U.S. patent application number 14/867816 was filed with the patent office on 2016-03-17 for targeted chromosomal mutagenesis using zinc finger nucleases.
The applicant listed for this patent is University of Utah Research Foundation. Invention is credited to Marina Bibikova, Dana Carroll, Gary Drews, Kent G. Golic, Mary M. Golic.
Application Number | 20160076045 14/867816 |
Document ID | / |
Family ID | 29250450 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160076045 |
Kind Code |
A1 |
Carroll; Dana ; et
al. |
March 17, 2016 |
TARGETED CHROMOSOMAL MUTAGENESIS USING ZINC FINGER NUCLEASES
Abstract
The present invention provides for a method or methods of
targeted genetic recombination or mutagenesis in a host cell or
organism, and compositions useful for carrying out the method. The
targeting method of the present invention exploits endogenous
cellular mechanisms for homologous recombination and repair of
double stranded breaks in genetic material. The present invention
provides numerous improvements over previous mutagenesis methods,
such advantages include that the method is generally applicable to
a wide variety of organisms, the method is targeted so that the
disadvantages associated with random insertion of DNA into host
genetic material are eliminated, and certain embodiments require
relatively little manipulation of the host genetic material for
success. Additionally, it provides a method that produces organisms
with specific gene modifications in a short period of time.
Inventors: |
Carroll; Dana; (Salt Lake
City, UT) ; Golic; Mary M.; (Salt Lake City, UT)
; Bibikova; Marina; (San Diego, CA) ; Drews;
Gary; (Salt Lake City, UT) ; Golic; Kent G.;
(Salt Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Utah Research Foundation |
Salt Lake City |
UT |
US |
|
|
Family ID: |
29250450 |
Appl. No.: |
14/867816 |
Filed: |
September 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13314490 |
Dec 8, 2011 |
9145565 |
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14867816 |
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10502565 |
Jul 22, 2004 |
8106255 |
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PCT/US03/02012 |
Jan 22, 2003 |
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13314490 |
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60351035 |
Jan 23, 2002 |
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Current U.S.
Class: |
435/419 |
Current CPC
Class: |
C12N 15/8257 20130101;
C12N 15/8213 20130101; C12N 15/902 20130101; C12N 9/22 20130101;
A01K 67/0339 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. Government has certain rights in the invention
based upon partial support by Grant R01 GM 58504.
Claims
1.-98. (canceled)
99. A plant cell comprising a mutation introduced by a Zinc Finger
Nuclease (ZFN) that binds to a target site in a chosen host
chromosomal target locus; wherein the mutation comprises an
insertion and/or deletion of genetic material surrounding the
target site.
100. The plant cell of claim 99, wherein the mutation is an
insertion of genetic material.
101. The plant cell of claim 99, wherein the mutation is a deletion
of genetic material.
102. The plant cell of claim 99, wherein the mutation is both a
deletion and an insertion of genetic material.
103. The plant cell of claim 99, wherein a donor DNA is inserted
into the chromosomal target locus.
104. The plant cell of claim 103, wherein the donor DNA provides a
gene sequence that encodes a product to be produced in the plant
cell.
105. The plant cell of claim 103, wherein the donor DNA provides a
gene sequence that encodes a pharmaceutical, hormone, protein,
nutriceutical or chemical.
106. The plant cell of claim 99, wherein the method further
comprises: selecting a zinc finger nuclease comprises a zinc finger
protein DNA binding domain capable and a non-specific DNA cleavage
domain capable of cleaving double-stranded DNA when operatively
linked to said DNA-binding domain.
107. The plant cell of claim 106, wherein the DNA binding domain is
comprised of three zinc fingers.
108. The plant cell of claim 106, wherein the zinc finger DNA
binding domain is a Cis2His2 zinc finger.
109. The plant cell of claim 106, wherein the cleavage domain is
from a Type II restriction endonuclease.
110. The plant cell of claim 109, wherein the Type II restriction
endonuclease is Fold.
111. The plant cell of claim 104, wherein the gene sequences
encodes one or more selectable markers.
112. The plant cell of claim 111, wherein the one or more
selectable markers provides positive selection for cells expressing
the marker.
113. The plant cell of claim 111, wherein the one or more
selectable markers provides negative selection for cells expressing
the marker.
114. The plant cell of claim 111, wherein the selectable marker
provides positive and negative selection for cells expressing the
marker.
115. The plant cell of claim 104, wherein the donor DNA comprises a
constitutively active or inducible promoter upstream of the gene
sequence that encodes a product to be produced in the plant cell.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present application is continuation of U.S. patent
application Ser. No. 10/502,565 file on Jul. 22, 2004, which is a
National Phase Application of International Application No.
PCT/US03/002012 filed on Jan. 22, 2003, which claims priority from
U.S. Provisional Patent Application No. 60/351,035 filed Jan. 23,
2002, which is hereby incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Gene targeting--the process of gene replacement by
homologous recombination or mutation--is a very useful, but
typically inefficient technique for introducing desired changes in
the genetic material of a host cell. Only when powerful selection
for the targeted product can be applied is recovery of the desired
alteration possible. A general method for improving the efficiency
of gene targeting would be valuable in many circumstances, as would
extension of this tool to a broader range of organisms.
[0004] It has been demonstrated in model experiments that
introduction of a double-strand break (DSB) in host DNA greatly
enhances the frequency of localized recombination. However, those
tests required insertion of a recognition site for a specific
endonuclease before cleavage could be induced. Similarly, in
Drosophila the DSBs produced by P-element excision are
recombinagenic, but require the P-element to preexist at the target
site.
[0005] Although previously demonstrated methods of genetic
transformation had been highly successful, transformation without
targeted recombination has also been accompanied by problems
associated with random insertion of the introduced DNA. Random
integration can lead to the inactivation of essential genes, or to
the aberrant expression of the introduced gene. Additional problems
associated with genetic transformation include mosaicism due to
multiple integrations, and technical difficulties associated with
generation of replication defective recombinant viral vectors.
[0006] Targeted genetic recombination or mutation of a cell or
organism is now possible because complete genomic sequences have
been determined for a number of organisms, and more sequences are
being obtained each day. Not only would the ability to direct a
mutation to a specific genetic locus greatly aid those studying the
function of particular genes, targeted genetic recombination would
also have therapeutic and agricultural applications. Methods of
targeted genetic recombination are needed that are more general,
efficient, and/or reproducible than currently available
techniques.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions and methods for
carrying out targeted genetic recombination or mutation. Any
segment of endogenous nucleic acid in a cell or organism can be
modified by the method of the invention as long as the sequence of
the target region, or portion of the target region, is known, or if
isolated DNA homologous to the target region is available.
[0008] In certain embodiments, the compositions and methods
comprise the transformation of a host organism by introducing a
nucleic acid molecule encoding a chimeric zinc finger nuclease into
a cell or organism and identifying a resulting cell or organism in
which a selected endogenous DNA sequence is cleaved and exhibits a
mutation.
[0009] In a preferred embodiment, such methods comprise selecting a
zinc finger DNA binding domain capable of preferentially binding to
a specific host DNA locus to be mutated; further selecting a
non-specific DNA cleavage domain capable of cleaving
double-stranded DNA when operatively linked to said binding domain
and introduced into the host cell; further selecting an inducible
promoter region capable of inducing expression in the host cell;
and further operatively linking DNA encoding the binding domain and
the cleavage domain and the inducible promoter region to produce a
DNA construct. The DNA construct is then introduced into a target
host cell and at least one host cell exhibiting recombination at
the target locus in the host DNA is identified. In a particular
embodiment, the DNA binding domain comprises the binding domains of
three Cis.sub.2His.sub.2 zinc fingers. In another embodiment, the
cleavage domain comprises a cleavage domain derived from the Type
II restriction endonuclease FokI. In one embodiment, an inducible
heat shock promoter is operatively linked to DNA encoding the
chimeric zinc finger nuclease.
[0010] Additional embodiments involve methods for targeted
insertion by homologous recombination of selected DNA sequences
(donor DNA). Donor DNA can comprise a sequence that encodes a
product to be produced in the host cell. Said product can be a
product produced for the benefit of the host cell or organism (for
example, gene therapy), or the product can be one that is produced
for use outside the host cell or organism (for example, the product
may be selected from, but not limited to, pharmaceuticals,
hormones, protein products used in the manufacture of useful
objects or devices, nutriceuticals, products used in chemical
manufacture or synthesis, etc.).
[0011] In a certain embodiment, the present invention is utilized
to disrupt a targeted gene in a somatic cell. Such gene may be
over-expressed in one or more cell types resulting in disease.
Disruption of such gene may only be successful in a low percentage
of somatic cells but such disruption may contribute to better
health for an individual suffering from disease due to
over-expression of such gene.
[0012] In another embodiment, the present invention can be utilized
to disrupt a targeted gene in a germ cell. Cells with such
disruption in the targeted gene can be selected for in order to
create an organism without function of the targeted gene. In such
cell the targeted gene function can be completely knocked out.
