NZ751577B2 - Methods And Compositions For Increasing Efficiency Of Targeted Gene Modification Using Oligonucleotide-Mediated Gene Repair - Google Patents
Methods And Compositions For Increasing Efficiency Of Targeted Gene Modification Using Oligonucleotide-Mediated Gene Repair Download PDFInfo
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- NZ751577B2 NZ751577B2 NZ751577A NZ75157714A NZ751577B2 NZ 751577 B2 NZ751577 B2 NZ 751577B2 NZ 751577 A NZ751577 A NZ 751577A NZ 75157714 A NZ75157714 A NZ 75157714A NZ 751577 B2 NZ751577 B2 NZ 751577B2
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Abstract
The invention provides to improved methods for the modification of genes in plant cells, and plants and seeds derived therefrom. More specifically, the invention relates to the increased efficiency of targeted gene mutation by combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms. In particular, the invention provides a method for introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target DNA sequence in a plant cell, comprising delivery of a GRON and a site-specific nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a CRISPR complex, and meganuclease into a plant cell. The GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to attract the cell's gene repair system to the site where the mismatched base-pair(s) is created, and is degraded after designated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutation into the target DNA sequence and the plant cell is non-transgenic following the introduction. The GRON comprises one or more alterations from conventional RNA and DNA nucleotides at the 5' or 3' end. availability of components of the target cell gene repair mechanisms. In particular, the invention provides a method for introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target DNA sequence in a plant cell, comprising delivery of a GRON and a site-specific nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a CRISPR complex, and meganuclease into a plant cell. The GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to attract the cell's gene repair system to the site where the mismatched base-pair(s) is created, and is degraded after designated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutation into the target DNA sequence and the plant cell is non-transgenic following the introduction. The GRON comprises one or more alterations from conventional RNA and DNA nucleotides at the 5' or 3' end.
Description
(12) Granted patent specificaon (19) NZ (11) 751577 (13) B2
(47) Publicaon date: 2021.12.24
(54) Methods And Composions For Increasing Efficiency Of Targeted Gene Modificaon Using
Oligonucleode-Mediated Gene Repair
(51) Internaonal Patent Classificaon(s):
A01H 1/00 A01H 5/00
(22) Filing date: (73) Owner(s):
2014.03.14 CIBUS US LLC
CIBUS EUROPE B.V.
(23) Complete specificaon filing date:
3.14 (74) t:
Wrays Pty Ltd
(62) Divided out of 711145
(72) Inventor(s):
(30) Internaonal Priority Data: SCHOPKE, Christian
US 61/801,333 3.15 SAUER, Noel, Joy
PEARCE, James
SEGAMI, Rosa, E.
MOZORUK, Jerry
GOCAL, Gregory, F.w.
BEETHAM, Peter, R.
(57) Abstract:
The invenon provides to ed methods for the modificaon of genes in plant cells, and
plants and seeds derived therefrom. More cally, the invenon relates to the increased
efficiency of targeted gene mutaon by combining gene repair oligonucleodes with approaches
that enhance the availability of components of the target cell gene repair isms. In
parcular, the invenon provides a method for introducing a gene repair oligonucleobase (GRON)-
mediated mutaon into a target DNA sequence in a plant cell, comprising delivery of a GRON
and a site-specific nuclease selected from the group ng of a zinc finger nuclease (ZFN),
a CRISPR complex, and meganuclease into a plant cell. The GRON hybridizes at the target DNA
sequence to create a mismatched base-pair(s), which acts as a signal to aract the cell's gene
repair system to the site where the mismatched base-pair(s) is created, and is degraded aer
751577 B2 designated nucleode(s) within the target DNA sequence is ted by the cell's gene repair
system such that the plant cell introduces the GRON-mediated mutaon into the target DNA
sequence and the plant cell is ansgenic following the introducon. The GRON comprises one
or more alteraons from convenonal RNA and DNA nucleodes at the 5' or 3' end.
METHODS AND COMPOSITIONS FOR INCREASING EFFICIENCY OF
ED GENE MODIFICATION USING OLIGONUCLEOTIDE—NLEDIATED
GENE REPAIR
The present application claims priority to US. Provisional Patent Application
61/801,333 filed March 15, 2013, which is hereby incorporated by nce.
FIELD OF THE INVENTION
This invention generally relates to novel methods to e the efficiency of the
targeting of modifications to c locations in c or other nucleotide sequences.
Additionally, this invention relates to target DNA that has been modified, mutated or marked
by the approaches disclosed herein. The invention also relates to cells, tissue, and organisms
which have been modified by the invention's methods.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid
the reader in understanding the ion and is not admitted to describe or constitute prior art
to the present invention.
The modification of genomic DNA is central to advances in biotechnology, in general,
and biotechnologically based medical advances, in particular. nt methods for site—
directed genomic ations are desirable for research and possibly for gene therapy
applications. One approach es triplex—forming oligonucleotides (TFO) which bind as
third strands to duplex DNA in a sequence—specific manner, to mediate directed mutagenesis.
Such TFO can act either by delivering a tethered mutagen, such as psoralen or chlorambucil
(Havre et al., Proc Nat’l Acad Sci, USA. 90:7879—7883, 1993; Havre et al., J Virol 67:7323-
7331, 1993; Wang et al., Mol Cell Biol 15: 1759~1768, 1995; Takasugi et al., Proc Nat’l Acad
Sci, USA. 88:5602-5606, 1991; Belousov et al., Nucleic Acids Res 25:3440—3444, 1997), or
by binding with sufficient affinity to provoke error—prone repair (Wang eta1., Science 271
:802—805, 1996).
r strategy for genomic modification involves the induction of homologous
recombination n an exogenous DNA fragment and the targeted gene. This approach
has been used successfully to target and disrupt selected genes in mammalian cells and has
enabled the tion of transgenic mice carrying specific gene knockouts chi et al.,
Science 244: 1288-1292, 1989; US. Pat. No. 4,873,191 to Wagner). This approach, however,
relies on the transfer of selectable markers to allow
isolation of the desired recombinants. Without selection, the ratio of homologous to non-
homologous ation of transfected DNA in typical gene transfer experiments is low,
usually in the range of 1:1000 or less (Sedivy et al., Gene Targeting, W. H. Freeman and
Co., New York, 1992). This low efficiency of homologous integration limits the utility of
gene transfer for experimental use or gene therapy. The frequency of homologous
ination can be enhanced by damage to the target site from UV irradiation and
selected carcinogens (Wang et al., Mol Cell Biol 8:196—202, 1988) as well as by site—
ic endonucleases (Sedivy et a1, Gene Targeting, W. H. n and Co., New
York, 1992; Rouet et al., Proc Nat’l Acad Sci, U.S.A. 91:6064-6068, 1994', Sega] et al.,
Proc Nat’l Acad Sci, USA. 92:806-810, 1995). In addition, DNA damage induced by
triplex—directed psoralen dducts can ate recombination within and between
extrachromosomal vectors (Segal et al., Proc Nat’l Acad Sci, USA. 92:806—810, 1995;
Faruqieta1., Mol Cell Biol 16:6820-6828, 1996; US. Pat. No. 5,962,426 to Glazer).
Other work has helped to define parameters that influence recombination in
mammalian cells. In general, linear donor fragments are more recombinogenic than their
circular counterparts (Folger eta1., Mol Cell Biol 2:1372-1387, 1982). Recombination is
also influenced by the length of uninterrupted homology between both the donor and
target sites, with short fragments appearing to be ineffective ates for ination
(Rubnitz et al., Mol Cell Biol —2258, 1984). Nonetheless, several recent efforts
have focused on the use of short fragments of DNA or DNA/RNA hybrids for gene
correction. (Kunzelmann et al., Gene Ther 3:859—867, 1996).
The sequence~specific binding properties of TFO have been used to r a
series of different molecules to target sites in DNA. For example, a diagnostic method
for examining triplex interactions utilized TFO coupled to Fe—EDTA, a DNA cleaving
agent (Moser et al., Science 238:645~650, 1987). Others have linked biologically active
demonstrated
enzymes like micrococcal nuclease and ococcal nuclease to TFO and
site—specific cleavage of DNA (Pei et al., Proc Nat’l Acad Sci USA. 87:9858—9862,
1990; Landgraf et al., Biochemistry 33:10607—10615, 1994). Furthermore, site—directed
DNA damage and mutagenesis can be achieved using TFO conjugated to either psoralen
(Havre et al., Proc Nat’l Acad Sci USA. 9—7883, 1993; Takasurgi et al., Proc
Natl Acad Sci U.S.A. 2—5606, 1991) or alkylating agents (Belousov et al., Nucleic
Acids Res 25:3440—3444, 1997; Posvic eta1., J Am Chem Soc 112:9428-9430, 1990).
PCT/U82014/029566
WIPO Patent Application WO/2001/025460 describes methods for mutating a
target DNA sequence of a plant that include the steps of (l) electroporating into a
microspore of the plant a recombinagenic oligonucleobase that contains a first
gous region that has a sequence identical to the sequence of at least 6 base pairs of
a first fragment of the target DNA sequence and a second homologous region which has a
of the
sequence identical to the sequence of at least 6 base pairs of a second fragment
target DNA sequence, and an intervening region which contains at least 1 nucleobase
heterologous to the target DNA sequence, which intervening region connects the first
homologous region and the second homologous region; (2) culturing the pore to
produce an embryo; and (3) producing from the embryo a plant having a on located
between the first and second fragments of the target DNA sequence, e. g., by culturing the
microspore to produce a somatic embryo and regenerating the plant from the embryo. In
s embodiments of the ion, the recombinagenic oligonucleobase is an MDON
and each of the homologous regions contains an RNA segment of at least 6 RNA—type
nucleotides; the intervening region is at least 3 nucleotides in length; the first and or
second RNA segment contains at least 8 contiguous stituted ribonucleotides.
One of the major goals of ical research is the targeted modification of
the genome. As noted above, although methods for delivery of genes into mammalian
cells are well developed, the frequency of modification and/or gous recombination
is limited (Hanson et al., Mol Cell Biol 15:45-51 1995). As a result, the modification of
genes is a time consuming process. Numerous methods have been plated or
ted to enhance modification and/or recombination between donor and genomic
DNA. However, the present techniques often exhibit low rates of modification and/or
ination, or istency in the modification and/or recombination rate, thereby
hampering research and gene therapy technology.
SUMMARY OF THE INVENTION
The present invention provides novel s and compositions for
improving the efficiency of the targeting of modifications to specific locations in genomic
or other nucleotide sequences. As described hereinafter, nucleic acids which direct
specific changes to the genome may be combined with s approaches to enhance the
availability of components of the natural repair systems present in the cells being targeted
for modification.
PCT/USZOl4/029566
In a first aspect, the ion relates to methods for introducing a gene repair
oligonueienbase (GROW-mediated mutation into. a target deoxyribonucleic acid (DNA)
cell under
ce in a plant cell. The methods comprise, inter alia, culturing the plant
conditions that increase one or more cellular DNA repair processes prior to, and/or
coincident with, delivery of a GRON into the plant cell; and/or ry of a GRON into
the plant cell greater than 55 bases in length, the GRON optionally comprising two or
more mutation sites for introduction into the target DNA.
In certain embodiments, the conditions that increase one or more cellular DNA
repair ses comprise one or more of: introduction of one or more sites into the
GRON or into the plant cell DNA that are s for base excision repair, introduction of
one or more sites into the GRON or into the plant cell DNA that are targets for non—
homologous end joining, uction of one or more sites into the GRON or into the
plant cell DNA that are targets for microhomology—mediated end joining, uction of
one or more sites into the GRON or into the plant cell DNA that are targets for
homologous recombination, and introduction of one or more sites into the GRON or into
the plant cell DNA that are targets for pushing repair.
As described after, GRONs for use in the present invention can
comprises one or more of the following alterations from conventional RNA and DNA
nucleotides:
one or more abasic nucleotides;
one or more 8’oxo dA and/or 8’oxo dG nucleotides;
a reverse base at the 3’ end thereof;
one or more 2’ O—methyl nucleotides;
one or more 2’O—methyl RNA nucleotides at the 5’ end thereof, and preferably 2, 3, 4, 5,
6, 7, 8, 9, 10, or more;
an intercalating dye;
a 5’ terminus cap;
a backbone modification selected from the group consisting of a phosphothioate
modification, a methyl phosphonate modification, a locked nucleic acid (LNA)
PCT/U82014/029566
modification, a O —(2~methoxyethyl) (MOE) modification, a di PS modification, and a
peptide nucleic acid (PNA) modification;
one or more intrastrand crosslinks;
one or more cent dyes conjugated o, eably at the 5’ or 3’ end of the
GRON; and
one or more bases which increase hybridization energy. This list is not meant to be
limiting.
As described after, in certain embodiments GRON quality and
conversion efficiency may be ed by synthesizing all or a portion of the GRON
using nucleotide multimers, such as dimers, trimers, tetramers, etc improving its purity.
In certain embodiments, the target deoxyribonucleic acid (DNA) sequence is
within the plant cell genome. The plant cell may be non-transgenic or transgenic, and the
target DNA sequence may be a transgene or an endogenous gene of the plant cell.
In n embodiments, the conditions that increase one or more cellular DNA
repair processes comprise introducing one or more compounds which induce single or
double DNA strand breaks into the plant cell prior to or coincident with delivering the
GRON into the plant cell. Exemplary nds are bed hereinafter.
The methods and compositions described herein are applicable to plants
generally. By way of example only, a plant species may be selected from the group
consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley,
rice, alfafa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana,
melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field
pea, faba bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field
beans, poplar, pine, eucalyptus, grape, , triticale, alfalfa, rye, oats, turf and forage
grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant,
marigold, lotus, cabbage, daisy, carnation, tulip, iris, and lily. These may also apply in
whole or in part to all other biological systems including but not limited to ia, fungi
and mammalian cells and even their organelles (e.g., mitochondria and chloroplasts).
In certain embodiments, the methods further comprise regenerating a plant having a
mutation introduced by the GRON from the plant cell, and may comprise collecting seeds
from the plant.
In related aspects, the present invention relates to plant cells comprising a genomic
modification introduced by a GRON according to the methods bed herein, a plant
sing a genomic modification introduced by a GRON according to the methods
described herein, or a seed comprising a genomic ation introduced by a GRON
according to the methods described herein.
Other embodiments of the invention will be apparent from the following detailed
description, exemplary embodiments, and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts BFP to GFP sion mediated by phosphothioate (PS) labeled
GRONs g 3 PS moieties at each end of the GRON) and 5'Cy3/ 3'idC labeled GRONs.
Fig. 2 s GRONs sing RNA/DNA, referred to herein as "Okazaki
Fragment GRONs."
[0022a] Fig 3 depicts the native complex and the chimera reproduced from Cong ez‘ al.,
(2013) e, Vol. 339 (6120), pp 819—823.
[0022b] Fig 4 depicts a schematic of the expression vector for chimeric chNA.
DETAILED DESCRIPTION OF THE ION
Developed over the past few years, targeted genetic modification mediated by
oligonucleotides has been shown to be a le technique for use in the specific alteration
of short stretches ofDNA to create deletions, short insertions, and point mutations. These
methods involve DNA pairing/annealing, followed by a DNA repair/recombination event.
First, the nucleic acid s with its complementary strand in the double—stranded DNA in a
located
process mediated by cellular protein factors. This annealing creates a centrally
mismatched base pair (in the case of a point mutation), resulting in a structural perturbation
that most likely stimulates the endogenous protein machinery to te the second step in the
repair process: site—specific modification of the chromosomal sequence and even their
organelles (e.g., mitochondria and chloroplasts). This newly introduced mismatch induces the
DNA repair machinery to perform a second repair event, g to the final on of the
target site. The present methods improve these methods by providing novel approaches which
increase the
WC 2014/144951 PCT/USZOI4/029566
bility of DNA repair components, thus increasing the efficiency and reproducibility
of gene —mediated modifications to targeted nucleic acids.
Definitions
To facilitate understanding of the invention, a number of terms are defined
below.
“Nucleic acid sequence,3’ ‘4nucleotide sequence” and “polynucleotide
sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin which may be
single— or double—stranded, and ent the sense or antisense strand.
As used herein, the terms “oligonucleotides” and “oligomers” refer to a
nucleic acid sequence of at least about 10 nucleotides and as many as about 201
nucleotides, preferably about 15 to 30 nucleotides, and more ably about 20—25
nucleotides, which can be used as a probe or amplimer.
The terms “DNA-modifying molecule” and “DNA—modifying reagent” as used
herein refer to a molecule which is capable of recognizing and ically binding to a
nucleic acid sequence in the genome of a cell, and which is capable of modifying a target
nucleotide sequence within the genome, wherein the ition and specific g of
the difying molecule to the nucleic acid sequence is protein—independent. The
term “protein-independent” as used herein in connection with a difying
molecule means that the DNA-modifying molecule does not require the ce and/or
activity of a protein and/or enzyme for the recognition of, and/or specific g to, a
nucleic acid sequence. DNA—modifying molecules are exemplified, but not limited to
triplex g oligonucleotides, peptide nucleic acids, polyamides, and oligonucleotides
which are intended to promote gene conversion. The DNA—modifying molecules of the
invention are distinguished from the prior art‘s nucleic acid sequences which are used for
homologous recombination [Wong & Capecchi, Molec. Cell. Biol. 7:2294—2295, 1987] in
that the prior art‘s nucleic acid sequences which are used for homologous recombination
are protein-dependent. The term “protein—dependent” as used herein in connection with a
molecule means that the molecule requires the presence and/or activity of a protein and/or
enzyme for the recognition of, and/or specific binding of the molecule to, a nucleic acid
sequence. Methods for determining whether a DNA—modifying le requires the
presence and/or activity of a protein and/or enzyme for the recognition of, and/or specific
WO 44951 ZOI4/029566
binding to, a nucleic acid sequence are within the skill in the art [see, e. g., Dennis et al.
Nucl. Acids Res. 27:47344742, 1999]. For example, the DNA—modifying molecule may
be ted in. vitro with the nucleic acid sequence in the absence of any proteins and/or
enzymes. The detection of specific binding between the DNA-modifying molecule and
the nucleic acid sequence demonstrates that the difying molecule is protein-
independent. On the other hand, the absence of specific binding between the DNA-
modifying molecule and the nucleic acid sequence demonstrates that the DNA—modifying
molecule is protein-dependent and/or requires additional factors.
