NZ765592B2 - Methods and compositions for targeted genetic modifications and methods of use - Google Patents
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- NZ765592B2 NZ765592B2 NZ765592A NZ76559215A NZ765592B2 NZ 765592 B2 NZ765592 B2 NZ 765592B2 NZ 765592 A NZ765592 A NZ 765592A NZ 76559215 A NZ76559215 A NZ 76559215A NZ 765592 B2 NZ765592 B2 NZ 765592B2
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Abstract
Methods and compositions are provided for generating targeted genetic modifications on the Y chromosome or a challenging target locus. Compositions include an in vitro culture comprising an XY pluripotent and/or totipotent animal cell (i.e., XY ES cells or XY iPS cells) having a modification that decreases the level and/or activity of an Sry protein; and, culturing these cells in a medium that promotes development of XY F0 fertile females. Such compositions find use in various methods for making a fertile female XY non-human mammal in an F0 generation. ecreases the level and/or activity of an Sry protein; and, culturing these cells in a medium that promotes development of XY F0 fertile females. Such compositions find use in various methods for making a fertile female XY non-human mammal in an F0 generation.
Description
METHODS AND COMPOSITIONS FOR TARGETED
GENETIC MODIFICATIONS AND METHODS OF USE
This application is a divisional application from New Zealand Patent Application Number
728561, the entire disclosures of which are incorporated herein by reference.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
62/017,582, filed June 26, 2014, and of U.S. Provisional Application No. 62/017,627, filed
June 26, 2014, each of which is hereby incorporated herein in its entirety by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED
AS A TEXT FILE VIA EFS WEB
The official copy of the sequence listing is submitted electronically via EFS-Web
as an ASCII formatted sequence listing with a file named 463545SEQLIST.TXT, created on
June 25, 2015, and having a size of 14 kilobytes, and is filed concurrently with the
specification. The ce listing ned in this ASCII formatted document is part of the
specification and is herein incorporated by reference in its entirety.
FIELD
The invention relates to the methods and itions for maintaining or
ing pluripotent and/or totipotent cells and s and compositions for generating cell
populations and transgenic animals.
BACKGROUND
s due to unique structural es of the Y chromosome, conventional gene
targeting strategies in mouse embryonic stem cells to generate mutations on the Y-linked
genes have had d success. Therefore, often the understanding of the functions of
murine Y-linked genes is limited to insights gained from studies of mice that carry
spontaneous deletions, random gene traps insertions or autosomal transgenes. Methods are
needed to improve the y to target a c locus on the Y chromosome.
The Sry protein (sex-determining region Y) is the key regulator of male sex
determination in placental mammals. The Sry gene, also known as the Testis ining
Factor (TDF), resides on the Y chromosome. Sry is thought to be a transcription factor that
binds DNA through its High Mobility Group (HMG) domain. Expression of the mouse Sry
gene is restricted to the l ridge in a narrow time window around day 11 of embryonic
development; both Sry mRNA and protein are detected. Sufficient Sry must be made within
this time window to convert the bipotential l ridge toward the male testis forming
program while inhibiting the female program of ovary development. In adult testes a ar
Sry transcript is ed but not the Sry protein. Mutations in the Sry gene that cause the
production of an inactive Sry protein or that alter the timing and strength of gene expression
can cause male to female sex reversal, resulting in animals that have an X and a Y
chromosome but are ically female. So-called XY females are often sterile or have a
low fertility. Being able to control sex ination by regulation of the Sry would have
great value in the production of genetically modified animals.
SUMMARY
A method for making an XY embryonic stem (ES) cell line capable of producing a
fertile XY female non-human mammal in an F0 generation is provided. The method
comprises: (a) modifying a non-human mammalian XY embryonic stem (ES) cell to have a
modification that decreases the level and/or activity of an Sry protein; and, (b) culturing the
ed ES cell line under conditions that allow for making an ES cell line e of
producing a fertile XY female non-human mammal in an F0 generation.
A method for making a fertile XY female non-human mammal in an F0
generation is also provided. The method comprises: (a) introducing the non-human
mammalian XY ES cell made by the above method having a modification that decreases the
level and/or activity of an Sry protein into a host embryo; (b) gestating the host embryo; and,
(c) obtaining an F0 XY female non-human , wherein upon attaining sexual maturity
the F0 XY female non-human mammal is fertile. In one embodiment, the female XY F0 non-
human mammal is fertile when crossed to a wild type mouse. In specific embodiments, the
wild type mouse is C57BL/6.
In one embodiment, the non-human mammalian XY ES cell is from a rodent. In a
specific embodiment, the rodent is a mouse. In one embodiment, the mouse XY ES cell is
derived from a 129 strain. In one ment, the mouse XY ES cell is a VGFl mouse ES
cell. In one embodiment, the mouse XY ES cell ses a Y chromosome derived from the
129 strain. In one embodiment, the mouse XY ES cell is from a C57BL/6 strain. In another
embodiment the rodent is a rat or a hamster.
In some embodiments, the decreased level and/or activity of the Sry protein
results from a genetic modification in the Sry gene. In some such methods, the genetic
modification in the Sry gene comprises an insertion of one or more nucleotides, a deletion of
one or more nucleotides, a substitution of one or more nucleotides, a knockout, a knockin, a
replacement of an endogenous nucleic acid sequence with a homologous, heterologous, or
orthologous nucleic acid sequence, or a combination thereof.
In the methods provided herein, the targeted c modification can comprises
an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof. In
another embodiment, the targeted c modification is on an autosome.
In some embodiments, the modification of the Sry gene comprises an insertion of
a selectable marker and/or a reporter gene operably linked to a er active in the non-
human mammalian ES cell. In some embodiments, the the cation of the Sry gene
comprises an insertion of a reporter gene operably linked to the endogenous Sry promoter. In
a specific embodiment, the reporter gene encodes the reporter protein LacZ.
In one embodiment, the culturing step comprises culturing the non-human
mammalian XY ES cell in a medium sing a base medium and supplements suitable for
maintaining the non-human mammalian ES cell in culture, n the medium is a low-
osmolality medium. In one embodiment, the molality medium exhibits an osmolality
from about 200 mOsm/kg to less than about 329 g. In other embodiments the low-
osmolality medium exhibits one or more of the following characteristic: a conductivity of
about 11 mS/cm to about 13 mS/cm; a salt of an alkaline metal and a halide in a
tration of about 50 mM to about 110 mM; a carbonic acid salt concentration of about
l7mM to about 30 mM; a total ne metal halide salt and carbonic acid salt concentration
of about 85mM to about 130 mM; and/or a combination of any two or more thereof.
In some ments, upon introduction of the non-human mammalian XY ES
cells into a host embryo and following gestation of the host embryo, at least 80% at least
85%, at least 90%, or at least 95% of the F0 non-human mammals are XY females which
upon ing sexual maturity the F0 XY female non-human mammal is fertile.
In one embodiment, the non-human mammalian XY ES cell comprises a target
genomic locus on the Y chromosome comprising a recognition site for a nuclease agent, and
wherein the nuclease agent induces a nick or double-strand break at the recognition site. Such
a method can r comprise exposing the ES cell to the nuclease agent in the presence of a
targeting vector comprising an insert polynucleotide, wherein ing exposure to the
se agent and the targeting vector, the ES cell is modified to contain the insert
polynucleotide. In one embodiment, the nuclease agent is an mRNA encoding a nuclease. In
specific embodiments, the nuclease agent is (a) a zinc finger nuclease (ZEN); (b) is a
Transcription Activator-Like Effector Nuclease (TALEN); or (C) a clease. In other
embodiments, the nuclease agent comprises a Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA). In such
s, the guide RNA (gRNA) comprises (a) a Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) RNA (chNA) that targets the first recognition site; and (b) a
trans-activating CRISPR RNA (trachNA). In some cases, the recognition site is immediately
flanked by a Protospacer nt Motif (PAM) sequence. In one embodiment, the Cas
protein is Cas9.
Also provided is an in vitro culture comprising the non-human mammalian XY ES
cell line according to any of the methods provided herein.
An in vitro culture is provided and ses (a) a non-human mammalian XY
embryonic stem (ES) cell having a modification that decreases the level and/or activity of an
Sry n; and, (b) a medium comprising a base medium and supplements suitable for
maintaining the man mammalian ES cell in culture. In one ment, the base
medium exhibits an osmolality from about 200 mOsm/kg to less than about 329 mOsm/kg. In
other embodiments, the base medium exhibits one or more of the following characteristic: a
conductivity of about 11 mS/cm to about 13 mS/cm; a salt of an alkaline metal and a halide in
a concentration of about 50 mM to about 110 mM; a carbonic acid salt concentration of about
l7mM to about 30 mM; a total ne metal halide salt and carbonic acid salt tration
of about 85mM to about 130 mM; and/or a combination of any two or more thereof. In one
embodiment, the non-human mammalian XY ES cell is from a rodent. In one embodiment,
the rodent is a mouse or a rat. In one embodiment, the mouse XY ES cell is a VGFl mouse
ES cell. In one embodiment, the rodent is a rat or a hamster. In one embodiment, the
decreased level and/or activity of the Sry n is from a c modification in the Sry
gene. In one embodiment, the genetic modification in the Sry gene ses an insertion of
one or more tides, a deletion of one or more nucleotides, a substitution of one or more
nucleotides, a knockout, a n, a replacement of an endogenous nucleic acid sequence
with a heterologous nucleic acid sequence or a ation thereof. In one embodiment, the
non-human mammalian ES cell comprises one, two, three or more targeted genetic
modifications. In one embodiment, the targeted genetic modification comprises an insertion,
a deletion, a knockout, a knockin, a point mutation, or a combination thereof. In one
embodiment, the targeted genetic modification comprises at least one insertion of a
heterologous polynucleotide into the genome of the XY ES cell. In one embodiment, the
targeted genetic modification is on an autosome. In one embodiment, the base medium
exhibits 50 i 5 mM NaCl, 26 i 5 mM carbonate, and 218 i 22 mOsm/kg. In one
embodiment, the base medium exhibits about 3 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, and 218 g. In one embodiment, the base medium exhibits 87 i 5 mM
NaCl, 18 i 5 mM carbonate, and 261 i 26 mOsm/kg. In one embodiment, the base medium
exhibits about 5.1 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 261 g. In one
embodiment, the base medium exhibits 110 i 5 mM NaCl, 18 i 5 mM carbonate, and 294 i
29 mOsm/kg. In one embodiment, the base medium exhibits about 6.4 mg/mL NaCl, 1.5
mg/mL sodium bicarbonate, and 294 mOsm/kg. In one embodiment, the base medium
exhibits 87 i 5 mM NaCl, 26 i 5 mM carbonate, and 270 i 27 mOsm/kg. In one
embodiment, the base medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, and 270 mOsm/kg. In one embodiment, the base medium exhibits 87 i 5 mM
NaCl, 26 i 5 mM carbonate, 86 i 5 mM glucose, and 322 i 32 mOsm/kg. In one
embodiment, the base medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, 15.5 mg/mL glucose, and 322 mOsm/kg. In one embodiment, upon introduction
of the non-human mammalian XY ES cells into a host embryo and ing gestation of the
host embryo, at least 80% of the F0 non-human mammals are XY females which upon
attaining sexual maturity the F0 XY female man mammal is fertile.
