for filamentous fungi for several reasons.
is there. First, active and easy-to-manipulate endogenous transposons have a very broad spectrum.
No mention was made of filamentous fungi. Second, homologous recombination is a DNA trait
Frequency of conversion, compared to heterologous (illegal or ectopic) recombination
Is low. The DNA construct introduced during the ectopic recombination will have its homologous genomic sequence
Recombination at various sites throughout this genome without recombination
available. Therefore, the result of homologous recombination was
Containing site-directed mutants such as gene knockouts (KOs)
Against a large number of background strains containing ectopic (heterologous) recombination events.
I have to separate. Finally, a large homologous chromosomal DNA region (> 1000 bp)
) Is required to guide homologous recombination. Therefore, many standard combinations
Recombinant DNA method (DNA digestion with restriction enzymes, isolation of DNA fragments, plasmid
Ligation into vector, E. coli E. coli. coli transformation and fungal colony screen
Is required to assemble the single gene KO vector construct.
This requirement is an obstacle for efficient automation.       [0011]   Filamentous fungi are a large and diverse group in the kingdom Fungi. They are land bio
Chemical, vitamin, enzyme and pharmaceutical industrial
As a host for production, as a degrading and spoiling substance, and as a plant and
As a human and animal pathogen, with serious effects on human health. This group of organisms
Usually, a distantly related simple substance such as yeast Saccharomyces cerevisiae
It is thought to be distinctly different from cellular fungi. This distinction is based on the growth morphology (single cell
Multicellular filamentous hyphae for budding and metabolism (eg aerobic with strictly speaking filamentous fungi)
, Whereas S.D. cerevisiae is facultative anaerobic)
It is obvious. Systematically analyze the function of all filamentous fungal genes
Sorting is a very new and valuable information about these important organisms.
Information will be provided.       [0012]   The present invention provides for the systematic variation of essentially all genes in an organism, particularly a filamentous fungus.
Technology enabled by promoting homologous recombination of all genes of the organism
And provide the material. Homologous recombination occurs when one of the cells
Large insert vector libraries containing multiple genes (eg, cosmid or BA)
C). Gene mutations in cosmids use transposons
Get up. In this way, genomic variants can be produced rapidly and on a large scale. Ko
With large insert vector constructs such as sumids, each side of the inserted transposon
Large flanking DNA sequences that are homologous to This franc
King DNA sequences use primers based on the inserted transposon.
The large flanking DNA sequences described above are particularly homologous recombination.
Promotes homologous recombination in species that have previously been considered to be less efficient. Kosumi
The use of transposons to insert DNA into DNA has never been achieved.
Are not available and are recommended by commercially available transposon manufacturers.
Not recommended. Thus, the present invention addresses the above problems of homologous recombination of difficult species.
New methods and materials to solve the problem, rapid, large-scale production of genomic variants,
As well as routine sequencing of mutated genes. The present invention relates to
Identification of all genes and analysis of corresponding genomic variants
And assigning functions to each of these genes will enable the industrialization of both.
I do.       [0013]   (Summary of the Invention)   The present invention promotes site-directed homologous recombination in an organism to produce mutants
Wherein the method comprises: 1) Construction of a large insert vector library containing one or more large insert vectors
Each of said large insert vectors comprises a DNA fragment;
Is a multi-gene from the target organism and the first to function for selection in fungi
Including selectable markers, 2) constructing a second vector comprising a transposable element, said transposable element
Is a nucleic acid encoding a second selectable marker flanked by inverted repeats on each side.
An otide sequence, wherein said selectable marker is selected in fungi and target organisms.
The inverted repeats are bifunctional for selection, and the inverted repeats are
Functional as a binding site, 3) The transposable element is randomly present in each large insert of the large insert library.
The plasmid vector to produce large insert vectors transferred to
Step 2 in the presence of a transposase specific for said inverted repeat on the
Incubating the product of step 1) with the product of step 1); 4) amplifying the disturbed large insert vector generated as a result of step 3);
5) Disrupting the large insert vector produced by step 4) into the target host cell
Introducing into the cell, and 6) A second selectable marker for the successful homologous recombination obtained by step 5)
Choosing by, including.       [0014]   In a preferred embodiment of the present invention, the large insert vector library
Library or BAC library, and more preferably
Ri.   In a preferred embodiment of the present invention, the transposable element and the transposase are
, Himar1, AT-2, GPS-1 or GPS-2 systems.   The most preferred embodiment of the present invention is a filamentous fungus, especially Magnaporthe gri.
For homologous recombination in sea.   Other aspects of the invention include kits for performing the methods of the invention.
. Also included are libraries produced in this way.       [0015]   The present invention promotes site-directed homologous recombination in an organism to produce mutants.
A method for producing, said method comprising: 1) Construction of a large insert vector library containing one or more large insert vectors
Each of said large insert vectors comprises a DNA fragment;
Is a multi-gene from the target organism and the first to function for selection in fungi
Including selectable markers, 2) constructing a second vector comprising a transposable element, said transposable element
Is a nucleic acid encoding a second selectable marker flanked by inverted repeats on each side.