“Triplex g oligonucleotide” (TFO) is defined as a sequence of DNA or
RNA that is capable of binding in the major grove of a duplex DNA or RNA helix to
form a triple helix. Although the TFO is not d to any particular , a red
length of the TFO is 200 nucleotides or less, more preferably 100 nucleotides or less, yet
more preferably from 5 to 50 nucleotides, even more preferably from 10 to 25
nucleotides, and most preferably from 15 to 25 nucleotides. Although a degree of
sequence specificity between the TFO and the duplex DNA is necessary for formation of
the triple helix, no particular degree of specificity is ed, as long as the triple helix is
capable of forming. se, no specific degree of avidity or affinity between the TF0
and the duplex helix is required as long as the triple helix is capable of forming. While
not intending to limit the length of the nucleotide sequence to which the TFO specifically
binds in one embodiment, the nucleotide sequence to which the TFO specifically binds is
from 1 to 100, more preferably from 5 to 50, yet more ably from 10 to 25, and most
preferably from 15 to 25, nucleotides. Additionally, “triple helix” is defined as a double-
helical nucleic acid with an oligonucleotide bound to a target sequence within the double—
helical nucleic acid, The “double—helical” c acid can be any double—stranded
c acid including double—stranded DNA, double—stranded RNA and mixed duplexes
of DNA and RNA. The double—stranded nucleic acid is not limited to any particular
length. However, in red embodiments it has a length of greater than 500 bp, more
preferably r than 1 kb and most preferably greater than about 5 kb. In many
applications the double—helical nucleic acid is cellular, genomic nucleic acid. The triplex
forming oligonucleotide may bind to the target sequence in a parallel or anti-parallel
manner.
“Peptide Nucleic Acids,” “polyamides” or “PNA” are nucleic acids wherein
the phosphate backbone is replaced with an N-aminoethylglycine—based polyamide
structure. PNAs have a higher affinity for complementary nucleic acids than their l
counter parts following the Watson—Crick base—pairing rules. PNAs can form highly
stable triple helix ures with DNA of the following stoichiometry: (PNA)2.DNA.
gh the peptide c acids and polyamides are not limited to any particular
length, a preferred length of the peptide nucleic acids and polyamides is 200 nucleotides
or less, more ably 100 nucleotides or less, and most preferably from 5 to 50
nucleotides long. While not intending to limit the length of the nucleotide sequence to
which the peptide nucleic acid and polyamide specifically binds, in one embodiment, the
nucleotide sequence to which the peptide nucleic acid and polyamide specifically bind is
from 1 to 100, more preferably from 5 to 50, yet more preferably from 5 to 25, and most
preferably from 5 to 20, nucleotides.
The term “cell” refers to a single cell. The term “cells” refers to a population
of cells. The population may be a pure population sing one cell type. Likewise,
the population may comprise more than one cell type. In the present invention, there is
no limit on the number of cell types that a cell population may comprise.
The term “synchronize” or “synchronized,” when referring to a sample of
cells, or “synchronized cells” or “synchronized cell population” refers to a plurality of
cells which have been d to cause the population of cells to be in the same phase of
the cell cycle. It is not necessary that all of the cells in the sample be synchronized. A
small percentage of cells may not be synchronized with the majority of the cells in the
sample. A preferred range of cells that are synchronized is between lO—lOO%. A more
preferred range is between ISO—100%. Also, it is not ary that the cells be a pure
population of a single cell type. More than one cell type may be contained in the sample.
In this regard, only one of cell types may be synchronized or may be in a different phase
of the cell cycle as compared to another cell type in the sample.
The term “synchronized cell” when made in reference to a single cell means
that the cell has been manipulated such that it is at a cell cycle phase which is different
from the cell cycle phase of the cell prior to the manipulation. atively, a
“synchronized cell” refers to a cell that has been manipulated to alter (i.e., increase or
se) the duration of the cell cycle phase at which the cell was prior to the
manipulation when compared to a control cell (e.g., a cell in the absence of the
manipulation).
2014/029566
The term “cell cycle” refers to the physiological and morphological
ssion of changes that cells undergo when ng (i.e. proliferating). The cell
7) (6
cycle is generally ized to be composed of phases termed “interphase, prophase,”
“metaphase,” “anaphase,” and “telophase”. Additionally, parts of the cell cycle may be
termed “M is),” “S (synthesis),” “G0,” “G1 (gap 1)” and “G2 (gap2)”.
Furthermore, the cell cycle includes periods of progression that are intermediate to the
above named phases.
The term “cell cycle inhibition” refers to the cessation of cell cycle
progression in a cell or population of cells. Cell cycle inhibition is usually induced by
re of the cells to an agent (chemical, naceous or otherwise) that interferes
with aspects of cell physiology to prevent continuation of the cell cycle.
“Proliferation” or “cell growth” refers to the ability of a parent cell to divide
into two daughter cells ably thereby resulting in a total increase of cells in the
population. The cell population may be in an organism or in a culture apparatus.
The term “capable of modifying DNA” or “DNA modifying means” refers to
procedures, as well as endogenous or exogenous agents or reagents that have the ability to
induce, or can aid in the induction of, s to the nucleotide sequence of a targeted
t of DNA. Such changes may be made by the deletion, addition or substitution of
one or more bases on the targeted DNA segment. It is not necessary that the DNA
sequence changes confer functional s to any gene encoded by the targeted
sequence. Furthermore, it is not necessary that changes to the DNA be made to any
particular portion or percentage of the cells.
The term “nucleotide sequence of st” refers to any nucleotide sequence,
the manipulation of which may be deemed desirable for any reason, by one of ordinary
skill in the art. Such nucleotide sequences include, but are not limited to, coding
sequences of structural genes (e. g., reporter genes, selection marker genes, oncogenes,
drug resistance genes, growth s, etc), and non—coding regulatory sequences that do
not encode an mRNA or protein product (e. g., promoter sequence, enhancer sequence,
polyadenylation sequence, termination sequence, regulatory RNAs such as miRNA, etc.).
“Amino acid sequence,’3 Npolypeptide sequence,3’ (4peptide sequence” and
“peptide” are used interchangeably herein to refer to a sequence of amino acids.
PCT/USZOl4/029566
“Target sequence,” as used herein, refers to a double-helical nucleic acid
comprising a sequence preferably greater than 8 nucleotides in length but less than 201
nucleotides in length. In some embodiments, the target sequence is preferably between 8
to 30 bases. The target sequence, in general, is defined by the nucleotide sequence on one
of the strands on the double-helical c acid.
As used herein, a “purine—rich sequence” or “polypurine sequence” when
made in reference to a nucleotide sequence on one of the strands of a double—helical
nucleic acid sequence is defined as a contiguous sequence of nucleotides wherein greater
than 50% of the nucleotides of the target sequence contain a purine base. However, it is
preferred that the purine—rich target sequence n greater than 60% purine
nucleotides, more preferably greater than 75% purine nucleotides, next most preferably
greater than 90% purine nucleotides and most preferably 100% purine nucleotides.
As used herein, a “pyrimidine-rich sequence” or “polypyrimidine sequence”
when made in reference to a nucleotide sequence on one of the strands of a —helical
nucleic acid ce is defined as a contiguous sequence of tides n greater
that 50% of the nucleotides of the target ce contain a pyrimidine base. However, it
is preferred that the pyrimidine—rich target sequence contain r than 60% pyrimidine
nucleotides and more preferably greater than 75% pyrimidine nucleotides. In some
ments, the sequence contains preferably greater than 90% pyrimidine nucleotides
and, in other embodiments, is most ably 100% pyrimidine nucleotides.
A “variant” of a first nucleotide sequence is defined as a nucleotide sequence
which differs from the first nucleotide sequence (e. g., by having one or more deletions,
insertions, or substitutions that may be detected using hybridization assays or using DNA
sequencing). Included within this definition is the detection of alterations or
cations to the genomic sequence of the first nucleotide sequence. For example,
hybridization assays may be used to detect (1) alterations in the pattern of restriction
enzyme fragments capable of hybridizing to the first nucleotide sequence when
comprised in a genome (i.e., RFLP analysis), (2) the ity of a selected portion of the
first nucleotide sequence to hybridize to a sample of genomic DNA which contains the
first nucleotide sequence (e.g., using allele-specific oligonucleotide ), (3) er
or unexpected ization, such as hybridization to a locus other than the normal
chromosomal locus for the first tide sequence (e. g., using fluorescent in situ
ZOI4/029566
hybridization (FISH) to metaphase chromosomes s, etc). One example of a variant
is a mutated wild type sequence.
The terms “nucleic acid” and “unmodified nucleic acid” as used herein refer to
any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine,
and e). The term “modified nucleic acid” refers to a nucleic acid whose structure
is altered relative to the structure of the unmodified nucleic acid. Illustrative of such
modifications would be replacement covalent modifications of the bases, such as
tion of amino and ring nitrogens as well as saturation of double bonds.
As used herein, the terms “mutation” and ication” and grammatical
equivalents thereof when used in reference to a nucleic acid sequence are used
interchangeably to refer to a deletion, insertion, substitution, strand break, and/or
introduction of an adduct. A “deletion” is defined as a change in a nucleic acid sequence
in which one or more tides is absent. An “insertion” or “addition” is that change in
a nucleic acid sequence which has ed in the addition of one or more nucleotides. A
“substitution” results from the replacement of one or more tides by a molecule
which is a different molecule from the replaced one or more nucleotides. For e, a
nucleic acid may be replaced by a different c acid as exemplified by replacement of
a e by a cytosine, adenine, guanine, or uridine. Pyrimidine to pyrimidine (e. g. C to
T or T to C nucleotide substitutions) or purine to purine (e.g. G to A or A to G nucleotide
substitutions) are termed transitions, whereas pyrimidine to purine or purine to pyrimidine
(e. g. G to T or G to C or A to T or A to C) are termed transversions. Alternatively, a
nucleic acid may be replaced by a modified nucleic acid as exemplified by ement of
a thymine by thymine glycol. Mutations may result in a mismatch. The term tch”
refers to a non—covalent interaction between two nucleic acids, each nucleic acid residing
on a different polynucleic acid sequence, which does not follow the base—pairing rules.
For example, for the partially complementary ces 5’-AGT—3’ and 5’-AAT—3’, a GA
mismatch (a transition) is present. The terms “introduction of an adduct” or “adduct
formation” refer to the covalent or non~c0valent linkage of a molecule to one or more
nucleotides in a DNA sequence such that the linkage results in a reduction (preferably
from 10% to 100%, more preferably from 50% to 100%, and most preferably from 75%
to 100%) in the level of DNA replication and/or ription.
The term “strand break” when made in reference to a double stranded nucleic
acid sequence includes a single—strand break and/or a double—strand break. A single—
PCT/USZOI4/029566
strand break (a nick) refers to an interruption in one of the two s of the double
stranded c acid sequence. This is in contrast to a double—strand break which refers
to an interruption in both strands of the double stranded nucleic acid sequence. Strand
breaks may be introduced into a double ed nucleic acid sequence either directly
(e. g., by ionizing ion or treatment with certain chemicals) or indirectly (e.g., by
enzymatic incision at a nucleic acid base).
The terms “mutant cell” and “modified cell” refer to a cell which contains at
least one modification in the cell‘s genomic sequence.
The term “portion” when used in reference to a nucleotide sequence refers to
fragments of that tide ce. The nts may range in size from 5
nucleotide residues to the entire nucleotide sequence minus one nucleic acid e.
DNA molecules are said to have “5’ ends” and “3’ ends” because
mononucleotides are reacted to make oligonucleotides in a manner such that the 5’
phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor
in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is
referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a
mononucleotide pentose ring. An end of an ucleotide is referred to as the “3’ end”
if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring.
As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete
elements are ed to as being “upstream” or 5' of the “downstream” or 3’ elements.
This terminology reflects that transcription proceeds in a 5’ to 3’ direction along the DNA
strand. The promoter and enhancer elements which direct transcription of a linked gene
are generally located 5’ or upstream of the coding region. However, enhancer elements
can exert their effect even when located 3’ of the promoter element and the coding region.
Transcription termination and polyadenylation signals are located 3’ or downstream of the
coding region.
The term binant DNA molecule” as used herein refers to a DNA
molecule which is comprised of segments of DNA joined together by means of molecular
biological techniques.
The term “recombinant protein” or “recombinant polypeptide” as used herein
refers to a protein le which is expressed using a recombinant DNA molecule.
PCT/U82014/029566
As used herein, the terms “vector” and “vehicle” are used interchangeably in
reference to nucleic acid molecules that transfer DNA segment(s) from one cell to
another.
The terms “in le combination,9’ C"in operable order” and “operably
linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that
a nucleic acid molecule capable of ing the transcription of a given gene and/or the
synthesis of a desired protein molecule is produced The terms also refer to the linkage of
amino acid sequences in such a manner so that a functional protein is produced.
The term “transfection” as used herein refers to the introduction of foreign
DNA into cells. Transfection may be accomplished by a variety of means known to the
art including calcium phosphate-DNA co—precipitation, DEAE—dextran—mediated
transfection, polybrene~mediated transfection, electroporation, microinjection, liposome
fusion, lipofectin, protoplast fusion, retroviral ion, biolistics (i.e., particle
bombardment) and the like.
As used herein, the terms ementary” or “complementarity” are used in
reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms
that refer to a sequence of nucleotides) related by the base—pairing rules. For example, the
sequence GT—3’,” is complementary to the ce “5’—ACTG~3’.”
Complementarity can be “partia ” or “tota ”. “Partial” complementarity is where one or
more nucleic acid bases is not matched according to the base pairing rules. “Total” or
“complete” mentarity between nucleic acids is where each and every c acid
base is matched with another base under the base pairing rules. The degree of
complementarity between nucleic acid strands may have significant effects on the
efficiency and strength of hybridization between nucleic acid strands. This may be of
particular importance in amplification ons, as well as detection methods which
depend upon binding between nucleic acids. For the sake of ience, the terms
“polynucleotides” and “oligonucleotides” include molecules which include nucleosides.
The terms ogy” and “homologous” as used herein in reference to
tide sequences refer to a degree of mentarity with other nucleotide
When
sequences. There may be partial homology or complete homology (i.e., ty).
used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic
clone, the term “substantially homologous” refers to any nucleic acid sequence (e.g.,
probe) which can ize to either or both s of the double—stranded nucleic acid
sequence under conditions of low stringency as described above. A nucleotide sequence
which is partially complementary, i.e., “substantially homologous,” to a nucleic acid
sequence is one that at least lly inhibits a completely complementary sequence from
hybridizing to a target nucleic acid sequence The inhibition of hybridization of the
completely complementary sequence to the target sequence may be examined using a
hybridization assay (Southern or Northern blot, solution hybridization and the like) under
conditions of low stringency. A ntially homologous sequence or probe will
e for and inhibit the binding (i.e., the hybridization) of a completely homologous
sequence to a target sequence under conditions of low stringency. This is not to say that
ions of low stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to one another be a
specific (i.e., selective) interaction. The absence of non—specific binding may be tested by
the use of a second target sequence which lacks even a partial degree of complementarity
(e. g., less than about 30% identity); in the absence of non—specific binding the probe will
not hybridize to the second mplementary target.
Low stringency conditions comprise conditions equivalent to binding or
hybridization at 68° C. in a solution consisting of SXSSPE (43.8 g/l NaCl, 6.9 g/l
NaHZPO4-HZO and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x
Denhardt's reagent (50X Denhardt‘s ns per 500 ml: 5 g Ficoll (Type 400,
Pharmacia), 5 g BSA (Fraction V; Sigma» and 100 ug/ml denatured salmon sperm DNA
followed by washing in a solution comprising 2.0xSSPE, 0.1% SDS at room ature
when a probe of about 100 to about 1000 nucleotides in length is employed.
In addition, conditions which promote hybridization under conditions of high
stringency (e. g., increasing the ature of the hybridization and/or wash steps, the use
of formamide in the hybridization on, etc.) are well known in the art. High
stringency conditions, when used in reference to nucleic acid hybridization, comprise
conditions equivalent to binding or hybridization at 68°C. in a solution consisting of
SXSSPE, 1% SDS, SXDenhardt's reagent and 100 ug/ml denatured salmon sperm DNA
followed by g in a solution comprising 0.l><SSPE and 0.1% SDS at 68°C. when a
probe of about 100 to about 1000 nucleotides in length is employed.
It is well known in the art that us equivalent conditions may be
employed to comprise low stringency conditions; factors such as the length and nature
PCT/USZOI4/029566
(DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base
ition, present in solution or immobilized, etc.) and the tration of the salts
and other components (e.g., the presence or absence of formamide, dextran sulfate,
polyethylene glycol), as well as components of the hybridization solution may be varied
to generate conditions of low stringency hybridization different from, but equivalent to,
the above listed conditions.
The term “equivalent” when made in reference to a hybridization condition as
it relates to a hybridization condition of interest means that the hybridization condition
and the hybridization condition of interest result in hybridization of nucleic acid
sequences which have the same range of percent (%) homology. For example, if a
hybridization condition of interest s in hybridization of a first nucleic acid sequence
with other c acid sequences that have from 50% to 70% homology to the first
nucleic acid sequence, then another hybridization condition is said to be lent to the
hybridization condition of interest if this other hybridization condition also results in
hybridization of the first nucleic acid sequence with the other nucleic acid sequences that
have from 50% to 70% homology to the first nucleic acid sequence.
As used herein, the term “hybridization” is used in reference to the pairing of
mentary nucleic acids using any process by which a strand of nucleic acid joins
with a complementary strand through base pairing to form a hybridization complex.
Hybridization and the th of hybridization (i.e., the strength of the association
between the nucleic acids) is ed by such factors as the degree of complementarity
between the nucleic acids, stringency of the conditions involved, the Tm of the formed
hybrid, and the G:C ratio within the nucleic acids.
As used herein the term “hybridization complex” refers to a complex formed
between two nucleic acid sequences by virtue of the formation of hydrogen bounds
between complementary G and C bases and between complementary A and T bases; these
en bonds may be further stabilized by base stacking ctions. The two
complementary nucleic acid sequences hydrogen bond in an rallel configuration. A
hybridization x may be formed in solution (e.g., Cot or Rot analysis) or between
one c acid sequence present in solution and r nucleic acid sequence
immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as
employed in rn and Northern blotting, dot blotting or a glass slide as employed in
in situ hybridization, including FISH (fluorescent in situ hybridization».