Further provided is a method for making a fertile female XY non-human mammal
in an F0 generation, sing: (a) culturing a donor non-human mammalian XY
nic stem (ES) cell having a modification that decreases the level and/or activity of an
Sry protein in a medium comprising a base medium and supplements suitable for maintaining
the non-human ian ES cell in culture, (b) introducing the donor XY non-human
mammalian ES cell into a host embryo; (c) gestating the host embryo; and, (d) ing an
F0 XY female non-human mammal, wherein upon attaining sexual maturity the F0 XY
female non-human mammal is fertile. In one ment, the medium ts an osmolality
from about 200 mOsm/kg to less than about 329 mOsm/kg. In other embodiments, the
medium exhibits a characteristic comprising one or more of the following: a conductivity of
about 11 mS/cm to about 13 mS/cm; a salt of an alkaline metal and a halide in a
concentration of about 50mM to about 110 mM; a carbonic acid salt concentration of about
17 mM to about 30 mM; a total alkaline metal halide salt and ic acid salt concentration
of about 85 mM to about 130 mM; and/or a combination of any two or more thereof; In one
embodiment, the non-human mammalian XY ES cell is from a . In one embodiment,
the rodent is a mouse or a rat. In one embodiment, the mouse XY ES cell is a VGFl mouse
ES cell. In one embodiment, the rodent is a rat or a hamster. In one embodiment, the
WO 00805
decreased level and/or activity of the Sry protein is from a genetic modification in the Sry
gene. In one ment, the genetic modification in the Sry gene comprises an insertion of
one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more
nucleotides, a knockout, a knockin, a replacement of an endogenous c acid sequence
with a heterologous nucleic acid sequence or a combination thereof. In one embodiment, the
non-human mammalian ES cell comprises one, two, three or more targeted genetic
modifications. In one embodiment, the targeted genetic modification comprises an insertion,
a deletion, a knockout, a knockin, a point mutation, or a combination thereof. In one
embodiment, the targeted c cation comprises at least one insertion of a
logous polynucleotide into a genome of the XY ES cell. In one embodiment, the
ed genetic modification is on an autosome. In one embodiment, the base medium
exhibits 50 i 5 mM NaCl, 26 i 5 mM carbonate, and 218 i 22 mOsm/kg. In one
embodiment, the base medium exhibits about 3 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, and 218 mOsm/kg. In one embodiment, the base medium exhibits 87 i 5 mM
NaCl, 18 i 5 mM carbonate, and 261 i 26 mOsm/kg. In one embodiment, the base medium
exhibits about 5.1 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 261 mOsm/kg. In one
embodiment, the base medium exhibits 110 i 5 mM NaCl, 18 i 5 mM carbonate, and 294 i
29 mOsm/kg. In one embodiment, the base medium exhibits about 6.4 mg/mL NaCl, 1.5
mg/mL sodium onate, and 294 mOsm/kg. In one embodiment, the base medium
exhibits 87 i 5 mM NaCl, 26 i 5 mM carbonate, and 270 i 27 mOsm/kg. In one
ment, the base medium ts about 5.1 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, and 270 mOsm/kg. In one embodiment, n the base medium exhibits 87 i
mM NaCl, 26 i 5 mM carbonate, 86 i 5 mM glucose, and 322 i 32 g. In one
embodiment, wherein the base medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium
bicarbonate, 15.5 mg/mL glucose, and 322 mOsm/kg.
Further provided are methods of producing a transgenic non-human mammal
homozygous for a targeted genetic mutation in the F1 generation comprising: (a) crossing an
F0 XY fertile female having a decreased level and/or activity of the Sry protein with a cohort
clonal g, derived from the same ES cell clone, F0 XY male non-human mammal,
wherein the F0 XY fertile female non-human mammal and the F0 XY male non-human
mammal each is heterozygous for the genetic mutation; ) obtaining an F1 progeny
mouse that is homozygous for the genetic modification.
A method for modifying a target genomic locus on the Y chromosome in a cell is
also provided and comprises (a) providing a cell comprising a target genomic locus on the Y
some comprising a recognition site for a nuclease agent, (b) introducing into the cell
(i) the nuclease agent, wherein the nuclease agent induces a nick or double-strand break at
the first recognition site; and, (ii) a first targeting vector comprising a first insert
polynucleotide flanked by a first and a second homology arm ponding to a first and a
second target site located in sufficient proximity to the first recognition site; and, (c)
identifying at least one cell comprising in its genome the first insert polynucleotide integrated
at the target genomic locus. In one embodiment, a sum total of the first homology arm and
the second gy arm is at least 4kb but less than 150kb. In one embodiment, the length
of the first homology arm and/or the second homology arm is at least 400 bp but less than
1000 bp. In r embodiment, the length of the first homology arm and/or the second
homology arm is from about 700 bp to about 800 bp.
Further provided is a method for modifying a target genomic locus on the Y
some in a cell is provided and ses: (a) providing a cell comprising a target
genomic locus on the Y chromosome comprising a recognition site for a nuclease agent, (b)
introducing into the cell a first targeting vector comprising a first insert polynucleotide
flanked by a first and a second homology arm corresponding to a first and a second target
site; and, (c) identifying at least one cell comprising in its genome the first insert
polynucleotide integrated at the target genomic locus. In one embodiment, the length of the
first homology arm and/or the second homology arm is at least 400 bp but less than 1000 bp.
In another embodiment, the length of the first homology arm and/or the second homology
arm is from about 700 bp to about 800 bp. In one embodiment, the cell is a mammalian cell.
In one embodiment, the mammalian cell is a non-human cell. In one embodiment, the
mammalian cell is from a . In one embodiment, the rodent is a rat, a mouse or a
hamster. In one embodiment, the cell is a otent cell. In one embodiment, the
mammalian cell is an induced pluripotent stem (iPS) cell. In one embodiment, the pluripotent
cell is a non-human embryonic stem (ES) cell. In one embodiment, the otent cell is a
rodent embryonic stem (ES) cell, a mouse embryonic stem (ES) cell or a rat embryonic stem
(ES) cell. In one embodiment, the nuclease agent is an mRNA encoding a nuclease. In one
embodiment, the nuclease agent is a zinc finger se (ZFN). In one embodiment, the
nuclease agent is a Transcription Activator-Like Effector Nuclease (TALEN). In one
ment, the se agent is a meganuclease. In some embodiments, the nuclease
agent comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-
associated (Cas) protein and a guide RNA . In such a method the guide RNA (gRNA)
can comprise (a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
RNA (chNA) that targets the first recognition site; and (b) a activating CRISPR RNA
(trachNA). In one embodiment, the first or the second recognition sites are immediately
flanked by a Protospacer Adjacent Motif (PAM) sequence. In some embodiments, the Cas
protein is Cas9.
In some embodiments, the modification comprises a deletion of an endogenous
nucleic acid sequence. In some embodiments, the deletion ranges from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to
about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about
100 kb to about 150 kb, or from about 150 kb to about 200 kb, from about 200 kb to about
300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about
500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,
from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb. In a specific
embodiment, the deletion is at least 500 kb. In one embodiment, the cell is a ian cell.
In one embodiment, the mammalian cell is a man cell. In one embodiment, the
mammalian cell is from a rodent. In one embodiment, the rodent is a rat, a mouse or a
hamster. In one embodiment, the cell is a pluripotent cell. In one embodiment, the
mammalian cell is an induced pluripotent stem (iPS) cell. In one embodiment, the pluripotent
cell is a non-human embryonic stem (ES) cell. In one embodiment, the pluripotent cell is a
rodent embryonic stem (ES) cell, a mouse embryonic stem (ES) cell or a rat embryonic stem
(ES) cell. In some embodiments, the se agent comprises a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA
(gRNA). In such a method the guide RNA (gRNA) can comprise (a) a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) RNA (chNA) that targets the first
ition site; and (b) a trans-activating CRISPR RNA (trachNA). In one embodiment,
the first or the second recognition sites are immediately flanked by a Protospacer Adjacent
Motif (PAM) sequence. In some embodiments, the Cas n is Cas9. In one embodiment,
the nuclease agent is a zinc finger nuclease (ZFN). In one embodiment, the nuclease agent is
a Transcription tor-Like Effector Nuclease (TALEN). In one embodiment, the nuclease
agent is a meganuclease.
Methods for modifying the Y chromosome comprising exposing the Y
chromosome to a Cas protein and a CRISPR RNA in the ce of a large ing vector
(LTVEC) comprising a nucleic acid sequence of at least 10 kb and comprises following
exposure to the Cas protein, the CRISPR RNA, and the LTVEC, the Y chromosome is
modified to contain at least 10 kb nucleic acid sequence. The LTVEC can comprise a nucleic
acid sequence of at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at
least 70 kb, at least 80 kb, or at least 90 kb. In other embodiments, the LTVEC comprises a
nucleic acid ce of at least 100 kb, at least 150 kb, or at least 200 kb.
Further provided is a method for modifying a target genomic locus on the Y
chromosome, comprising: (a) providing a mammalian cell comprising the target genomic
locus on the Y chromosome, wherein the target genomic locus comprises a guide RNA
(gRNA) target sequence; (b) introducing into the ian cell: (i) a large targeting vector
) comprising a first nucleic acid flanked with targeting arms gous to the
target genomic locus, wherein the LTVEC is at least 10 kb; (ii) a first expression construct
comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein,
and (iii) a second expression construct comprising a second promoter operably linked to a
third nucleic acid encoding a guide RNA (gRNA) comprising a nucleotide sequence that
izes to the gRNA target sequence and a trans-activating CRISPR RNA NA),
wherein the first and the second promoters are active in the mammalian cell; and (c)
identifying a modified mammalian cell comprising a targeted genetic modification at the
target genomic locus on the Y chromosome. In other ments, the LTVEC is at least 15
kb, at least 20 kb, at least 30kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at
least 80 kb, or at least 90 kb. In other embodiments, the LTVEC is at least 100 kb, at least
150 kb, or at least 200 kb. In one embodiment, the ian cell is a non-human
mammalian cell. In one embodiment, the mammalian cell is a fibroblast cell. In one
embodiment, the mammalian cell is from a rodent. In one embodiment, the rodent is a rat, a
mouse, or a hamster. In one ment, the mammalian cell is a pluripotent cell. In one
embodiment, the pluripotent cell is an induced pluripotent stem (iPS) cell. In one
embodiment, the otent cell is a mouse embryonic stem (ES) cell or a rat embryonic
stem (ES) cell. In one embodiment, the pluripotent cell is a developmentally restricted
human itor cell. In one embodiment, the Cas protein is a Cas9 protein. In one
embodiment, the gRNA target sequence is immediately flanked by a pacer Adjacent
Motif (PAM) sequence. In one embodiment, the sum total of 5’ and 3’ homology arms of the
LTVEC is from about 10 kb to about 150 kb. In one embodiment, the sum total of the 5’ and
the 3’ homology arms of the LTVEC is from about 10 kb to about 20 kb, from about 20 kb to
about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about
80 kb to about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb.
In one embodiment, the targeted genetic modification comprises: (a) a replacement of an
endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid
2015/038001
sequence; (b) a on of an endogenous nucleic acid sequence; (c) a deletion of an
endogenous nucleic acid sequence, wherein the deletion ranges from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to
about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about
100 kb to about 150 kb, or from about 150 kb to about 200 kb, from about 200 kb to about
300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about
500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,
from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (d) insertion of an
exogenous nucleic acid sequence; (e) insertion of an ous nucleic acid sequence
ranging from about 5kb to about 10kb, from about 10 kb to about 20 kb, from about 20 kb to
about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about
80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150 kb to about 200
kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300
kb to about 350 kb, or from about 350 kb to about 400 kb; (f) insertion of an exogenous
c acid sequence comprising a homologous or an orthologous nucleic acid sequence; (g)
insertion of a chimeric nucleic acid sequence comprising a human and a non-human nucleic
acid sequence; (h) insertion of a conditional allele flanked with site-specific recombinase
target sequences; (i) ion of a selectable marker or a reporter gene operably linked to a
third promoter active in the ian cell; or (1') a combination thereof. In one
embodiment, the target genomic locus comprises (i) a 5’ target sequence that is homologous
to a 5’ homology arm; and (ii) a 3’ target sequence that is homologous to a 3’ homology arm.
In one embodiment, the 5’ target sequence and the 3’ target sequence is separated by at least
kb but less than 3 Mb. In one embodiment, the 5’ target sequence and the 3’ target
sequence is separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at
least 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but less than
80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb, or at
least 150 kb but less than 200 kb, at least about 200 kb but less than about 300 kb, at least
about 300 kb but less than about 400 kb, at least about 400 kb but less than about 500 kb, at
least about 500 kb but less than about le, at least about 1 Mb but less than about 1.5 Mb, at
least about 1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than about 2.5 Mb,
or at least about 2.5 Mb but less than about 3 Mb. In one ment, the first and the
second expression constructs are on a single nucleic acid molecule. In one embodiment, the
target genomic locus comprises the Sry locus.
Further provided is a method for ed genetic modification on the Y
chromosome of a non-human animal, comprising: (a) ing a genomic locus of interest
on the Y chromosome of a man pluripotent cell ing to the methods described
herein, thereby producing a genetically modified non-human pluripotent cell sing a
targeted genetic modification on the Y chromosome; (b) introducing the modified non-
human pluripotent cell of (a) into a non-human host embryo; and gestating the non-human
host embryo comprising the ed pluripotent cell in a surrogate mother, wherein the
surrogate mother produces F0 progeny comprising the targeted genetic modification, wherein
the targeted genetic modification is capable of being transmitted through the germline. In
one embodiment, the genomic locus of interest comprises the Sry locus.
s and compositions are provided for generating targeted genetic
modifications on the Y chromosome. Compositions include an in vitro culture comprising an
XY pluripotent and/or tent animal cell (i.e., XY ES cells or XY iPS cells) having a
modification that decreases the level and/or activity of an Sry protein; and, ing these
cells in a medium that es development of XY F0 fertile females. Such compositions
find use in various methods for making a fertile female XY non-human mammals in an F0
generation.