An otide sequence, wherein said selectable marker is selected in fungi and target organisms.
The inverted repeats are bifunctional for selection, and the inverted repeats are
Functional as a binding site, 3) The transposable element is randomly present in each large insert of the large insert library.
The plasmid vector to produce large insert vectors transferred to
Step 2 in the presence of a transposase specific for said inverted repeat on the
Incubating the product of step 1) with the product of step 1); 4) amplifying the disturbed large insert vector generated as a result of step 3);
5) Disrupting the large insert vector produced by step 3) or 4)
Introducing into the target host cell; and 6) A second selectable marker for the successful homologous recombination obtained by step 5)
Choosing by, including.       [0016]   Therefore, the present invention promotes site-directed homologous recombination in an organism to regulate
This is a method to introduce mutation at a certain site. Homologous recombination is two homologous sequences
DNA fragments exchange part or fragments of the full length of the similar DNA sequence due to a transfer event.
It happens when you change. In particular, the two parts of DNA involved in homologous recombination or
The fragments are preferably the termini or distal regions of the DNA portion or fragment to be exchanged.
Have similar or nearly identical DNA sequences. Mutation is an organism or
Or the sequence of nucleotide bases in genetic material (DNA) of cells or
Alteration, either exclusion, addition, deletion or other disruption.
It happens. Modifications to the DNA sequence have the same meaning, that is,
Is to modify the ability to produce a normal type of protein, as a result,
The organism or cell itself is modified. Such modified organisms or cells are
Called a variant. The most preferred mutations for the purposes of the present invention are those containing modified DNA.
Mutations that cause a decrease or increase in the biological activity of the gene product of the gene. Especially suitable
A variant is a gene product that is lost due to a null mutation in the gene or loss of translation of the gene.
A knockout mutant that inevitably destroys activity. Such mutations are
Another piece of DNA is inserted into the DNA of the gene, and the inserted DNA is replaced with the normal gene.
Disrupts the translation of or produces incompletely translated forms, resulting in biological
Ensure that inactive or no gene products are produced
It happens by doing.       [0017]Construction of large insert vectors and vector libraries   The first step in the method of the present invention is the construction of a large insert vector containing a piece of DNA.
Yes, said DNA comprises multiple genes and an amplification host, preferably fungi, from an organism.
Including a first selectable marker that functions for selection in. Such a large insertion
Somatic vectors include cosmids, λ vectors, and preferably bacterial artificial chromosomes (BACs).
), Preferably a large plasmid containing an inserted DNA fragment of 5-10 kb.
Any large vector, including large plasmids that are rasmids (ie, replication
Possible double-stranded DNA). Construction of cosmids or BACs is preferred. In bacteria
The markers that function for selection in and the sources of said markers are
Known in the art, ampicillin (Amp), tetracycline (Tet),
Chloramphenicol, kanamycin (Kan) and the like. Large insert vector
Construction begins with a vector, a self-replicating DNA unit into which DNA fragments are inserted.
Vectors contain operable genes, including origins of replication, binding sites, restriction sites, etc.
Have. Preferably, the origin of replication is compatible with cells for amplification, such as E. coli, and
Functional in it, but not compatible with the host organism that is the target of homologous recombination or
Not functional in it. Suitable first choice for large insert framework
A marker and a large DNA fragment from the organism targeted for homologous recombination are added.
The large fragment contains at least one entire gene from the target organism, but more preferably.
Suitably, it is a plurality of genes. Since genes are typically about 3 kbp in length,
The large fragment is preferably at least 5-10 kbp in length, more preferably
Suitably over 10 kbp. The length of the DNA fragment inserted into the vector is long
The more mutated the gene is, the longer the adjacent DNA will be and will promote homologous recombination.
Would.       [0018]   The construction of many large insert vectors from a single organism has resulted in thousands of
The total D of an organism inserted as random inserts into different vectors
A gene library containing NA is generated. The most useful libraries are complete
Genes, preferably genomic inserts of the size required to encompass multiple genes
Are those containing. Gene library construction is not so frequent within DNA.
It relies on the use of restriction enzymes, such as EcoRI, which do not produce a frequent truncated end. So
At this time, when it is suspected that the desired genes are too long, incomplete digestion D
It may be preferable to use NA. In addition, long genomic inserts can
Can also be obtained by physically shearing the DNA of the target organism. Few
At least 5 kilobase pairs (kbp) of genomic fragments were inserted into the plasmid.
It is reproduced very stably. Larger fragments are packaged inside the mature particles.
Requires a 22 kbp DNA insert to be tailored and is specifically tailored
Proliferates stably inside the λ phage system. Exogenous DNA, even large fragments
Can be stably inserted into cosmid vectors that can hold 40 to 50 kbp
. Therefore, using cosmids, for example,
It would be possible to obtain a duplicated coverage of the entire genome of the organism in the species. And
For example, in many fungal genomes, 3000 large insert vectors are
It will provide about a three-fold coverage of the entire genome of the organism.       [0019]   In another aspect of the invention, the large insert vector has vector operable genes.