[0013] In another embodiment, the present invention can be utilized
to enhance expression of a particular gene by the insertion of a
control element into a somatic cell. Such a control element may be
selected from a group consisting of, but not limited to, a
constitutively active, inducible, tissue-specific or development
stage-specific promoters. Such control element may be targeted to a
chromosomal locus where it will effect expression of a particular
gene that is responsible for a product with a therapeutic effect in
such a cell or the host organism. The present invention may further
provide for the insertion of donor DNA containing a gene encoding a
product that, when expressed, has a therapeutic effect on the host
cell or organism. An example of such a therapeutic method would be
to use the targeted genetic recombination of the present invention
to effect insertion into a pancreatic cell of an active promoter
operatively linked to donor DNA containing an insulin gene. The
pancreatic cell containing the donor DNA would then produce
insulin, thereby aiding a diabetic host. Additionally, donor DNA
constructs could be inserted into a crop genome in order to effect
the production of a pharmaceutical relevant gene product. A gene
encoding a pharmaceutical useful protein product, such as insulin
or hemoglobin, functionally linked to a control element, such as a
constitutively active, inducible, tissue-specific or development
stage-specific promoters, could be inserted into a host plant in
order to produce a large amount of the pharmaceutically useful
protein product in the host plant. Such protein products could then
be isolated from the plant. Alternatively, the above-mentioned
methods can be utilized in a germ cell.
[0014] The present invention can be utilized in both somatic and
germ line cells to effect alteration at any chromosomal target
locus.
[0015] Methods of the present invention are applicable to a wide
range of cell types and organisms. The present invention can apply
to any of the following cells, although the methods of the
invention are not limited to the cells or organisms herein listed:
A single celled or multicellular organism; an oocyte; a gamete; a
germline cell in culture or in the host organism; a somatic cell in
culture or in the host organism; an insect cell, including an
insect selected from the group consisting of Coleoptera, Diptera,
Hemiptera, Homoptera, Hymenoptera, Lepidoptera, or Orthoptera,
including a fruit fly, a mosquito and a medfly; a plant cell,
including a monocotyledon cell and a dicotyledon cell; a mammalian
cell, including but not limited to a cell selected from the group
consisting of mouse, rat, pig, sheep, cow, dog or cat cells; an
avian cell, including, but not limited to a cell selected from the
group consisting of chicken, turkey, duck or goose cells; or a fish
cell, including, but not limited to zebrafish, trout or salmon
cells.
[0016] Many alterations and variations of the invention exist as
described herein. The invention is exemplified for targeted genetic
recombination in the insect, Drosophila and the plant, Arabidopsis.
In Drosophila and Arabidopsis, the nucleotide sequence is known for
most of the genome. Large segments of genomic sequences from other
organisms are becoming known at a fast pace. The elements necessary
to carry out the methods of the present invention as herein
disclosed can be adapted for application in any cell or organism.
The invention therefore provides a general method for targeted
genetic recombination in any cell or organism.
[0017] Table 1: Illustrates the number of germline mutants
recovered by crossing males exposed to a heat shock with attached-X
[C(1)DX] females and females from the heat shock to FM6 (y) males
in accordance with an embodiment of the present invention. The
percent of all the heat-shocked parents screened that gave at least
one germline mutant is shown in parentheses in the # Giving y
column. The total number of mutant flies recovered is given in the
Total y column and also expressed as a percent of all candidate
offspring (in parentheses). The number of mutant offspring per fly
varied from 1 to 15. The ND data are from M. Bibikova et al. (2002)
Genetics 161: 1169-1175 which is hereby incorporated by
reference.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to methods and compositions
for carrying out targeted genetic recombination or mutation. In
contrast to previously known methods for targeted genetic
recombination, the present invention is efficient and inexpensive
to perform and is adaptable to any cell or organism. Any segment of
double-stranded nucleic acid of a cell or organism can be modified
by the method of the present invention. The method exploits both
homologous and non-homologous recombination processes that are
endogenous in all cells.
[0019] The method of the present invention provides for both
targeted DNA insertions and targeted DNA deletions. The method
involves transformation of a cell with a nucleic acid construct
minimally comprising DNA encoding a chimeric zinc finger nuclease
(ZFN). In a particular embodiment, the method further involves
transforming a cell with a nucleic acid construct comprising donor
DNA. Other schemes based on these general concepts are within the
scope and spirit of the invention, and are readily apparent to
those skilled in the art.
[0020] The present invention can be utilized in both somatic and
germ cells to conduct genetic manipulation at a particular genetic
locus.
[0021] In a particular embodiment, the present invention is
utilized to disrupt a gene in a somatic cell wherein that gene is
over-expressing a product and/or expressing a product that is
deleterious to the cell or organism. Such gene may be
over-expressed in one or more cell types resulting in disease.
Disruption of such gene by the methods of the present invention may
contribute to better health for an individual suffering from
disease due to expression of such gene. In other words, disruption
of genes in even a small percentage of cells can work to decrease
expression levels in order to produce a therapeutic effect.
[0022] In another embodiment, the present invention can be utilized
to disrupt a gene in a germ cell. Cells with such disruption in a
particular gene can be selected for in order to create an organism
without function of such gene. In such cell the gene can be
completely knocked-out. The absence of function in this particular
cell can have a therapeutic effect.
[0023] In another embodiment, the present invention can be utilized
to enhance expression of a particular gene by the insertion of a
control element into a somatic cell. Such control element may be a
constitutively active, inducible or development stage-specific
promoter. It may also be a tissue-specific promoter capable of
effecting expression only in particular cell types. Such control
element may be placed in such a manner to effect expression of a
particular gene that is responsible for a product with a
therapeutic effect in such a cell.
[0024] The present invention may further provide for the insertion
of donor DNA encoding a gene product that, when constitutively
expressed, has a therapeutic effect. An example of this embodiment
would be to insert such DNA constructs into an individual suffering
from diabetes in order to effect insertion of an active promoter
and donor DNA encoding the insulin gene in a population of
pancreatic cells. This population of pancreatic cells containing
the exogenous DNA would then produce insulin, thereby aiding the
diabetic patient. Additionally, such DNA constructs could be
inserted into crops in order to effect the production of
pharmaceutically-relevant gene products. Genes for protein
products, such as insulin, lipase or hemoglobin, could be inserted
into plants along with control elements, such as constitutively
active or inducible promoters, in order to produce large amounts of
these pharmaceuticals in a plant. Such protein products could then
be isolated from the plant. Transgenic plants or animals may be
produced with this method through a nuclear transfer technique
(McCreath, K. J. et al. (2000) Nature 405: 1066-1069; Polejaeva, I.
A. et al., (2000) Nature 407: 86-90). Tissue or cell-type specific
vectors may also be employed for providing gene expression only in
the cells of choice.
[0025] Alternatively, the above-mentioned methods can be utilized
in a germ cell in order to select cells where insertion has
occurred in the planned manner in order for all subsequent cell
divisions to produce cells with the desired genetic change.
[0026] As used herein, the cells in which genetic manipulation
occurs and an exogenous DNA segment or gene has been introduced
through the hand of man are called recombinant cells. Therefore,
recombinant cells are distinguishable from naturally occurring
cells which do not contain a recombinantly introduced exogenous DNA
segment or gene. Recombinant cells include those having an
introduced cDNA or genomic gene, and also include genes positioned
adjacent to a heterologous promoter not naturally associated with
the particular introduced gene.
[0027] To express a recombinant encoded protein or peptide, whether
mutant or wild-type, in accordance with the present invention one
would prepare an expression vector that comprises isolated nucleic
acids under the control of, or operatively linked to, one or more
promoters, which may be inducible, constitutively active or tissue
specific, for example. To bring a coding sequence "under the
control of a promoter, one positions the 5' end of the
transcription initiation site of the transcriptional reading frame
generally between about 1 and about 50 nucleotides "downstream"
(i.e., 3') of the chosen promoter. The "upstream" promoter
stimulates transcription of the DNA and promotes expression of the
encoded recombinant protein. This is the meaning of "recombinant
expression" in this context.
[0028] Ways of effecting protein expression are well known in the
art. One skilled in the art is capable of expression a protein of
his or her choice in accordance with the present invention.
[0029] The methods of the present invention can be applied to whole
organisms or in cultured cells or tissues or nuclei, including
those cells, tissues or nuclei that can be used to regenerate an
intact organism, or in gametes such as eggs or sperm in varying
stages of their development. Because DSBs stimulate mutagenic
repair in essentially all cells or organisms, cleavage by ZFNs may
be used in any cells or organisms. The methods of the present
invention can be applied to cells derived any organism, including
but not limited to insects, fungi, rodents, cows, sheep, goats,
chickens, and other agriculturally important animals, as well as
other mammals, including, but not limited to dogs, cats and
humans.