BRIEF DESCRIPTION OF THE S
provides a schematic of the CRISPR Cas9/gRNA targeting the mouse Sry
gene. VG-l (SEQ ID NO:10); VG-2 (SEQ ID NO:1 l); VG-3 (SEQ ID NO:12). The primers
and probes indicated in are provided in SEQ ID NOS: 13-29.
provides a schematic of targeting the Sry gene with TALEN and CRISPR
using a lacZ reporter gene. The Sry gene was targeted with both a LTVEC and a short-armed
vector TVEC) having homology arms r than a LTVEC in order to avoid
challenging loci on the Y chromosome.
illustrates LacZ expression in s.
provides a schematic of a large deletion of greater than 500 kb on the Y
chromosome mediated by ZFNs or by CRISPR guide RNAs in combination with Cas9 DNA
endonuclease.
A, B, and C provides the sequencing confirmation of the large Y
chromosome deletion in various clones. A is the sequencing result for clone l-D5. The
Kdm5 Up and Uspy9 down sequence is provided in SEQ ID NO:30; l-D5 l500F (SEQ ID
NO:3l); l-D5 1000R (SEQ ID NO:32); is the sequencing result for clone 5-C4. The
2015/038001
Kdm5 Up and Uspy9 down sequence is provided in SEQ ID NO:33; l500F (SEQ ID NO:34);
1000R (SEQ ID NO:35); 1000F (SEQ ID N036); and is the sequencing result for
clone 6-Al2. The Kde Up and Uspy9 down sequence is provided in SEQ ID NO:37; l500F
(SEQ ID N038); 1000R (SEQ ID ; 1000F (SEQ ID NO:40); 1500R (SEQ ID
NO:4l). The boxed regions in and represent micro-homology regions.
DETAILED DESCRIPTION
DEFINITIONS
The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein,
include polymeric forms of amino acids of any length, including coded and non-coded amino
acids and chemically or biochemically modified or derivatized amino acids. The terms also
include rs that have been modified, such as ptides having modified peptide
backbones.
The terms “nucleic acid” and “polynucleotide,” used interchangeably herein,
include polymeric forms of nucleotides of any length, including ribonucleotides,
deoxyribonucleotides, or s or modified versions thereof. They e -, -
and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and
polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified,
biochemically modified, non-natural, or derivatized nucleotide bases. For simplicity, nucleic
acid size may be referred to in bp whether the nucleic acid is in double-or single-stranded
form, in the latter case, the bp being those formed if and when the single-stranded nucleic
acid is duplexed with its exactly complementary strand.
“Codon optimization” generally includes a process of ing a nucleic acid
sequence for ed expression in particular host cells by replacing at least one codon of
the native sequence with a codon that is more frequently or most frequently used in the genes
of the host cell while maintaining the native amino acid sequence. For example, a nucleic
acid encoding a Cas n can be modified to substitute codons having a higher frequency
of usage in a given yotic or eukaryotic cell, including a ial cell, a yeast cell, a
human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a
hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid
sequence. Codon usage tables are readily available, for example, at the “Codon Usage
Database.” These tables can be adapted in a number of ways. See Nakamura et a1. (2000)
Nucleic Acids Research 28:292. Computer algorithms for codon optimization of a particular
sequence for expression in a particular host are also available (see, e. g., Gene Forge).
“Operable linkage” or being “operably linked” includes osition of two or
more components (e.g., a promoter and another ce element) such that both components
on normally and allow the possibility that at least one of the components can mediate a
function that is exerted upon at least one of the other components. For example, a promoter
can be operably linked to a coding sequence if the promoter controls the level of transcription
of the coding sequence in response to the presence or absence of one or more transcriptional
regulatory factors.
“Complementarity” of nucleic acids means that a nucleotide sequence in one
strand of nucleic acid, due to orientation of its nucleobase groups, forms hydrogen bonds with
r sequence on an opposing nucleic acid strand. The complementary bases in DNA are
typically A with T and C with G. In RNA, they are typically C with G and U with A.
Complementarity can be perfect or substantial/sufficient. Perfect complementarity between
two nucleic acids means that the two nucleic acids can form a duplex in which every base in
the duplex is bonded to a complementary base by -Crick pairing. "Substantial" or
"sufficient" complementary means that a sequence in one strand is not completely and/or
perfectly complementary to a sequence in an opposing strand, but that sufficient bonding
occurs between bases on the two strands to form a stable hybrid complex in set of
hybridization ions (e.g., salt tration and temperature). Such conditions can be
predicted by using the sequences and standard mathematical calculations to predict the Tm of
hybridized strands, or by empirical determination of Tm by using routine methods. Tm
includes the temperature at which a population of hybridization xes formed n
two nucleic acid s are 50% denatured. At a temperature below the Tm, formation of a
hybridization complex is favored, whereas at a temperature above the Tm, melting or
tion of the strands in the hybridization complex is favored. Tm may be estimated for a
nucleic acid having a known G+C content in an aqueous l M NaCl on by using, e. g.,
Tm=81.5+0.4l(% G+C), although other known Tm computations take into account nucleic
acid ural characteristics.
dization condition" includes the cumulative environment in which one
nucleic acid strand bonds to a second nucleic acid strand by complementary strand
interactions and hydrogen bonding to produce a hybridization complex. Such conditions
include the chemical components and their concentrations (e. g., salts, chelating agents,
formamide) of an aqueous or organic solution containing the nucleic acids, and the
temperature of the mixture. Other factors, such as the length of tion time or reaction
chamber dimensions may contribute to the environment. See, e. g., Sambrook et al.,
Molecular Cloning, A Laboratory Manual, nd ed., pp. 1.90-1.91, .51, 1 1.47-
1157 (Cold Spring Harbor tory Press, Cold Spring Harbor, N.Y., 1989).
Hybridization requires that the two nucleic acids contain complementary
sequences, although mismatches between bases are possible. The conditions appropriate for
hybridization between two nucleic acids depend on the length of the nucleic acids and the
degree of complementation, variables well known in the art. The greater the degree of
complementation between two nucleotide sequences, the greater the value of the melting
ature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations
between nucleic acids with short stretches of complementarity (e.g. complementarity over 35
or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides) the
position of mismatches becomes important (see Sambrook et al., supra, 1.8).
Typically, the length for a izable nucleic acid is at least about 10 nucleotides.
Illustrative minimum lengths for a hybridizable nucleic acid e at least about 15
nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25
nucleotides, and at least about 30 nucleotides. Furthermore, the temperature and wash
solution salt concentration may be adjusted as necessary according to factors such as length
of the region of complementation and the degree of complementation.
The sequence of polynucleotide need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize
over one or more segments such that intervening or adjacent segments are not ed in the
hybridization event (e. g., a loop structure or hairpin structure). A polynucleotide (e. g.,
gRNA) can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or
100% sequence complementarity to a target region within the target nucleic acid sequence to
which they are targeted. For example, a gRNA in which 18 of 20 nucleotides are
complementary to a target region, and would therefore specifically hybridize, would
represent 90% complementarity. In this example, the remaining noncomplementary
tides may be clustered or interspersed with complementary nucleotides and need not be
contiguous to each other or to complementary nucleotides.
Percent complementarity n particular stretches of nucleic acid sequences
within nucleic acids can be determined routinely using BLAST programs (basic local
alignment search tools) and PowerBLAST programs known in the art (Altschul et a1. (1990)
J. M01. Biol. 3-410; Zhang and Madden (1997) Genome Res. 7:649-656) or by using
the Gap program nsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, Madison Wis.), using default settings, which
uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).
The methods and compositions provided herein employ a variety of different
components. It is recognized throughout the description that some components can have
active variants and fragments. Such components include, for example, Cas proteins, CRISPR
RNAs, trachNAs, and guide RNAs. Biological activity for each of these components is
described ere herein.
"Sequence identity" or "identity" in the context of two polynucleotides or
ptide sequences makes reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison window. When
percentage of sequence identity is used in reference to proteins it is recognized that residue
ons which are not identical often differ by conservative amino acid tutions, where
amino acid residues are substituted for other amino acid residues with similar al
properties (e. g., charge or hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to t for the vative nature of the
substitution. Sequences that differ by such conservative substitutions are said to have
"sequence similarity" or "similarity." Means for making this ment are well known to
those of skill in the art. Typically, this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the percentage sequence identity.
Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution is given a score between zero
and 1. The scoring of conservative substitutions is calculated, e. g., as implemented in the
program E (Intelligenetics, Mountain View, California).
ntage of ce identity" includes the value determined by comparing
two optimally aligned sequences over a comparison window, n the portion of the
polynucleotide sequence in the comparison window may comprise additions or deletions (i.e.,
gaps) as compared to the reference sequence (which does not comprise additions or ons)
for optimal alignment of the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or amino acid e occurs in
both sequences to yield the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of comparison, and multiplying the
result by 100 to yield the percentage of sequence identity.
Unless otherwise stated, sequence identity/similarity values include the value
obtained using GAP Version 10 using the following parameters: % identity and % similarity
for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the
nwsgapdna.cmp scoring matrix; % ty and % similarity for an amino acid sequence
using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any
equivalent program thereof. "Equivalent program" includes any sequence comparison
program that, for any two sequences in question, generates an alignment having cal
nucleotide or amino acid residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version 10.
Compositions or methods “comprising” or “including” one or more d
elements may e other elements not specifically d. For example, a composition
that “comprises” or “includes” a protein may n the n alone or in combination with
other ingredients.
Designation of a range of values includes all integers within or ng the range,
and all subranges defined by integers within the range.
Unless otherwise apparent from the context, the term ” encompasses values
within a standard margin of error of measurement (e. g., SEM) of a stated value.
The singular forms of the articles “a,” “an,” and “the” include plural references
unless the context clearly dictates otherwise. For example, the term “a Cas protein” or “at
least one Cas protein” can include a plurality of Cas proteins, including mixtures thereof.
1. Methods and itions to Make a Fertile Female XY Animal in an F0 tion
Methods for making non-human animals from donor ES cells and host embryos
are known. Donor ES cells are selected for n characteristics that enhance the ability of
the cells to populate a host embryo and thus contribute in part or in substantial part to an
animal formed by the donor ES cells and the host embryo. The animal formed may be male
or , based in large part on the genotype of the ES cell (e. g., XY or XX).
The majority of ES cell lines for making enic animals have a male XY
genotype. Because of the dominance of the Y chromosome in mammalian sex determination,
when XY ES cells are introduced into a blastocyst host embryo and gestated, they nearly
always produce in the first generation (F0) phenotypically male animals that are chimeras,
i.e., that contain cells derived from the male donor ES cell (XY) and cells derived from the
host embryo, which can be either male (XY) or female (XX). XY ES cells, when introduced
into an 8-cell host embryo by the VelociMouse method and gestated, can produce in the first
generation (F0) phenotypically male animals that are fully derived from the XY ES cells.
W0201 1/156723 provides methods and itions which employ a culture
media for maintaining XY donor cells in culture such that after introduction of the XY donor
cells into a host embryo and gestation in a suitable host, fertile XY female animals are
produced in the F0 population. Such compositions find use in making Fl progeny that are
homozygous for the given targeted genetic modification.
The instant application provides methods and compositions that employ a
ation of XY donor cells having a modification that decreases the level and/or activity
of the Sry protein in combination with a culture media that promotes the tion of
anatomically , fertile and fecund, XY F0 females. Such s and compositions
allow for making a fertile female XY non-human animal in an F0 tion. The
combination of XY ES cells having a modification that decreases the level and/or activity of
the Sry n in combination with the culture media described herein icantly increases
the percentage of fertile female XY progeny in the F0 tion. Methods for the
ent male to female sex sion are valuable to the domestic animal industry. For
example, female calves are much more valuable to the dairy cattle industry than males. The
same is true for y. For breeding purposes, whether it be cattle or hogs or sheep, it is
preferred to breed many females to only a few bulls, boars, or rams. Thus, the various
methods provided herein find use in various commercially important breeding industries.
Methods and compositions are also provided for making a XY embryonic stem
(ES) cell line capable of producing a fertile XY female non-human mammal in an F0
generation t culturing in a feminizing media. In such methods, the XY ES cell line
having a modification that decreases the level and/or activity of an Sry protein can produce
an ES cell line capable of producing a fertile XY female non-human mammal in an F0
generation in the absence of a feminizing media provided elsewhere herein (e. g., by culturing
in a base medium, such as DMEM, described elsewhere herein).