As used herein, the term “Tm” is used in reference to the “melting
temperature.” The melting temperature is the temperature at which a population of
double—stranded nucleic acid les becomes half iated into single strands. The
equation for calculating the Tm of c acids is well known in the art. As indicated by
standard references, a simple estimate of the Tm value may be calculated by the equation:
Tm=8l.5+0.4l(% G+C), when a nucleic acid is in aqueous on at l M NaCl (see e.g.,
on and Young, tative Filter Hybridization, in Nucleic Acid
Hybridization,1985). Other references include more sophisticated computations which
take structural as well as sequence characteristics into account for the calculation of T111.
As used herein the term “stringency” is used in nce to the conditions of
temperature, ionic strength, and the presence of other compounds such as organic
solvents, under which nucleic acid hybridizations are conducted. “Stringency” lly
occurs in a range from about Tm°C. to about 20°C. to 25 OC. below Tm. As will be
understood by those of skill in the art, a ent ization can be used to identify or
detect identical polynucleotide sequences or to fy or detect similar or related
polynucleotide sequences.
The terms “specific binding,” “binding specificity,” and grammatical
equivalents thereof when made in reference to the binding of a first nucleotide sequence
to a second nucleotide sequence, refer to the preferential interaction between the first
nucleotide sequence with the second nucleotide sequence as compared to the interaction
between the second nucleotide sequence with a third tide sequence. Specific
binding is a ve term that does not require absolute specificity of binding; in other
words, the term “specific binding” does not require that the second nucleotide sequence
interact with the first nucleotide sequence in the absence of an interaction n the
second nucleotide sequence and the third nucleotide sequence. Rather, it is ient that
the level of interaction between the first nucleotide sequence and the second nucleotide
sequence is greater than the level of interaction between the second nucleotide sequence
with the third nucleotide sequence. “Specific binding” of a first nucleotide sequence with
a second nucleotide sequence also means that the interaction between the first nucleotide
sequence and the second nucleotide sequence is dependent upon the presence of a
particular structure on or within the first nucleotide sequence; in other words the second
nucleotide sequence is recognizing and binding to a specific structure on or within the
first nucleotide sequence rather than to nucleic acids or to nucleotide sequences in
PCT/U82014/029566
general. For e, if a second tide sequence is specific for structure “A” that is
on or within a first nucleotide sequence, the presence of a third nucleic acid sequence
containing ure A will reduce the amount of the second nucleotide sequence which is
bound to the first nucleotide sequence.
As used herein, the term “amplifiable nucleic acid” is used in reference to
nucleic acids which may be amplified by any ication method. It is contemplated
that “amplifiable nucleic acid” will usually comprise e template.”
The terms “heterologous nucleic acid sequence” or ologous DNA” are
used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid
sequence to which it is not ligated in nature, or to which it is ligated at a different location
in nature. Heterologous DNA is not endogenous to the cell into which it is introduced,
but has been obtained from another cell. Generally, although not necessarily, such
heterologous DNA encodes RNA and proteins that are not normally produced by the cell
into which it is expressed. Examples of heterologous DNA e reporter genes,
transcriptional and translational regulatory sequences, selectable marker proteins (e.g.,
proteins which confer drug resistance), etc.
“Amplification” is defined as the production of additional copies of a c
acid sequence and is generally carried out using polymerase chain reaction logies
well known in the art (Dieffenbach C W and G S Dveksler (1995) PCR Primer, a
Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). As used , the
term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis US. Pat.
Nos. 4,683,195, and 4,683,202, hereby incorporated by reference, which describe a
method for increasing the concentration of a segment of a target ce in a mixture of
genomic DNA without cloning or purification. The length of the amplified segment of
the desired target sequence is determined by the ve positions of two ucleotide
primers with respect to each other, and therefore, this length is a controllable parameter.
By Virtue of the repeating aspect of the process, the method is referred to as the
“polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified
segments of the target sequence become the predominant sequences (in terms of
concentration) in the mixture, they are said to be “PCR amplified.”
With PCR, it is possible to amplify a single copy of a specific target sequence
in genomic DNA to a level detectable by l different methodologies (e.g.,
PCT/U82014/029566
hybridization with a labeled probe; incorporation of biotinylated s followed by
avidin—enzyme conjugate detection; incorporation of 32P—labeled deoxynucleotide
triphosphates, such as dCTP or dATP, into the amplified segment). In addition to
genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of
primer molecules. In particular, the ied segments created by the PCR process itself
are, themselves, efficient templates for subsequent PCR ications.
One such preferred method, particularly for commercial applications, is based
on the widely used TaqMan® real-time PCR logy, and combines —Specific
PCR with a Blocking reagent (ASB-PCR) to suppress ication of the wildype allele.
ASB-PCR can be used for ion of germ line or somatic mutations in either DNA or
RNA extracted from any type of tissue, including formalin-fixed paraffin—embedded
tumor specimens. A set of reagent design rules are developed enabling sensitive and
selective detection of single point substitutions, insertions, or ons against a
background of ype allele in thousand—fold or greater excess. (Morlan J, Baker J,
opi D Mutation Detection by Real—Time PCR: A Simple, Robust and Highly
Selective Method. PLoS ONE 4(2): e4584, 2009)
The terms “reverse transcription polymerase chain reaction” and “RT—PCR”
refer to a method for reverse transcription of an RNA sequence to generate a mixture of
cDNA sequences, ed by increasing the concentration of a desired segment of the
transcribed cDNA sequences in the mixture without cloning or purification. lly,
RNA is e transcribed using a single primer (e. g., an oligo—dT primer) prior to PCR
amplification of the desired segment of the transcribed DNA using two primers.
As used herein, the term “primer” refers to an oligonucleotide, whether
ing naturally as in a purified restriction digest or produced synthetically, which is
capable of acting as a point of initiation of synthesis when placed under conditions in
which synthesis of a primer extension product which is complementary to a nucleic acid
strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as
DNA polymerase and at a suitable temperature and pH). The primer is preferably single
stranded for maximum efficiency in amplification, but may alternatively be double
stranded. If double stranded, the primer is first treated to te its strands before being
used to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of
extension products in the presence of the ng agent. The exact lengths of the primers
PCT/USZOI4/029566
will depend on many factors, including temperature, source of primer and the use of the
method.
As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence
of nucleotides), whether occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is e of hybridizing to
another ucleotide of interest. A probe may be single—stranded or double-stranded.
Probes are useful in the detection, identification and isolation of ular gene
sequences. It is contemplated that any probe used in the present invention will be d
with any ter molecule,” so that it is detectable in any detection system, including,
but not limited to enzyme (e. g., ELISA, as well as enzyme~based histochemical assays),
fluorescent, ctive, and luminescent systems. It is not intended that the present
invention be limited to any particular detection system or label.
As used herein, the terms “restriction endonucleases” and “restriction
enzymes” refer to bacterial enzymes, each of which cut or nick double or single—stranded
DNA at or near a specific tide sequence, for example, an endonuclease domain of a
type HS restriction endonuclease (e. g., Fold) can be used, as taught by Kim et al., 1996,
Proc. Nat’l. Acad. Sci. USA, 6:1 156-60).
As used herein, the term “an oligonucleotide having a nucleotide sequence
encoding a gene” means a nucleic acid sequence comprising the coding region of a gene,
i.e. the nucleic acid ce which encodes a gene product. The coding region may be
t in either a CDNA, genomic DNA or RNA form. When present in a DNA form,
the oligonucleotide may be single—stranded (i.e., the sense strand) or double—stranded.
Additionally “an ucleotide having a nucleotide sequence encoding a gene” may
include suitable control elements such as enhancers, ers, splice junctions,
polyadenylation signals, etc. if needed to permit proper initiation of transcription and/or
correct processing of the y RNA ript. Further still, the coding region of the
present invention may contain endogenous enhancers, splice junctions, intervening
sequences, polyadenylation signals, etc.
Transcriptional control signals in eukaryotes comprise “enhancer” elements.
Enhancers consist of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (Maniatis, T. et al., Science 236: 1237, 1987).
Enhancer ts have been isolated from a variety of eukaryotic sources including
genes in plant, yeast, insect and mammalian cells and s. The selection of a
ular enhancer depends on what cell type is to be used to express the protein of
interest.
The presence of “splicing signals” on an expression vector often results in
higher levels of expression of the recombinant transcript. Splicing signals mediate the
removal of introns from the primary RNA transcript and consist of a splice donor and
acceptor site (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, New York, pp. 16.7—16.8, 1989). A commonly
used splice donor and acceptor site is the splice junction from the 168 RNA of SV40.
Efficient expression of recombinant DNA sequences in eukaryotic cells
requires expression of signals directing the efficient termination and polyadenylation of
the resulting transcript. Transcription termination s are generally found
downstream of the polyadenylation signal and are a few hundred nucleotides in length.
The term “poly A site” or “poly A ce” as used herein denotes a DNA ce
which s both the termination and enylation of the t RNA transcript.
Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking
a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an
expression vector may be “heterologous” or “endogenous.” An endogenous poly A
signal is one that is found naturally at the 3’ end of the coding region of a given gene in
the genome. A heterologous poly A signal is one which is isolated from one gene and
placed 3’ of another gene.
The term “promoter,” ter element” or “promoter sequence” as used
herein, refers to a DNA sequence which when placed at the 5’ end of (Le, precedes) an
oligonucleotide sequence is capable of controlling the transcription of the oligonucleotide
sequence into mRNA. A promoter is typically located 5' (i.e., upstream) of an
oligonucleotide sequence whose transcription into mRNA it controls, and provides a site
for specific binding by RNA polymerase and for initiation of transcription.
The term “promoter activity” when made in nce to a nucleic acid
sequence refers to the ability of the nucleic acid sequence to initiate transcription of an
oligonucleotide ce into mRNA.
The term “tissue specific” as it applies to a promoter refers to a promoter that
is capable of ing selective expression of an oligonucleotide sequence to a specific
W0 2014/144951 PCT/USZOl4/029566
type of tissue in the relative absence of expression of the same oligonucleotide in a
ent type of . Tissue specificity of a promoter may be evaluated by, for
example, operably linking a reporter gene to the promoter sequence to generate a er
construct, introducing the reporter construct into the genome of a plant or an animal such
that the reporter construct is integrated into every tissue of the resulting transgenic
animal, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein,
or the activity of a protein d by the reporter gene) in different tissues of the
transgenic plant or . Selectivity need not be absolute. The detection of a greater
level of expression of the reporter gene in one or more s relative to the level of
expression of the reporter gene in other tissues shows that the promoter is ic for the
tissues in which greater levels of expression are detected.
The term “cell type specific” as applied to a promoter refers to a promoter
which is capable of ing selective expression of an oligonucleotide sequence in a
specific type of cell in the relative absence of expression of the same oligonucleotide
sequence in a ent type of cell within the same tissue. The term “cell type specific”
when applied to a er also means a promoter capable of promoting selective
expression of an oligonucleotide in a region within a single . Again, selectivity
need not be absolute. Cell type specificity of a promoter may be assessed using methods
well known in the art, e. g., immunohistochemical staining as described herein. Briefly,
tissue sections are embedded in in, and paraffin sections are reacted with a primary
antibody which is specific for the polypeptide product encoded by the oligonucleotide
sequence whose expression is controlled by the promoter. As an alternative to paraffin
ning, samples may be cryosectioned. For example, sections may be frozen prior to
and during sectioning thus avoiding potential interference by residual paraffin. A labeled
(e.g., peroxidase conjugated) secondary dy which is specific for the primary
antibody is allowed to bind to the sectioned tissue and specific g detected (e.g.,
with avidin/biotin) by microscopy.
The terms “selective expression, 9’ (Gselectively express” and grammatical
equivalents thereof refer to a comparison of relative levels of expression in two or more
regions of interest. For example, “selective expression” when used in connection with
tissues refers to a ntially r level of expression of a gene of interest in a
particular tissue, or to a substantially greater number of cells which express the gene
within that tissue, as compared, respectively, to the level of expression of, and the number
of cells sing, the same gene in another tissue (i.e., selectivity need not be absolute).
Selective expression does not require, although it may include, expression of a gene of
interest in a particular tissue and a total absence of expression of the same gene in r
. Similarly, “selective expression” as used herein in reference to cell types refers to
a substantially r level of expression of, or a ntially greater number of cells
which express, a gene of interest in a particular cell type, when compared, respectively, to
the expression levels of the gene and to the number of cells sing the gene in another
cell type.
The term “contiguous” when used in reference to two or more nucleotide
sequences means the nucleotide sequences are ligated in tandem either in the absence of
intervening sequences, or in the presence of intervening sequences which do not comprise
one or more control elements.
As used herein, the terms ic acid molecule encoding,7’ eotide
encoding,” “DNA sequence encoding” and “DNA encoding” refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of
these deoxyribonucleotides determines the order of amino acids along the polypeptide
(protein) chain. The DNA sequence thus codes for the amino acid sequence.
The term “isolated” when used in relation to a nucleic acid, as in “an isolated
oligonucleotide” refers to a nucleic acid sequence that is separated from at least one
contaminant nucleic acid with which it is ordinarily associated in its natural source.
Isolated nucleic acid is nucleic acid present in a form or setting that is different from that
in which it is found in nature. In contrast, olated nucleic acids are nucleic acids
such as DNA and RNA which are found in the state they exist in nature. For example, a
given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to
neighboring genes; RNA sequences, such as a ic mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous other mRNAs which
encode a multitude of proteins. However, isolated nucleic acid encoding a polypeptide of
interest includes, by way of example, such c acid in cells ordinarily expressing the
polypeptide of interest where the c acid is in a chromosomal or hromosomal
location different from that of natural cells, or is otherwise flanked by a different nucleic
acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may
be t in single~stranded or double-stranded form. Isolated nucleic acid can be
readily identified (if desired) by a variety of techniques (e.g., hybridization, dot blotting,
W0 2014/144951
etc). When an isolated nucleic acid or oligonucleotide is to be utilized to express a
protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the
oligonucleotide may be single—stranded). Alternatively, it may n both the sense and
anti-sense strands (i.e., the oligonucleotide may be double—stranded).
As used herein, the term “purified” or “to purify” refers to the removal of one
or more (undesired) components from a sample. For example, where recombinant
polypeptides are expressed in bacterial host cells, the ptides are purified by the
removal of host cell ns thereby increasing the percent of recombinant ptides
in the sample.
As used herein, the term antially purified” refers to molecules, either
nucleic or amino acid sequences, that are removed from their natural nment,
isolated or separated, and are at least 60% free, preferably 75% free and more preferably
90% free from other components with which they are naturally associated. An “isolated
polynucleotide” is, ore, a ntially purified polynucleotide.
As used herein the term “coding region” when used in reference to a structural
gene refers to the nucleotide sequences which encode the amino acids found in the
nascent polypeptide as a result of translation of a mRNA le. The coding region is
bounded, in eukaryotes, on the 5’ side generally by the nucleotide t “ATG” which
encodes the initiator methionine and on the 3’ side by one of the three triplets which
specify stop codons (i.e., TAA, TAG, TGA).
By "coding sequence" is meant a sequence of a nucleic acid or its
complement, or a part thereof, that can be transcribed and/or translated to produce the
mRNA for and/or the polypeptide or a nt thereof. Coding sequences include exons
in a genomic DNA or immature y RNA transcripts, which are joined together by
the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the
complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
By "non~coding sequence" is meant a sequence of a nucleic acid or its
complement, or a part thereof that is not transcribed into amino acid in Vivo, or where
tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences
include both intron sequences in genomic DNA or immature primary RNA ripts,
and gene—associated sequences such as promoters, enhancers, silencers, etc.
PCT/U82014/029566
As used herein, the term “structural gene” or “structural nucleotide sequence”
refers to a DNA sequence coding for RNA or a protein which does not control the
expression of other genes. In contrast, a “regulatory gene” or “regulatory sequence” is a
structural gene which encodes products (e. g., transcription factors) which control the
expression of other genes.
As used herein, the term “regulatory element” refers to a genetic element
which controls some aspect of the expression of nucleic acid sequences. For example, a
promoter is a regulatory element which facilitates the initiation of transcription of an
operany linked coding region. Other regulatory ts e splicing s,
polyadenylation s, ation signals, etc.
As used herein, the term “peptide transcription factor binding site” or
“transcription factor binding site” refers to a nucleotide sequence which binds protein
transcription factors and, thereby, controls some aspect of the expression of nucleic acid
sequences. For example, Sp—l and APl (activator protein 1) binding sites are examples of
peptide transcription factor binding sites.
As used herein, the term “gene” means the deoxyribonucleotide ces
sing the coding region of a structural gene. A “gene” may also e non-
translated sequences located adjacent to the coding region on both the 5’ and 3’ ends such
that the gene corresponds to the length of the full-length mRNA. The sequences which
are located 5’ of the coding region and which are present on the mRNA are referred to as
’ non-translated sequences. The sequences which are located 3’ or downstream of the
coding region and which are present on the mRNA are referred to as 3’ non—translated
sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A
genomic form or clone of a gene contains the coding region interrupted with non—coding
sequences termed ns” or “intervening regions” or “intervening sequences.” Introns
are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns are removed or
“spliced out” from the nuclear or primary transcript; introns therefore are absent in the
messenger RNA (mRNA) ript. The mRNA functions during translation to specify
the ce or order of amino acids in a nascent polypeptide.
In addition to containing s, genomic forms of a gene may also include
ces located on both the 5’ and 3’ end of the sequences which are present on the
PCT/U82014/029566
RNA transcript. These sequences are ed to as “flanking” ces or regions
(these flanking sequences are located 5’ or 3’ to the non—translated sequences present on
the mRNA transcript). The 5’ g region may contain regulatory sequences such as
promoters and enhancers which control or nce the transcription of the gene. The 3’
flanking region may contain sequences which direct the termination of transcription, post-
transcriptional cleavage and polyadenylation.
A “non—human animal” refers to any animal which is not a human and
includes vertebrates such as rodents, man primates, ovines, bovines, ruminants,
rphs, porcines, caprines, equines, canines, felines, aves, etc. Preferred non-human
animals are selected from the order Rodentia. “Non—human animal” additionally refers to
amphibians (e.g. Xenopus), reptiles, insects (e.g. Drosophila) and other non-mammalian
animal species.