[0030] Additionally, the compositions and methods of the present
invention may be used in plants. It is contemplated that the
compositions and methods can be used in any variety of plant
species, such as monocots or dicots. In certain embodiments, the
invention can be used in plants such as grasses, legumes, starchy
staples, Brassica family members, herbs and spices, oil crops,
ornamentals, woods and fibers, fruits, medicinal plants, poisonous
plants, corn, cotton, castor bean and any other crop specie. In
alternative embodiments, the invention can be used in plants such
as sugar cane, wheat, rice, maize, potato, sugar beet, cassava,
barley, soybean, sweet potato, oil palm fruit, tomato, sorghum,
orange, grape, banana, apple, cabbage, watermelon, coconut, onion,
cottonseed, rapeseed and yam. In some embodiments, the invention
can be used in members of the Solanaceae specie, such as tobacco,
tomato, potato and pepper. In other embodiments, the invention can
be used in poisonous ornamentals, such as oleander, any yew specie
and rhododendron. In a particular embodiment, the Brassica specie
is Arabidopsis.
[0031] Grasses include, but are not limited to, wheat, maize, rice,
rye, triticale, oats, barley, sorghum, millets, sugar cane, lawn
grasses and forage grasses. Forage grasses include, but are not
limited to, Kentucky bluegrass, timothy grass, fescues, big
bluestem, little bluestem and blue gamma. Legumes include, but are
not limited to, beans like soybean, broad or Windsor bean, kidney
bean, lima bean, pinto bean, navy bean, wax bean, green bean,
butter bean and mung bean; peas like green pea, split pea,
black-eyed pea, chick-pea, lentils and snow pea; peanuts; other
legumes like carob, fenugreek, kudzu, indigo, licorice, mesquite,
copaifera, rosewood, rosary pea, senna pods, tamarind, and
tuba-root; and forage crops like alfalfa. Starchy staples include,
but are not limited to, potatoes of any species including white
potato, sweet potato, cassava, and yams. Brassica, include, but are
not limited to, cabbage, broccoli, cauliflower, brussel sprouts,
turnips, collards, kale and radishes. Oil crops include, but are
not limited to, soybean, palm, rapeseed, sunflower, peanut,
cottonseed, coconut, olive palm kernel. Woods and fibers include,
but are not limited to, cotton, flax, and bamboo. Other crops
include, but are not limited to, quinoa, amaranth, tarwi,
tamarillo, oca, coffee, tea, and cacao.
DEFINITIONS
[0032] For the purposes of the present invention, the following
terms shall have the following meanings:
[0033] As used herein, the term "targeted genetic recombination"
refers to a process wherein recombination occurs within a DNA
target locus present in a host cell or host organism. Recombination
can involve either homologous or non-homologous DNA. One example of
homologous targeted genetic recombination would be cleavage of a
selected locus of host DNA by a zinc finger nuclease (ZFN),
followed by homologous recombination of the cleaved DNA with
homologous DNA of either exogenous or endogenous origin. One
example of non-homologous targeted genetic recombination would be
cleavage of a selected locus of host DNA by a ZFN, followed by
non-homologous end joining (NHEJ) of the cleaved DNA.
[0034] As used herein, the terms "host cell" or "host organism" or,
simply, "target host", refer to a cell or an organism that has been
selected to be genetically transformed to carry one or more genes
for expression of a function used in the methods of the present
invention. A host can further be an organism or cell that has been
transformed by the targeted genetic recombination or mutation
methods of the present invention.
[0035] The term "target" or "target locus" or "target region"
refers herein to the gene or DNA segment selected for modification
by the targeted genetic recombination method of the present
invention. Ordinarily, the target is an endogenous gene, coding
segment, control region, intron, exon or portion thereof, of the
host organism. However, the target can be any part or parts of the
host DNA.
[0036] For the purposes of the present invention, the term "zinc
finger nuclease" or "ZFN" refers to a chimeric protein molecule
comprising at least one zinc finger DNA binding domain effectively
linked to at least one nuclease capable of cleaving DNA.
Ordinarily, cleavage by a ZFN at a target locus results in a double
stranded break (DSB) at that locus.
[0037] For the purposes of the present invention, the term "marker"
refers to a gene or sequence whose presence or absence conveys a
detectable phenotype to the host cell or organism. Various types of
markers include, but are not limited to, selection markers,
screening markers and molecular markers. Selection markers are
usually genes that can be expressed to convey a phenotype that
makes an organism resistant or susceptible to a specific set of
environmental conditions. Screening markers can also convey a
phenotype that is a readily observable and distinguishable trait,
such as Green Fluorescent Protein (GFP), GUS or beta-galactosidase.
Molecular markers are, for example, sequence features that can be
uniquely identified by oligonucleotide probing, for example RFLP
(restriction fragment length polymorphism), or SSR markers (simple
sequence repeat).
[0038] As used herein, the term "donor" or "donor construct" refers
to the entire set of DNA segments to be introduced into the host
cell or organism as a functional group. The term "donor DNA" as
used herein refers to a DNA segment with sufficient homology to the
region of the target locus to allow participation in homologous
recombination at the site of the targeted DSB.
[0039] For the purposes of the present invention, the term "gene"
refers to a nucleic acid sequence that includes the translated
sequences that encode a protein ("exons"), the untranslated
intervening sequences ("introns"), the 5' and 3' untranslated
region and any associated regulatory elements.
[0040] For the purposes of the present invention, the term
"sequence" means any series of nucleic acid bases or amino acid
residue, and may or may not refer to a sequence that encodes or
denotes a gene or a protein. Many of the genetic constructs used
herein are described in terms of the relative positions of the
various genetic elements to each other. For the purposes of the
present invention, the term "adjacent" is used to indicate two
elements that are next to one another without implying actual
fusion of the two elements. Additionally, for the purposes of the
present invention, "flanking" is used to indicate that the same,
similar, or related sequences exist on either side of a given
sequence. Segments described as "flanking" are not necessarily
directly fused to the segment they flank, as there can be
intervening, non-specified DNA between a given sequence and its
flanking sequences. These and other terms used to describe relative
position are used according to normal accepted usage in the field
of genetics.
[0041] For the purposes of the present invention, the term
"recombination," is used to indicate the process by which genetic
material at a given locus is modified as a consequence of an
interaction with other genetic material. For the purposes of the
present invention, the term "homologous recombination" is used to
indicate recombination occurring as a consequence of interaction
between segments of genetic material that are homologous, or
identical. In contrast, for purposes of the present invention, the
term "non-homologous recombination" is used to indicate a
recombination occurring as a consequence of interaction between
segments of genetic material that are not homologous, or identical.
Non-homologous end joining (NHEJ) is an example of non-homologous
recombination.
[0042] Moreover, for the purposes of the present invention, the
term "a" or "an" entity refers to one or more than one of that
entity; for example, "a protein" or "an nucleic acid molecule"
refers to one or more of those compounds, or at least one compound.
As such, the terms "a" or "an", "one or more" and "at least one"
can be used interchangeably herein. It is also to be noted that the
terms "comprising," "including," and "having" can be used
interchangeably. Furthermore, a compound "selected from the group
consisting of refers to one or more of the compounds in the list
that follows, including mixtures (i.e. combinations) of two or more
of the compounds. According to the present invention, an isolated
or biologically pure compound is a compound that has been removed
from its natural milieu. As such, "isolated" and "biologically
pure" do not necessarily reflect the extent to which the compound
has been purified. An isolated compound of the present invention
can be obtained from its natural source, can be produced using
molecular biology techniques or can be produced by chemical
synthesis.
[0043] Zinc Finger Nucleases
[0044] A zinc finger nuclease (ZFN) of the present invention is a
chimeric protein molecule capable of directing targeted genetic
recombination or targeted mutation in a host cell by causing a
double stranded break (DSB) at the target locus. A ZFN of the
present invention includes a DNA-binding domain and a DNA-cleavage
domain, wherein the DNA binding domain is comprised of at least one
zinc finger and is operatively linked to a DNA-cleavage domain. The
zinc finger DNA-binding domain is at the N-terminus of the chimeric
protein molecule and the DNA-cleavage domain is located at the
C-terminus of said molecule.
[0045] A ZFN as herein described must have at least one zinc
finger. In a preferred embodiment a ZFN of the present invention
would have at least three zinc fingers in order to have sufficient
specificity to be useful for targeted genetic recombination in a
host cell or organism. A ZFN comprising more than three zinc
fingers is within the scope of the invention. A ZFN having more
than three zinc fingers, although more time-consuming to construct,
would have progressively greater specificity with each additional
zinc finger. In a particular embodiment, the DNA-binding domain is
comprised of three zinc finger peptides operatively linked to a DNA
cleavage domain.