A. Animal XY Cells Having a Modification that Decreases the Level and/or Activity
of an Sry Protein
Various compositions and methods are provided herein which comprise various
XY pluripotent and/or totipotent cells from an animal. The term potent cell” as used
herein includes an undifferentiated cell that possesses the ability to develop into more than
one differentiated cell types. Such pluripotent and/or totipotent XY cells can be, for example,
an nic stem (ES) cell or an induced pluripotent stem (iPS) cell. The term onic
stem cell" or "ES cell" as used herein includes an embryo-derived totipotent or pluripotent
cell that is capable of contributing to any tissue of the ping embryo upon introduction
into an embryo.
The term "animal," in reference to cells, pluripotent and/or totipotent cells, XY
cells, ES cells, iPS cells, donor cells and/or host embryos, includes mammals, fishes, and
birds. Mammals include, e. g., humans, non-human primates, monkey, ape, cat dog, horse,
bull, deer, bison, sheep, rodents (e. g., mice, rats, hamsters, guinea pigs), livestock (e. g.,
bovine s, e. g., cows, steer, etc.; ovine species, e. g., sheep, goats, etc.; and porcine
species, e.g., pigs and boars). Birds include, e.g., chickens, turkeys, ostrich, geese, ducks, etc.
Domesticated animals and agricultural animals are also included. The phrase "non-human
animal," in reference to cells, XY cells, ES cells, donor cells and/or host embryos, excludes
humans.
In specific embodiments, the pluripotent cell is a human XY ES cell, a human XY
iPS cell, a human adult XY ES cell, a pmentally restricted human itor ES cell, a
non-human XY ES cell, a non-human XY iPS cell, a rodent XY ES cell, a rodent XY iPS
cell, a mouse XY ES cell, a mouse XY iPS cell, a rat XY ES cell, a rat XY iPS cell, a hamster
XY ES cell, a hamster XY iPS cell, a monkey XY ES cell, a monkey XY iPS cell, an
agricultural mammal XY ES cell, an agricultural XY iPS cell, a domesticated mammal XY
ES cell, or a domesticated XY iPS cell. Moreover, the XY ES cell or the XY iPS cell can be
from an inbred strain, a hybrid strain or an outbred strain. It is further recognized that the
pluripotent and/or totipotent XY cells can comprise an XYY karyotype or an XXY
karyotype.
Mouse pluripotent and/or totipotent cells (i.e., XY ES cells or XY iPS cells) can
be from a 129 , a C57BL/6 strain, a mix of 129 and C57BL/6, a BALB/c strain, or a
Swiss Webster strain. In a specific embodiment, the mouse is 50% 129 and 50% 6.
In one embodiment, the mouse is a 129 strain selected from the group consisting of a strain
that is 129P1
, 129P2, 129P3, 129X1 , 129S1 (e.g., 129S1/SV, Svlm), 129S2, 129S4,
129S5, 129S9/SvaH, 129S6 vaTac), 129S7, 129S8, 129T1 , 129T2. See, for
example, Festing et al. (1999) Mammalian Genome 10:836). In one embodiment, the mouse
is a C57BL , and in a specific embodiment is from C57BL/A, C57BL/An, C57BL/GrFa,
C57BL/Kal_wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/6NTac,
C57BL/10, C57BL/10ScSn, 10Cr, or C57BL/Ola. In a specific embodiment, the
mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In
another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of
aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6
(129/SvaTac) strain. In some embodiments, the mouse XY ES cell comprises a Y
chromosome derived from the 129 strain.
In yet another embodiment, the XY mouse ES cell is a VGF1 mouse ES cell.
VGF1 (also known as F1H4) mouse ES cells were derived from hybrid embryos produced by
crossing a female C57BL/6NTac mouse to a male 129S6/SvaTac mouse. Therefore, VGF1
ES cells contain a Y chromosome from 129S6/SvaTac mouse. See, for example, Auerbach,
W. et a1. (2000) ishment and chimera analysis of 129/Sva- and C57BL/6-derived
mouse nic stem cell lines. Biotechniques 29, 1024—1028, 1030, 1032, herein
incorporated by reference in its ty.
A rat pluripotent and/or totipotent cell (i.e., XY ES cell or XY iPS cell) can be
from any rat strain, including but not d to, an ACI rat strain, a Dark Agouti (DA) rat
strain, a Wistar rat , a LEA rat strain, a Sprague Dawley (SD) rat strain, or a r rat
strain such as Fisher F344 or Fisher F6. Rat otent and/or totipotent cells (i.e., XY ES
cells or XY iPS cells) can also be obtained from a strain derived from a mix of two or more
strains recited above. In one embodiment, the rat pluripotent and/or totipotent cell (i.e., XY
ES cell or XY iPS cell) is derived from a strain selected from a DA strain and an ACI strain.
In a ic embodiment, the rat pluripotent and/or totipotent cell (i.e., XY ES cell or XY
iPS cell) is derived from an ACI strain. The ACI rat strain is characterized as having black
agouti, with white belly and feet and an RT] haplotype. Such strains are ble from a
variety of sources including Harlan Laboratories. In other embodiments, the various rat
pluripotent and/or totipotent cell (i.e., XY ES cell or XY iPS cell) are from a Dark Agouti
(DA) rat strain, which is characterized as having an agouti coat and an RT] haplotype.
Such rats are available from a variety of sources including Charles River and Harlan
tories. In a further embodiment, the rat pluripotent and/or totipotent cells (i.e., XY ES
cells or XY iPS cells) are from an inbred rat strain. In specific embodiments the rat ES cell
line is from an ACI rat and comprises the ACI.G1 rat ES cell. In another ment, the rat
ES cell line is from a DA rat and comprises the DA.2B rat ES cell line or the DA.2C rat ES
cell line. See, for example, US. Utility Application No. 14/185,703, filed on February 20,
2014 and herein orated by reference in its entirety.
In various embodiments, the pluripotent and/or totipotent cell (i.e., XY ES cell or
XY iPS cell), the donor cell and/or the host embryo are not from one or more of the
following: Akodon spp., Myopus spp., Microtus spp., Talpa spp. In various embodiments, the
donor cell and/or the host embryo are not from any species of which a normal wild-type
characteristic is XY female ity. In various embodiments, where a genetic modification
is present in the pluripotent and/or totipotent cell (i.e., XY ES cell or XY iPS cell), the donor
cell or the host embryo, the genetic modification is not an XYY or XXY, a Tdy-negative sex
reversal, Tdy-positive sex reversal, an X0 modification, an aneuploidy, an fgf9'/' genotype, or
a SOX9 modification.
The pluripotent and/or totipotent XY cells (i.e., an XY ES cell or an XY iPS cell)
employed in the s and itions have a genetic modification that results in a
decreased level and/or activity of the Sry protein. The “Sex Determining Region Y” protein
or the “Sry” protein is a transcription factor that is a member of the high mobility group
(HMG)-box family of DNA-binding proteins. Sry is the testis-determining factor that
initiates male sex ination. The sequence of the Sry protein from a variety of organisms
is known, ing from mouse (Accession No. Q05738); rat (GenBank: CAA61882.1)
human (Accession No. Q05066); cat (Accession No. Q67C50), and horse (Accession No.
P36389), each of which is herein incorporated by reference.
In general, the level and/or ty of the Sry protein is decreased if the protein
level and/or the activity level of the Sry protein is statistically lower than the protein level of
Sry in an appropriate control cell that has not been genetically modified or mutagenized to
inhibit the expression and/or activity of the Sry protein. In specific embodiments, the
tration and/or activity of the Sry protein is decreased by at least 1%, 5%, 10%, 20%,
%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a control cell which has not been
modified to have the decreased level and/or activity of the Sry protein.
A “subject cell” is one in which a genetic alteration, such as a c
modification disclosed herein has been effected, or is a cell which is descended from a cell so
altered and which comprises the alteration. A “control” or “control cell” provides a nce
point for measuring s in phenotype of the subject cell. In one ment, a control
cell is as closely matched as possible with the cell with reduced Sry activity except it lacks
the genetic modification or mutation ing in the reduced activity (for example, the
respective cells can originate from the same cell line). In other ces, the control cell may
comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material
for the genetic tion which resulted in the subject cell; (b) a cell of the same genotype as
the starting material but which has been genetically modified with a null construct (i.e. with a
construct which has no known effect on the trait of interest, such as a construct comprising a
marker gene); (c) a cell which is a non-genetically modified progeny of a subject cell (i.e.,
the l cell and the t cell originate from the same cell line); (d) a cell genetically
identical to the subject cell but which is not exposed to conditions or stimuli that would
induce expression of the gene of st; or (e) the t cell itself, under conditions in
which the genetic modification does not result in an alteration in expression of the
polynucleotide of interest.
The sion level of the Sry polypeptide may be measured directly, for
example, by assaying for the level of the Sry ptide in the cell or sm, or
indirectly, for example, by measuring the activity of the Sry polypeptide. Various methods
for determining the ty of the Sry protein are known. See, Wang et a1. (2013) Cell
153:910-918, Mandalos et a1. (2012) PLOS ONE 7:e45768:1-9, and Wang et al. (2013) Nat
Biotechnol. 31:530-532, each of which is herein incorporated by reference.
In other instances, cells having the targeted genetic modification that reduces the
activity and/or level of the Sry polypeptide are selected using methods that include, but are
not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or ypic
analysis. Such cells are then employed in the various methods, compositions and kits
described herein.
A targeted genetic modification can comprise a targeted alteration to a
polynucleotide of interest including, for example, a targeted alteration to a target genomic
locus on the Y chromosome, a targeted alteration to the Sry gene, or a ed alteration to
other desired polynucleotides. Such targeted modifications include, but are not limited to,
additions of one or more nucleotides, deletions of one or more nucleotides, substitutions of
one or more nucleotides, a knockout of the polynucleotide of interest or a n thereof, a
knock-in of the polynucleotide of st or a portion thereof, a replacement of an
endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or a
combination thereof. In specific embodiments, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more
nucleotides are changed to form the targeted genomic modification.
A decrease in the level and/or activity of the Sry protein can result from a genetic
modification in the Sry gene (i.e., a genetic modification in a regulatory region, the coding
region, and/or introns etc). Such genetic modifications include, but are not d to,
additions, deletions, and substitutions of nucleotides into the genome. Such genetic
modifications can include an alteration of the Sry gene, ing, for example, an insertion
of one or more nucleotides into the Sry gene, a deletion of one or more tides from the
Sry gene, a substitution of one or more nucleotides in the Sry gene, a knockout of the Sry
gene or a portion thereof, a knockin of the Sry gene or a portion thereof, a replacement of an
endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or a
combination thereof. Thus, in specific embodiments, the activity of an Sry polypeptide may
be reduced or eliminated by disrupting the gene encoding the Sry polypeptide. In specific
embodiments, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more tides are changed in the Sry
gene. Various methods can be used to generate the additional targeted genetic modification.
See, for e, Wang et a1. (2013) Cell 153:910-918, Mandalos et a1. (2012) PLOS ONE
7:e45768:1-9, and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is herein
incorporated by reference. In addition, the various methods described herein to modify
genomic locus on the Y chromosome can be used to uce targeted genetic modification
to the Sry gene.
In other embodiments, the activity and/or level of the Sry polypeptide is reduced
or eliminated by introducing into the cell a polynucleotide that inhibits the level or activity of
the Sry polypeptide. The polynucleotide may inhibit the sion of the Sry polypeptide
directly, by preventing translation of the Sry messenger RNA, or indirectly, by encoding a
polypeptide that inhibits the ription or translation of the gene encoding an Sry protein.
In other ments, the activity of Sry polypeptide is reduced or eliminated by introducing
into the cell a sequence encoding a polypeptide that inhibits the activity of the Sry
polypeptide.
In one embodiment, the XY pluripotent and/or totipotent cells (i.e., XY ES cell or
XY iPS cell) comprise a conditional Sry allele that reduces the ty and/or level of the Sry
protein. A “conditional Sry allele” es a modified Sry gene designed to have the
decreased level and/or activity of the Sry protein at a desired developmental time and/or
within a d tissue of interest. Reduced level and/or activity can be compared with a
control cell lacking the modification giving rise to the ional allele, or in the case of
reduced activity at a desired developmental time with preceding and/or following times, or in
the case of a desired tissue, with a mean activity of all tissues. In one embodiment, the
conditional Sry allele comprises a ional null allele of Sry that can be switch off at a
desired developmental time point and/or in specific tissues. Such a conditional allele can be
used to create fertile XY females derived from any gene-targeted clone. As described
elsewhere , such a method enables the creation of a desired homozygous c
modification in the F1 generation. Such methods provide a quick look at the phenotype
without having to breed to the F2 generation.