As used herein, the term “transgenic” refers to an sm or cell that has
DNA derived from another organism inserted into which becomes integrated into the
genome either of somatic and/or germ line cells of the plant or animal. A “transgene”
means a DNA sequence which is partly or entirely heterologous (i.e., not present in
nature) to the plant or animal in which it is found, or which is homologous to an
endogenous sequence (Le, a sequence that is found in the animal in nature) and is
inserted into the plant’ or animal's genome at a location which differs from that of the
naturally occurring ce. enic plants or animals which include one or more
enes are within the scope of this invention. Additionally, a “transgenic” as used
herein refers to an animal that has had one or more genes ed and/or “knocked out”
(made non—functional or made to function at reduced level, Le, a “knockout” mutation)
by the invention‘s methods, by homologous recombination, TFO mutation or by similar
processes. For example, in some embodiments, a transgenic organism or cell includes
inserted DNA that includes a foreign promoter and/or coding region.
A formed cell” is a cell or cell line that has acquired the ability to grow
in cell culture for multiple generations, the ability to grow in soft agar, and/or the y
to not have cell growth ted by o-cell contact. In this regard, transformation
refers to the introduction of foreign genetic material into a cell or organism.
Transformation may be accomplished by any method known which permits the successful
introduction of nucleic acids into cells and which results in the expression of the
introduced nucleic acid. “Transformation” includes but is not limited to such methods as
PCT/U82014/029566
transfection, microinjection, oporation, nucleofection and lipofection (liposome—
ed gene transfer). Transformation may be accomplished through use of any
expression vector. For example, the use of baculovirus to introduce foreign nucleic acid
into insect cells is contemplated. The term “transformation” also includes methods such
as P—element mediated germline transformation of whole insects. Additionally,
transformation refers to cells that have been transformed naturally, usually through
genetic mutation.
As used herein “exogenous” means that the gene encoding the protein is not
ly expressed in the cell. Additionally, “exogenous” refers to a gene transfected
into a cell to augment the normal (i.e. natural) level of expression of that gene.
A peptide sequence and nucleotide sequence may be “endogenous” or
“heterologous” (i.e., gn”). The term enous” refers to a sequence which is
naturally found in the cell into which it is introduced so long as it does not contain some
modification relative to the naturally-occurring sequence. The term “heterologous” refers
to a sequence which is not endogenous to the cell into which it is uced. For
example, heterologous DNA includes a nucleotide ce which is ligated to, or is
manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in
nature, or to which it is ligated at a different location in nature. Heterologous DNA also
includes a nucleotide ce which is naturally found in the cell into which it is
introduced and which contains some modification relative to the lly—occurring
sequence. Generally, although not necessarily, heterologous DNA encodes heterologous
RNA and heterologous proteins that are not normally produced by the cell into which it is
introduced. Examples of heterologous DNA include reporter genes, transcriptional and
translational regulatory sequences, DNA sequences which encode selectable marker
proteins (e.g., proteins which confer drug resistance), etc.
ucts
The nucleic acid molecules disclosed herein (e.g., site specific nucleases, or
guide RNA for CRISPRs) can be used in the production of inant nucleic acid
constructs. In one embodiment, the nucleic acid molecules of the present disclosure can
be used in the preparation of nucleic acid constructs, for example, sion cassettes for
expression in the plant of interest. This expression may be transient for instance when the
construct is not integrated into the host genome or ined under the control offered
PCT/USZOI4/029566
by the promoter and the position of the construct within the host’s genome if it becomes
integrated.
Expression cassettes may include regulatory sequences ly linked to the
site specific nuclease or guide RNA sequences sed herein. The cassette may
additionally contain at least one additional gene to be co—transformed into the organism.
Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
The nucleic acid constructs may be provided with a plurality of restriction
sites for insertion of the site specific nuclease coding sequence to be under the
transcriptional regulation of the regulatory regions. The nucleic acid constructs may
additionally contain nucleic acid molecules encoding for selectable marker genes.
Any promoter can be used in the production of the nucleic acid constructs.
The promoter may be native or ous, or n or heterologous, to the plant host
c acid sequences disclosed herein. Additionally, the promoter may be the natural
sequence or alternatively a synthetic sequence. Where the promoter is “foreign” or
“heterologous” to the plant host, it is intended that the promoter is not found in the native
plant into which the promoter is introduced. As used herein, a chimeric gene comprises a
coding sequence operably linked to a ription initiation region that is heterologous to
the coding sequence.
The site directed nuclease ces disclosed herein may be sed using
heterologous promoters.
Any promoter can be used in the preparation of constructs to control the
expression of the site ed nuclease sequences, such as promoters ing for
constitutive, tissue~preferred, inducible, or other promoters for expression in plants.
Constitutive promoters e, for example, the core promoter of the Rsyn7 promoter
and other constitutive promoters disclosed in W0 99/43 838 and U.S. Patent No.
6,072,050; the core CaMV 35S promoter (Odell et al. Nature 313:810-812; 1985); rice
actin (McElroy et al., Plant Cell 2:163-171, 1990); ubiquitin (Christensen et al., Plant
Mol. Biol. 12:619—632, 1989 and Christensen et al., Plant Mol. Biol. 182675—689, 1992);
pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); MAS n et al., EMBO J.
322723—2730, 1984); ALS promoter (US. Patent No. 5,659,026), and the like. Other
constitutive promoters include, for example, US. Patent Nos. 149; 144;
,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 142; and 6,177,611.
PCT/USZOl4/029566
Tissue~preferred promoters can be utilized to direct site directed nuclease
expression within a particular plant tissue. Such tissue—preferred ers include, but
are not limited to, leaf-preferred promoters, root—preferred promoters, seed—preferred
ers, and stem-preferred promoters. Tissuetpreferred promoters e Yamamoto
et al., Plant J. 12(2):255—265, 1997; Kawamata et al., Plant Cell Physiol. 38(7):792—803,
1997; Hansen et al., Mol. Gen Genet. 254(3):337—343, 1997; Russell et al., Transgenic
Res. 6(2):157—168, 1997; Rinehart et al., Plant Physiol. 1 12(3):1331~134l, 1996; Van
Camp et al., Plant Physiol. 1 12(2):525-535, 1996; Canevascini et al., Plant Physiol.
112(2): 513—524, 1996; Yamamoto et al., Plant Cell Physiol. 35(5):773—778, 1994; Lam,
Results Prob]. Cell Differ. 20:181—196, 1994; Orozco et al. Plant Mol Biol. 23(6):1129~
1138, 1993; Matsuoka et al., Proc Nat’l. Acad. Sci. USA 90(20):9586- 9590, 1993; and
Guevara—Garcia et al., Plant J. 4(3):495~505, 1993.
The nucleic acid constructs may also include ription termination regions.
Where ription ations s are used, any termination region may be used in
the preparation of the nucleic acid ucts. For e, the termination region may
be derived from another source (i.e., foreign or heterologous to the promoter). Examples
of termination regions that are available for use in the constructs of the present disclosure
include those from the Ti—plasmid of A. tzmzefaciens, such as the ne synthase and
nopaline synthase termination regions. See also Guerineau et al., Mol. Gen. Genet.
1-144, 1991; Proudfoot, Cell 642671-674, 1991; Sanfacon et al., Genes Dev.
:141~149, 1991; Mogen et al., Plant Cell 2: 1261—1272, 1990; Munroe et al., Gene
91:151—158, 1990; Ballas et al., Nucleic Acids Res. 17:7891—7903, 1989; and Joshi et al.,
Nucleic Acid Res. 15:9627—9639, 1987.
In conjunction with any of the aspects, embodiments, methods and/or
compositions disclosed herein, the nucleic acids may be optimized for increased
expression in the transformed plant. That is, the nucleic acids encoding the site directed
nuclease proteins can be synthesized using plant-preferred codons for improved
expression. See, for example, Campbell and Gowri, (Plant Physiol. 92: 1-11, 1990) for a
discussion of host—preferred codon usage. Methods are available in the art for
synthesizing plant-preferred genes. See, for example, US. Patent Nos. 5,380,831, and
,436,391, and Murray et al., Nucleic Acids Res. 17:477—498, 1989.
In addition, other sequence modifications can be made to the nucleic acid
sequences sed herein. For example, additional ce modifications are known
2014/029566
to enhance gene expression in a cellular host. These include elimination of sequences
encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like
repeats, and other such well-characterized sequences that may be deleterious to gene
expression. The SC content of the sequence may also be adjusted to levels average for a
target cellular host, as calculated by reference to known genes expressed in the host cell.
In addition, the sequence can be modified to avoid predicted hairpin secondary mRNA
structures.
Other nucleic acid sequences may also be used in the ation of the
constructs of the present disclosure, for example to enhance the expression of the site
directed nuclease coding sequence. Such nucleic acid sequences include the introns of
the maize Adhl, intronl gene (Callis et al., Genes and Development 121183-1200, 1987),
and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize
Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al., Nucleic Acid Res.
:8693-871 l, 1987; and Skuzeski et al., Plant Mol. Biol. 15:65—79, 1990). The first
intron from the shrunken~l locus of maize has been shown to increase sion of
genes in chimeric gene constructs. US. Pat. Nos. 5,424,412 and 874 se the
use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol.
1062929939, 1994) also have shown that introns are useful for regulating gene
expression on a tissue specific basis. To further e or to ze site directed
se gene sion, the plant expression vectors disclosed herein may also contain
DNA sequences containing matrix attachment regions (MARS). Plant cells transformed
with such modified expression systems, then, may exhibit overexpression or constitutive
expression of a nucleotide sequence of the sure.
The expression constructs disclosed herein can also include nucleic acid
sequences capable of directing the expression of the site directed nuclease sequence to the
chloroplast. Such nucleic acid sequences include plast targeting sequences that
encodes a chloroplast transit peptide to direct the gene product of interest to plant cell
plasts. Such transit peptides are known in the art. With respect to chloroplast-
targeting sequences, “operably linked” means that the nucleic acid sequence encoding a
transit e (i.e., the chloroplast-targeting sequence) is linked to the site directed
nuclease nucleic acid molecules sed herein such that the two sequences are
contiguous and in the same reading frame. See, for example, Von Heijne et al., Plant
Mol. Biol. Rep. 126, 1991; Clark eta1., J. Biol. Chem. 264:17544—17550, 1989;
WO 44951 PCT/USZOl4/029566
Della—Cioppa et al., Plant Physiol. 84:965-968, 1987; Romer et al., Biochem. Biophys.
Res. Connnun. 14—1421, 1993; and Shah et al., Science 233:478—481, 1986.
] Chloroplast targeting sequences are known in the art and e the
chloroplast small subunit of ribulose—1,5—bisphosphate carboxylase (Rubisco) (de Castro
Silva Filho et al., Plant Mol. Biol. 302769480, 1996; Schnell et al., J. Biol. Chem.
266(5):3335-3342, 1991); 5— (enolpyruvyl)shikimate—3phosphate synthase (EPSPS)
(Archer eta1., J. Bioenerg. Biomemb. 22(6):789~810, 1990); tryptophan synthase (Zhao et
al., J. Biol. Chem. 270(1 1):6081— 6087, 1995); plastocyanin (Lawrence eta1., J. Biol.
Chem. 272(33):20357~20363, 1997); chorismate synthase (Schmidt eta1., J. Biol. Chem.
268(3 6):27447-27457, 1993); and the light harvesting chlorophyll a/b binding protein
(LHBP) (Lamppa et al., J. Biol. Chem. 263:14996—14999, 1988). See also Von Heijne et
al., Plant Mol. Biol. Rep. 9:104~126, 1991; Clark eta1., J. Biol. Chem. 264:1754447550,
1989; Della—Cioppa et al., Plant Physiol. —968, 1987; Romer eta1., Biochem.
Biophys. Res. Commun. 196:1414—1421, 1993; and Shah et al., Science 233 81,
1986.
] In conjunction with any of the aspects, embodiments, methods and/or
compositions disclosed herein, the nucleic acid constructs may be prepared to direct the
expression of the mutant site directed se coding sequence from the plant cell
chloroplast. Methods for transformation of chloroplasts are known in the art. See, for
example, Svab eta1., Proc. Nat’l. Acad. Sci. USA 87:8526-8530, 1990; Svab and Maliga,
Proc. Nat’l. Acad. Sci. USA 90:913-917. 1993; Svab and Maliga, EMBO J. 122601—606,
1993. The method relies on particle gun delivery of DNA containing a selectable marker
and ing of the DNA to the plastid genome h homologous recombination.
Additionally, plastid transformation can be accomplished by transactivation of a silent
plastid-borne transgene by tissue—preferred expression of a nuclear—encoded and plastid—
directed RNA polymerase. Such a system has been reported in McBride et al. Proc.
Nat’l. Acad. Sci. USA 91:7301-7305, 1994.
The nucleic acids of interest to be targeted to the chloroplast may be optimized
for expression in the chloroplast to account for differences in codon usage between the
plant nucleus and this organelle. In this manner, the nucleic acids of interest may be
sized using clfloroplast-preferred codons. See, for example, US. Patent No.
,3 , herein incorporated by reference.
PCTfUSZOl4/029566
The nucleic acid constructs can be used to transform plant cells and regenerate
transgenic plants comprising the site directed nuclease coding sequences. Numerous
plant transformation vectors and methods for transforming plants are available. See, for
example, US. Patent No. 6,753,458, An, G. eta1., Plant l, 81 :301—305, 1986; Fry,
J. et al., Plant Cell Rep. 6:321—325, 1987; Block, M., Theor. Appl Genet. 76:767—774,
1988; Hinchee et al., Stadler. Genet. 03212.203~212, 1990; Cousins et al., Aust. J.
Plant l. 18:481—494, 1991; Chee, P. P. and Slightom, J. L., 18:255~260,
1992; Christou et al., Trends. Biotechnol. 10:239—246, 1992; uin eta1.,
Bio/Technol. 10:309-3 14, 1992; Dhir eta1., Plant Physiol. 99:81—88, 1992; Casas et al.,
Proc. Nat’l. Acad Sci. USA 90:11212—11216, 1993; ou, P., In. Vitro Cell. Dev.
lant 29le 19—124, 1993; Davies, et al., Plant Cell Rep. 12:180—183, 1993; Dong, J.
A. and Mc Hughen, A., Plant Sci. -148, 1993; Franklin, C. I. and Trieu, T. N.,
Plant. Physiol. 102:167, 1993; Golovkin et al., Plant Sci. 90:41-52, 1993; Guo Chin Sci.
Bull. 38:2072—2078; Asano, et al., Plant Cell Rep. 13, 1994; Ayeres N. M. and Park, W.
D., Crit. Rev. Plant. Sci. 13:219—239, 1994; Barcelo eta1., Plant. J. 5:583—592, 1994;
Becker, eta1., Plant. J. 307, 1994; Borkowska eta1., Acta. Physiol Plant. 16:225-
230, 1994; Christou, P., Agro. Food. Ind. Hi Tech. 5:17—27, 1994; Eapen et al., Plant Cell
Rep. 13:582—586, 1994; Hartman et al., Bio-Technology 12:919923, 1994; Ritala et al.,
Plant. Mol. Biol. -325, 1994; and Wan, Y. C. and Lemaux, P. 6., Plant Physiol.
10413748, 1994. The constructs may also be transformed into plant cells using
homologous recombination.
The term “wild-type” when made in reference to a peptide sequence and
nucleotide sequence refers to a peptide sequence and nucleotide sequence
(locus/gene/allele), respectively, which has the characteristics of that peptide sequence
and nucleotide sequence when isolated from a naturally occurring source. A wild—type
peptide sequence and nucleotide sequence is that which is most frequently observed in a
tion and is thus arbitrarily designated the “normal” or “wild—type” form of the
peptide ce and nucleotide sequence, respectively. "Wild-type" may also refer to
the sequence at a ic. nucleotide. position or positions, or the sequence at a particular
codon position or positions, or the sequence at a particular amino acid position or
positions.
“Consensus sequence” is d as a sequence of amino acids or nucleotides
that contain identical amino acids or nucleotides or functionally equivalent amino acids or
PCT/U82014/029566
nucleotides for at least 25% of the sequence. The identical or functionally equivalent
amino acids or nucleotides need not be contiguous.
The term ica” as used herein refers to plants of the ca genus.
Exemplary Brassica species include, but are not limited to, B. carinata, B. te, B.
fruticulosa, B. juncea, B. napus, B. narinosa, B. nigra, B. oleracea, B. perviridis, B. rapa
(syn B. campestris), B. rupestris, B. septiceps, and B. tournefortii.
A nucleobase is a base, which in certain preferred ments is a purine,
pyrimidine, or a derivative or analog thereof. Nucleosides are nucleobases that n a
pentosefuranosyl moiety, e. g., an optionally substituted riboside or xyriboside.
Nucleosides can be linked by one of several linkage es, which may or may not
contain phosphorus. Nucleosides that arc linked by unsubstituted phosphodiester
linkages are termed nucleotides. The term "nucleobase" as used herein includes peptide
nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as
nucleosides and nucleotides.
An oligonucleobase is a polymer comprising nucleobases; preferably at least a
portion of which can hybridize by Watson—Crick base pairing to a DNA having the
complementary sequence. An oligonucleobase chain may have a single 5' and 3‘
terminus, which are the te nucleobases of the polymer. A particular
oligonucleobase chain can contain nucleobases of all types. An oligonucleobase
compound is a compound sing one or more oligonucleobase chains that may be
complementary and hybridized by Watson-Crick base pairing. Ribo—type nucleobases
include pentosefuranosyl containing nucleobases wherein the 2’ carbon is a ene
substituted with a hydroxyl, alkyloxy or halogen. ibo-type nucleobases are
bases other than ribo—type nucleobases and include all nucleobases that do not
contain a pentosefuranosyl moiety.
In n embodiments, an oligonucleobase strand may include both
oligonucleobase chains and segments or regions of oligonucleobase chains. An
oligonucleobase strand may have a 3' end and a 5' end, and when an oligonucleobase
strand is coextensive with a chain, the 3' and 5' ends of the strand are also 3' and 5' termini
of the chain.
PCT/USZOl4/029566
The term ” gene repair oligonucleobase" as used herein denotes
oligonucleobases, including mixed duplex oligonucleotides, non~nucleotide containing
molecules, single stranded oligodeoxynucleotides and other gene repair molecules.