[0046] The zinc finger domain of the present invention can be
derived from any class or type of zinc finger. In a particular
embodiment, the zinc finger domain comprises the Cis.sub.2His.sub.2
type of zinc finger that is very generally represented, for
example, by the zinc finger transcription factors TFIIIA or Sp1. In
a preferred embodiment, the zinc finger domain comprises three
Cis.sub.2His.sub.2 type zinc fingers. The DNA recognition and/or
the binding specificity of a ZFN can be altered in order to
accomplish targeted genetic recombination at any chosen site in
cellular DNA. Such modification can be accomplished using known
molecular biology and/or chemical synthesis techniques. (see, for
example, M. Bibikova et al. (2002) Genetics 161: 1169-1175). ZFNs
comprising zinc fingers having a wide variety of DNA recognition
and/or binding specificities are within the scope of the present
invention.
[0047] The ZFN DNA-cleavage domain is derived from a class of
non-specific DNA cleavage domains, for example the DNA-cleavage
domain of a Type II restriction enzyme. In a particular embodiment
the DNA-cleavage domain is derived from the Type 11 restriction
enzyme, FokI.
[0048] In a preferred embodiment, a ZFN comprises three
Cis.sub.2His.sub.2 type of zinc fingers, and a DNA-cleavage domain
derived from the type II restriction enzyme, FokI According to this
preferred embodiment, each zinc finger contacts 3 consecutive base
pairs of DNA creating a 9 bp recognition sequence for the ZFN DNA
binding domain. The DNA-cleavage domain of the preferred embodiment
requires dimerization of two ZFN DNA-cleavage domains for effective
cleavage of double-stranded DNA. (See, for example, J. Smith et
al., (2000) Nucleic Acids Res. 28: 3361-3369). This imposes a
requirement for two inverted recognition (target DNA) sites within
close proximity for effective targeted genetic recombination. If
all positions in the target sites are contacted specifically, these
requirements enforce recognition of a total of 18 base pairs of
DNA. There may be a space between the two sites. The space between
recognition sites for ZFNs of the present invention may be
equivalent to 6 to 35 bp of DNA. The region of DNA between the two
recognitions sites is herein referred to as the "spacer".
[0049] A linker, if present, between the cleavage and recognition
domains of the ZFN comprises a sequence of amino acid residues
selected so that the resulting linker is flexible. Or, for maximum
target site specificity, linkerless constructs are made. A
linkerless construct has a strong preference for binding to and
then cleaving between recognition sites that are 6 bp apart.
However, with linker lengths of between 0 and 18 amino acids in
length, ZFN-mediated cleavage occurs between recognition sites that
are between 5 and 35 bp apart. For a given linker length, there
will be a limit to the distance between recognition sites that is
consistent with both binding and dimerization. (M. Bibikova et al.
(2001) Mol. Cell. Biol. 21: 289-287). In a preferred embodiment,
there is no linker between the cleavage and recognition domains,
and the target locus comprises two nine nucleotide recognition
sites in inverted orientation with respect to one another,
separated by a six nucleotide spacer.
[0050] In order to target genetic recombination or mutation
according to a preferred embodiment of the present invention, two 9
bp zinc finger DNA recognition sequences must be identified in the
host DNA. These recognition sites will be in an inverted
orientation with respect to one another and separated by about 6 bp
of DNA. ZFNs are then generated by designing and producing zinc
finger combinations that bind DNA specifically at the target locus,
and then linking the zinc fingers to a cleavage domain of a Type II
restriction enzyme.
Targeted Genetic Recombination or Mutation
[0051] The method of the present invention can be used for targeted
genetic recombination or mutation of any cell or organism. Minimum
requirements include a method to introduce genetic material into a
cell or organism (either stable or transient transformation),
sequence information regarding the endogenous target region, and a
ZFN construct or constructs that recognizes and cleaves the target
locus. According to some applications of the present invention, for
example homologous recombination, donor DNA may also be
required.
[0052] According to another application of the present invention,
DNA encoding an identifiable marker will also be included with the
DNA construct. Such markers may include a gene or sequence whose
presence or absence conveys a detectable phenotype to the host cell
or organism. Various types of markers include, but are not limited
to, selection markers, screening markers and molecular markers.
Selection markers are usually genes that can be expressed to convey
a phenotype that makes an organism resistant or susceptible to a
specific set of environmental conditions. Screening markers can
also convey a phenotype that is a readily observable and
distinguishable trait, such as Green Fluorescent Protein (GFP),
beta-glucuronidase (GUS) or beta-galactosidase. Markers may also be
negative or positive selectable markers. In a particular
embodiment, such negative selectable marker is codA. Molecular
markers are, for example, sequence features that can be uniquely
identified by oligonucleotide probing, for example RFLP
(restriction fragment length polymorphism), or SSR markers (simple
sequence repeat).
[0053] The efficiency with which endogenous homologous
recombination occurs in the cells of a given host varies from one
class of cell or organism to another. However the use of an
efficient selection method or a sensitive screening method can
compensate for a low rate of recombination. Therefore, the basic
tools for practicing the invention are available to those of
ordinary skill in the art for a wide range and diversity of cells
or organisms such that the successful application of such tools to
any given host cell or organism is readily predictable. The
compositions and methods of the present invention can be designed
to introduce a targeted mutation or genetic recombination into any
host cell or organism. The flexibility of the present invention
allows for genetic manipulation in order to create genetic models
of disease or to investigate gene function.
[0054] The compositions and methods of the present invention can
also be used to effect targeted genetic recombination or mutation
in a mammalian cell. In addition, a ZFN can be designed to cleave a
particular gene or chromosomal locus, which is then injected into
an isolated embryo prior to reimplantation into a female.
ZFN-mediated DNA cleavage can occur either in the presence or
absence of donor DNA. Off-springs can then be screened for the
desired genetic alteration.
[0055] The compositions and methods of the present invention can
also be used accomplish germline gene therapy in mammals. In one
embodiment, ZFNs could be designed to target particular genes of
interest. Eggs and sperm could be collected and in-vitro
fertilization performed. At the zygote stage, the embryo could be
treated with both a ZFN designed to target a particular sequence
and a donor DNA segment carrying a sequence without the deleterious
mutation. The embryo could then be returned to a female or a
uterine alternative for the rest of the gestational period. In a
particular embodiment, for example, the deleterious gene is the
common cystic fibrosis (CF) allele delta F508. ZFNs and donor DNA
are used according to the methods of the present invention in order
to alleviate disease caused by a mutant gene. According to the
method, eggs and sperm from known carrier parents are collected and
in-vitro fertilized. After in-vitro fertilization, the zygote could
be injected with ZFNs designed to target the delta F508 allele, and
with donor DNA carrying the wild-type allele. The transformed
zygote could then be reimplanted into the mother. With the
compositions and methods of the present invention, such gene
replacement would allow the offspring and all descendants to be
free of the CF mutation.
[0056] In another embodiment, homologous recombination can be used
as follows. First, a site for integration is selected within the
host cell. Sequences homologous to the integration site are then
included in a genetic construct, flanking the selected gene to be
integrated into the genome. Flanking, in this context, simply means
that target homologous sequences are located both upstream (5') and
downstream (3') of the selected gene. These sequences should
correspond to some sequences upstream and downstream of the target
gene. The construct is then introduced into the cell, thus
permitting recombination between the cellular sequences and the
construct.
[0057] As a practical matter, the genetic construct will normally
act as far more than a vehicle to insert the gene into the genome.
For example, it is important to be able to select for recombinants
and, therefore, it is common to include within the construct a
selectable marker gene. The marker permits selection of cells that
have integrated the construct into their genomic DNA. In addition,
homologous recombination may be used to "knock-out" (delete) or
interrupt a particular gene. Thus, another approach for inhibiting
gene expression involves the use of homologous recombination, or
"knockout technology". This is accomplished by including a mutated
or vastly deleted form of the heterologous gene between the
flanking regions within the construct. Thus, it is possible, in a
single recombinational event, to (i) "knock out" an endogenous
gene, (ii) provide a selectable marker for identifying such an
event and (iii) introduce a transgene for expression.
[0058] The frequency of homologous recombination in any given cell
is influenced by a number of factors. Different cells or organisms
vary with respect to the amount of homologous recombination that
occurs in their cells and the relative proportion of homologous
recombination that occurs is also species-variable. The length of
the region of homology between donor and target affects the
frequency of homologous recombination events, the longer the region
of homology, the greater the frequency. The length of the region of
homology needed to observe homologous recombination is also species
specific. However, differences in the frequency of homologous
recombination events can be offset by the sensitivity of selection
for the recombinations that do occur. It will be appreciated that
absolute limits for the length of the donor-target homology or for
the degree of donor-target homology cannot be fixed but depend on
the number of potential events that can be scored and the
sensitivity of the selection for homologous recombination events.
Where it is possible to screen 10.sup.9 events, for example, in
cultured cells, a selection that can identify 1 recombination in
10.sup.9 cells will yield useful results. Where the organism is
larger, or has a longer generation time, such that only 100
individuals can be scored in a single test, the recombination
frequency must be higher and selection sensitivity is less
critical.