In a miting ment, the conditional Sry allele is a multifunctional
allele, as described in US 2011/0104799, which is incorporated by reference in its entirety.
WO 00805
In specific embodiments, the conditional allele comprises: (a) an actuating sequence in sense
orientation with respect to transcription of a target gene, and a drug selection cassette (DSC)
in sense or antisense orientation; (b) in nse orientation a nucleotide sequence of interest
(NSI) and a conditional by inversion module (COIN, which utilizes an exon-splitting intron
and an invertible genetrap-like module; see, for example, US 2011/0104799, which is
incorporated by reference in its entirety); and (c) recombinable units that recombine upon
re to a first recombinase to form a conditional allele that (i) lacks the actuating
sequence and the DSC, and (ii) contains the NSI in sense orientation and the COIN in
antisense orientation.
The ional allele of the Sry gene can be generated in any cell type, and is not
limited to an XY pluripotent and/or totipotent cell. Such cells types along with non-limiting
methods to target a genomic locus on the Y chromosome are discussed in further detail
elsewhere herein.
As sed elsewhere herein, the pluripotent and/or totipotent XY cell (i.e., an
XY ES cell or an XY iPS cell) having genetic modification that decreases the level and/or
activity of the Sry protein can further comprise at least one additional targeted genetic
modification to a cleotide of interest. The at least one additional targeted genetic
modification can comprise a substitution of one or more nucleic acids, a replacement of an
endogenous nucleic acid sequence with a heterologous nucleic acid sequence, a knockout,
and a knock-in. The additional targeted genetic modification can be on the Y chromosome,
the X chromosome or on an autosome. Various methods can be used to te the
additional targeted genetic modification, including employing ing plasmids and large
ing s as discussed elsewhere . See, also, [[820080092249,
WG/1999/005266A2, USEOOL’EQIWZ’éS’O, WO/2008/017234Al, and US Patent No. 7,612,250,
each of which is herein incorporated by nce, for methods related to nuclear transfer. In
addition, the various methods described herein to modify genomic locus on the Y
chromosome (i.e., the Sry gene) can also be used to introduce targeted genetic modifications
to polynucleotides of st that are not located on the Y chromosome.
B. Mediafor Culturing the Pluripotent and/or tent XY Cells Having a
Modification that Decreases the Level and/or Activity ofan Sry Protein
The culture media employed in the various methods and compositions that
promote XY e female in the F0 generation is such that it maintains the pluripotent and/or
totipotent cells (i.e., ES cell, iPS cells, XY ES cells, XY iPS cells, etc.). The terms
WO 00805
“maintain77 ll
, maintaining" and enance" refer to the stable preservation of at least one
or more of the characteristics or phenotypes of pluripotent and/or totipotent cells described
herein (including ES cells or iPS cells). Such phenotypes can include maintaining
pluripotency and/or tency, cell logy, gene expression profiles and the other
functional characteristics of the cells. The terms “maintain”, "maintaining" and
"maintenance" can also encompass the propagation of cells, or an increase in the number of
cells being cultured. The terms further contemplate culture conditions that permit the cells to
remain pluripotent, while the cells may or may not continue to divide and increase in number.
In some embodiments, the XY cells having the genetic cation that s
the level and/or activity of the Sry protein are maintained by culturing in any base medium
known in the art (e. g., DMEM) that is suitable for use (with added supplements) in growing
or maintaining the pluripotent and/or totipotent cells (i.e., ES cell, iPS cells, XY ES cells, XY
iPS cells, etc.) in culture. In such cases, the ed XY ES cells have the potential to
develop into fertile female animals but still retain pluripotency and/or totipotency, such that
the cells can be implemented into a ent embryo and give rise to a fertile female
progeny.
In other embodiments, XY cells having the genetic modification that reduces the
level and/or activity of the Sry protein are maintained by culturing in a medium as further
defined below for ient time that some of the cells convert to KY cells with the potential
to develop into fertile female animals but still retain pluripotency and/or totipotency, such
that the cells can be implemented into a recipient embryo and give rise to a fertile female
progeny.
The medium employed to maintain the XY otent and/or totipotent cells (i.e.,
XY ES cells, XY iPS cells, etc.) having the genetic modification that reduces the level and/or
activity of the Sry protein promotes the development of XY F0 fertile females. Thus,
culturing in such a medium increases the number of XY F0 fertile females that are obtained
when compared to culturing in an appropriate control medium (such as, for example, one
based on DMEM). Thus, an increased number of XY F0 fertile females can comprise at least
%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of
the F0 man animals (following uction of the non-human animal XY ES cells
into a host embryo and gestation of the host embryo) are XY females and which upon
attaining sexual maturity the F0 XY female non-human animal is fertile.
The phrase "base medium" or "base media" includes, for example, a base medium
known in the art (e. g., DMEM) that is suitable for use (with added supplements) in growing
or maintaining the pluripotent and/or totipotent cells (i.e., ES cell, iPS cells, XY ES cells, XY
iPS cells, etc.) in culture. Base media suitable for making a fertile XY female (i.e., alt
DMEM" or “low-osmolality medium”) differs from base media typically used to maintain ES
cells in culture. For purposes of discussing base media in general, base media that are not
suitable for making fertile XY females are described in this section as "DMEM" and in Table
1 (e. g., typical DMEM media). For purposes of discussing base media suitable for making
e XY females, the phrase "low-salt DMEM" or smolality DMEM” is used.
ences between base media typically used to maintain pluripotent and/or totipotent cells
in culture (e. g., DMEM) and base media suitable for making fertile XY females (e. g., "low-
salt DMEM") are articulated . The phrase "low-salt DMEM" is used for convenience;
suitable DMEM for making fertile XY females exhibits characteristics not limited to "low-
salt," but es those bed herein. For example, the DMEM shown in Table 1 can be
made suitable for making fertile XY females by altering the sodium chloride and/or sodium
bicarbonate concentrations as ed for herein, which will also result in a different
osmolality and a different conductivity as compared with the DMEM shown in Table 1. An
example of base medium is Dulbeco's Modified Eagle's Medium (DMEM), in various forms
(e. g., Invitrogen DMEM, Cat. No. 1 1971 -025) (Table 1). A suitable low-salt DMEM is
available commercially as KO-DMEMTM (Invitrogen Cat. No. 10829-018). Base medium is
typically supplemented with a number of supplements known in the art when used to
in cells in culture for use as donor cells. Such supplements are indicated as
"supplements" or "+ supplements" in this disclosure.
Table 1 : DMEM Base Media for Maintaining or Culturing Pluripotent and/or Totipotent
Cells
Component Mg/L mM
Glycine 30 0.4
L-Arginine0HCI 84 0.398
ine02HCI 63 0.201
L-Glutamine 584 4
L—Histidine-HCI-H2O 42 0.2
L—Isoleucine 105 0.802
L-Leucine 105 0.802
L-Lysine0HCI 146 0.798
ionine 30 0.201
L-Phenylalanine 66 0.4
L-Serine 42 0.4
L—Threonine 95 0.798
L-Tryptophan 16 0.0784
sine disodium salt dihydrate 104 0.398
L—Valine 94 0.803
e de 4 0.0286
D-Calcium pantothenate 4 8.39 X 10'3
Folic Acid 4 9.07 x 10'3
Niacinamide 4 0.0328
Pyridoxine°HCI 4 0.0196
Riboflavin 0.4 1.06 x 10'3
Thiamine°HCI 4 0.0119
i-Inositol 7.2 0.04
Calcium Chloride (CaClz) (anhydrous) 200 1.8
Ferric Nitrate (Fe(N03)3.9HzO) 0.1 2.48 x 10-4
Magnesium Sulfate (MgSO4) (anhyd.) 97.67 0.814
Potassium Chloride (KCI) 400 5.33
D-Glucose (Dextrose) 4500 25
Phenol Red 15 0.0399
NaCL/NaHCO3 Content of DMEM
Sodium Bicarbonate (NaHCOg) 3700 44.05
Sodium Chloride (NaCl) 6400 110.34
NaCl/NaHCO3 Content of Low-salt
DMEM
Sodium Bicarbonate (NaHCOg) <3700 <44.05
Sodium Chloride (NaCl) <6400 4
The term "supplements" or the phrase "+ supplements," es elements added
to base medium for growing or maintaining otent and/or totipotent cells (i.e., XY ES
cell or XY iPS cells) in culture, e. g., for maintaining pluripotency or totipotency of donor
cells in e. For example, media supplements suitable for growing or maintaining
pluripotent and/or totipotent cells in culture include, but are not limited to, fetal bovine serum
(FBS), glutamine, antibiotic(s), penicillin and streptomycin (e. g., penstrep), pyruvate salts
(e. g., sodium pyruvate), nonessential amino acids (e. g., MEM NEAA), 2-mercaptoethanol,
and Leukemia Inhibitory Factor (LIF).
WO 00805
In one embodiment, the base medium comprises one or more ments suitable
for maintaining pluripotent cells in culture, including for example, XY ES cells or XY iPS
cells having a reduced capacity to contribute to the male sex determination developmental
program after injection into an embryo and intrauterine transfer to a surrogate mother mouse.
In a ic embodiment, the one or more supplements suitable for maintaining
the pluripotent cell in culture are PBS (90 ml 5L base medium), glutamine (2.4
mmoles/0.5 L base medium), sodium te (0.6 mmoles/0.5L base medium), nonessential
amino acids (< 0.1 mmol/0.5 L base medium), 2-mercaptoethanol, LIF, and one or more
antibiotics.
In other embodiments, the media for maintaining pluripotent cells in culture,
including for example, XY ES cells or XY iPS cells having a d capacity to contribute
to the male sex determination developmental program after injection into an embryo and
intrauterine transfer to a surrogate mother mouse, comprises about 500 ml of base medium in
which the following supplements are added: about 90 ml FBS (e.g., Hylcone FBS Cat. No.
SH30070.03), about 2.4 millimoles of glutamine (e. g., about 12 ml of a 200 mM glutamine
solution, e. g., Invitrogen Cat. No. 081, penicillin:streptomycin (e. g., 60,000 units of
Penicillin G sodium and 60 mg of streptomycin sulfate, with about 51 mg of NaCl; e. g., about
6 ml. of Invitrogen pennstrep, Cat. No. 122), about 0.6 millimoles of sodium pyruvate
(e. g., 6 ml. of 100 mM sodium pyruvate, Invitrogen Cat. No. 1 1360-070), about 0.06
millimoles of nonessential amino acids (e. g., about 6 ml. of MEM NEAA, e. g., MEM NEAA
from Invitrogen Cat. No. 1 1 140-050), about 1.2 ml. 2-mercaptoethanol, and about 1.2
micrograms of LIF (e.g., about 120 microliters of a 106 units/mL LIF preparation; e. g., about
120 microliters of Millipore ESGROTM-LIF, Cat. No. ESGl 107). When composing base
media for maintaining XY ES or XY iPS cells for making e XY females, typically the
same supplements in about the same s are employed, but the composition of the base
medium will differ (from DMEM, e.g., from the medium described in the table above) and
the ence(s) correspond to the difference(s) taught herein.
In some embodiments, supplements include Wnt-conditioned media, e.g., Wnt-3a
conditioned media.
In one embodiment, the pluripotent cell, including for example, an XY ES cell or
an XY iPS cell having a reduced capacity to contribute to the male sex determination
developmental program after injection into an embryo and intrauterine er to a surrogate
mother mouse, is maintained in an in vitro e in a medium comprising base medium and
supplements, wherein the base medium exhibits one or more of the following characteristics:
(a) an osmolality from about 200 mOsm/kg to less than about 329 mOsm/kg; (b) a
tivity of about 11 mS/cm to about 13 mS/cm; (C) a salt of an alkaline metal and a
halide in a concentration of about 50mM to about 110 mM; (d) a ic acid salt
concentration of about 17mM to about 30 mM; (e) a total alkaline metal halide salt and
ic acid salt concentration of about 85mM to about 130 mM; and/or (f) a combination
of any two or more thereof. In other embodiments, the XY pluripotent and/or totipotent cells
(i.e., XY ES cell or XY iPS cell) is ined in an in vitro culture in a media as bed
in WO201 1/156723, herein incorporated by reference in its entirety.