As used herein the term "codon" refers to a sequence of three adjacent
nucleotides (either RNA or DNA) constituting the genetic code that determines the
insertion of a specific amino acid in a polypeptide chain during protein synthesis or the
signal to stop protein synthesis. The term "codon" is also used to refer to the
corresponding (and complementary) sequences of three nucleotides in the messenger
RNA into which the original DNA is transcribed.
As used herein, the term "homology" refers to sequence similarity among
proteins and DNA. The term ”homology" or "homologous" refers to a degree of identity.
There may be l homology or complete homology. A lly homologous sequence
is one that has less than 100% sequence identity when compared to r ce.
“Heterozygous" refers to having different s at one or more genetic loci in
homologous chromosome segments. As used herein "heterozygous” may also refer to a
sample, a cell, a cell population or an organism in which different alleles at one or more
genetic loci may be detected. zygous samples may also be determined Via methods
known in the art such as, for example, nucleic acid sequencing. For example, if a
sequencing electropherogram shows two peaks at a single locus and both peaks are
roughly the same size, the sample may be characterized as heterozygous. Or, if one peak
is smaller than r, but is at least about 25% the size of the larger peak, the sample
may be characterized as heterozygous. In some embodiments, the smaller peak is at least
about 15% of the larger peak. In other ments, the smaller peak is at least about
% of the larger peak. In other embodiments, the smaller peak is at least about 5% of
the larger peak. In other embodiments, a l amount of the smaller peak is detected.
As used herein, ”homozygous" refers to having cal alleles at one or more
genetic loci in homologous some segments. ygous" may also refer to a
sample, a cell, a cell tion or an organism in which the same alleles at one or more
genetic loci may be detected. Homozygous samples may be determined Via methods
known in the art, such as, for example, nucleic acid sequencing. For example, if a
sequencing electropherogram shows a single peak at a particular locus, the sample may be
termed "homozygous" with respect to that locus.
PCT/U82014/029566
The term "hemizygous" refers to a gene or gene segment being present only
once in the genotype of a cell or an sm because the second allele is deleted. As used
herein "hemizygous" may also refer to a sample, a cell, a cell population or an organism
in which an allele at one or more genetic loci may be detected only once in the pe.
The term ”zygosity status" as used herein refers to a sample, a cell population,
or an organism as appearing heterozygous, homozygous, or hemizygous as determined by
testing methods known in the art and described herein. The term ”zygosity status of a
nucleic acid" means determining whether the source of nucleic acid appears
heterozygous, homozygous, or hemizygous. The "zygosity status" may refer to
differences in a single tide in a sequence. In some methods, the zygosity status of a
sample with respect to a single mutation may be categorized as homozygous wild~type,
heterozygous (i.e., one wi1d~type allele and one mutant allele), homozygous mutant, or
hemizygous (i.e., a single copy of either the wild-type or mutant allele).
As used herein, the term "RTDS" refers to The Rapid Trait pment
SystemTM (RTDS) ped by Cibus. RTDS is a pecific gene modification
system that is effective at making precise changes in a gene sequence without the
incorporation of foreign genes or control sequences.
The term "about“ as used herein means in quantitative terms plus or minus
%. For example, ”about 3%" would encompass 2.733% and "about 10%" would
encompass 9—1 1%.
Repair Oligonucleotides
This invention generally relates to novel methods to improve the efficiency of
the targeting of modifications to specific locations in genomic or other nucleotide
ces. Additionally, this invention s to target DNA that has been ed,
mutated or marked by the ches disclosed herein. The invention also s to
cells, tissue, and organisms which have been modified by the invention‘s methods. The
present invention builds on the development of compositions and methods related in part
to the successful conversion system, the Rapid Trait Development System (RTDSTM,
Cibus US LLC).
RTDS is based on altering a targeted gene by utilizing the cell's own gene
repair system to specifically modify the gene ce in situ and not insert foreign DNA
and gene expression control sequences. This procedure effects a precise change in the
PCT/U82014/029566
genetic sequence while the rest of the genome is left unaltered. In contrast to
conventional transgenic GMOs, there is no integration of n genetic material, nor is
any foreign genetic material left in the plant. The changes in the genetic sequence
introduced by RTDS are not randomly inserted. Since affected genes remain in their
native location, no random, uncontrolled or adverse pattern of expression occurs.
The RTDS that effects this change is a chemically sized ucleotide
which may be composed of both DNA and modified RNA bases as well as other chemical
moieties, and is designed to hybridize at the targeted gene location to create a mismatched
base—pair(s). This mismatched base-pair acts as a signal to attract the cell's own natural
gene repair system to that site and correct (replace, insert or delete) the designated
nucleotide(s) within the gene. Once the tion process is complete the RTDS
molecule is degraded and the dified or repaired gene is expressed under that
gene‘s normal endogenous control mechanisms.
The methods and compositions disclosed herein can be practiced or made with
gene repair oligonucleobases" (GRON) having the conformations and chemistries as
described in detail below. The " gene repair oligonucleobases" as contemplated herein
have also been described in published scientific and patent literature using other names
including "recombinagenic oligonucleobases;” ”RNA/DNA ic oligonucleotides;"
"chimeric oligonucleotides;” ”mixed duplex oligonucleotides" ); ”RNA DNA
oligonucleotides (RDOS);" " H II II H
gene targeting oligonucleotides; genoplasts; single
stranded modified oligonucleotides;" "Single stranded oligodeoxynucleotide onal
vectors" (SSOMVS); "duplex mutational vectors;" and ”heteroduplex mutational vectors."
The gene repair oligonucleobase can be introduced into a plant cell using any method
commonly used in the art, including but not limited to, microcarriers (biolistic delivery),
microfibers, hylene glycol (PEG)-mediated uptake, oporation, and
microinjection.
In one embodiment, the gene repair oligonucleobase is a mixed duplex
oligonucleotides (MDON) in which the RNA-type nucleotides of the mixed duplex
oligonucleotide are made RNase ant by replacing the 2‘~hydroxyl with a fluoro,
chloro or bromo functionality or by placing a substituent on the 2‘—O. Suitable
substituents include the substituents taught by the Kmiec II. Alternative substituents
include the substituents taught by US. Pat. No. 71 l (Sproat) and the substituents
taught by patent ations EP 629 387 and EP 679 657 (collectively, the Martin
PCT/USZOl4/029566
ations), which are hereby incorporated by reference. As used herein, a 2'~fluor0,
chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a T— OH
substituted with a substituent described in the Martin Applications or Sproat is termed a
”T~ Substituted Ribonucleotide." As used herein the term ”RNA—type nucleotide" means
a T— yl or 2 '—Substituted Nucleotide that is linked to other nucleotides of a mixed
duplex oligonucleotide by an unsubstituted odiester linkage or any of the non—
natural linkages taught by Kmiec I or Kmiec 11. As used herein the term ribo-type
nucleotide" means a nucleotide having a T—H, which can be linked to other nucleotides of
a gene repair oligonucleobase by an tituted odiester linkage or any of the
non-natural linkages taught by Kmiec I or Kmiec II.
In a particular embodiment of the t invention, the gene repair
oligonucleobase is a mixed duplex oligonucleotide (MDON) that is linked solely by
tituted phosphodiester bonds. In alternative embodiments, the linkage is by
substituted phosphodiesters, phosphodiester derivatives and non—phosphorus-based
linkages as taught by Kmiec II. In yet r embodiment, each RNA—type nucleotide in
the mixed duplex oligonucleotide is a 2 ‘—Substituted Nucleotide. Particular preferred
embodiments of 2‘-Substituted cleotides are 2'-fluoro, T— methoxy, 2‘-propyloxy,
yloxy, 2'—hydroxylethyloxy, 2'~methoxyethyloxy, T— fluoropropyloxy and 2’—
trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2'-
Substituted cleotides are 2‘~fluoro, 2‘-methoxy, 2'—methoxyethyloxy, and 2'-
allyloxy substituted nucleotides. In another embodiment the mixed duplex
oligonucleotide is linked by unsubstituted phosphodiester bonds,
Although mixed duplex oligonucleotides (MDONs) having only a single type
of 2'- substituted pe nucleotide are more conveniently synthesized, the methods of
the invention can be practiced with mixed duplex oligonucleotides having two or more
types of RNA-type nucleotides. The function of an RNA segment may not be affected by
an interruption caused by the introduction of a deoxynucleotide between two RNA-type
trinucleotides, accordingly, the term RNA segment encompasses terms such as
”interrupted RNA segment." An uninterrupted RNA segment is termed a contiguous RNA
segment. In an alternative embodiment an RNA segment can n alternating RNase-
resistant and unsubstituted 2'—OH nucleotides. The mixed duplex oligonucleotides
preferably have fewer than 100 nucleotides and more preferably fewer than 85
nucleotides, but more than 50 nucleotides. The first and second strands are Watson—Crick
base paired. In one embodiment the strands of the mixed duplex oligonucleotide are
ntly bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so
that the first and second strands are segments of a single oligonucleotide chain having a
single 3' and a single 5' end. The 3‘ and 5' ends can be protected by the addition of a
"hairpin cap" whereby the 3‘ and 5‘ terminal nucleotides are Watson-Crick paired to
adjacent tides. A second hairpin cap can, additionally, be placed at the junction
between the first and second strands distant from the 3' and 5' ends, so that the Watson—
Crick pairing between the first and second strands is ized.
The first and second strands n two regions that are homologous with two
fragments of the target gene, i.e., have the same sequence as the target gene. A
homologous region contains the nucleotides of an RNA segment and may contain one or
more DNA-type nucleotides of ting DNA segment and may also contain DNA~
type nucleotides that are not within the ening DNA t. The two regions of
gy are separated by, and each is adjacent to, a region having a sequence that
differs from the sequence of the target gene, termed a "heterologous region." The
heterologous region can contain one, two or three mismatched nucleotides. The
mismatched nucleotides can be contiguous or alternatively can be separated by one or two
nucleotides that are homologous with the target gene. Alternatively, the heterologous
region can also contain an ion or one, two, three or of five or fewer nucleotides.
Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the
sequence of the target gene only by the deletion of one, two, three, or five or fewer
nucleotides from the mixed duplex oligonucleotide. The length and position of the
heterologous region is, in this case, deemed to be the length of the deletion, even though
no nucleotides of the mixed duplex oligonucleotide are within the heterologous region.
The distance n the fragments of the target gene that are complementary to the two
homologous regions is identical to the length of the heterologous region where a
substitution or substitutions is intended. When the heterologous region contains an
insertion, the gous regions are thereby separated in the mixed duplex
ucleotide farther than their complementary homologous fragments are in the gene,
and the converse is applicable when the heterologous region encodes a deletion.
The RNA ts of the mixed duplex oligonucleotides are each a part of a
homologous region, i.e., a region that is identical in sequence to a fragment of the target
gene, which segments together preferably contain at least 13 RNA~type nucleotides and
WO 44951 PCT/USZOl4/029566
preferably from 16 to 25 RNA~type tides or yet more ably 18-22 pe
nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the
homology regions are separated by and adjacent to, i.e., "connected by" an intervening
DNA segment. In one embodiment, each tide of the heterologous region is a
nucleotide of the ening DNA segment. An intervening DNA segment that contains
the heterologous region of a mixed duplex oligonucleotide is termed a "mutator segment."
In another embodiment of the present invention, the gene repair
oligonucleobase (GRON) is a single stranded oligodeoxynucleotide mutational vector
(SSOMV), which is disclosed in International Patent Application PCT/USOO/23457,
US. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 which is incorporated by reference in
its entirety. The sequence of the SSOMV is based on the same principles as the
mutational vectors described in US. Pat. Nos. 325; 5,871,984; 5,760,012;
,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in
International Publication Nos. W0 98/49350; W0 99/07865; W0 99/58723; W0
99/5 8702; and W0 99/40789. The sequence of the SSOMV contains two regions that are
gous with the target sequence separated by a region that contains the desired
c alteration termed the mutator region. The mutator region can have a sequence
that is the same length as the sequence that separates the homologous s in the target
sequence, but having a different sequence. Such a mutator region can cause a
substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to
each other, while the regions in the target gene having the same sequence are separated by
one, two or more nucleotides. Such an SSOMV causes a deletion from the target gene of
the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene
that is identical to the homologous regions may be adjacent in the target gene but
separated by one, two, or more nucleotides in the sequence of the SSOMV. Such an
SSOMV causes an insertion in the sequence of the target gene.
The nucleotides of the SSOMV are deoxyn'bonucleotides that are linked by
unmodified phosphodiester bonds except that the 3' terminal and/or 5‘ terminal
internucleotide linkage or alternatively the two 3' terminal and/or 5' terminal
internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an
internucleotide e is the e between nucleotides of the SSOMV and does not
e the linkage between the 3' end nucleotide or 5‘ end nucleotide and a blocking
substituent. In a specific ment the length of the SSOMV is between 21 and 55
PCTfUSZOl4/029566
deoxynucleotides and the lengths of the homology regions are, accordingly, a total length
of at least 20 deoxynucleotides and at least two homology regions should each have
lengths of at least 8 deoxynucleotides.
The SSOMV can be designed to be complementary to either the coding or the
non— coding strand of the target gene. When the desired mutation is a substitution of a
single base, it is preferred that both the mutator nucleotide and the targeted nucleotide be
a pyrimidine. To the extent that is consistent with achieving the desired functional result,
it is preferred that both the mutator nucleotide and the targeted nucleotide in the
complementary strand be pyrimidines. Particularly preferred are SSOMVs that encode
transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with
a C or T nucleotide in the complementary strand.
ing efficiency
The present invention describes a number of approaches to increase the
effectiveness of conversion of a target gene using repair oligonucleotides, and which may
be used alone or in ation with one another. These include:
1. Introducing modifications to the repair oligonucleotides which t
DNA repair ery to the targeted tch) site.
A. uction of one or more abasic sites in the oligonucleotide (e. g., within
bases, and more preferably with 5 bases of the d mismatch site)
generates a lesion which is an ediate in base excision repair (BER), and
which attracts BER machinery to the vicinity of the site. targeted for
sion by the repair oligonucleotide. dSpacer (abasic furan) modified
ucleotides may be prepared as described in, for example, Takeshita et
al., J. Biol. Chem, 171—79, 1987.
B. Inclusion of compounds which induce single or double strand breaks,
either into the oligonucleotide or together with the oligonucleotide, generates a
lesion which is repaired by non—homologous end joining (NHEJ),
microhomology-mediated end joining (MMEJ), and homologous
recombination. By way of example,the bleomycin family of antibiotics, zinc
fingers, FokI (or any type 118 class of restriction enzyme) and other nucleases
may be covalently coupled to the 3’ or 5’ end of repair ucleotides, in
order to introduce double strand breaks in the vicinity of the site targeted for
conversion by the repair oligonucleotide. The bleomycin family of antibiotics
are DNA cleaving glycopeptides include bleomycin, zeocin, phleomycin,
tallysomycin, pepleomycin and others.
C. Introduction of one or more 8’oxo dA or dG incorporated in the
oligonucleotide (e.g., within 10 bases, and more preferably with 5 bases of the
desired mismatch site) generates a lesion which is similar to lesions created by
reactive oxygen species. These lesions induce the so—called “pushing repair”
system. See, e.g., Kim et a1., , J. Biochem. Mol. Biol. 371657—62, 2004.
2. Increase stability of the repair oiigonucleotides:
Introduction of a reverse base (idC) at the 3’ end of the oligonucleotide to
create a 3’ blocked end on the repair oligonucieotide.
Introduction of one or more Z’O-methyl nucleotides or bases which increase
hybridization energy (see, e.g., W02007/O73 149) at the 5' and/or 3’ of the
repair oiigonucleotide.
Introduction of a ity of Z’O-methyl RNA nucleotides at the 5’ end of the
repair ucleotide, leading into DNA bases which provide the desired
mismatch site, thereby creating an Okazaki Fragment—like nucleic acid
structure.
Conjugated (5’ or 3’) intercalating dyes such as acridine, psoralen, um
e and Syber stains.
Introduction of a 5’ us cap such as a T/A clamp, a cholesterol moiety,
SIMA (HEX), riboC and amidite.
Backbone modifications such as othioate, 2’-O methyl, methyl
onates, locked nucleic acid (LNA), MOE(methoxyethy1), di PS and
peptide c acid (PNA).
inking of the repair oligonucleotide, eg, with intrastrand crosslinking
reagents agents such as cisplatin and mitomycin C.
Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5, Cy3B, CyS
and DY647.
PCT/U82014/029566
3. Increase hybridization energy of the repair oligenucieotide h
incorporation of bases which increase hybridization energy (see, e.g,
W02007/073149).
4. Increase the quality of repair oligonucleotide synthesis by using nucleotide
multimers (dimers, trimers, tetramers, etc.) as building blocks for synthesis. This
results in fewer coupling steps and easier separation of the full length products
from building blocks.
. Use of long repair oligonucleotides (i.e., greater than 55 nucleotides in
length, ably between 75 and 300 nucleotides in length, more preferably at
least 100 nucleotides in length, still more preferably at least 150 nucleotides in
length, and most preferably at least 200 nucleotides in length), preferably with two
or more mutations targeted in the repair oligonucleotide.
Examples of the ing ches are provided in the following table
Table 1. GRON chemistries to be tested.
fliige type Metiiticatiens
‘ mods ’l‘lA clamp TIA clamp
Backbone modifications Phosphothioate PS
{morcaiating dyes 5' Acridine ’3' idC e, MC.