[0059] The method of the present invention dramatically increases
the efficiency of homologous recombination in the presence of
extrachromosomal donor DNA (see Examples). The invention can be
most readily carried out in the case of cells or organisms that
have rapid generation times or for which sensitive selection
systems are available, or for organisms that are single-celled or
for which pluripotent cell lines exist that can be grown in culture
and which can be regenerated or incorporated into adult organisms.
Rapid generation time is the advantage demonstrated for the fruit
fly, Drosophila, in the present invention. The plant cells,
Arabidopsis are one example of pluripotent cells that can be grown
in culture then regenerated or incorporated into an intact
organism. These cells or organisms are representative of their
respective classes and the description demonstrates how the
invention can be applied throughout those classes. It will be
understood by those skilled in the art that the invention is
operative independent of the method used to transform the organism.
Further, the fact that the invention is applicable to such
disparate organisms as plants and insects demonstrates the
widespread applicability of the invention to living organisms
generally.
Nucleic Acid Delivery
[0060] Transformation can be carried out by a variety of known
techniques which depend on the particular requirements of each cell
or organism. Such techniques have been worked out for a number of
organisms and cells, and can be adapted without undue
experimentation to all other cells. Stable transformation involves
DNA entry into cells and into the cell nucleus. For single-celled
organisms and organisms that can be regenerated from single-cells
(which includes all plants and some mammals), transformation can be
carried out in in vitro culture, followed by selection for
transformants and regeneration of the transformants. Methods often
used for transferring DNA or RNA into cells include forming DNA or
RNA complexes with cationic lipids, liposomes or other carrier
materials, micro-injection, particle gun bombardment,
electroporation, and incorporating transforming DNA or RNA into
virus vectors. Other techniques are well known in the art.
[0061] Examples of Some Delivery Systems Useful in Practicing the
Present Invention
[0062] Liposomal Formulations:
[0063] In certain broad embodiments of the invention, the oligo- or
polynucleotides and/or expression vectors containing ZFNs and,
where appropriate, donor DNA, may be entrapped in a liposome.
Liposomes are vesicular structures characterized by a phospholipid
bilayer membrane and an inner aqueous medium. Multilamellar
liposomes have multiple lipid layers separated by aqueous medium.
They form spontaneously when phospholipids are suspended in an
excess of aqueous solution. The lipid components undergo
self-rearrangement before the formation of closed structures and
entrap water and dissolved solutes between the lipid bilayer. Also
contemplated are cationic lipid-nucleic acid complexes, such as
lipofectamine-nucleic acid complexes. Lipids suitable for use
according to the present invention can be obtained from commercial
sources. Liposomes used according to the present invention can be
made by different methods and such methods are known in the art.
The size of the liposomes varies depending on the method of
synthesis.
[0064] Microinjection:
[0065] Direct microinjection of DNA into various cells, including
egg or embryo cells, has also been employed effectively for
transforming many species. In the mouse, the existence of
pluripotent embryonic stem (ES) cells that are culturable in vitro
has been exploited to generate transformed mice. The ES cells can
be transformed in culture, then micro-injected into mouse
blastocysts, where they integrate into the developing embryo and
ultimately generate germline chimeras. By interbreeding
heterozygous siblings, homozygous animals carrying the desired gene
can be obtained.
[0066] Adenoviruses:
[0067] Human adenoviruses are double-stranded DNA tumor viruses
with genome sizes of approximate 36 Kb. As a model system for
eukaryotic gene expression, adenoviruses have been widely studied
and well characterized, which makes them an attractive system for
development of adenovirus as a gene transfer system. This group of
viruses is easy to grow and manipulate, and they exhibit a broad
host range in vitro and in vivo. In lyrically infected cells,
adenoviruses are capable of shutting off host protein synthesis,
directing cellular machineries to synthesize large quantities of
viral proteins, and producing copious amounts of virus.
[0068] Particular advantages of an adenovirus system for delivering
DNA encoding foreign proteins to a cell include (i) the ability to
substitute relatively large pieces of viral DNA with foreign DNA;
(ii) the structural stability of recombinant adenoviruses; (iii)
the safety of adenoviral administration to humans; and (iv) lack of
any known association of adenoviral infection with cancer or
malignancies; (v) the ability to obtain high titers of recombinant
virus; and (vi) the high infectivity of adenovirus.
[0069] In general, adenovirus gene transfer systems are based upon
recombinant, engineered adenovirus which is rendered
replication-incompetent by deletion of a portion of its genome,
such as E1, and yet still retains its competency for infection.
Sequences encoding relatively large foreign proteins can be
expressed when additional deletions are made in the adenovirus
genome. For example, adenoviruses deleted in both the E1 and E3
regions are capable of carrying up to 10 kB of foreign DNA and can
be grown to high titers in 293 cells.
[0070] Other Viral Vectors as Expression Constructs.
[0071] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from, for example,
vaccinia virus, adeno-associated virus (AAV), and herpes viruses
may be employed. Defective hepatitis B viruses, may be used for
transformation of host cells. In vitro studies show that the virus
can retain the ability for helper-dependent packaging and reverse
transcription despite the deletion of up to 80% of its genome.
Potentially large portions of the viral genome can be replaced with
foreign genetic material. The hepatotropism and persistence
(integration) are particularly attractive properties for
liver-directed gene transfer. The chloramphenicol acetyltransferase
(CAT) gene has been successfully introduced into duck hepatitis B
virus genome in the place of the viral polymerase, surface, and
pre-surface coding sequences. The defective virus was cotransfected
with wild-type virus into an avian hepatoma cell line, and culture
media containing high titers of the recombinant virus were used to
infect primary duckling hepatocytes. Stable CAT gene expression was
subsequently detected.
[0072] Non-Viral Methods.
[0073] Several non-viral methods are contemplated by the present
invention for the transfer into a host cell of DNA constructs
encoding ZFNs and, when appropriate, donor DNA. These include
calcium phosphate precipitation, lipofectamine-DNA complexes, and
receptor-mediated transfection. Some of these techniques may be
successfully adapted for in vivo or ex vivo use.
[0074] In one embodiment of the invention, the expression construct
may simply consist of naked recombinant DNA. Transfer of the
construct may be performed by any of the DNA transfer methods
mentioned above which physically or chemically permeabilize the
cell membrane. For example, polyomavirus DNA in the form of CaPO4
precipitates was successfully injected into liver and spleen of
adult and newborn mice which then demonstrated active viral
replication and acute infection. In addition, direct
intraperitoneal injection of CaPO4 precipitated plasmid expression
vectors results in expression of the transfected genes.
[0075] Transformation of Plants:
[0076] Transformed plants are obtained by a process of transforming
whole plants, or by transforming single cells or tissue samples in
culture and regenerating whole plants from the transformed cells.
When germ cells or seeds are transformed there is no need to
regenerate whole plants, since the transformed plants can be grown
directly from seed. A transgenic plant can be produced by any means
known in the art, including but not limited to Agrobacterium
tumefaciens-mediated DNA transfer, preferably with a disarmed T-DNA
vector, electroporation, direct DNA transfer, and particle
bombardment. Techniques are well-known to the art for the
introduction of DNA into monocots as well as dicots, as are the
techniques for culturing such plant tissues and regenerating those
tissues. Regeneration of whole transformed plants from transformed
cells or tissue has been accomplished in most plant genera, both
monocots and dicots, including all agronomically important
crops.
Screening for Mutations
[0077] Methods for genetic screening to accurately detect mutations
in genomic DNA, cDNA or RNA samples may be employed, depending on
the specific situation. A number of different methods have been
used to detect point mutations, including denaturing gradient gel
electrophoresis ("DGGE"), restriction enzyme polymorphism analysis,
chemical and enzymatic cleavage methods, and others. The more
common procedures currently in use include direct sequencing of
target regions amplified by PCR.TM. and single-strand conformation
polymorphism analysis ("SSCP"). SSCP relies upon the differing
mobilities of single-stranded nucleic acid molecules of different
sequence on gel electrophoresis. Techniques for SSCP analysis are
well known in the art.
[0078] Another method of screening for point mutations is based on
RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA
heteroduplexes. As used herein, the term "mismatch" is defined as a
region of one or more unpaired or mispaired nucleotides in a
double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This
definition thus includes mismatches due to insertion/deletion
mutations, as well as single and multiple base point mutations.
EXAMPLES
[0079] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Induction of Targeted Mutations
Zinc Finger Design
[0080] A pair of ZFNs were designed and constructed for a
chromosomal target locus in the yellow (y) gene of Drosophila. Zinc
fingers generally bind preferentially to G-rich regions of DNA, and
extensive study has been performed of fingers that bind all
5'-GNN-3' triplets (Segal et al. (1999) PNAS USA 96: 2758-2763).