In one embodiment, the base medium is a low-salt DMEM. In a specific
embodiment, the low-salt DMEM has a NaCl concentration of 85-130 mM. In one
embodiment, the base medium is a low osmolality DMEM. In a specific ment, the
low osmolality DMEM has an osmolality of 250-310 mOsm/kg. In one embodiment, the
base medium is a low tivity DMEM. In a specific embodiment, the low conductivity
DMEM has a conductivity of 11 -13 mS/cm.
In other embodiments, the base medium exhibits an osmolality of no more than
about 320, 310, 300, 290, 280, 275, 270, 260, 250, or 240 mOsm/kg. In one embodiment, the
base medium or the medium comprising the base medium and the supplements exhibits an
osmolality of no more than about 240-320, 250-310, 275-295, or 260-300 g. In a
specific embodiment, the base medium or the medium sing the base medium and the
ments exhibits an osmolality of about 270 mOsm/kg.
In other embodiments, the base medium exhibits a conductivity of no more than
about 10.0, 10.5, 1 1.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, or 14.0 mS/cm. In one embodiment, the
base medium exhibits a conductivity of no more than about 10-14 mS/cm or 1 1 -13 mS/cm.
In a specific embodiment, the base medium exhibits a conductivity of about 12-13 mS/cm.
In a specific embodiment, the base medium exhibits a conductivity of about 12-13
mS/cm and an osmolality of about 260-300 mOsm/kg. In a further specific ment, the
base medium comprises sodium chloride at a concentration of about 90 mM NaCl. In a
further specific embodiment, the concentration of sodium chloride is about 70-95 mM. In a
further specific embodiment, the base medium comprises sodium bicarbonate at a
concentration of less than about 35 mM. In a further specific embodiment, the concentration
of sodium bicarbonate is about 20-30 mM.
In one embodiment, the base medium exhibits a concentration of a salt of an
alkaline metal and a halide of no more than about 100 mM. In one embodiment, the salt of
the alkaline metal and the halide is NaCl. In one embodiment, the concentration of the salt of
the ne metal and halide is no higher than 90, 80, 70, 60, or 50 mM. In one embodiment,
the tration in the base medium of the salt of the alkaline metal and halide is about 60-
105, 70-95, or 80-90 mM. In a specific embodiment, the concentration is about 85 mM.
In one embodiment, the base medium exhibits a concentration of a salt of carbonic
acid. In one embodiment, the salt of carbonic acid is a sodium salt. In one embodiment, the
sodium salt is sodium bicarbonate. In one embodiment, the concentration of carbonic acid
salt in the base medium is no higher than 40, 35, 30, 25, or 20 mM. In one embodiment the
concentration of carbonic acid salt in the base medium is about 10-40, in another embodiment
about 20-30 mM. In a ic embodiment, the concentration is about 25 or 26 mM. In still
other embodiments, the sodium bicarbonate concentration is about 26 mM, about 18 mM,
about 18 mM to about 26 mM or about 18 mM to about 44 mM.
In one embodiment, the sum of the concentration of the salt of the alkaline metal
and halide and the salt of carbonic acid in the base medium is no more than 140, 130, 120,
110, 100, 90, or 80 mM. In one embodiment, the sum of the concentration of the salt of the
alkaline metal and halide and the salt of carbonic acid in the base medium is about 80-140,
, 90-120, 95-120, or 100-120 mM. In a specific ment, the sum of the
concentration of the salt of the alkaline metal and halide and the salt of carbonic acid in the
base medium is about 115 mM.
In one embodiment, the molar ratio of the salt of the alkaline metal and halide and
the salt of ic acid is higher than 2.5. In one ment, the ratio is about 2.6-4.0, 2.8-
3.8, 3-3.6, or 3.2-3.4. In one embodiment, the ratio is 3.3-3.5. In a specific embodiment, the
ratio is 3.4.
In one embodiment, the base medium exhibits an osmolality of about 250-310
mOsm/kg, and a concentration of a salt of an alkaline metal and a halide of about 60-105
mM. In a further embodiment, the base medium has a concentration of a salt of carbonic acid
of about 20-30 mM. In a further embodiment, the sum of the concentrations of the salt of an
alkaline metal and halide and the salt of carbonic acid is about 80-140 mM. In a further
embodiment, the conductivity of the base medium is about 12-13 mS/cm.
In one embodiment, the base medium comprises about 50 i 5 mM NaCl and about
26 i 5 mM carbonate, with an osmolality of about 218 i 22 mOsm/kg. In a specific
embodiment, the base medium comprises about 3 mg/mL NaCl and 2.2 mg/mL sodium
bicarbonate, with an osmolality of about 218 mOsm/kg.
In another embodiment, the base medium comprises about 87 i 5 mM NaCl and
about 18 i 5 mM, with an lity of about 261 i 26 mOsm/kg. In a specific embodiment,
the base medium ses about 5.1 mg/mL NaCl and about 1.5 mg/mL sodium
bicarbonate, with an osmolality of about 261 mOsm/kg.
In another ment, the base medium comprises about 110 i 5 mM NaCl and
about 18 i 5 mM carbonate, with an osmolality of about 294 i 29 mOsm/kg. In a specific
embodiment, the base medium comprises about 6.4 mg/mL NaCl and about 1.5 mg/mL
sodium bicarbonate, with an osmolality of about 294 mOsm/kg.
In another embodiment, the base medium exhibits about 87 i 5 mM NaCl and
about 26 i 5 mM carbonate, with an osmolality of about 270 i 27 mOsm/kg. In a specific
ment, the base medium exhibits about 5.1 mg/mL NaCl and about 2.2 mg/mL sodium
onate, with an osmolality of about 270 mOsm/kg.
In another embodiment, the base medium comprises about 87 i 5 mM NaCl,
about 26 i 5 mM carbonate, and about 86 i 5 mM e, with an osmolality of about 322 i
32 mOsm/kg. In a specific ment, the base medium comprises about 5.1 mg/mL NaCl,
about 2.2 mg/mL sodium bicarbonate, and about 15.5 mg/mL glucose, with an osmolality of
about 322 mOsm/kg.
Additional base media that can be employed in the various methods and
compositions disclosed herein include, a base medium comprising 50 i 5 mM NaCl and 26 i
mM carbonate, with an osmolality of 218 i 22 mOsm/kg. In a particular embodiment, the
base medium comprises about 3 mg/mL NaCl and 2.2 mg/mL sodium bicarbonate, with an
osmolality of about 218 mOsm/kg.
In other embodiments, the base medium comprises 50 i 5 mM NaCl and 26 i 5
mM carbonate, with an osmolality of 218 i 22 mOsm/kg. In a specific embodiment, the base
medium comprises about 3 mg/mL NaCl and 2.2 mg/mL sodium bicarbonate, with an
osmolality of about 218 g.
In other embodiments, high glucose DMEM media (LifeTech) with NaHC03
concentrations as sed herein, including, about 44mM, 26mM or 18mM, were
supplemented with 0.1mM nonessential amino acids, 1mM sodium pyruvate, 0.1mM 2-
mercaptoethanol, 2mM L-glutamine, 50ug/ml each penicillin and streptomycin (LifeTech),
% FBS (Hyclone), and 2000U/ml LIF (Millipore).
C. Methodfor Making Targeted Genetic Modifications
Various methods for making targeted genetic modifications that decrease the level
and/or the activity of the Sry protein can be used. For example, in one instance, the ed
genetic cation s a system that will generate a targeted genetic modification via
a homologous recombination event. In other instances, the animal cell can be modified using
se agents that te a single or double strand break at a targeted genomic location.
The single or double-strand break is then repaired by the non-homologous end joining
pathway (NHEJ). Such systems find use, for example, in generating targeted loss of function
c modifications. Non-limiting methods for generating such targeted genetic
modification are discussed in detail elsewhere herein, including, for example, the use of
targeting ds, small targeting vectors (smallTVECs) or large targeting s. See,
also, Wang et al. (2013) Cell 153:910-918, Mandalos et al. (2012) PLOS ONE 7:e45768:1-9,
and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is herein incorporated by
reference.
It is recognized that in specific embodiments, the targeted genetic modification of
the Sry gene and/or the targeted genetic modification of any other polynucleotide of interest
can occur while the pluripotent cell (i.e., ES cell) is being ined in the culture media
described herein (e.g. a medium that promotes the development of XY F0 fertile s).
Alternatively, the targeted genetic modification of the Sry gene and/or any other
polynucleotide of interest can occur while the pluripotent cell (i.e., ES cell) is being
maintained in different culture media, and subsequently erred to the culture media
disclosed herein (e. g. a medium that promotes the development of XY F0 fertile females).
D. Method of Culturing and ining a Pluripotent and/0r T0tip0tent Cell In
Culture
A method for maintaining or ing an XY otent and/or totipotent cell
(i.e., an XY ES cell or an XY iPS cell) in an in vitro culture is provided, wherein the cell
comprises a modification that decreases the level and/or activity of an Sry protein and the cell
is maintained in an in vitro culture under conditions described herein. Such methods of
maintaining or culturing an XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an
XY iPS cell) in an in vitro culture is such as to promote an increase in the number XY F0
fertile female animals upon the introduction of the non-human animal XY ES cells into a host
embryo and following ion of the host embryos.
While any media disclosed herein can be employed for such maintaining or
culturing methods, one non-limiting example, includes culturing in a medium comprising a
base medium and supplements suitable for maintaining or culturing the XY pluripotent and/or
totipotent cell (i.e., an XY ES cell or an XY iPS cell) in e, wherein the base medium or
the medium comprising the base medium and the supplements exhibits an osmolality from
about 200 mOsm/kg to less than about 329 mOsm/kg.
In some embodiments, the base medium or the medium comprising the base
medium and the supplements exhibits one or more of the following teristic: a
conductivity of about 11 mS/cm to about 13 mS/cm; a salt of an ne metal and a halide in
a concentration of about 50mM to about 110 mM; a carbonic acid salt concentration of about
l7mM to about 30 mM; a total alkaline metal halide salt and carbonic acid salt concentration
of about 85mM to about 130 mM; and/or a combination of any two or more thereof.
In one embodiment, the method comprises maintaining or culturing the XY
pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) in a suitable culture
medium that comprises a base medium and supplements, wherein the base medium or the
medium comprising the base medium and the supplements comprises an osmolality of about
240-320 mOsm/kg, a conductivity of about 10-14 mS/cm, an alkaline metal halide salt
concentration of about 50-105 mM, a salt of carbonic acid concentration of 10-40 mM, and/or
a combined ne metal salt and carbonic acid salt concentration of about 80-140 mM. In
one embodiment, the XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS
cell) is maintained in the medium (with supplements for maintaining ES cells) for a period of
l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, or 13 days, or 2 weeks, 3 weeks, or 4 weeks prior to
introduction into a host embryo. In a specific embodiment, the XY otent and/or
totipotent cell (i.e., an XY ES cell or an XY iPS cell) is maintained in the medium (low-salt
base medium with supplements for maintaining ES cells) for about 2-4 weeks prior to
introduction into the host embryo.
In r ment, the XY pluripotent and/or tent cell (i.e., an XY ES
cell or an XY iPS cell) is maintained in a medium with a low-salt base medium for at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, 2 weeks, 3 weeks, or 4 weeks prior to introducing the
donor cell into a host . In a specific embodiment, the XY pluripotent and/or totipotent
cell (i.e., an XY ES cell or an XY iPS cell) is maintained in a medium with a low-salt base
medium at least 2-4 weeks prior to introduction of the cell into the host embryo.
In another embodiment, the XY pluripotent and/or totipotent cell (i.e., an XY ES
cell or an XY iPS cell) is maintained (e. g., frozen) in a medium that promotes XY fertile F0
females and the donor cell is thawed in and maintained in the medium that promotes XY
fertile F0 females for at least 1, 2, 3, or 4 or more days before ucing the XY pluripotent
and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) into the host . In a
specific embodiment, the XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an KY
iPS cell) is passaged at least once in a medium that promotes XY fertile F0 females, the cell
is frozen in the medium that promotes XY e F0 females, and the cell is thawed in a
medium that es XY fertile F0 females and grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
or 13 days, 2 weeks, 3 weeks, 4 weeks, or more prior to introduction into the host embryo.
In still another embodiment, the XY otent and/or totipotent cell (i.e., an XY
ES cell or an XY iPS cell) is maintained in the medium that promotes XY fertile F0 females
for a period of one, two, three, or four days prior to introduction into a host embryo. In one
embodiment, the XY pluripotent and/or tent cell (i.e., an XY ES cell or an XY iPS cell)
is maintained in the medium that promotes XY fertile F0 females for a period of 3 days.