Ukasaki fragments A
C323 replacements. DYSW
Faeilitators ZOE/it?» oiigos designed 5’ E't'BMe
and 3' of the converting
01ng
Abasic Abasic site placed. in Abasic 2
varioua locations 5' and 3’ to
the ting base. 44 mar
Assist Assist approach {3373i itiC on one. none on
rlap: the. other:
2 oiigos: l with illnyidC,
l unmodified repair oligo
Assist Assist approach only make the 'utm'iodified
No overlap: oiigc
2 aligns: l with Cy3fidC, i.
unmodified repair oligo
PCTfU82014/029566
{Riga iym fiam
Abasic THE“ site placed in various- "i‘ea‘rahydmfman (dspacer)
ons 5‘ and 3‘ to the
converting base. 44:1161‘
Backhanfi modifications 9 Z’QMe
’1‘rin‘mrs 1%ch amidites, C523. idC
g repair S'oxo GA” 5’ {3511 MC
} fishing “pair‘1 ‘ '
x v3 : B‘OXO (EA, 5' C1313, idC
Double Strand braak Bieomycln
Cmasfinker Cisplatin
Crflsglizfizer Mitomycin C
Facilitators super bases 5’ and 3' of Ci amim dA and. 2., £315 a "if
converting oligo
Super s Z'amixm CL 5' (3373, MC
Supcsr (fligos Cathie T, 5' C312”), idC
Super oligos 7-deaza A, 5’ (338, MC
Super obi 30.9 aza {if Cy3, idC
Super 01125133 pre-pzmyl (iii, 5‘ (13,73, idC
Emercaiaiing dyes 5' Panama/’3' idC Psmaien, idC
intercala‘ring dyes 5' Eihidium bmmific
inflamed:1:ng (lyes 5' Sybez‘ stains
‘ mods 5' Chfl- ’3' MC Chit-fiestero}
{Ecume- mutation Long, {:Iigo (:3 GO heme-s) w/ Unhnfiwn
2 mutation
’ mods ’JR ' SEMA HEX/B‘iciC SEMI-‘5 HEX, idC
Backbene modifications KC? Methyl phosphonates
Backbone: modificatiom ENA
Backbone. modifications MUE (methgxyethyl)
PCT/USZOl4/029566
{Riga type b’iedificafiens
Cy3 replacements I Cy'fi 5
{13,13 repiacements CyS
Backbone modifications di PS
' mods riboC for branch my:
Backbone cations PNA
€313 mpiacements ‘i‘l’Y64?
' mods 5' branch symmetric branch
amidite/iL‘iC
The ing modifications may also include known nucleotide modifications
such as methylation, 5’ intercalating dyes, modifications to the 5’ and 3’ ends, ne
modifiications, crosslinkers, cyclization and 'caps‘ and substitution of one or more of the
lly occurring nucleotides with an analog such as inosine. Modifications of
nucleotides include the addition of acridine, amine, biotin, cascade blue, cholesterol,
Cy3@, Cy5 @, Cy5.5@ Daboyl, digoxigenin, dinitrophenyl, Edans, 6—FAM, scein,
3'— glyceryl, HEX, 0, IRD—SOO, JOE, phosphate psoralen, rhodamine, ROX, thiol
(SH), spacers, TAMRA, TET, AMCA—S", SE, BODIPY°, Marina Blue@, Pacific Blue@,
Oregon Green@, Rhodamine Green@, Rhodamine Red@, Rhodol Green@ and Texas
Red@. Polynucleotide backbone modifications e methylphosphonate, 2‘-OMe-
methylphosphonate RNA, orothiorate, RNA, RNA. Base modifications
include 2-amino-dA, Z—aminopun'ne, 3'- (ddA), 3‘dA (cordycepin), 7—deaza—dA, 8-Br—dA,
8— oxo—dA, N6-Me—dA, abasic site (dSpacer), biotin dT, 2‘—OMe-5Me—C, 2'-OMe—
propynyl-C, 3'- (5—Me—dC), 3'- (ddC), 5—Br—dC, c, C, S—F-dC, carboxy—dT,
convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, 7—deaza~
dG, 8—Br—dG, 8— oxo-dG, O6~Me~dG, S6-DNP—dG, 4-methyl~indole, 5-nitroindole, 2'—
OMe—inosine, 2‘—dl, 06— phenyl-dl, 4—methy1—indole, 2'—deoxynebularine, S—nitroindole, 2-
aminopurine, dP (purine ue), dK (pyrimidine analogue), 3—nitropyrrole, 2-thio—dT,
4-thio-dT, biotin-dT, carboxy~dT, 04—Me—dT, 04~triazol dT, 2'—OMe—propynyl—U, 5—Br—
dU, 2‘-dU, 5—F—dU, 5-l-dU, 04-tn'azol dU. Said terms also encompass peptide nucleic
acids (PNAS), a DNA analogue in which the backbone is a pseudopeptide consisting of
N— (2—aminoethyl)—glycine units rather than a sugar. PNAS mimic the behavior of DNA
PCT/U82014/029566
and bind complementary nucleic acid strands. The neutral backbone of PNA results in
stronger binding and greater specificity than normally achieved. In addition, the unique
chemical, al and biological ties of PNA have been exploited to produce
powerful biomolecular tools, nse and antigene agents, molecular probes and
biosensors.
Oligonucleobases may have nick(s), gap(s), modified nucleotides such as
modified oligonucleotide backbones, abasic nucleotides, or other chemical moieties. In a
further embodiment, at least one strand of the oligonucleobase includes at least one
additional modified nucleotide, e.g., a 2’—O-methyl modified nucleotide such as a MOE
(methoxyethyl), a nucleotide having a 5’-phosphorothioate group, a terminal nucleotide
linked to a cholesteryl tive, a 2’—deoxy~2’—fluoro modified nucleotide, a 2’-de0xy—
modified nucleotide, a locked nucleotide, an abasic tide (the nucleobase is missing
or has a hydroxyl group in place thereof (see, e. g., Glen Research,
http://www.glenresearch.com/GlenReports/GRZl —14.html)), a 2’—amino-modified
nucleotide, a 2’—alkyl-modified nucleotide, a lino nucleotide, a phosphoramidite,
and a non-natural base comprising nucleotide. Various salts, mixed salts and free acid
forms are also included.
Preferred modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphoro—dithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl onates including 3’-alkylene
phosphonates, ylene phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3’-amino phosphoramidate and
aminoalkylphosphoramidates, phosphoramidates, thionoalkyl—phosphonates,
thionoalkylphosphotriesters, selenophosphates and phosphates having normal 3’-5’
linkages, 2’—5’ linked analogs of these, and those having inverted polarity wherein one or
more internucleotide linkages is a 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage. red
oligonucleotides having ed polarity comprise a single 3' to 3’ linkage at the 3’—most
intemucleotide linkage Le. a single inverted nucleoside residue which may be abasic (the
nucleobase is missing or has a hydroxyl group in place thereof). The most common use
of a linkage inversion is to add a 3‘-3‘ linkage to the end of an antisense oligonucleotide
with a phosphorothioate backbone. The 3'—3' e further stabilizes the antisense
oligonucleotide to exonuclease degradation by creating an oligonucleotide with two 5'—
OH ends and no 3‘—OH end. Linkage inversions can be introduced into specific locations
WO 44951 PCT/U82014/029566
during oligonucleotide synthesis through use of "reversed phosphoramidites". These
reagents have the phosphoramidite groups on the 5‘—OH position and the dimethoxytrityl
(DMT) protecting group on the 3‘—OH position. Normally, the DMT protecting group is
on the 5'—OH and the phosphoramidite is on the 3'~OH.
Examples of modified bases include, but are not limited to, 2—an1inopurine, 2’-
amino—butyryl pyrene—uridine, nouridine, 2’—deoxyuridine, 2’—fluoro~cytidine, 2’—
fluoro-uridine, 2,6—diaminopurine, —uridine, o-uridine, 5-fluoro—cytidine, 5—
ridine, 5—indo—uridine, 5—methy1—cytidine, inosine, N3—methyl-uridine, 7~deaza—
guanine, 8-aminohexyl-amino—adenine, 6-thi0-guanine, 4—thio-thymine, 2~thio—thymine,
—i0do—uridine, -cytidine, 8—bromo—guanine, o—adenine, 7—deaza-adenine, 7-
diaza—guanine, 8—oxo-guanine, 5,6~dihydro—uridine, and 5—hydroxymethyl—u1idine. These
synthetic units are commercially available; (for example, purchased from Glen Research
Company) and can be incorporated into DNA by chemical synthesis.
Examples of modification of the sugar moiety are xylation, 2’—
fluorination, and arabanosidation, however, it is not to be construed as being d
o. Incorporation of these into DNA is also possible by chemical synthesis.
Examples of the 5’ end modification are 5’—amination, 5’-biotinylation, 5’—
fluoresceinylation, 5'—tetrafluoro-fluoreceinyaltion, 5’—thionation, and 5’~dabsylation,
however it is not to be construed as being limited thereto.
Examples of the 3’ end modification are 3’-amination, 3’—biotinylation, 2,3-
dideoxidation, 3’-thionation, 3’—dabsylation, 3’—carboxylation, and 3’—cholesterylation,
however, it is not to be construed as being d thereto.
In one preferred embodiment, the oligonucleobase can contain a 5' blocking
substituent that is attached to the 5 ' terminal carbons through a linker. The chemistry of
the linker is not critical other than its length, which should preferably be at least 6 atoms
long and that the linker should be flexible. A variety of non—toxic substituents such as
biotin, terol or other steroids or a non-intercalating cationic fluorescent dye can be
used. Particularly preferred reagents to make oligonucleobases are the reagents sold as
Cy3TM and Cy5TM by Glen Research, Sterling Va. (now GE Healthcare), which are
d phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3',3'—
ethyl N,N'—isopropyl substituted nocarbocyanine and indodicarbocyanine
dyes, respectively. Cy3 is particularly preferred. When the indocarbocyanine is N—
PCT/U82014/029566
oxyalkyl substituted it can be conveniently linked to the 5' terminal of the
eoxynucleotide as a phosphodiester with a 5' terminal phosphate. When the
cially available Cy3 phosphoramidite is used as directed, the resulting 5‘
modification consists of a blocking substituent and linker together which are a N—
ypropyl, N'—phosphatidylpropy1 ,3'—tetramethy1indomonocarbocyanine.
Other dyes contemplated include Rhodamine6G, Tetramethylrhodamine, Sulforhodamine
101, Merocyanine 540, Att0565, Att0550 26, Cy3.5, Dy547, Dy548, Dy549, Dy554,
Dy555, Dy556, Dy560, mStrawberry and mCherry.
In a preferred embodiment the indocarbocyanine dye is tetra substituted at the
3 and 3' positions of the indole rings. Without limitations as to theory these substitutions
prevent the dye from being an alating dye. The identity of the substituents at these
positions is not critical.
The oligo designs herein described might also be used as more efficient donor
templates in combination with other DNA editing or recombination logies
ing, but not limited to, gene targeting using pecific homologous recombination
by zinc finger nucleases, Transcription Activator~Like Effector Nucleases (TALENs) or
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRS).
The present invention generally relates to methods for the efficient
modification of genomic cellular DNA and/or recombination of DNA into the genomic
DNA of cells. Although not limited to any ular use, the methods of the present
invention are useful in, for example, introducing a modification into the genome of a cell
for the purpose of determining the effect of the modification on the cell. For example, a
modification may be introduced into the nucleotide sequence which encodes an enzyme
to ine r the cation alters the enzymatic activity of the enzyme, and/or
determine the location of the enzyme's tic region. Alternatively, the cation
may be introduced into the coding sequence of a DNA-binding protein to determine
whether the DNA binding activity of the protein is altered, and thus to delineate the
particular DNA-binding region within the protein. Yet another alternative is to introduce
a modification into a ding regulatory sequence (e.g., promoter, enhancer,
regulatory RNA sequence (miRNA), etc.) in order to determine the effect of the
modification on the level of expression of a second sequence which is operably linked to
the non~coding regulatory sequence. This may be desirable to, for example, define the
particular sequence which possesses regulatory activity.
WO 44951 PCT/U82014/029566
One gy for producing targeted gene disruption is through the generation
of single strand or double strand DNA breaks caused by site—specific endonucleases.
Endonucleases are most often used for targeted gene disruption in organisms that have
traditionally been refractive to more conventional gene targeting methods, such as algae,
plants, and large animal models, including . For example, there are currently
human clinical trials ay involving zinc finger ses for the treatment and
tion of HIV infection. Additionally, endonuclease engineering is currently being
used in attempts to disrupt genes that produce undesirable phenotypes in crops.
The homing endonucleases, also known as meganucleases, are sequence
specific endonucleases that generate double strand breaks in genomic DNA with a high
degree of icity due to their large (e.g., >14 bp) ge sites. While the specificity
of the homing endonucleases for their target sites allows for precise targeting of the
induced DNA breaks, homing endonuclease cleavage sites are rare and the probability of
finding a naturally occuiring Cleavage site in a targeted gene is low.
One class of artificial endonucleases is the zinc finger endonucleases. Zinc
finger endonucleases combine a non—specific cleavage domain, typically that of Fold
endonuclease, with zinc finger protein domains that are engineered to bind to specific
DNA sequences. The r structure of the zinc finger endonucleases makes them a
versatile platform for delivering site—specific double~strand breaks to the genome. One
limitation of the zinc finger endonucleases is that low specificity for a target site or the
presence of multiple target sites in a genome can result in off-target cleavage events. As
FokI endonuclease cleaves as a dimer, one gy to prevent rget cleavage events
has been to design zinc finger domains that bind at adjacent 9 base pair sites.
TALENs are targetable nucleases are used to induce single- and double—strand
breaks into ic DNA sites, which are then repaired by mechanisms that can be
exploited to create sequence alterations at the cleavage site.
] The fundamental building block that is used to er the DNA—binding
region of TALENs is a highly conserved repeat domain derived from naturally occurring
TALES encoded by Xanthomonas spp. proteobacteria. DNA binding by a TALEN is
mediated by arrays of highly conserved 33—35 amino acid repeats that are flanked by
additional TALE—derived domains at the amino-terminal and carboxy-terminal ends of the
repeats.
WO 44951 PCT/U82014/029566
These TALE repeats specifically bind to a single base of DNA, the identity of
which is determined by two hypervariable residues typically found at positions 12 and 13
of the , with the number of repeats in an array corresponded to the length of the
desired target nucleic acid, the identity of the repeat selected to match the target nucleic
acid sequence. The target nucleic acid is preferably between 15 and 20 base pairs in order
to maximize selectivity of the target site. Cleavage of the target nucleic acid typically
occurs within 50 base pairs of TALEN binding. Computer programs for TALEN
recognition site design have been described in the art. See, e.g., Cermak et al., Nucleic
Acids Res. 2011 July; : e82.
()nce designed to match the desired target sequence, TALENS can be
expressed recombinantly and introduced into protoplasts as exogenous proteins, or
expressed from a plasmid within the protoplast.
Another class of artificial endonucleases is the engineered meganucleases.
Engineered homing endonucleases are generated by modifying the specificity of existing
homing endonucleases. In one approach, variations are introduced in the amino acid
ce of naturally occurring homing endonucleases and then the resultant engineered
homing endonucleases are screened to select onal ns which cleave a targeted
binding site. In another approach, chimeric homing endonucleases are engineered by
combining the recognition sites of two ent homing cleases to create a new
recognition site composed of a half site of each homing endonuclease.
Other DNA—modifying molecules may be used in targeted gene
recombination. For example, peptide c acids may be used to induce modifications
to the genome of the target cell or cells (see, e.g., US. Pat. No. 5,986,053, to Ecker,
herein orated by reference). In brief, synthetic tides comprising, at least, a
partial peptide backbone are used to target a homologous genomic nucleotide ce.
Upon binding to the double-helical DNA, or through a mutagen ligated to the peptide
nucleic acid, modification of the target DNA sequence and/or recombination is d to
take place. Targeting specificity is determined by the degree of sequence homology
between the targeting sequence and the genomic sequence.
] Furthermore, the present invention is not limited to the particular methods
which are used herein to execute modification of genomic sequences. Indeed, a number
of methods are contemplated. For example, genes may be targeted using triple helix
PCT/U82014/029566
forming oligonucleotides (TFO). TFOs may be generated synthetically, for example, by
PCR or by use of a gene synthesizer apparatus. onally, TFOs may be isolated from
genomic DNA if suitable natural sequences are found. TFOs may be used in a number of
ways, including, for example, by tethering to a mutagen such as, but not limited to,
en or chlorambucil (see, e. g., Havre et al., Proc Nat’l Acad Sci, USA. 90:7879—
7883, 1993; Havre et al., J Virol 67:7323—7331, 1993; Wang et al., Mol Cell Biol
:1759-1768, 1995; Takasugi et al., Proc Nat’l Acad Sci, USA. 88:5602—5606, 1991;
Belousov et al., Nucleic Acids Res 25:3440—3444, 1997). Furthermore, for example,
TFOs may be tethered to donor duplex DNA (see, e.g., Chan et al., J Biol Chem
272:11541—11548, 1999). TFOS can also act by binding with sufficient affinity to
provoke error—prone repair (Wang et al., Science 271 05, 1996).
The invention‘s methods are not limited to the nature or type of DNA~
modifying reagent which is used. For example, such DNA-modifying reagents release
radicals which result in DNA strand breakage. Alternatively, the reagents alkylate DNA
to form adducts which would block replication and transcription. In another alternative,
the reagents generate crosslinks or molecules that inhibit cellular enzymes leading to
strand . Examples of DNA—modifying reagents which have been linked to
oligonucleotides to form TFOS include, but are not limited to, carbazoles,
napthalene diimide (NDI), transplatin, bleomycin, analogues of cyclopropapyrroloindole,
and phenanthodihydrodioxins. In particular, indolocarbazoles are topoisomerase I
inhibitors. tion of these enzymes results in strand breaks and DNA n adduct
formation [Arimondo et al., Bioorganic and Medicinal Chem. 8, 777, 2000]. NDI is a
photooxidant that can oxidize guanines which could cause mutations at sites of guanine
residues , et al., Biochemistry, 39, 6190, 2000]. Transplatin has been shown to
react with DNA in a triplex target when the TFO is linked to the t. This reaction
causes the formation of DNA adducts which would be mutagenic [Columbier, et al.,
Nucleic Acids Research, 24: 4519, 1996]. Bleomycin is a DNA breaker, widely used as a
radiation c. It has been linked to oligonucleotides and shown to be active as a
breaker in that format [Sergeyev, c Acids Research 23, 4400, 1995; Kane, et al.,
mistry, 34, 16715, 1995]. Analogues of cyclopropapyrroloindole have been linked
to TFOs and shown to alkylate DNA in a triplex target sequence. The alkylated DNA
would then contain al adducts which would be mutagenic [Lukhtanov, et al.,
Nucleic Acids Research, 25, 5077, 1997]. Phenanthodihydrodioxins are masked quinones
that release radical species upon photoactivation. They have been linked to TFOs and
have been shown to introduce breaks into duplex DNA on photoactivation [Bendinskas et
al., jugate Chem. 9, 555, 1998].