Because the binding sites must be in an inverted orientation with
respect to each other for effective cleavage by ZFNs (Bibikova et
al. (2001) Mol. Cell. Biol. 21: 289-297), the chromosomal target
locus of Drosophila (y) was searched for inverted recognition
sequences of the form (NNC).sub.3 . . . (GNN).sub.3. Such a site
was identified in exon 2 with a 6-bp separation between the
component 9-mer recognition sites, which is the optimal spacer for
specific recognition and cleavage by ZFNs that have no added linker
or spacer between the binding and cleavage domains (M. Bibikova et
al. (2001) Mol. Cell. Biol. 21: 289-287). The specific recognition
sequences of the two ZFNs are described in Bibikova et al. 2002,
Genetics 161: 1169-1175. DNAs encoding zinc fingers that recognize
the DNA sequences, 5'-GCGGATGCG-3' (SEQ ID NO: 1) and
5'-GCGGTAGCG-3' (SEQ ID NO: 2), were obtained from Drs. David Segal
and Carlos Barbas (Scripps Research Institute, La Jolla, Calif.)
(Segal, D. J, et al. (1999) PNAS 96: 2758-2763). The DNAs encoding
the zinc fingers were then modified using mutagenic PCR primers,
and two sets of three zinc fingers each were produced: one,
referred to as yA that recognizes one of the component 9-mers of
the y gene target (5'-GTG-GATGAG-3' (SEQ ID NO: 3)), and another,
referred to as yB, that recognizes the other component 9-mer of the
y gene target (5'-GCGGTAGGC-3' (SEQ ID NO: 4)). Two fingers were
modified in yA, but only one in yB. DNA encoding each of the
resulting 3-finger sets of zinc fingers were both cloned in frame
with the FokI DNA cleavage domain in the pET15b expression plasmid,
with no intervening linker DNA between the DNA recognition and
cleavage domains. Both chimeric ZFN proteins were expressed,
purified by Ni-affinity chromatography, and tested for cleavage
activity in vitro by methods described previously (Smith, J., et
al. (2000) Nucleic Acids Res. 28, 3361-3369; and Bibikova, M., et
al. (2001) Mol. Cell. Biol. 21, 289-297), using the pS/G plasmid
(Geyer, P. K. & Corces, V. G. (1987) Genes Dev. 1, 996-1004),
which carries the complete y gene. Together the two ZFNs made a
single double stranded break (DSB) at the expected site in a
10.7-kb plasmid DNA carrying the y gene.
P Element Vectors and Transformation of Fly Larvae.
[0081] The yA and yB ZFN coding sequences were then cloned
separately behind the Drosophila Hsp70 heat shock promoter by
insertion of ZFN DNA between the BamHI and SalI sites of a modified
phsp70 plasmid (Petersen, R. B. & Lindquist, S. (1989) Cell.
Regul. 1, 135-149). A fragment carrying the heat shock promoter and
ZFN DNA sequences was excised by partial HindIII and complete Apal
digestion and cloned between these same endonuclease sites in the
commercially available cloning vector, pBluescript. After
verification of the sequence of the insert, it was excised by
digestion with NotI and inserted into the ry+P element vector pDM30
(Mismer, D. & Rubin, G. M. (1987) Genetics 116, 565-578). The
resulting yA and yB plasmids were injected separately into v ry
embryos, along with the P-transposase expression plasmid
p.pi.25.1wc, and eclosing adults were mated to screen for ry+
germline transformants. The ry+ insertion was mapped to a specific
chromosome for multiple independent transformants with each ZFN.
Both balanced and homozygous stocks were created for several lines
carrying yA and yB without viability problems in most cases. Genes
for the two ZFNs were brought together (as described in the
Examples below) with appropriate crosses of mature flies, and the
offspring were heat shocked 4 days after the initiation of mating
by immersing the glass vials containing the flies in a water bath
at 35.degree. for one hour. As adults eclosed they were screened
for evidence of somatic y mutations. Control vials from crosses
involving each nuclease separately were subjected to the heat
shock, and yA+yB flies that had not been heat shocked were also
screened.
Recovery of Germline Mutants.
[0082] All flies emerging from the heat shock protocol and carrying
both the yA and yB nucleases were mated to reveal potential
germline mutations. Males were crossed with 2 or 3 attached-X
[C(1)DX] females, and the resulting male offspring screened for
yellow body color. Females were crossed with 2 or 3 y (FM6) males,
and the resulting offspring of both genders screened. Mutants were
identified and all of them were males that had originated from male
parents. These identified mutant male offspring were then crossed
to C(1)DX females to produce additional progeny carrying the same
mutation.
DNA Analysis.
[0083] The presence or absence of the target DNA was identified by
DNA analysis. Individual flies were homogenized in 100 .mu.l of a
1:1 mixture of phenol and grind, buffer (7 M urea, 2% SDS, 10 mM
Tris, pH 8.0, 1 mM EDTA, 0.35 M NaCl) preheated to 60.degree.. Each
sample was extracted with 50 pi of chloroform, the organic phase
back-extracted with 100 .mu.l of grind buffer, and the combined
aqueous phases re-extracted with 50 .mu.l of chloroform. DNA was
precipitated with ethanol and re-dissolved in 20 .mu.l of 10 mM
Tris, pH 8.5. A 600-bp DNA fragment was amplified by PCR with
primers flanking the yA+yB recognition site. The primers were
called YF2 (5'ATTCCTTGTGTCCAAAATAATGAC-3' (SEQ ID NO: 5)) and YR3
(5'-AAAATAGGCATATGCATCATCGC3' (SEQ ID NO: 6)) For the larger
deletions, YR3 was used in combination with a more distant
sequence, YF1 (5'ATTTTG-TACATATGTTCTTAAGCAG-3' (SEQ ID NO: 7)).
Amplified fragments were recovered after gel electrophoresis, and
DNA sequences were determined at the University of Utah DNA
Sequencing Core Facility with an ABI3700 capillary sequencer and
the YR3 primer.
Induction of Targeted y Mutations Resulting from Double Stranded
Breaks and Nonhomologous End Joining
[0084] The levels of expression of yA induced at 37.degree. were
found, in several independent transformants, to be lethal when
applied at larval and embryonic stages. Moderating the heat shock
to 35.degree. allowed survival of a good proportion of the
yA-carrying flies. The yB ZFN did not affect viability at any
temperature tested.
[0085] After individual flies carrying the yA and yB nucleases on
the same chromosome were crossed and their progeny heat-shocked,
offspring demonstrating y mosaic, as well as germline mutations
were observed in male offspring. In males (except following DNA
replication), only simple religation or NHEJ would be available to
repair the damage after a DSB. In Drosophila, as in many other
eukaryotes, NHEJ frequently produces deletions and/or insertions at
the joining site. Since the DSB is targeted to protein coding
sequences in y+, most such alterations would lead to frame-shifts
or to deletion of essential codons, which can lead to a phenotype
of patches of y mutant tissue.
[0086] Somatic yellow mosaics were identified in multiple yA+yB
males. Most of the patches were in the distal abdominal cuticle and
bristles, but some examples in leg, wing and scutellar bristles
were also observed. No other phenotypic defects have been seen on a
regular basis. The frequency of somatic mosaics was quite high. In
pooled data from crosses involving a number of independent yA and
yB lines, 105 of 228 candidate males (46%) showed obvious y
patches. For some yA+yB combinations the frequency was greater than
80%. No yellow mosaics were observed in controls with a single
nuclease or without heat shock. This indicates that the yA+yB ZFNs
are capable of inducing somatic mutations at their designated
target.
Characterization of Germline y Mutations.
[0087] To isolate germline y mutations, all yA+yB males from
several heat shock experiments were crossed to females carrying an
attached-X chromosome [C(1)DX/Y], in order to produce male
offspring that were known to only receive their father's X
chromosome. In total, 228 male fathers yielded 5,870 sons; 26 of
the male off-spring, from 13 different fathers, were clearly y
throughout their entire bodies. Thus, 5.7% of the yA+yB male
fathers produced at least one germline mutant. Of the 13 fathers, 6
had been identified as having y somatic patches, while the other 7
appeared to be entirely y+in diagnostic features. No y flies were
isolated among 7050 progeny of 125 heat-shocked yA+yB females
crossed to y males. The ZFNs appear to be effective in inducing
mutations via NHEJ most efficiently in the male germline.
[0088] DNA was isolated from the 13 fathers identified above and 5
additional males in order to analyze each of them for the presence
of the target DNA. A 600-bp fragment including the expected
cleavage site was amplified by PCR. In three of the 18 male flies,
the binding site for one of the primers had been deleted, and a new
primer had to be generated in order to accomplish amplification.
This new primer was located at a more distant location. Sequence
analysis of all fragments revealed unique alterations precisely at
the target site. Nine of the sequenced mutants had simple
deletions; five had deletions accompanied by insertions; and three
were simple, short duplications. Three of the deletions extended
for hundreds of bps to one side of the target and these were the
three samples that required a new primer design. These are exactly
the types of mutations that were expected to result from NHEJ after
cleavage by the yA+yB ZFNs, and they are very similar to those
produced after P element excision. Some of the frameshift y
mutations created a stop codon within a short distance of the
alteration, while one inserted an asparagine codon into the normal
reading frame.
Targeted Cleavage and Mutagenesis.