Not isolated large DNA fragments. Such DNA fragments are
Restriction enzyme digestion of nome DNA, physical shearing of genomic DNA, genomic DNA section
PCR amplification or homologous recombination as described above
Can be obtained by any of the methods used to obtain large DNA fragments.
You. The large fragment contains at least one whole gene from the target organism, and more preferably.
Suitably, it contains multiple genes. Genes typically have a length of about 3 kbp
Therefore, the large fragment is preferably 5-10 kbp, more preferably 10 kbp.
bp. The longer the length of the DNA fragment inserted into the vector,
Adjacent DNA that promotes homologous recombination will likely be longer
. Collecting many large DNA fragments from an organism results in a large DNA fragment library.
Will be obtained. Such a large DNA fragment library can be handled.
A large number of large DNA fragments can be obtained that cover multiple genomes of the whole organism.
You.       [0020]Construction of transposable elements   Another step of the present invention is a second vector, preferably a plasmid containing a transposable element.
Wherein the transposable element is flanked by inverted repeats on each side.
A nucleotide sequence encoding a second selectable marker, wherein said selectable marker is
The marker is bifunctional for selection in fungi and fungi,
Orthorepeats function as binding sites for the transposase. Built
The vector does not need to be large and, therefore, is preferably a plasmid vector.
Or approximately plasmid size. This vector is a self-replicating vector
Does not need to contain all of the sex genes; uses primers and polymerases
Can be produced by other amplification techniques. The vector comprises a transpo
A bifunctional selectable marker flanked on each side by inverted repeats that bind the enzyme
Will contain a car. Transposons encode the enzyme transposase
Naturally appearing as DNA sequences that are adjacent to the gene for the transposase.
Recognize and cleave the DNA at the abutting site. Integrase is a transpo
It is another form of a zase. The recognition sites or binding sites for transposase
Classes are called inverted repeats. Elements that can be transposed as such
Is activated and facilitates excision and excision from one position in the DNA.
Produce an enzyme that facilitates the insertion of the inserted DNA into another site. Prior art
As noted, the insertion location should be “hot spots” and site specific.
Can be. In the present invention, the bifunctional marker is inserted between the inverted repeats described above.
And usually produces live transposase by the perturbed gene,
Usually inactivated.       [0021]   Transposable elements can usually be obtained from appropriate sources using restriction enzymes.
When appropriate, a bifunctional selectable marker is used to determine the binding for the appropriate transposon.
As long as the insertion does not disturb the inverted repeats that are the joining site,
It can be inserted into a positionable element. Some of the above transposable elements are publicly available in the art.
And Himar1 (Lampe et al., (1998) Genetics 14).
9: 179-187), AT-2 (Perkin Elmer; Devine et al.).
(1997) Genome Res. 7: 551-563), GPS-1 (Ne
w England Biolabs) or GPS-2 (New Engla)
nd Biolabs).       [0022]   Suitable bifunctional selectable markers are useful for amplified organisms and homologous recombination.
Selectable markers that function in both target organisms. Preferred embodiment of the present invention
In some cases, a bifunctional selectable marker allows for selection in bacteria and in filamentous fungi.
To be effective, the amplified organism is a fungus and the target organism for homologous recombination.
Is a filamentous fungus. Preferred selectable markers for the present invention include
Mycin, argB, pyr-4. Separately, bifunctional selection possible
Functional markers also include two separate selectable markers, one of which is the amplified organism
In the target organism, and others in the target organism for homologous recombination.
Will do.       [0023]Transferring transposable elements randomly into large insert vectors   Construction of the large insert vector library and transposable elements as described above.
After construction, the transposable element is transferred in vitro into the DNA of the large insert vector.
They can be mixed or injected with a suitable transposase so that
Can incubate. Large Insert Vectors in Large Insert Vector Library
Activating the transposase to cause the transposable element to transpose into the fragment.
Conditions for accomplishing this include using an appropriate transposase.
You. A suitable transposase is the inverted repeat arrangement used in the transposable element.
A transposase specific for binding and excision of DNA in a row. this is,
Usually within the boundaries of the inverted repeat in its normal, unmutated condition
Is a transposase that is encoded by DNA that normally occurs in some parts. In the present invention
Useful particularly suitable transposases and their corresponding transposable elements
Are Himar1 Mariner transposons (Lampe et al. (1998) Gen.
etics 149: 179-187; Lampe et al. (1996) EMBO J.
. 15: 5470-5479), AT-2 (Perkin Elmer Cor).
position) and GPS-1 and GPS-2 (New England)
d Biolabs). The suitable transposase is a large insert vector
Library and displaceable elements for a sufficient time and at a sufficient temperature
So that dislocations occur. The appropriate temperature is the temperature at which the enzymes
It does not unfold or significantly change the folding structure of the formation
Suitably from about 15 ° C to about 45 ° C, more preferably from about 20 ° C to about 3 ° C.