In one embodiment, the XY pluripotent and/or totipotent cell (i.e., an XY ES cell
or an XY iPS cell) is maintained the medium that es XY fertile F0 females before
introduction into the host embryo for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, 2
weeks, 3 weeks, or 4 weeks or more. In a specific embodiment, the donor cell is maintained
in the medium that promotes XY fertile F0 females for at least a week before introduction
into the host embryo. In a specific embodiment, the XY pluripotent and/or tent cell
(i.e., an XY ES cell or an XY iPS cell) is maintained in the medium that es XY fertile
F0 females for 2-4 weeks before introduction into the host .
Thus, a method for maintaining or culturing an XY pluripotent and/or totipotent
cell (i.e., an XY ES cell or an XY iPS cell) in culture is provided, wherein the cell is
ined under conditions that promote or favor development of a female XY animal
following introduction of the XY cell into a host embryo and following gestation in a suitable
female host.
In one aspect, a method for maintaining or culturing a donor XY pluripotent
and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) in culture is provided, under
conditions as bed herein, wherein following introduction of the donor XY ES cell into a
host embryo to form a F0 embryo and gestation of the F0 embryo in a suitable animal, the F0
embryo develops into an F0 animal that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or greater KY and is a female which, upon attaining
sexual maturity, is fertile.
E. Generating F0 s and F1 Progeny Having A Targeted Genetic Modification
The various methods and compositions employing the XY pluripotent and/or
totipotent cell (i.e., an XY ES cell or an XY iPS cell) having a decreased level and/or activity
of Sry protein provided herein can be used to generate a genetically modified animal.
Various s for introducing genetic modifications are discussed in detail elsewhere
herein.
i. Methodfor Making a Fertile Female XYNon-Human Animal in an F0
tion
A method for making a fertile female XY non-human animal in an F0 generation
is provided. Such methods comprise: (a) maintaining or culturing a donor man
animal XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) having a
modification that ses the level and/or activity of an Sry protein in a medium that
promotes the development of XY fertile female ES cells; (b) introducing the donor XY non-
human animal XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell)
into a host embryo; (c) gestating the host embryo; and, (d) obtaining an F0 XY female non-
human animal, wherein upon attaining sexual maturity the F0 XY female non-human animal
is fertile. In specific embodiments, the donor non-human animal XY donor cell can comprise
at least one onal targeted genetic cation in a polynucleotide of interest. Such
modifications are discussed in detail elsewhere herein.
The XY ES cells having a modification that ses the level and/or ty of
an Sry protein can be maintained without a low-salt medium and can develop into an XY
fertile female.
In some embodiments, the medium that promotes the development of XY fertile
F0 female animals can comprise a low-salt based medium which comprises a base medium
and supplements suitable for maintaining or culturing the non-human mammalian ES cell in
culture, wherein the low-salt base medium exhibits a characteristic comprising one or more of
the following: an osmolality from about 200 mOsm/kg to less than about 329 mOsm/kg; a
conductivity of about 11 mS/cm to about 13 mS/cm; a salt of an alkaline metal and a halide in
a concentration of about 50 mM to about 110 mM; a carbonic acid salt concentration of about
17 mM to about 30 mM; a total alkaline metal halide salt and ic acid salt concentration
of about 85 mM to about 130 mM; and/or a combination of any two or more thereof.
In other embodiments, such methods for making a fertile female XY non-human
animal in an F0 generation can be performed using the s disclosed herein including,
but not limited to, (a) a base medium comprising 50 i 5 mM NaCl, 26 i 5 mM carbonate,
and 218 i 22 mOsm/kg; (b) a base medium comprising about 3 mg/mL NaCl, 2.2 mg/mL
sodium bicarbonate, and 218 mOsm/kg; (c) a base medium comprising 87 i 5 mM NaCl, 18
i 5 mM carbonate, and 261 i 26 mOsm/kg; (d) a base medium comprising about 5.1 mg/mL
2015/038001
NaCl, 1.5 mg/mL sodium bicarbonate, and 261 mOsm/k; (e) a base medium comprises 110 i
mM NaCl, 18 i 5 mM carbonate, and 294 i 29 mOsm/kg; (f) a base medium ses
about 6.4 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 294 mOsm/kg; (g) a base
medium comprises 87 i 5 mM NaCl, 26 i 5 mM carbonate, and 270 i 27 mOsm/kg; (h) a
base medium comprises about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 270
g; (i) a base medium comprises 87 i 5 mM NaCl, 26 i 5 mM carbonate, 86 i 5 mM
glucose, and 322 i 32 mOsm/kg; and/or (1') a base medium comprises about 5.1 mg/mL NaCl,
2.2 mg/mL sodium bicarbonate, 15.5 mg/mL glucose, and 322 mOsm/kg.
The cally ed XY pluripotent and/or tent cell (i.e., an XY ES cell
or an XY iPS cell) having a modification that decreases the level and/or activity of an Sry
protein and having been cultured in the medium that promotes the development of XY F0
fertile females can be implanted into a host embryo. Cells that have been implanted into a
host embryo are referred to herein as “donor cells.” In specific embodiments, the genetically
modified XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) is from
the same strain as the host embryo or from a ent strain as the host embryo. se,
the surrogate mother can be from the same strain as the genetically modified XY pluripotent
and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) and/or the host embryo, or the
surrogate mother can be from a different strain as the genetically modified XY pluripotent
and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell) and/or the host embryo. In one
embodiment, the XY donor cell is implanted into an XX host embryo.
A variety of host embryos can be employed in the methods and compositions
disclosed herein. In some embodiments, the XY pluripotent and/or tent cells (i.e., the
XY ES cell or the XY iPS cell) having the targeted genetic modification resulting in a
decreased level and/or activity of the Sry protein are introduced into a pre-morula stage
embryo from a corresponding organism, e. g., an 8-cell stage embryo. See, e.g., US
7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1, all of which are
incorporated by reference herein in their entireties. In other embodiments, the donor ES cells
may be implanted into a host embryo at the 2-cell stage, 4-cell stage, 8-cell stage, 16-cell
stage, 32-cell stage, or 64-cell stage host embryo. In another embodiment, the host embryo is
a blastocyst. In one embodiment, the host embryo is in a stage selected from a pre-blastocyst
, a rula stage, a morula stage, an uncompacted morula stage, and a compacted
morula stage. In one embodiment, when ing a mouse embryo, the host embryo stage
is selected from a Theiler Stage 1 (TSl), a TS2, a TS3, a TS4, a TS5, and a TS6,
, with
reference to the Theiler stages described in Theiler (1989) "The House Mouse: Atlas of
Mouse Development," Springer-Verlag, New York. In a specific embodiment, the Theiler
Stage is selected from TSl, TS2, TS3, and a TS4. In one ment, the host embryo
comprises a zona pellucida, and the donor cell is an XY ES cell that is introduced into the
host embryo through a hole in the zona pellucida, while in other embodiments, the host
embryo is a zona-less embryo. In yet other specific ments, the morula-stage host
embryo is aggregated.
Nuclear er techniques can also be used to generate the genetically ed
animals. Briefly, s for nuclear transfer include the steps of: (l) enucleating an oocyte;
(2) isolating a donor cell or nucleus to be combined with the enucleated oocyte; (3) inserting
the cell or nucleus into the enucleated oocyte to form a reconstituted cell; (4) implanting the
reconstituted cell into the womb of an animal to form an embryo; and (5) allowing the
embryo to develop. In such methods oocytes are generally ved from ed animals,
although they may be isolated also from either oviducts and/or s of live animals.
Oocytes can be matured in a variety of medium known to those of ordinary skill in the art
prior to enucleation. Enucleation of the oocyte can be performed in a number of manners
well known to those of ordinary skill in the art. Insertion of the donor cell or nucleus into the
enucleated oocyte to form a reconstituted cell is y by microinjection of a donor cell
under the zona pellucida prior to fusion. Fusion may be induced by application of a DC
electrical pulse across the contact/fusion plane (electrofusion), by exposure of the cells to
fusion-promoting chemicals, such as polyethylene glycol, or by way of an inactivated virus,
such as the Sendai virus. A reconstituted cell is typically activated by electrical and/or non-
ical means before, during, and/or after fusion of the nuclear donor and recipient oocyte.
Activation methods include electric pulses, chemically induced shock, penetration by sperm,
increasing levels of divalent cations in the oocyte, and reducing phosphorylation of ar
proteins (as by way of kinase inhibitors) in the oocyte. The ted reconstituted cells, or
embryos, are typically cultured in medium well known to those of ordinary skill in the art and
then transferred to the womb of an animal. See, for example, US20080092249,
WO/l999/005266A2, US20040177390, WO/2008/017234Al, and US Patent No. 250,
each of which is herein incorporated by reference.
The host embryo comprising the genetically modified XY pluripotent and/or
totipotent cell (i.e., an XY ES cell or an XY iPS cell) having the decreased level and/or
activity of the Sry protein is incubated until the blastocyst stage and then implanted into a
surrogate mother to produce an F0 animal. Animals bearing the genetically modified
genomic locus can be identified via cation of allele (MOA) assay as bed herein.
In one embodiment, the host embryo comprising the cally modified XY
pluripotent and/or totipotent cells (i.e., an XY ES cell or an XY iPS cell) having the
decreased level and/or activity of the Sry protein is maintained in a medium that promotes the
development of XY fertile female ES cells (i.e., a low-salt base medium) for one, two, three,
or four or more days prior to implantation in a suitable host. Such methods provide for
favoring the generation of an F0 fertile female animal.
In one embodiment, the cultured host embryo is implanted into a surrogate
mother, and the cultured host embryo is gestated in the surrogate .
In ic ments, upon introduction of the non-human animal XY
pluripotent and/or totipotent cells (i.e., an XY ES cell or an XY iPS cell) into a host embryo
and ing gestation of the host embryo, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%,
75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the F0
non-human animals are XY females which upon attaining sexual maturity the F0 XY female
non-human mammal is fertile.
Further provided is an F0 embryo comprising an inner cell mass having at least
one heterologous stem cell comprising an XY ES cell or XY iPS cell having a targeted
genetic modification that decreases the level and/or activity of the Sry n.
The various methods described herein to generate a e female XY non-human
animal in an F0 generation can employ XY pluripotent and/or totipotent cells (i.e., an XY ES
cell or an XY iPS cell) having (1) the genetic modification to reduce the level and/or ty
of the Sry polypeptide; and, in specific embodiments, (2) one or more onal targeted
genetic modification in a polynucleotide of interest. As outlined elsewhere herein, at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional ed genetic modifications can be made in the
XY pluripotent and/or totipotent cell (i.e., an XY ES cell or an XY iPS cell). In such
instances, the F0 e female XY non-human animal can comprises one or more of these
additional targeted genetic modifications.
In other embodiments, the F0 e female XY non-human animal produces l, 2,
3, 4, 5, 6, 7, 8, or 9 litters during its lifetime. In one embodiment, the F0 fertile female XY
non-human animal produces at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 offspring per litter. In one
embodiment, the F0 fertile female XY non-human animal es about 4-6 offspring per
litter. In one embodiment, the F0 fertile female XY non-human animal produces 2-6 litters,
wherein each litter has at least 2, 3, 4, 5, or 6 offspring. In one embodiment, at least about
%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or
100% of the offspring are XY fertile female offspring.
A method for generating a rodent litter (i.e., a mouse or a rat litter) is also
ed and comprises introducing an XY otent and/or totipotent donor cell (i.e., an
XY donor ES cell or XY donor iPS cell) having the decreased level and/or activity of Sry
protein prepared according to the methods set forth herein into host embryos, gestating the
embryos in a suitable segregate mother, and obtaining F0 progeny that comprises at least one
XY female rodent that upon reaching sexual maturity is a fertile XY female rodent. In one
embodiment, the tage of F0 XY female rodents born that upon reaching sexual
maturity are fertile is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
75%, 80%, 85%, 95% or 100%.
In other embodiments, the F0 y produced from such methods are about 3%,
about 10% or more, or about 63% or more derived from the genetically modified donor XY
cell.
The methods and itions provided herein allow for at least 1%, 3%, 5%,
%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater of
the F0 animals to have the targeted genetic modification (i.e., the decrease in Sry protein
level and/or activity and/or the targeted genetic modification to a polynucleotide of interest)
to transmit the genetic modification to the F1 progeny.
In one embodiment, the F0 generation female XY non-human animal and/or the
male XY non-human animal is at least 90%, 92%, 94%, 96%, 98%, 99%, or 99.8% derived
from the donor cell. In one embodiment, the F0 female XY man animal and/or the F0
male XY non-human animal has a coat color that is 100% derived from the donor cell.