[0089] This example demonstrated that ZFNs can be designed to
produce DSBs in target chromosomal locus in an exemplary genome in
order to produce a permanent genetic alteration. The frequency of
observed somatic mutation was quite high, and the real number of
somatic mosaics may be even higher, since y mutations have no
effect on many visible features. This was corroborated by the
recovery of germline mutations from phenotypically y+ parents.
[0090] In this particular Example, germline mutations were
recovered only in males and at a lower frequency than somatic
mosaics.
Example 2
ZFN-Induced Double Stranded Breaks Stimulate Targeted Genetic
Recombination in the Presence of Homologous Donor DNA
Zinc Finger and Donor DNA Design
[0091] A pair of ZFNs were designed and constructed for a
chromosomal target locus in the yellow (y) gene of Drosophila as
described in Example 1.
[0092] In order to make an identifiable donor DNA for the
Drosophila gene, y, the yA and yB recognition sites for the zinc
fingers were replaced with two in-frame stop codons and an XhoI
site. These changes were introduced by amplification with PCR
primers carrying the desired sequence. Relative to the wild type y,
21 bp were deleted leaving only 3 bp of the yA recognition site,
and a 9 bp replacement inserted the two in-frame stop codons and
inserted the XhoI site. This mutant (yM) carries a total of 8 kb of
homology to the y locus. It was inserted into a P element vector
and introduced into the fly genome. The yM sequence is flanked by
recognition sites for the FLP recombinase (FRT) and the
meganuclease I-SceI to permit excision and linearization of the
donor. Generating a linear extrachromosomal donor DNA in situ by
this means has been shown to enhance its effectiveness in
recombination (Y. S. Rong and K. G. Golic, Science 288, 2013-2018
(2000)).
Experimental Design
[0093] The design of the targeted genetic recombination experiment
is as follows: The y.sup.+ target lies on the X chromosome. The
transgenes for the yA and yB ZFNs are on one chromosome 2, while
those for FLP and/or I-SceI (when present) are on the other
chromosome 2. The donor DNA (yM) is located on chromosome 3 in a
p-element vector that also carries the white gene (W.sup.+). Each
of these inserted genes is under the control of a Drosophila HSP70
promoter. Upon heat-shock induction, the ZFNs will cut their target
at y. This broken chromosome can be restored to wild type, or it
can acquire a y mutation either by NHEJ or by homologous
recombination. When neither FLP nor I-SceI is present, the donor
remains integrated. When FLP is expressed, the donor is excised as
an extrachromosomal circle. When I-SceI is also expressed, it
converts the donor to an ends-out linear molecule which can
recombine with the cleaved target locus. Experiments were also
performed with linear donor only in the absence of yA and yB (and
therefor without cleavage of the target).
[0094] Larvae carrying single copies of these introduced DNA
components were heat-shocked at 35.degree., for one hour, 0-4 days
after egg laying. The experiment contained five groups as
exemplified below:
[0095] ND, no donor: yA+yB only;
[0096] ID, integrated donor: yA+yB+donor, no FLP or I-SceI;
[0097] CD, circular extrachromosomal donor: yA+yB+FLP+donor;
[0098] LD, linear extrachromosomal donor:
yA+yB+FLP+I-SceI+donor;
[0099] DO, linear donor only: FLP+I-SceI+donor, but no ZFNs.
[0100] Adults emerging from the heat shock protocol were crossed to
reveal germline y mutations. The frequencies of germline y
mutations resulting from the heat-shock treatment are shown in
Table 1 in column 3. The frequencies of mutation rose in both males
and females in the presence of the donor and the frequency
increased further with extrachromosomal and linear DNA. With linear
extrachromosomal DNA, nearly 20% of males and 14% of females
yielded at least one mutant offspring.
[0101] The y mutations were propagated in further crosses,
chromosomal DNA was recovered. The frequency of germline y mutants
and the proportion due to either NHEJ or homologous recombination
with the donor DNA was determined by PCR amplification of 600 bp of
DNA including the target region of the y gene followed by XhoI
digestion of the amplified product. Products of homologous
recombination between donor and target were recognized by XhoI
digestion of the PCR fragment; some of these and many of the
XhoI-resistant products were sequenced. The latter showed small
deletions and/or insertions and occasionally larger deletions, all
of which are characteristic of NHEJ.
[0102] The fourth column of Table 1 reports the recovery of
germline mutants as a percentage of all offspring. The fractions of
those mutations resulting from either NHEJ or homologous
recombination with the donor rose as the donor DNA became more
effective at participating in homologous recombination: linear
donor DNA being more effective than circular donor DNA, which was
more effective than integrated donor DNA. The integrated donor,
located on chromosome 3, was not very effective in serving as a
template for repair of the break at y and the majority of recovered
mutations were due to NHEJ. The circular donor was much more
effective and approximately 1/3 of all mutations were determined to
be due to gene replacements. With the linear donor, more than 2% of
all sons of males were mutant, and 63% of these were products of
homologous recombination. In the female germline 73% of y mutations
were homologous replacements. Target cleavage by chimeric ZFNs
stimulates targeted genetic recombination substantially, and the
most effective way to integrate donor DNA into a host organism's
genome is with linear donor DNA.
[0103] The ZFN-induced targeted genetic recombination results
differ from those obtained without targeted cleavage in several
respects. First, induced mutations were found in both the male and
female germlines, while only females had yielded good frequencies
in previous trials by other researchers. Apparently the presence of
a DSB in the target activates recombination processes in males that
are not efficient on intact chromosomes. The lower targeting
frequencies observed in females may reflect the possibility of
repairing the break by recombination with an uncut homologous X
chromosome. Second, the overall frequency of induced mutations was
about 10-fold higher in males in the linear DNA and circular DNA
experiments than was seen earlier at y in females with an ends-in
donor: approximately 1/50 gametes, compared to 1/500 gametes. Even
in the female germline, the frequency of ZFN-induced mutations was
1/200 gametes, and 3/4 of these were gene replacements. Thus, the
presence of a homologue donor does not preclude interaction with
the extrachromosomal donor. Third, deletions and insertions due to
NHEJ were also observed, in addition to the targeted homologous
recombinants. Such products were not expected nor observed in the
absence of target cleavage.
Example 3
Expression of Chimeric ZFNs in Arabidopsis in Order to Stimulate
Induction of Targeted Mutations
[0104] Experimental Design The method of the present invention will
be used to target and knock out the Arabidopsis TRANSPARENT TESTA
GLABRA1 gene (TTG1, gene number AT5G24520 (GenBank number
AJ133743). An EST for this gene has been sequenced (GenBank numbers
F20055, F20056). The gene encodes a protein containing WD40 repeats
(Walker et al. (1999) Plant Cell 11, 1337-1349).
[0105] Two chimeric DNA constructs will be generated consisting of
(1) nucleic acid sequence encoding the promoter region from the
Arabidopsis HSP18. 2 gene and (2) nucleic acid sequence encoding
zinc finger proteins specific for the TTG1 gene operatively linked
to a nucleic acid sequence encoding a non-specific endonuclease.
The HSP18.2 promoter will confer expression in Arabidopsis and gene
expression will be controlled by heat-shocking the resulting
plants. The chimeric genes will be referred to as HS::ZnTTG1 A and
HS::ZnTTG1B. These two genes can be incorporated into the same
Agrobacterium vector.
[0106] All of our experiments will be carried out using the model
genetic organism Arabidopsis thaliana, because of a number of
desirable features of this system including small size, small
genome, and fast growth. A ttg1 mutant has a distinctive phenotype,
making it an excellent exemplary model. For instance, ttg1 mutants
are glabrous and mutant plants lack trichomes on leaves and stems.
Trichomes are hair-like outgrowths from the epidermis.
[0107] Additionally, ttg1 mutant are defective in flavonoid
production. Flavonoids are a complex class of compounds including
purple anthocyanin pigments and tannins TTG1 protein positively
regulates synthesis of the enzyme dihydroflavonol reductase, which
is required for production of both anthocyanins and tannins
(Shirley et al. (1995) Plant Journal 8: 659-671; Pelletier and
Shirley (1996) Plant Physiology 111: 339-345).
[0108] These ttg1 mutants also have a transparent testa or seed
coat. In wild type, the seed coat (inner layer of the inner
integument) has dense, brown tannin and ttg1 mutants lack this
pigment. As a consequence, the seed coat of seed collected from
ttg1 mutants are transparent, and seed collected from ttg1 mutants
are yellow because the yellow embryos show through the transparent
seed coat.
[0109] These ttg1 mutants also lack anthocyanins. In wild type,
seedlings, stems, and leaves produce reddish/purple anthocyanin
pigments, particularly under stress. These pigments are absent in
ttg1 mutants.
[0110] Additionally, ttg1 mutants produce extra root hairs. In wild
type, root hairs are produced only from trichoblast cells. In ttg1
mutants, by contrast, root hairs are produced by both trichoblast
cells and atrichoblast cells. The result is a root that appears
more hairy (Galway et al. (1994) Developmental Biology 166,
740-754).