0 ° C., most preferably about room temperature. Incubation length
Of the expected amount of cover in the large insert vector library and the target vector
Transposable with the donor vector, depending on the ratio of the donor vector
And the target vector is a large insert vector. Suitable donors:
The target ratio is about 1: 1 and a suitable length of incubation time is about 12 hours.
Less than one hour, preferably about one hour.       [0024]   Thus, each large insert vector of the large insert vector library,
It will have a transposable element inserted somewhere in the large fragment DNA. This
The result is a large insert vector that has been disrupted at one or more positions in the large fragment.
Will. Large insert vectors also contain large fragment DNA only, as described above.
The collection of large fragment DNAs may be a large fragment DNA library.
Can be produced. If the insertion occurs within the gene of the large fragment, a mutation appears,
Preferably, a knockout mutation will occur. If Large Insert Vector Library
Is large enough that after exposure to transposase and transposable elements
The resulting library will contain mutations of essentially all genes of the target organism.
Would.       [0025]Amplification of disturbed large insert vectors   Disrupted large insert vectors generated by the invention or disturbed
The large DNA fragments can then be optionally amplified. Large inserts other than large DNA fragments
Amplification for the vectors is carried out by transferring the disrupted large insert vectors to a suitable host.
After transformation into a suitable host. this
Such a suitable host can be any transformable cell or organism.
However, a preferred amplification host is a fungus, more preferably E. coli. Preferably,
An accommodation compatible with the replication and operation genes of the perturbed large insert vector.
The Lord will be chosen. The host may also be a first choice for a large insert vector.
It would be such as to allow for the selection of transformants by markers. Transformation method
Methods include methods for selecting transformed hosts and harvesting amplification vectors.
Both are well known to those skilled in the art. Other methods of amplification, including PCR, are also particularly useful for large inserts.
It can be used when the vector is a large DNA fragment.       [0026]Evaluation of randomness   When a transposable element is inserted into the large insert vector of the large insert library
Randomness (integrity) is determined by preparing cosmid DNA and transposon
By performing DNA sequencing as indicated by primers at both ends of the
Worth it. If inserts are widely distributed, perfect sequencing of hundreds of thousands of inserts
However, it covers most of the genome, and many genes have multiple trans
It should have a poson insert (alleles). In fact, transposon insertion
Randomization on one or several cosmids and by restriction digestion and sequencing
Will be evaluated by       [0027]Homologous recombination in host organisms   Disrupted large insert vectors containing large DNA fragments can cause homologous recombination.
Can be inserted into the target host cell. Large insert vectors into target host cells
Transformation methods for ters are known in the art. Especially suitable for filamentous fungi
Transformation methods involve the electroporation of fungal protoplasts under appropriate conditions.
Or calcium-mediated transformation. Suitable conditions include, for example, T
albot et al. (1993) Plant Cell 5: 1575-1590.
It is listed. The host organism can then be grown and a second selectable marker
To select the successfully transformed host cells using a suitable medium.
Propagate with. Confirmation of homologous recombination uses various methods known in the art.
Can be achieved. These include Southern blotting, PCR and restriction enzyme
Analysis.       [0028]   A preferred target host cell is a filamentous fungus, most preferably Magnaporthe.
grisea, which is the causative agent of rice blast. Blast is the Earth
Serious threat to the world's tropical rice cultivation, feeding more than one-third of the population
(Ou (1980) Plant Disease 64:43
9-445). An additional 13 million tonnes of rice are added each year due to the rapid growth of the rice consuming population.
Needed to get better (Lampe, (1994) Forewar)
d, Rice Blast Disease, Manilla, Philip
pins, CAB International, ix-x). However, 15
More than 7 million tons of rice lost from rice blast between 1975 and 1990
This is equivalent to 11 to 30% of global rice production (B
aker et al., (1997) Science 276: 726-733).       [0029]   Pathogen M. grisea is a crossbreeding Ascomycet (Ascomycet)
e), which is also the case for many forest grasses and wheat, barley and
It is pathogenic to economically important cereal crops, including ret. Rice blast disease true
The fungus invades the above-ground parts of rice, and in the case of severe epidemics, large oval foci
The whole surface can be swallowed. Spore-forming foci are responsible for this infection,
Can be extended to panicles. This infection cycle is easy under controlled conditions
It can be reproduced and the disease process can be studied in more detail.       [0030]   The method of the present invention also includes the components necessary to perform the method of the present invention, as well as
They can be packaged as a kit containing instructions for their use. like this
The kit comprises a transposable element, a transposase adapted to the transposable element.
Insert vector used to construct a large and large insert vector library
Could be included in whole or in part. This kit (could)
Also, suitable selectable markers separately or incorporated into the large insert vector described above.