Other methods of inducing modifications and/or recombination are
contemplated by the present invention. For example, another embodiment involves the
ion of homologous recombination between an exogenous DNA fragment and the
targeted gene (see e. g., hi et al., Science 244:1288~1292, 1989) or by using peptide
nucleic acids (PNA) with affinity for the targeted site. Still other methods e
Dervan et al.,
sequence specific DNA recognition and targeting by polyamides (see e. g.,
Curr Opin Chem Biol 3:688—693, 1999; mistry 38:2143~2151, 1999) and the use
nucleases with site specific activity (e. g., zinc finger proteins, TALENs, Meganucleases
and/0r CRISPRS).
The present invention is not limited to any particular frequency of
modification and/or recombination. The invention’s methods result in a frequency of
modification in the target nucleotide sequence of from 0.2% to 3%. Nonetheless, any
frequency (i.e., between 0% and 100%) of modification and/or recombination is
plated to be within the scope of the present invention. The frequency of
modification and/or recombination is dependent on the method used to induce the
modification and/0r recombination, the cell type used, the specific gene ed and the
DNA mutating reagent used, if any. Additionally, the method used to detect the
modification and/or recombination, due to limitations in the detection method, may not
detect all occurrences of modification and/or recombination. Furthermore, some
modification and/or recombination events may be silent, giving no detectable indication
that the modification and/or recombination has taken place. The inability to detect silent
modification and/or recombination events gives an artificially low estimate of
cation and/or recombination. Because of these reasons, and , the invention is
not limited to any particular modification and/or recombination ncy. In one
ment, the frequency of modification and/or recombination is between 0.01% and
100%. In r embodiment, the frequency of modification and/or recombination is
between 0.01 % and 50%. In yet another embodiment, the ncy of modification
and/0r recombination is between 0.1% and 10%. In still yet another embodiment, the
frequency of modification and/or recombination is between 0.1% and 5%.
PCT/U82014/029566
The term “frequency of mutation” as used herein in reference to a population
of cells which are treated with a DNA—modifying molecule that is e of introducing
a mutation into a target site in the cells‘ genome, refers to the number of cells in the
treated population which contain the mutation at the target site as compared to the total
number of cells which are treated with the difying molecule. For example, with
respect to a population of cells which is treated with the difying molecule TFO
tethered to psoralen which is designed to introduce a mutation at a target site in the cells'
genome, a frequency of mutation of 5% means that of a total of 100 cells which are
treated with TFO-psoralen, 5 cells contain a mutation at the target site.
Although the present invention is not limited to any degree of precision in the
modification and/or recombination of DNA in the cell, it is contemplated that some
embodiments of the present invention require higher degrees of precision, depending on
the desired result. For example, the specific sequence s required for gene repair
(e. g., particular base changes) require a higher degree of precision as compared to
producing a gene ut wherein only the disruption of the gene is necessary. With the
methods of the t invention, achievement of higher levels of precision in
modification and/or homologous recombination techniques is greater than with prior art
methods.
Delivery of Gene Repair Oligonucleobases into Plant Cells
Any commonly known method used to transform a plant cell can be used for
delivering the gene repair oligonucleobases. rative methods are listed below. The
present invention plates many methods to ect the cells with the DNA—
modifying reagent or reagents. Indeed, the present invention is not limited to any
particular . Methods for the introduction of DNA modifying reagents into a cell
or cells are well known in the art and e, but are not limited to, microinjection,
electroporation, passive adsorption, calcium phosphate—DNA co—precipitation, DEAE—
dextran—mediated transfection, polybrene—mediated transfection, liposome ,
lipofectin, nucleofection, protoplast fusion, retroviral infection, biolistics (i.e., particle
bombardment) and the like.
The use of metallic microcarriers (microspheres) for introducing large
fragments of DNA into plant cells having cellulose cell walls by projectile penetration is
well known to those d in the nt art (henceforth biolistic delivery). US. Pat.
WO 44951 2014/029566
Nos. 4,945,050; 5,100,792 and 5,204,253 describe l techniques for selecting
microcarriers and devices for projecting them.
Specific conditions for using microcaniers in the s of the present
invention are described in International Publication WO 99/07865. In an illustrative
technique, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60 mg/mL)
2.5 M CaClz and 0.1 M dine are added in that order; the mixture gently agitated,
e.g., by vortexing, for 10 s and then left at room ature for 10 minutes,
whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and
resuspended in 100% ethanol. Good results can be obtained with a concentration in the
adhering solution of 8-10 ng/uL microcarriers, 14-17 ug/mL mixed duplex
oligonucleotide, 1.1—1.4 M CaClz and 18—22 mM spermidine. Optimal results were
observed under the conditions of 8 ug/uL microcarriers, 16.5 ug/mL mixed duplex
oligonucleotide, 1.3 M CaClz and 21 mM spermidine.
Gene repair ucleobases can also be introduced into plant cells for the
practice of the present invention using microfibers to penetrate the cell wall and cell
membrane. US. Pat. No. 5,302,523 to Coffee et a1 describes the use of silicon carbide
fibers to facilitate transformation of sion maize cultures of Black Mexican Sweet.
Any mechanical que that can be used to uce DNA for transformation of a
plant cell using microfibers can be used to deliver gene repair oligonucleobases for
utation.
An illustrative technique for microfiber delivery of a gene repair
oligonucleobase is as follows: Sterile microfibers (2 pg) are suspended in 150 nL of plant
culture medium containing about 10 ug of a mixed duplex oligonucleotide. A suspension
culture is allowed to settle and equal volumes of packed cells and the sterile
fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are
applied immediately or with a delay of up to about 120 h as is appropriate for the
particular trait.
In an alternative embodiment, the gene repair oligonucleobases can be
delivered to the plant cell by electroporation of a protoplast derived from a plant part.
The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf,
according to techniques well known to those skilled in the art. See, e.g., Gallois et a1,
1996, in Methods in lar Biology 55:89—107, Humana Press, Totowa, N.J.; Kipp et
W0 2014/144951 2014/029566
al., 1999, in s in Molecular Biology 133:213—221, Humana Press, Totowa, NJ 4
The protoplasts need not be cultured in growth media prior to electroporation. Illustrative
conditions for electroporation are 3. timele. sup.5 protoplasts in a total volume of 0.3
mL with a concentration of gene repair oligonucleobase of between 0.6—4 ng/mL.
In an alternative embodiment, nucleic acids are taken up by plant protoplasts
in the presence of the membrane-modifying agent polyethylene glycol, according to
ques well known to those skilled in the art. In another alternative embodiment, the
gene repair oligonucleobases can be delivered by injecting it with a microcapillary into
plant cells or into protoplasts.
In an alternative embodiment, nucleic acids are embedded in microbeads
composed of calcium alginate and taken up by plant protoplasts in the presence of the
membrane-modifying agent hylene glycol (see, e.g., Sone et al., 2002, Liu et al.,
2004).
In an alternative ment, nucleic acids forzen in water and introduced
into plant cells by bombardment in the form of microparticles (see, e.g., Gilmore, 1991,
US. Patent 5,219,746; Brinegar et al.).
] In an alternative embodiment, nucleic acids attached to nanoparticles are
introduced into intact plant cells by incubation of the cells in a suspension containing the
nanoparticlethe (see, e.g., Pasupathy et al., 2008) or by delivering them into intact cells
h particle bomardment or into protoplasts by co—incubation (see, e.g., Torney et al.,
2007).
In an ative embodiment, nucleic acids complexed with penetrating
peptides and delivered into cells by co—incubation (see, e.g., Chugh et al., 2008, WO
2008148223 A1; Eudes and Chugh.
In an alternative embodiment, nucleic acids are uced into intact cells
through electroporation (see, e. g., He et al., 1998, US 2003/0115641 A1, Dobres et al.).
] In an alternative embodiment, nucleic acids are red into cells of dry
embryos by soaking them in a solution with nucleic acids (by soaking dry embryos in
(see, e.g., Topfer et al., 1989, Senaratna etal., 1991 ).
Selection of Plants
PCT/U82014/029566
In various embodiments, plants as disclosed herein can be of any species of
dicotyledonous, monocotyledonous or gymnospermous plant, including any woody plant
species that grows as a tree or shrub, any herbaceous species, or any species that produces
edible fruits, seeds or vegetables, or any species that produces colorful or aromatic
. For e, the plant maybe selected from a species of plant from the group
consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, ,
rice, alfafa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana,
melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, ea, field
field
pea, faba bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale,
beans, poplar, pine, eucalyptus, grape, citrus, tn'ticale, alfalfa, rye, oats, turf and forage
grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant,
marigold, lotus, e, daisy, carnation, tulip, iris, lily, and nut producing plants insofar
as they are not already specifically mentioned.
Plants and plant cells can be tested for resistance or nce to an herbicide
using ly known s in the art, e.g., by growing the plant or plant cell in the
ce of an herbicide and measuring the rate of growth as compared to the growth rate
in the absence of the herbicide.
As used herein, substantially normal growth of a plant, plant organ, plant
tissue or plant cell is defined as a growth rate or rate of cell on of the plant, plant
organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least
75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant
tissue or plant cell expressing the wild—type AHAS protein.
As used herein, substantially normal development of a plant, plant organ, plant
tissue or plant cell is defined as the occurrence of one or more development events in the
plant, plant organ, plant tissue or plant cell that are substantially the same as those
ing in a corresponding plant, plant organ, plant tissue or plant cell expressing the
wild—type protein.
In certain embodiments plant organs provided herein include, but are not
limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos,
cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores,
pollen, pollen tubes, ovules, s and fruits, or sections, slices or discs taken
therefrom. Plant tissues e, but are not limited to, callus tiSSues, ground tissues,
PCT/U52014/029566
vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root
tissues, gall tissues, plant tumor tissues, and reproductive tissues. Plant cells include, but
are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and
lasts.
Plants are substantially "tolerant" to a relevant herbicide when they are
subjected to it and provide a dose/response curve which is shifted to the right when
compared with that provided by rly ted non-tolerant like plant. Such
dose/response curves have ”dose" plotted on the X—axis and ”percentage kill”, "herbicidal
effect", etc., plotted on the y—axis. Tolerant plants will require more ide than non-
tolerant like plants in order to produce a given herbicidal effect. Plants that are
substantially "resistant" to the ide exhibit few, if any, necrotic, lytic, chlorotic or
other lesions, when subjected to herbicide at concentrations and rates which are typically
employed by the agrochemical community to kill weeds in the field. Plants which are
resistant to an herbicide are also tolerant of the herbicide.
Generation of plants
Tissue culture of various tissues of plant species and regeneration of plants
therefrom is known. For example, the propagation of a canola cultivar by tissue culture is
described in any of the following but not limited to any of the following: Chuong et al.,
"A Simple Culture Method for ca tyls Protoplasts," Plant Cell Reports 424—
6, 1985; Barsby, T. L., et al., "A Rapid and Efficient ative Procedure for the
Regeneration of Plants from Hypocotyl Protoplasts of Brassica " Plant Cell
Reports (Spring, 1996); Kartha, K., et al., "In vitro Plant Formation from Stem Explants
of Rape,” Physiol. Plant, ~220, 1974; Narasimhulu, S., et al., ”Species Specific
Shoot Regeneration Response of Cotyledonary ts of Brassicas," Plant Cell Reports
(Spring 1988); n, E., "Microspore Culture in Brassica," Methods in Molecular
Biology, Vol. 6, Chapter 17, p. 159, 1990.
Further reproduction of the variety can occur by tissue culture and
regeneration. Tissue culture of various tissues of soybeans and regeneration of plants
therefrom is well known and widely published. For example, nce may be had to
Komatsuda, T. et al., ”Genotype X Sucrose Interactions for Somatic Embryogenesis in
Soybeans," Crop Sci. 3l1333—337, 1991; Stephens, P. A., et al., "Agronomic Evaluation
of Tissue-Culture~Derived n Plants,” Theor. Appl. Genet. 82:633—635, 1991;
PCT/USZOI4/029566
Komatsuda, T. et a1., "Maturation and Germination of Somatic Embryos as Affected by
Sucrose and Plant Growth tors in ns Glycine gracilis Skvortz and e
max (L) Merr." Plant Cell, Tissue and Organ e, 28:103-113, 1992; Dhir, S. et al.,
”Regeneration of Fertile Plants from Protoplasts of Soybean (Glycine max L. ;
Genotypic Differences in Culture Response,” Plant Cell Reports 11:285-289, 1992;
Pandey, P. et al., "Plant Regeneration from Leaf and Hypocotyl Explants of Glycine
wightii (W. and A.) VERDC. var. longicauda," Japan J. Breed. 4221-5, 1992; and Shetty,
K., et al., "Stimulation of In, Vitro Shoot Organogenesis in Glycine max (Merrill) by
Allantoin and Amides," Plant Science 81:245-251, 1992. The disclosures of US Pat.
No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and US. Pat. No. 5,008,200 issued
Apr. 16, 1991 to Ranch et al., are hereby incorporated herein in their entirety by
reference.
EXAMPLES
Example 1: GRON length
Sommer et al., (M01 Biotechnol. 33:115—22, 2006) describes a reporter system
for the detection of in viva gene conversion which relies upon a single nucleotide change
to convert n blue and green fluorescence in green cent protein (GFP)
ts. This reporter system was adapted for use in the following ments using
Arabidopsis thaliana as a model species in order to assess efficiency of GRON
conversion following cation of the GRON length.
In short, for this and the subsequent examples an Arabidopsis line with
multiple copies of a blue fluorescent protein gene was created by methods known to those
skilled in the art (see, e. g., Clough and Brent, 1998). Root-derived meristematic tissue
cultures were established with this line, which was used for protoplast isolation and
culture (see, e.g., Mathur et al., 1995). GRON delivery into protoplasts was ed
through polyethylene glycol (PEG) mediated GRON uptake into protoplasts. A method
using a 96-well format, similar to that described by similar to that described by Fujiwara
and Kate (2007) was used. In the following the protocol is briefly described. The
volumes given are those applied to dual wells of a 96—well dish.
W0 2014/144951
1. Mix 6.25 pl of GRON (80 pM) with 25 pl ofArabidopsis BFP transgenic
root meristematic tissue—derived protoplasts at 5x106 cells/ml in each well of a 96 well
plate.
2. 31.25 pl of a 40% PEG solution was added and the protoplasts were mixed.
3. Treated cells were incubated on ice for 30 min.
4. To each well 200 pl of W5 solution was added and the cells mixed.
5. The plates were allowed to incubate on ice for 30 min allowing the
protoplasts to settle to the bottom of each well.
6. 200 pl of the medim above the settled lasts was removed.
7. 85 pl of culture medium (MSAP, see Mathur et al., 1995) was added.
8. The plates were incubated at room temperate in the dark for 48 hours. The
final concentration of GRON after adding culture medium is 8 pM.
Forty eight hours after GRON delivery samples were analyzed by flow
cytometry in order to detect protoplasts whose green and yellow fluorescence is different
from that of control protoplasts (BFPO tes rgeting GRONS with no change
compared to the EFF target; C is the coding strand design and NC is the non—coding
strand design). A single C to T nucleotide difference (coding strand) or G to A nucleotide
targeted on (non-coding strand) in the center of the BFP4 les. The green
fluorescence is caused by the introduction of a targeted mutation in the EFF gene,
resulting in the synthesis of GFP. The results are shown in Figure 1.
The ing table shows the sequence of exemplary 10l—mer and 201—mer
BFP4/NC 5’—3PS/ 3’—3PS GRONS designed for the conversion of a blue fluorescent
protein (BFP) gene to green fluorescence. (3P8 indicates 3 othioate linkages at
each of the 5’ and 3’ oligo ends).
PCT/U82014/029566
[11112131 Tame. 1:
CG C
TAG GTG AAG GTG WC. ACG AGG GTG GGC CAG (IKA. ACG GGC AGC‘. TTG CCG
,2/(/nnnnnnn/zn”
”4”,,”0,» ”EGG GTG AAG OTC: GTC‘. AC‘GAGT: G’Cz‘C: “SGC CAG GGC .ACG CvGC‘. AGCI'1"1'13 CCG /
. 5:inAT GA[\ (ii/A G1Ct\1-j '1J*IA
If«,,.,,.,I/,»-»,” \“Axsxxxsxx \s\\\~\\\\\\\\\\\rltlr((rrlrtrr
IGTGC.
”Mu/”flflauu’, \\\\‘\s\\\\K\\\\\\\\\‘\\\\\k\\\/a\ururr
Cl GAAGAAC‘fl‘CG’TG
ACGAGCATGUCKC'AGGGLIACGKISCAiCTTL:CC(J(_11C3GTG(AGATGAACTTCAGULvTC'A
Cm1-N“CM““w““mmmm‘u“
1'30’Ni" 20‘ ~m‘ct l,»~;-»~:»,~ GAAGA A GTKIGTGC
A(‘:{"1"'1Gf"‘C:GTC:Cz'1GC'AGATG-AAC‘TTCAGGG'1C.AG" "1'GAAGG’1'GG’1'Z‘A
pp .4111wa Iri’rttzt’rttrtrrtrrllrlrrrrt(rtritrittir
u»»»:»»~»»4/:uu”:»»””» ,- .GCII'GACC{ITGAAGT CATCTGC/CCACCG
GCT-AAGC‘. CCCC‘:1GCC‘C".‘:’1( (:1I’CAC‘C‘C1CC:'1GACC‘ACC‘TIC‘ACCTACGCmCrlGCAC-I "
'V11C‘..IAC‘:(CGC'1ACC"(‘.C:AC.C'AC‘A'1GAACcC‘AGC‘ACGIAC1'1‘(‘.'1’1CAAG'1C‘C‘GC'CA1GCC.CGA
‘\(1{1( ‘ACG'1'CCAGGA GCGCACCAF *
:TGCCCTC; 31111" ACCCTiGTGACCACCTTCACCC‘11.1.1131.(J1Ci(.z‘\(ITC3C..