[0111] The ttg1 mutants also fail to produce mucilage in the outer
layer of the seed coat. Mucilage is a complex carbohydrate,
sometimes called slime that covers the seed coat. Lastly, the ttg1
mutants have altered dormancy and ttg1 seeds do not require drying
out or cold treatments to germinate.
[0112] The presence of all seven characteristics makes visual
screening for this mutant genotype an easy task.
Design of Zinc Fingers
[0113] The TTG1 gene was scanned for sequences of the form: NNY NNY
NNY RNN RNN RNN, where Y is either T or C, R is A or G, and N is
any base. This identified sequences comprised of triplets that are
initiated by an A or G in opposite orientation--i.e., on opposite
strands--and separated by exactly 6 bp. This has been shown to be a
preferred structure for zinc finger nuclease recognition and
cleavage (M. Bibikova et al. (2001) Mol. Cell. Biol. 21:
289-287).
[0114] The component triplets of the sequences identified in 1 were
then classified according to whether there were zinc fingers that
were known to bind them specifically. Two sites in TTG1 were
identified as potential ZFN binding and cleavage sites: 5'-TCC GGT
CAC AGA ATC GCC GTC GGA-3' (SEQ ID NO: 8), and 5'-ACT TCC TTC GAT
TGG AAC GAT GTA3' (SEQ ID NO: 9) (at nucleotide 406 in the TTG1
sequence).
[0115] Zinc finger nucleases comprising a binding domain designed
to bind the first of these sites will be constructed either by
oligonucleotide synthesis and extension (Segal, D. J. (2002)
Methods 26: 76-83), or by PCR with mutagenic primers (M. Bibikova
et al. (2002) Genetics 161:1169-1175). The resulting coding
sequences will be inserted into plasmids vectors in frame with the
FokI nuclease domain to create two ZFN coding sequences, ZnTTG1A
and ZnTTG1B. The encoded proteins will be expressed in E. coli and
partially purified(M. Bibikova et al. (2002) Genetics
161:1169-1175). The recovered ZFNs will be tested in vitro for the
ability to cleave plasmid DNA encoding the TTG1 gene. Success in
this assay will be evidenced by no cleavage by either ZFN alone,
but cleavage at the expected site by a mixture of the two ZFNs.
Transformation:
[0116] The HS::ZnTTG1A and HS::ZnTTG1B genes will be introduced
into the Arabidopsis genome using Agrobacterium-mediated
transformation. To do so, the HS::ZnTTG1A and B genes will be
inserted into an Agrobacterium T-DNA transformation vector
(pCAMBIA1380) that harbors a selectable hygromycin resistant
marker. A pCAMBIA HS::ZnTTG1 clone will then be introduced into
Agrobacterium cells using standard Agrobacterium transformation
procedures, and the HS::ZnTTG1A and HS::ZnTTG1B genes will then be
introduced into Arabidopsis plants using the standard floral dip
method. (See, Clough, S. and Bent, A (1999) Plant Journal 16:
735-743).
Induction of Expression of ZFNs in a Host Cell
[0117] Seeds from the T1 generation will be collected from the
dipped plants. In order to select for transformed seedlings, the T1
seeds will be germinated on agar plates containing the antibiotic
hygromycin. Approximately four days after germination, the plates
containing the germinated seedlings will be wrapped in plastic wrap
and immersed in 40.degree. C. water for two hours to induce
expression of the ZFN genes. At approximately two weeks following
germination, the hygromycin resistant transformed seedlings will be
transferred to dirt.
Screening for Gene-Targeting Event:
[0118] Screening Method 1: The HS::ZnTTG1 genes will be introduced
into wild-type Arabidopsis plants and the T1 plants will be heated
as described above. At 1-2 weeks following heat treatment, a sample
of tissue will be harvested from heat-treated plants and DNA
extracted from this tissue. PCR amplification using 20 bp primers
flanking the zinc finger target site (25 bp on each side of the
target site) will be utilized to determine if the HS::ZnTTG1 gene
is present. The PCR band from control plants that were not heat
treated should be approximately 90 bp in size. PCR bands from the
heat-treated plants should include smaller products than 90 bp that
result from the existence of deletions surrounding the zinc finger
target site. To verify the existence of small deletions, we will
clone and determine the DNA sequence of the smaller PCR
products.
[0119] Screening Method 2: The HS::ZnTTG1 A and HS::ZnTTG1 B genes
will be introduced into wild-type. Arabidopsis plants and the T1
plants will be heat-treated as described above. The T1 plants will
be grown to maturity, allowed to self pollinate, and T2 seeds will
be collected. The T2 seeds will be grown on agar plates and they
will be scored for seedling phenotypes including hairless leaves
(glabrous phenotype), brighter leaves (anthrocyanin minus
phenotype), and hairy roots, as described above. Mutant plants will
be transferred to dirt and grown further. Tissue from mutant plants
will be harvested and DNA extracted in preparation for
PCR--screening as described above. Briefly, PCR will be performed
with primers flanking the zinc finger target sites and samples
exhibiting approximately 90 bp products were not transformed,
whereas those exhibiting products less than 90 bp were transformed.
This is due to the existence of deletions surrounding the zinc
finger target site. Additionally, small insertions or much larger
deletions may be present around the zinc finger target site, as
well. To verify the existence of these occurrences, we will clone
and determine the DNA sequence of the smaller PCR products.
[0120] Screening Method 3:
[0121] The HS::ZnTTG1 A and HS::ZnTTG1 B genes will be introduced
into heterozygous ttg1 mutants (i.e., genotype ttg1/TTG1). The male
sterilel (ms1) plants will be introduced to the Agrobacterium
solution (note: the ms1 and ttg1 loci are linked, 6 cM apart on
chromosome 5). The dipped plants then will be pollinated with
pollen from homozygous ttg1-1 plants. The crossed plants will be
allowed to mature, the resultant T1/F1 seeds collected, and the
T1/F1 seeds allowed to germinate in the presence of hygromycin.
Surviving T1/F1 seedlings will contain the HS::ZnTTG1 transgene and
will be heterozygous at the ttg1 locus (i.e., genotype
MSI-ttg1-1/ms1-TTG1). The T1/F1 plants will be heat-shocked as
described above. In a subset of cells, the wild-type allele will be
knocked out, resulting in a sector of homozygous ttg1 (i.e.,
genotype ttg1-1/ttg1-ko) cells. These mutant sectors will be
detectable (and, thus, a targeted genetic recombination event) by
visualizing several phenotypes, such as hairless leaves (glabrous
phenotype), brighter leaves (anthocyanin minus phenotype), and
yellow seeds (transparent testa phenotype). Tissue will be
collected from mutant sectors and targeting verified using the
PCR-cloning-sequencing strategy discussed above. From the mutant
sectors, T2 seeds will be collected and grown into T2 plants. In
the T2 generation, the phenotype will be verified: plants
homozygous for the knockout allele (i.e., ttg1-ko) also will be
homozygous for the ms1 mutation and, thus, will be male sterile
(i.e., genotype ms1-ttg1-ko/ms1-ttg1-ko). Tissue from the double
mutants (phenotypically ttg1 and ms1) will be harvested and
verified for targeting using the PCR-cloning-sequencing strategy
discussed above.
[0122] All of the COMPOSITIONS, METHODS and APPARATUS disclosed and
claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in
terms of preferred embodiments, it will be apparent to those of
skill in the art that variations may be applied to the
COMPOSITIONS, METHODS and APPARATUS and in the steps or in the
sequence of steps of the methods described herein without departing
from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents that are both
chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
TABLE-US-00001 TABLE 1 Recovery of Germline y mutations 1 2 3 4
Donor # Screened # Giving y Total y Females: ND 125 0 (0%) 0 ID 188
9 (4.8%) 15 (0.16%) CD 309 31 (10%) 59 (0.38%) LD 503 68 (13.5%)
135 (0.54%) DO 158 1 (0.6%) 2 (0.02%) Males: ND 228 13 (5.7%) 24
(0.42%) ID 218 24 (11%) 40 (0.73%) CD 261 49 (19%) 104 (1.59%) LD
522 94 (18%) 292 (2.24%) DO 177 1 (0.6%) 1 (0.02%)
Sequence CWU 1
1
919DNAArtificialSynthetic Primer 1gcggatgcg
929DNAArtificialSynthetic Primer 2gcggtagcg 939DNADrosophilia
melanogaster 3gtggatgag 949DNADrosophila melanogaster 4gcggtaggc
9524DNAArtificialSynthetic Primer 5attccttgtg tccaaaataa tgac
24623DNAArtificialSynthetic Primer 6aaaataggca tatgcatcat cgc
23725DNAArtificialSynthetic Primer 7attttgtaca tatgttctta agcag
25824DNAArabidopsis thaliana 8tccggtcaca gaatcgccgt cgga
24924DNAArabidopsis thaliana 9acttccttcg attggaacga tgta 24
* * * * *