Class and transposable elements.       [0031]   The methods, kits and components of the present invention can be used for DNA fragments and similar DNs in host organisms.
A DNA fragment against a target organism which is very suitable for promoting homologous recombination of the A fragment.
Is useful for causing a mutation in the target organism by supplying
. In this way, systematic, high-yield, and site-
Phenotype mapping of many or essentially all genes in an organism
I do.       [0032]   After disturbing essentially all genes of the target organism, two collections
Is obtained. The first is a collection of annotated insertion vectors. this
Seek vectors that can be used to mutate various genomic regions
Can be included in the databases and searches used to do so.       [0033]   Second, a collection of fungal strains that will have insertion events at various genomic locations.
It is an option. Fungal strains can be of any desired phenotype (eg, plant as described in Example 5).
Loss of virulence), but at metabolite levels or gene expression
Current changes can also be screened. Phenotype screenin depending on fungal strain
Of animal pathogens, secreted metabolites (new as a result of pathway disruption)
Changes in low molecular weight metabolites) and changes in secreted protein levels (modification of secretory pathway)
Can be searched).       [0034]   Live from individually selected large insert vectors or genomic large insert vectors
The sequencing of a number of disruptive insert vector-events from any of the Lari
Will result in multiple alleles or "hits" in the same gene
U. This is where the insertion occurs in the gene and, for example, knockout
Such as mutants, decreased biological activity of gene products, decreased expression levels of gene products, etc.
Be able to determine which mutation type was introduced. This allows heredity
Phenotypic species predicted from gene mutation at a specific position in the child DNA sequence
Class predictions will be better done. This situation is for several reasons
Desirable.       [0035]   First, the acquisition of multiple alleles indicates that the knockout process is near saturation.
Suggesting that it is This means that the recovery of multiple alleles is
In mutagenesis projects suggesting that the target is saturated by constant mutagens
Is synonymous with       [0036]   The second reason for this desired outcome is that the multiple alleles have a broad phenotypic sequence.
Is made possible. For example, insertion near the amino terminus of a gene
Would be expected to result in a null (loss of function) phenotype. However,
Insertion into the promoter region, carboxy-terminal region or 3 'untranslated leader region
, Can result in different phenotypes. These phenotypes indicate the activity of the gene product
Reduced (for loss of function) or modified (increased, reduced or temporally matched)
No (mistimed)) expression (in case of promoter mutation)
. This sequence of possible phenotypes is strictly "knockout" or loss-of-function phenotype.
This is a clear advantage of the TAG-KO technology over the construction of the TAG. Multiple alleles
Make it easier to produce genes that result in products that play a rate-limiting role in various processes
Characteristic analysis.       [0037] Example   Example 1Construction of transposable plasmids containing selectable markers. A. Construction of the FRIGG transposon: a sample transposo named FRIGG
Built. pCB1636 is Aspergillus nidulans
  Under control of the trpC promoter and terminator (Mullaney et al., (1
985) Mol Gen Genet 199: 37-45), Hygromy
Contains the syn-B phosphotransferase (hph) bacterial gene (Gri
tz and Davies, (1983) Gene 25: 179-188).
. This plasmid is available at the Fungal Genetics Stock Center (Fun)
gal Genetics Stock Center) (Kansas Ci)
ty, MO). The following procedure is described in Sambrook et al. (1989) M.
olecular Cloning, a Laboratory Manual
, Cold Spring Harbor Laboratory Press
Was carried out according to Contains the trpC / hph gene from pCB1636
Repairs the SalI fragment to be repaired, cuts it with SamI, and dephosphorylates the transposon vector.
Ter skeleton pMM2611 (derivative of pMM26 (Lampe et al., (1998) G
eletics 149: 179-187) and the plasmid pLHPG
1 was obtained. Eligible E. coli XL1-BLUE cells (Stratagene)
Transformation with pLHPG1 was performed according to the manufacturer's instructions. Transformants are:
Ampicillin 100 μg / ml and Hygromycin B 35 μg / ml (S
igma Chem. LB agar (Sambrook et al. (1989) Mo)
rectangular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press)
Selected above. DNA from each of the transformants was obtained from Wizard (Wiza).
rd) Using a miniprep kit (Promega) according to the manufacturer's instructions
Was prepared. After restriction digestion of pLHPG1 with BglI, self-ligation was performed.
Causing the disappearance of the picillin resistance gene and FRIGG, Himar1 tiger
Transposon and hygromycin resistance genes containing transposons were obtained.
. Transformation of E. coli cells was performed as described above, with 35 μg / g hygromycin.
FRIGG containing colonies were selected on LB agar containing ml. Figure 1
4 shows the construction of FRIGG.       [0038] B. Construction of other transposons: Instead of pMM2611, the above AT-
2 (Perkin Elmer; Devine et al. (1997) Genome R
es. 7: 551-563), GPS-1 (New England Bio)
labs; www. neb. com) or GPS-2 (New England)
d Biolabs; www. neb. com) other than using transposable elements
Follows the method of Example 1A.       [0039]   Example 2Cosmid / BAC library containing fungal genes and selectable markers Construction   Sambrook et al. (1989) Molecular Cloning, a.