ACCC‘.JGAAGF‘T‘AKJ(: ..A _,'-‘1\ CG
C‘ZGCC
"§TC.AGCCGC’E'ACCCCGACC‘ACA"E:GAAGCAGC‘ACGACTTC1TCAAGTCCGCCATGCCCGA
AGGC1.ACGTCCIAGGA GC"CAC(IA’1‘*C‘* *T lrrlt7irritrlrrrl((lrrl(rrrrtlr(t
\“\“\“~\C““CCCC“C~C““““““Cfls“~““““~\~“~“~“““s“~“»“\““‘\WM~WM“~“~»“~\~“-»“
: PS 11111§8g€ (phosphothioate)
Exam 119 '3:i¢ Cs'awea‘ésian gates. 12: S’CVE/ 3’111C iabeied GRONS
1116 purpose of this series emf experiments 13 to e. the. encies of
phoaphothioate (PS) Jahaled GRONS g 3 PS moimies at each end of the GRON) to
111(35 ’Cy3l' 3 id( labeled (IRONS The. 5’(y1! 3’ir1C1-absiad GRONS have: a 5’ Cy3
flum‘ophore (amidiie) and a 3’ MC reverse. base: Efficiancy lag assessed using
Ciit-an'IIS1iiill 01' blue fluorescam pmtein (BF?) is green fluorescence
In 9:11 three. expariments, done either by PEG delivery 01' GRONS into
protoplasts in individual Falcon tubes (labeled ”Tubes") or in 96-well plates (1abeled “96
W811 dish”)> there \, 'as no signifiam. {1111L’10‘GLC bi-‘tw{n the different GRON 111011113111»
in B??? to GP? conversion-f1"cie:ncy as. datennined by cytimwiry (171g. 1) .
Exampie 3: {30111133115911between the flame? BFPAI‘NC S’-3PS/ 3’~3P$ GRGN and
()kazaki Fragment GRGNS
16} The pui‘poge of (his series 01' ments is to compare the sion
efficiencies of [he phosphofhioata (PS) labeled GRONS with 3P8 moietics at each and of
U: \G
SUBSTITUTE SHEET (RULE 26)
PCT/USZOI4/029566
the GRON to “Okazaki fragment GRONs” in the presence and absence of a member of
the bleomycin family, ZeocinTM (1 mg/ml) to induce DNA breaks. The design of these
GRONs are depicted in Fig. 2. GRONS were delivered into Ambidopsis BFP protoplasts
by PEG treatment and BFP to GFP conversion was determined at 24 h post treatment by
cytometry. Samples treated with zeocin (1 mg/ml) were incubated with zeocin for 90 min
on ice prior to PEG treatment.
In general the presence of zeocin (1 mg/ml) increased BFP to GFP conversion
as determined by cytometry (Table 2). In both the presence and absence of zeocin, the
NC Okazaki GRON containing one 2’—O Me group on the first RNA base at the 5’ end of
the GRON was more cious at ting BFP to GFP when compared to the NC
Okazaki GRON containing one 2’-O Me group on each of the first nine 5’ RNA bases
(Fig. 2 and Table 2).
In all experiments, there was no icant difference between the 4l-n1er
BFP4/NC 5’3PS/ 3’3PS and the 71—mer Okazaki Fragment BFP4/NC GRON that
contains one 5’ 2’-O me group on the first 5’ RNA base (denoted as BFP4 71-mer (1)
NC) in BFP to GFP conversion in both the ce or absence of 1 mg/ml of zeocin as
determined by cytometry (Fig. 2 and Table 2). It is ant to note that in the presence
of zeocin (and expected for bleomycin, phleomycin, tallysomycin, pepleomycin and other
members of this family of otics) that conversion s strand independent (i.e.,
both C and NC GRONs with the designs tested in these experiments display
approximately equal activity).
PCT/U82014/029566
{@219} Table. 2: Comparison of a rd GRON design with ()kazaki fragment
GRON designs. in the presence and absence of a giycopeptide antibiotic zeooin.
i Zeocin (+)
0001414 0.001061
0.001 ‘1:;» 0.00075
Exampie 4: Comparison betwgen iize 4141mm Tim-mar and 261-er BFPo‘i/NC S’-
35381 3’-SPS GRONS
The purpose. of this series of experiments. was to compare the conversion
efficissncies (in the. presence and absence of zeacin) of the at‘hioaie (PS) labeled
GRONS with 3P8 es at each and m" the GRON of different Iengihs: iii finer, 101-
mer and ZOE-mar Shawn in Table 1. Again, the pi'eSmice of zeocm {1 mg/ml) increased
BFP to G??? cmwersimx rates as determined by symmetry (Tame 5) The avaiiaii trend in
ail three expemnani‘s was linear with increasing NC GRON iength in both the. presence.
and absence of 2600111. Except fo: the. BFP-ii/N If101 and BFP-4/C/101 in the. presence
of 1600131, this had conversion rams that were (rinse. to cquai but iuwer than the 41mm: NC
GRQN. This is in contrast to all us experiments in which the EFF—414] coding and
SUBSTITUTE SHEET (RULE 26)
PCT/U52014/029566
nml-«ccydmg GRONS were usede wherein the nonucoding was always far superior its the
coding GRON. This asymn'letry in conversion frequency also applias 10 {he 201
GRONS uaed in this experimentai .
[002,211] Ta bk: 3:
Zeocin (+)
Exp.
Name
ham-35% 0.3225
API‘G66 0.713 0992
Mean 0.26825 0.4802857
d ‘ 01289(3-79
00795
.1 “'1‘- C.0"."8842-1L O.{‘13‘563 0.004390 711E
0.0015033 E
6.0557584 0.0110017 0.0032505 0OQSOO0.8
{0022?} Example 5: CRISPRS combined with GRONS to improve conversion in
plants.
[002231 Three design components must he considered when assembling a (TIRESPR
camplex: (32139, gRNA (guide. RNA) and the target region (proto~s_pacer in endogenous
target gene).
SUBSTITUTE SHEET (RULE 26)
Gas 9
— Transient expression of Cas9 gene from ococcus pyogenes codon optimized
for Arabidopsis or corn driven by 358 or corn ubiquidn respectively. Optimized
genes synthesized by Genewiz or DNA 2.0. NB must ensure no cryptic introns
are created.
- RBCSE9 terminator as per G1 155
- Single SV4O NLS (PKKRKV) as a C—terminal fusion
— The vector backbone would be as per ail our transient expression systems -
G1 155.
gRNA
— Propose to use a chimeric trachNA — pre—creRNA as per Le Cong et a1., 2013 and
Jinek et al., 2013. Note that LeCong et al. showed that the native full length tracr +
pre-chNA complex d much more ntly than the chimeric version. An
option therefore would be to make a chimera using the full length (89bp) trachNA. -
Sequence of gRNA ( (N)20 represents guide sequence). The bracketed sequence
comprises the full length 89bp form.
NNNNNNNNNNNNNNNGT'VI‘TAGAGCTI‘A(MA«K'I‘AGUAAEH’TAAAATA
AGCEC’I‘A(‘i'l‘('(3t’}ff’i‘:’\‘FGTT'(3’l"E’(iA1\AAA[\{E’l‘GAt’i’EY'}GRMTGAG'I’CGG’ETJG'I‘G
r1‘11'ii}
Figure 3 reproduced from Cong et al., shows the native complex and the
chimera.
[Text continued on page 64]
PCT/U82014/029566
— The gRN'A would he expressed under the AtUé RNA pol HI er in Arabia’opsis
{sequence given below). in com the 2mm RNA pol HI promoter could be used.
These choices are based on Wang; et at. 2008"
— RBCSEQ terminatm‘ as per @1155 or a String of T’s as per Wang ex (2]. 2.013. and the
one~eomponent approach shown below.
At U6 promoter sequence from Wang e: an?
{$62237} ’l’argetreglsm
— The guide ce specificity is d by the target region sequence. lniespeetive
of the choice. of model organism this will be the YGSH locus of BFP. A PAM (NGG)
sequence in the Vicinity of Y66H is the only design t‘esli‘ictlen. A150, including the
Y6<3H position in the '3’ 11;pr of the guide sequence (“seed ce") would mean.
that once repair has been achieved the site will. not get IS~C~11§.
Te gt}; ace ace tie ace cac ggc
VTTFTY
61 6'2 63 64 65 66 67
{$0228} A ct vector backbone item (5.1155 will be needed in order to enable co~
delivery of Can“) and gRNA. This problem will be circumvented with the. one—component
approach:
{@0229} One component approach
Le Cong e! a}. (2013) used a simplified appreach, expressing both the gRNA
and the C389 as a single transient construct, driven by the pol Ill U6 er, as outlined
below. In this. way, for a given crop, multiple. genes could be targeted by Simply
swapping in the guide insert sequence. We would replace the EFlcx promoter for one
suitable for the crop (pMAS for At, Ubi fer Zm). For the terminatnr we would use.
RBCSHQ. ’llhe $11.3 used in plants. would be a single C—temlinai SV40 as outlined above.
SUBSTITUTE SHEET (RULE 26)
Note that in the construct below a truncated gRNA is used where the tracer
RNA region is not included. The authors showed that in humans that this was less
effective at guiding the Cas9 that the full length version. It is therefore proposed that
the full length gRNA to be used here. y in a subsequent paper using CRISPRs
in yeast, DiCarlo et al. (2013) used the full length n. The cassette would be
cloned into a G1 155 background.
Figur 4 shows a schematic of the expression vector for chimeric chNA. The
guide sequence can be inserted between two BbsI sites using annealed
oligonucleotides. The vector already ns the partial direct repeat (gray) and
partial trachNA (red) sequences. WPRE, Woodchuck tis virus post
transcriptional regulatory element.
In Vivo assay
Transient option
— One approach to confirm target recognition and nuclease activity in planta would be
to emulate the YFP single stranded annealing assay which Zhang et al. (2013) used
for TALENS. The spacer sequence (target sequence) plus PAM would need to be
inserted into the YFP or equivalent gene.
- Transient option
— The TALEN - BFP system could be used as a control.
- Whilst the above approach would be an on-going tool for ing functionality
of a given CRISPR system for a given spacer sequence, proof of concept of the
activity of CRISPRS in plants would be to use the GFP system.
[Text continued on page 66]
PCT/U82014/029566
— Here the s used for FP could be co—transformed into At together with
G1155 and no GRON. If cutting were efficient enough, a reduction in GFP
expression could be apparent. This would likely require zation of plasmid
loading.
— Once activity is confirmed a genomic BFP target would be targeted with a Visual and
ce-based read-out.
In Vitro assay
— In order to rapidly confirm activity of a CRISPR system, an in vitro assay could be
used as per Jinek et a] 2012. Here a pre—made and purified S.pyogenes Cas9 is
incubated with synthesized gRNA and a plasmid ning the recognition ce.
Successful cleavage is analysed by gel electrophoresis to look for out plasmids.
Detailed protocol:
Plasmid DNA cleavage assay. Synthetic or in vino-transcribed trachNA and
chNA were pre—annealed prior to the on by heating to 95°C and slowly cooling
down to room temperature. Native or restriction digest—linearized plasmid DNA (300 ng
(~8 nM)) was incubated for 60 min at 37°C with purified Cas9 protein (50~500 nM) and
trachNAzchNA duplex (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM
HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM
MgC12. The reactions were stopped with 5X DNA loading buffer containing 250 mM
EDTA, resolved by 0.8 or 1% agarose gel electrophoresis and visualized by ethidium
bromide staining. For the Cas9 mutant cleavage assays, the reactions were stopped with
5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior to loading on
the agarose gel.
Trait targets in Crops
Given the flexibility of the CRISPR recognition sequence it is not difficult to
find potential pacer sequences as defined by a 3’ NGG PAM sequence.
ZmEPSPS
The e below shows a suitable protospacer sequence (yellow) and PAM
(blue) in order to create a DS break in the catalytic site of ZmEPSPS where mutations at
PCT/USZOl4/029566
the T97 and P101 are known to cause glyphosate tolerance. Subsequent oligo-mediated
repair (ODM) of the break would result in the desired changes.
T AM R P L T V A A V
act gca atg cgg cca ttg
] The table below gives the protospacer sequences of genes of interest in crops
of interest:
Eastssssrtastggtgsa..............
ggctgcagttactgctgct ,
.......................................................................................... Eggstgsaatta959593.....__.__.-_.-._
3ccastggagttagamete..-.........._.._E5
,.steststaasaesegft___-.__..-.....§'
sagttgstgtaest............ .i
ssagsagttasaatasst.,............ :
gtgcgcctcgctttgtcttgt
Eattttacaggtgtttacgcc
A limitation of the design constraints is that it is often hard to find a NGG
sequence within 12 bp of the nucleotide being altered by ODM. This is significant
because if this was the case, successful ODM would mean that subsequent cutting would
not be possible because the protospacer seed sequence would be altered. Jinek et a1.
(2012) showed this was detrimental to cutting efficiency.
References
LeCong et al 2013 e : vol. 339 no. 6121 pp. 819—823.
Jinek et a] 2012 Science. 337:816-21
Wang et a] 2008 RNA 14: 903—913
Zhang et al2013. Plant l. 161: 20—27
] One skilled in the art y iates that the present invention is well
adapted to carry out the objects and obtain the ends and advantages mentioned, as well as
those inherent therein. The examples provided herein are representative of preferred
embodiments, are exemplary, and are not intended as limitations on the scope of the
invention.
PCT/U82014/029566
It will be readily apparent to a person skilled in the art that varying
substitutions and modifications may be made to the invention disclosed herein without
departing from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative of
the levels of those of ry skill in the art to which the ion pertains. All patents
and publications are herein incorporated by reference to the same extent as if each
individual publication was ically and dually indicated to be incorporated by
reference.
The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not specifically
disclosed herein. Thus, for example, in each ce herein any of the terms
“comprising”, “consisting essentially of” and “consisting of” may be replaced with either
of the other two terms. The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no intention that in the use of such
terms and sions of excluding any equivalents of the features shown and described
or ns thereof, but it is recognized that s modifications are possible within the
scope of the invention claimed Thus, it should be understood that although the present
invention has been specifically disclosed by preferred embodiments and al features,
modification and variation of the concepts herein disclosed may be resorted to by those
skilled in the art, and that such modifications and variations are considered to be within
the scope of this invention as defined by the appended claims.
Other embodiments are set forth within the ing claims.
Claims (13)
1. A method for introducing a gene repair oligonucleobase (GRON)—mediated mutation into a target ibonucleic acid (DNA) sequence in the genome of a plant cell, comprising: delivering a GRON into the plant cell configured to mediate introduction of one or more targeted genetic changes within the DNA sequence in the plant cell genome, wherein the GRON ses a 3’ modifying substituent, a 5’ modifying substituent, or both a 3’ modifying substituent and a 5’ modifying substituent, and wherein the GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to attract the cell's gene repair system to the site where the mismatched base-pair(s) is created, and is degraded after ated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the ediated mutation into the target DNA sequence and the plant cell is non-transgenic following the introduction; culturing the plant cell under conditions that se one or more cellular DNA repair processes prior to or coincident with delivery of the GRON into the plant cell, wherein the conditions that increase one or more cellular DNA repair processes comprise introducing one or more site—specific ses which induce single stranded nicks or double stranded DNA strand breaks into the plant cell genome, wherein the one or more site—specific nucleases which induce single strand nicks or double DNA strand breaks are selected from the group consisting of: zinc finger endonucleases, CRISPR nucleases and meganucleases, and wherein the site—specific nuclease is designed to match the target DNA sequence; and selecting a plant cell comprising the one or more ed genetic changes within the DNA ce in the plant cell genome.
2. The method of claim 1, wherein the 3’ modifying substituent is present and is selected from the group consisting of a fluorescent dye, a reverse base, a 3’— dimethoxytriyl nucleotide, one or more phosphorothioate or phosphoamidate internucleotide linkages, one or more 2’—O—(2-methoxyethyl) nucleotides, and one or more 2’-O-methyl nucleotides.
3. The method of claim 1, wherein the 5’ modifying substituent is present and is ed from the group consisting of a fluorescent dye, one or more phosphorothioate or phosphoamidate internucleotide linkages, one or more 2’—O~(2—methoxyethyl) nucleotides, and one or more 2’—O—methyl nucleotides.
4. The method of claim 1, wherein the 3 ’ modifying substituent is present and is selected from the group consisting of a fluorescent dye, a reverse base, a 3’- dimethoxytrityl nucleotide, one or more phosphorothioate or phosphoamidate internucleotide linkages, one or more 2—methyoxyethyl) nucleotides, and one or more 2’—O—methyl nucleotides; and the 5’ modifying substituent is present and is ed from the group consisting of a fluorescent dye, one or more orothioate or phosphoamidate internucleotide linkages, one or more 2’—O—(2—methoxyethyl) nucleotides, and one or more 2’—O—methyl nucleotides.
5. The method of claim 4, wherein the 3’ modifying substituent ses one or more 2’-O-methyl nucleotides and the 5’ modifying substituent comprises one or more 2’—O—methyl nucleotides.
6. The method of claim 4, wherein the 3’ modifying substituent comprises a reverse base and the 5’ modifying substituent comprises a fluorescent dye.
7. The method of claim 4, wherein the 3 ’ modifying substituent comprises a reverse base and the 5’ modifying substituent comprises one or more 2’—O—(2- methoxyethyl) nucleotides.
8. The method of claim 4, n the 3 ’ modifying substituent comprises a reverse base and the 5’ modifying substituent comprises one or more 2’-O~methyl nucleotides.
9. The method of claim 4, wherein the 3 ’ modifying substituent ses one or more 2’~O—(2—methoxyethyl) tides and the 5’ modifying substituent comprises one or more 2’~O—(2—methoxyethyl) nucleotides.
10. The method of claim 1, wherein the DNA sequence is an endogenous gene in the plant cell genome.
11. The method of claim 1, wherein the plant cell is a cell from a plant selected from the group consisting of , sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, , sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, faba bean, lentils, turnip, rutabaga, l s, lupin, cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, mustard, cucumber, morning glory, , pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, and lily.
12. The method of claim 1, further comprising regenerating a plant having the mutation introduced by the GRON from the selected plant cell.
13. The method of claim 12, further comprising collecting seeds from the plant.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201361801333P | 2013-03-15 | 2013-03-15 | |
US61/801,333 | 2013-03-15 | ||
NZ711145A NZ711145B2 (en) | 2013-03-15 | 2014-03-14 | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
Publications (2)
Publication Number | Publication Date |
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NZ751577A NZ751577A (en) | 2021-08-27 |
NZ751577B2 true NZ751577B2 (en) | 2021-11-30 |
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