Laboratory Manual, Cold Spring Harbor
  As described in the Laboratory Press, cosmids and BAC
Construction of Ibrari was carried out. Adenaille of Magnaporthe grisea
Autocyclase (MAC1; Adachi and Hamer, (1998)
) The Plant Cell 10: 1361-1373)
The present invention was demonstrated by selecting an Amp-resistant cosmid, cos1f8. This and
Separately, the fungal artificial chromosome BAC 21D3 (Diaz-Perez et al. (1996)
Fung. Genet. Biol. 20: 280-288) large insert frame
Chloramphenicol was used as the first selectable marker
.       [0040]   Example 3Construction of cosmids using transposons inserted into fungal genes A. Transposition into Cosmid: Transposition onto the cosmid framework is performed by Lampe
(1996) EMBO J. et al. 15: 5470-5479.
And so on. A total of 20 μl of translocation buffer (10% glycerol, 25m
M HEPES (pH 7.9 at room temperature (RT)), 250 μg of acetylated BSA
2 mM DTT, 100 mM NaCl, 10 mM MgCl_TwoOr
MnCl_Two) Was mixed with the following components: 1: 1 molar amount of donor: target (100
ng FRIGG: 1 μg pCosmac1), 0.2 μM Himar1
Lansposase (Lampe et al. (1986) EMBO J. 15: 5470-
5479). The translocation mixture was incubated at RT for 30 minutes. Rearrangement reaction
With 80 μl of stop solution (50 mM Tris-HCl, pH 7.6, 0.5 mg
/ Ml proteinase K, 10 mM EDTA, 250 μg / ml yeast tRNA
Was stopped by adding to the rearrangement reaction. The mixture is left at 30 ° C. for 1 hour
Incubation. This DNA was converted to 50% v / v NH_FourOAc, 2
Precipitated with 00% v / v ethanol 95%, TE buffer (Sambr)
OK et al. (1989) Molecular Cloning, a Labora.
tory Manual, Cold Spring Harbor Labor
atory Press). 1 μl of this DNA solution is
E. coli TOP1 by electroporation according to the manufacturer's instructions.
OF (transformed into cells (InVitrogen). Cells were transformed with ampicillin.
L medium containing 50 μg / ml and 25 μg / ml hygromycin was inoculated and
Incubated at 7 ° C for 2 days. Confirm DNA recovery after transposition and precipitation
To inoculate an aliquot of cells into L medium containing 100 μg / ml ampicillin,
Incubated overnight at 37 ° C.       [0041] B. Transposition into BAC: transposition onto the BAC framework replaces cosmid
Rampe et al. (1998) EMBO J. 15: 5
Performed essentially as described in 470-5479. 20 μl total volume
Buffer (10% glycerol, 25 mM HEPES (pH 7 at room temperature (RT))
. 9), 250 μg of acetylated BSA, 2 mM DTT, 100 mM N
aCl, 10 mM MgCl_TwoOr MnCl_TwoIn), the following ingredients were mixed:
1: 1 molar amount of donor: target (GPS-1, GPS-2 or A from Example 1)
T-2 100 ng: 1 μg of BAC from Example 2) and 0.2 μ each
M GPS-1, GPS-2 or AT-2 transposase (Perkin
Elmer, New England Biolabs; www. neb. co
m). The translocation mixture was incubated at RT for 30 minutes. The rearrangement reaction,
80 μl of stop solution (50 mM Tris-HCl, pH 7.6, 0.5 mg /
ml proteinase K, 10 mM EDTA, 250 μg / ml yeast tRNA
It was stopped by adding to the rearrangement reaction. The mixture is left at 30 ° C. for 1 hour
Incubate. This DNA was converted to 50% v / v NH_FourOAc, 20
Precipitated with 0% v / v ethanol 95%, TE buffer (Sambro
Ok et al. (1989) Molecular Cloning, a Laboratory.
ory Manual, ColdSpring Harbor Laboratory
ory Press). 1 μl of this DNA solution was prepared
E. coli TOP10F by electroporation according to the manufacturer's instructions.
(Transformed into cells (InVitrogen).
Contact with an L medium containing 12.5 μg / ml and 50 μg / ml hygromycin
Seeds were incubated for 2 days at 37 ° C. DNA recovery after transposition and precipitation
Cell aliquots containing 100 μg / ml chloramphenicol to confirm
L medium was inoculated and incubated at 37 ° C. overnight.       [0042]   Example 4High yield preparation and confirmation of transposon insertion into fungal genes   Cosmid pCOSmac1 containing E. coli strains with transposon inserts were
And 100 μg / ml ampicillin in TB (Terrific Broth, S
(1989) Molecular Cloning, a La.
boratemanual, Cold Spring Harbor L
laboratory press) in a 96-well growth block containing 1.5 ml
I picked it up. Blocks are incubated at 37C overnight with shaking.
Was an option. E. coli cells are pelleted by centrifugation, and cosmids are
And an improved alkaline dissolution method (Marra et al. (1997) Genome Res. 7).
: 1072-1084). DNA quality is measured on agarose gel
Was checked by electrophoresis. Cosmids are primers (TCGCT
CTTGAAGGGAACTATAG; SEQ ID NO: 1) and commercial dideoxy sequence
Determination kits (Big Dye Terminators, Perkin El
(mer). The sequencing reactions were performed with the ABI377 DNA sequence.
Analyzed with a sequencer.       [0043]   DNA sequences flanking the insertion site were collected and used to construct a BLAST algorithm.
Rhythms (Altshul et al. (1997) Nucleic Acids Res
. 25: 3389-3402) using DNA and protein databases.
Was searched. At position 3573 of nucleotide 3M. grisea
Denylate cyclase gene (Adachi and Hamer, (1998)
FRIGG into The Plant Cell 10: 1361-1373)
Of single inserts were selected for further analysis. This cosmid was replaced with cpgfrmacF.
It was named RIGG01a05.       [0044]   Same as above except chloramphenicol was used for selection instead of Amp
The procedure was performed on BACs and appropriate primers were used. Multiple insertions
And was observed in BACs.       [0045]   Example 5Assembly of transposon alleles   From the selected cosmid or from the genomic cosmid library
By sequencing a large number of upcoming disruptive insert vector events,
Multiple alleles, or "hits", will be obtained. By this
Where in the gene the insertion occurs, and, for example, a knockout mutant,
What types of changes, such as decreased biological activity of the gene product and decreased expression level of the gene product?
You will be able to determine if a difference has occurred. This gives the gene DNA sequence
Phenotypic types predicted from gene mutations at specific locations in
Will be performed well.       [0046]   Example 6Cosmid DNA in Bacteria for Transformation of the Fungus Magnaporthe grisea Preparation   Cosmid DNA was prepared by the Qiagen midiprep method (Qiagen).
Prepared. Propagation and storage of Magnaporthe grisea strain Guy11
And transformations have been described previously (Talbot et al. (1993) T
he Plant Cell 5: 1575-1590). DNA
About 10 μg was used for each transformation and contained 1 × 10 6 protoplasts
. Transformation was performed using Talbot et al. (1993) The Plant Cell 5
: 1575-1590. Hygromycin resistance transformation
The body was prepared from oatmeal agar (Crawford et al. (1986) Genetic).
s114: 1111-1129). If argB or pyr-
If 4 selectable markers are used, argB or pyr-4 resistance transformation
The transformants are taken up on oatmeal agar.       [0047]   Example 7Confirmation of homologous integration of transposons into fungal genes   The in vivo disruption of the adenylate cyclase gene was determined by primers G
GCGTTGTGATCTGCAG; SEQ ID NO: 2 and GGCCAGGAAAC
TCCCAG; by PCR analysis using SEQ ID NO: 3 and
Loss of progeny) and appressorium formation (Adachi and Hamer, (1998)
), The phenotype showing The Plant Cell 10: 1361-1373).
Was confirmed at the molecular weight level.     [Sequence list] [Brief description of the drawings]   1 shows the construction of the transposable element FRIGG described in Example 1. FIG.                                                                   

[Procedural amendment] Submission of translation of Article 34 amendment of the Patent Cooperation Treaty [Submission date] March 10, 2001 (2001.3.1.10) [Procedure amendment 1] [Document name to be amended] Drawing Item name] Figure 1 [Correction method] Change [Contents of correction] [Figure 1]

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C U, CZ, DE, DK, EE, ES, FI, GB, GE , GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, L R, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, T R, TT, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Hummer, Rizbes             United States 27707 North Carola             Durham Dover Avenue, Ina             ー 3414 F term (reference) 4B024 AA20 CA04 DA11 EA06 FA10                       GA11 HA20

Claims (1)

Claims: 1. A method for producing mutants by promoting site-directed homologous recombination in an organism, the method comprising: 1) providing one or more large insert vectors; Constructing a large insert vector library comprising: each of said large insert vectors comprising a piece of DNA, said DNA piece comprising a plurality of genes from the target organism and a first selection functioning for selection in bacteria. 2) constructing a second vector comprising a transposable element, wherein said transposable element is a nucleotide sequence encoding a second selectable marker flanked by inverted repeats on each side. Wherein said selectable marker is bifunctional for selection in fungi and target organisms, and wherein said inverted repeats are binding sites for a transposase. 3) the transposable element is randomly transferred into each large insert of the large insert library to produce disturbed large insert vectors, Step 2 in the presence of a transposase specific for the inverted repeat of
Incubating the product of step 1) with the product of step 1); 4) amplifying the disturbed large insert vector resulting from step 3);
5) introducing the disrupted large insert vector produced by step 4) into a target host cell, and 6) selecting the successful homologous recombination obtained by step 5) with a second selectable marker. , including. 2. The method of claim 1, wherein said large insert vector library is a cosmid library. 3. The method according to claim 2, wherein said cosmid is cos1f8. 4. The method of claim 1, wherein said first selectable marker is Amp. 5. The method according to claim 5, wherein the transposable element and the inverted repeat sequence are Himar1.
2. The method according to claim 1, wherein the method is derived from a transposon. 6. The method of claim 1, wherein said second selectable marker is hygromycin. 7. The method of claim 5, wherein said transposase is Himar1 transposase. 8. The method of claim 1, wherein said target host cell is a filamentous fungus. 9. The method according to claim 8, wherein the filamentous fungus is Magnaporthe grisea. 10. The method according to claim 1, for producing mutants by promoting site-directed homologous recombination in a filamentous fungus, the method comprising: 1) a cosmid library containing one or more cosmids; Constructing the cosmids, wherein each of the cosmids comprises a piece of DNA, wherein the piece of DNA comprises a plurality of genes from a target organism and a first selectable marker that functions for selection in a bacterium; The selectable marker is Amp; 2) constructing a second vector comprising a transposable element, said transposable element comprising a nucleotide sequence encoding hygromycin flanked by inverted repeats on each side. Wherein the inverted repeats are Himar1 (mariner);
3) Himar such that the transposable element is randomly transferred into each cosmid insert of the cosmid library to produce disrupted cosmid vectors.
1) Incubating the product of step 2) with the product of step 1) in the presence of (mariner). 4) Transforming the disrupted cosmids into a bacterial host, Selecting the presence of a large insert with an insertion event; 5) introducing into the target host cell the disrupted cosmid produced by step 4); and 6) the successful homology obtained by step 5). Selecting the recombination with hygromycin. 11. The method of claim 1, wherein said large insert vector library is a BAC library. 12. The method of claim 11, wherein said BAC is BAC 21D3. 13. The method of claim 1, wherein said first selectable marker is chloramphenicol. 14. The method according to claim 14, wherein the transposable element and the inverted repeat are GPS-1
2. The method of claim 1, wherein the method is selected from the group consisting of, GPS-2 and AT-2. 15. The method of claim 11, wherein said second selectable marker is hygromycin. 14. The transposase of claim 1, wherein said transposase is GPS-1, GPS-2 and A.
15. The method of claim 14, wherein the method is selected from the group consisting of T-2. 15. The amplification comprising selecting the presence of a large insert having an insertion event using the first and second selectable markers following transformation of the transposed large insert into a host cell. The method of claim 1, wherein 16. The method of claim 10, wherein said target host cell is a filamentous fungus. 17. The method according to claim 16, wherein the filamentous fungus is Magnaporthe grisea. 18. The method according to claim 1, for promoting mutants by promoting site-directed homologous recombination in a filamentous fungus, the method comprising: 1) a BAC library containing one or more BACs. Constructing the BAC
Each of which comprises a piece of DNA, said piece of DNA comprising a plurality of genes from the target organism and first selectable markers that function for selection in fungi, wherein said first selectable marker is chloramphie. 2) constructing a second vector comprising a transposable element, said transposable element comprising a nucleotide sequence encoding hygromycin flanked by inverted repeats on each side; Oriented repeats include GPS-1, GPS-2 and AT-
2) 3) GPS-1, GP such that the transposable element is randomly transferred into each BAC insert of the BAC library to produce disturbed BAC vectors.
Incubating the product of step 2) with the product of step 1) in the presence of a transposase selected from the group consisting of S-2 and AT-2, 4) transforming the disrupted cosmids into a fungal host Selecting the presence of a large insert with an insertion event using chloramphenicol and hygromycin, 5) introducing the disrupted BAC produced by step 4) into the target host cell, And 6) selecting the successful homologous recombination obtained by step 5) with hygromycin. 19. The method of claim 1, wherein said large insert vector library is a large DNA fragment library. 20. A kit for promoting site-directed homologous recombination in an organism to produce mutants, said kit comprising: a) a large insert vector library comprising one or more large insert vectors. Each of said large insert vectors comprises a piece of DNA, said piece of DNA comprising a plurality of genes from the target organism and first selectable markers functioning for selection in fungi; b) transposition A second vector comprising a possible element, said transposable element comprising a nucleotide sequence encoding a second selectable marker flanked by inverted repeats on each side, said selectable marker being present in fungi and target organisms. Is bifunctional for selection in and the inverted repeats are functional as binding sites for a transposase; c) the transposable A transposase on a second vector specific for the inverted repeat, such that the active elements produce randomly disturbed large insert vectors that are randomly transferred into each large insert of the large insert library; d) Combining the components of a), b) and c) to obtain perturbed large insert vectors, and then incorporating the perturbed large insert vectors into a target organism for site-directed Instructions for obtaining the mutated target organism. 21. A large insert library produced by the method of claim 1.