In one embodiment, the non-human female XY animal in the F0 generation is a
rodent (i.e., a mouse or a rat) and has a coat color 100% derived from the donor cell. In one
embodiment, the non-human female XY non-human animal formed in the F0 generation is at
least 90%, 92%, 94%, 96%, 98%, or 99.8% derived from the XY donor cell. In one
embodiment, the non-human female XY animal in the F0 generation is about 100% derived
from the donor cell. In one embodiment, the contribution of a host embryo cell to the non-
human female XY animal in the F0 generation is ined by a quantitative assay that is
capable of detecting 1 cell in 2,000 (0.05%), and no tissue of the female XY animal is
positive for host embryo cell contribution.
ii. Various Methods ofBreeding the Female e XY F0 Generation
In ic embodiments, the resulting female fertile XY F0 generation derived
from the XY otent and/or totipotent cells (i.e., the XY ES cell or XY iPS cell) having
the genetic modification that ses the level and/or activity of the Sry protein is crossed
to an animal to obtain F1 generation offspring. In specific embodiments, the female fertile
XY F0 is crossed to a wild type animal. In one embodiment, the female XY F0 non-human
mammal is fertile when crossed to a wild type mouse. In specific embodiments, the wild type
mouse is C57BL/6. The F1 progeny can be genotyped using specific s and/or probes
to determine if the targeted genetic modification comprising the decreased level and/or
activity of the Sry protein is present. Moreover, if additional targeted genetic cations
were present in the F0 generation, the F1 progeny can be genotyped using specific primers
and/or probes that determine if such modifications are present. An appropriate Fl progeny
for a desired use can then be identified. In specific embodiments, Fl progeny lacking the
genetic cation that d the level and/or activity of the Sry n are selected. In
other embodiments, Fl progeny lacking the genetic modification that reduced the level and/or
activity of the Sry protein and which comprise at least one additional ed genetic
modification are selected.
In one non-limiting example, following genotyping with specific primers and/or
probes, Fl animals that are heterozygous for the targeted genetic modification to the
polynucleotide of interest and lacking the targeted modification that reduces the level and/or
activity of the Sry protein are crossed to one another. Such a cross produces an F2 progeny
that is homozygous for the genetically modified genomic locus of interest and does not
comprise the genetic cation to reduce Sry protein levels and/or activity.
Further provided is a method of producing a transgenic non-human animal
homozygous for a targeted genetic modification in the F1 generation. The method ses
(a) crossing an F0 XY fertile female non-human animal having a targeted genetic
modification that decreases the level and/or activity of the Sry protein with a F0 XY male
non-human animal, wherein the F0 XY fertile female non-human animal and the F0 XY male
non-human animal are each heterozygous for the same genetic modification of a
polynucleotide of interest, and (b) obtaining an F1 progeny that is homozygous for the
targeted genetic modification in the polynucleotide of interest. In a specific embodiment, the
F1 y selected are homozygous for the targeted genetic modification in the
polynucleotide of interest and lack the targeted c modification that ses the
activity and/or level of the Sry protein. Such methods can be employed to develop ng
pairs of non-human s, each fully derived from a donor ES cell or iPS cell, in the same
F0 generation.
Various methods can be employed to obtain the F0 animals described above. In
one non-limiting embodiment, an XY cell clone with a targeted modification in a
polynucleotide of interest on any chromosome is isolated. It is recognized that various
methods can be used to generate the targeted modification in the polynucleotide of interest.
In a second step, a targeted modification is introduced into the Sry gene such that the
modification ses the level and/or activity of the Sry protein. Such methods will further
employ culturing the XY ES cell in a media that promotes the development of XY F0 fertile
s, as describe in detail elsewhere herein. Methods of ed modification of the Sry
gene are disclosed in detail elsewhere herein and can comprise, for example, the use of a
targeting vector (including an LTVEC) either alone or in combination with a nuclease as
described ere herein (i.e., a Talen or CRISPR- or ZFN- system). A subclone is
isolated that comprises both the first targeted cation in the polynucleotide of interest
and the second targeted modification of the Sry gene that decreases the level and/or activity
of the Sry protein. Both the original XY clone with the targeted modification in the
polynucleotide of interest and the XY subclone comprising both the targeted modification to
the Sry gene and the polynucleotide of interest are introduced into te man host
embryos, as discussed ere . In specific embodiments, the non-human host
embryos comprise a pre-morula embryo (i.e., an 8 cell stage embryo). Each of the non-
human host embryos comprising the modified pluripotent cells is introduced into a surrogate
mother for gestation. Each of the surrogate mothers produces F0 progeny comprising the
targeted genome modification (i.e., an F0 XY male having the targeted modification in the
polynucleotide of interest and an F0 XY fertile female having the targeted modification in the
polynucleotide of interest and having the genetic cation that decreases the level and/or
activity of the Sry protein). In specific embodiments, each of the targeted c
modifications is e of being transmitted through the germline. Each of these F0 animals
are bred to one another, to generate an F1 animal sing a homozygous targeted
modification in the polynucleotide of interest. One-quarter of the F1 generation are expected
to be homozygous for the targeted modification in the polynucleotide of interest. Fl progeny
can be selected to retain the targeted cation to the Sry gene or the F1 progeny can be
selected to not retain the ed modification to the Sry gene.
In another embodiment, the introduction of the targeted modification of the Sry
gene employing a targeting vector (and, in specific embodiments, nucleases such as Talen,
Crispr, or an) can occur simultaneously with the vector targeting for the genetic
modification of the polynucleotide of interest. Such methods allow for the generation of an
XY ES cell having both a genetic modification that decreases the level and/or activity of the
Sry protein and further comprises the targeted modification to the polynucleotide of interest.
In one embodiment, the F1 generation progeny comprises a genome tely
derived from the donor ES cell. In other embodiments, the frequency of crosses of F0
generation male and F0 generation female mice that give rise to fully ES cell-derived mice is
100%.
II. Methods and Compositionsfor Modifying a nging Target Genomic Locus or a
Target Genomic Locus on the Y Chromosome
Methods and itions are provided that allow for modifying a target genomic
locus on the Y chromosome in a cell. Further provided are methods that allow for modifying
a “challenging” genomic locus. The term enging locus” includes a chromosomal
region that is difficult to target by conventional gene targeting strategies. Such loci can be
located on the Y chromosome, the X chromosome, or an autosome. In certain embodiments,
challenging loci are located within or in proximity to gene-poor, repeat-rich, and/or largely
heterochromatic chromosomal regions. See, e. g., Bernardini et al., Proc. Natl. Acad. Sci.
USA 111:7600-7605 (2014), herein incorporated by reference in its entirety for all purposes.
In certain ments, a challenging locus is located within or in proximity to somal
regions in which accessibility of the chromosomal DNA is limited by chromatin structure. In
certain embodiments, a challenging locus is within or in proximity to chromosomal regions
characterized by a high percentage of heterochromatin, such as at least about ~20%, at least
about ~30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70%
chromatin. In certain embodiments, a challenging locus is d within or in
proximity to chromosomal regions that have undergone ations and rearrangements or
that are characterized by the presence of repeats or inverted repeats. See, e. g., Gubbay et al.,
Proc. Natl. Acad. Sci. USA 89:7953-7957 (1992), herein incorporated by reference in its
entirety for all purposes.
The term “chromatin” includes nucleoprotein complexes which t and
organize cellular genetic al to contain it within cells. The term “heterochromatin”
includes s in the genome that are in a highly condensed state and are lly
transcriptionally silent. Heterochromatin is generally more y coiled and generally has
more repetitive DNA ces than euchromatin. The term “euchromatin” includes regions
in the genome characterized by more extended and less condensed chromatin domains that
are often transcriptionally active and accessible.
The term “exposing” includes using any method by which desired components are
brought into immediate proximity or direct contact.
Methods and compositions are provided that allow for modifying a challenging
target genomic locus or a target genomic locus on the Y chromosome in a cell. Perhaps due
to unique structural features of the Y some, conventional gene targeting strategies in
mouse embryonic stem cells to generate mutations on the Y-linked genes has had limited
success. Therefore, often the understanding of the functions of murine Y-linked genes is
limited to insights gained from studies of mice that carry spontaneous deletions, random gene
trap insertions or autosomal enes. Methods provided herein allow for the targeting of a
genomic locus on the Y chromosome by employing a targeting vector in the absence of or in
combination with a se agent.
Some such methods e a small targeting vector or smallTVEC. A
“smallTVEC” includes a targeting vector that comprises short homology arms. The length of
a homology arm on a smallTVEC can be from about 400-1000 bp. A homology arm of the
smallTVEC can be of any length that is sufficient to promote a homologous recombination
event with a corresponding target site, including for example, from about 400 bp to about 500
bp, from about 500 bp to about 600 bp, from about 600 bp to about 700 bp, from about 700
bp to about 800 bp, from about 800 bp to about 900 bp, or from about 900 bp to about 1000
bp. A preferred length of a homology arm on a smallTVEC is from about 700 bp to about 800
bp. In another embodiment, the sum total of 5’ and 3’ homology arms of the smallTVEC is
about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 7kb, 8kb, 9kb, about 0.5 kb to about 1
kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 3 kb, about 3
kb to about 4 kb, about 4 kb to about 5kb, about 5kb to about 6 kb, about 6 kb to about 7 kb,
about 8 kb to about 9 kb, or is at least 10 kb. In such methods, the short length of the
homology arms ses the targeting efficiency as compared to a targeting vector with
longer homology arms. Due to the nature of the Y some which has highly repetitive
sequences, the short arms of the smallTVECs allow for highly specific targeting on the Y
chromosome.
Methods are provided for modifying a target c locus on the Y chromosome
in a cell sing: (a) providing a cell comprising a target c locus on the Y
chromosome comprising a recognition site for a nuclease agent, (b) introducing into the cell a
first targeting vector comprising a first insert polynucleotide d by a first and a second
homology arm corresponding to a first and a second target site; and (c) identifying at least
one cell comprising in its genome the first insert polynucleotide integrated at the target
genomic locus on the Y chromosome. In specific embodiments, the sum total of the first
homology arm and the second homology arm of the targeting vector is about 0.5 kb, 1 kb, 1.5
kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 7kb, 8kb, 9kb, about 0.5 kb to about 1 kb, about 1 kb to about
1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4
kb to about 5kb, about 5kb to about 6 kb, about 6 kb to about 7 kb, about 8 kb to about 9 kb,
or is at least 10 kb or at least 10 kb and less than 150 kb. In some embodiments, a
VEC is employed. In specific ments, an LTVEC is employed. Similar methods
can be performed when targeting a challenging target genomic locus. In one non-limiting
ment, such methods are performed employing the culture media that promotes the
development of XY F0 fertile females disclosed herein and y generating XY F0 fertile
female s. In other instance, the methods described herein are employed to produce a
targeted genetic modification in the Sry gene, as discussed elsewhere herein.
r provided are methods for modifying a target genomic locus on the Y
some in a cell sing: (a) providing a cell comprising a target genomic locus on
the Y chromosome comprising a recognition site for a nuclease agent, (b) introducing into the
cell (i) the nuclease agent, wherein the nuclease agent s a nick or double-strand break
at the first recognition site; and, (ii) a first targeting vector comprising a first insert
polynucleotide flanked by a first and a second homology arm corresponding to a first and a
second target site located in sufficient proximity to the first recognition site; and (c)
identifying at least one cell comprising in its genome the first insert polynucleotide integrated
at the target genomic locus on the Y some. In specific embodiments, the sum total of
the first homology arm and the second homology arm of the targeting vector is about 0.5 kb,
1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 7kb, 8kb, 9kb, about 0.5 kb to about 1 kb, about 1 kb
to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4
kb, about 4 kb to about 5kb, about 5kb to about 6 kb, about 6 kb to about 7 kb, about 8 kb to
about 9 kb, or is at least 10 kb or at least 10 kb and less than 150 kb. In some embodiments, a
smallTVEC is employed. In specific embodiments, an LTVEC is employed. r methods
can be med when targeting a challenging target genomic locus. In one non-limiting
embodiment, such methods are performed employing the culture media that promotes the
development of XY F0 fertile females disclosed herein and thereby ting XY F0 fertile
female animals. In other instance, the methods bed herein are employed to produce a
targeted genetic modification in the Sry gene, as discussed elsewhere herein.
It is recognized that the various methods disclosed herein to generate a targeted
modification in a genomic locus of the Y chromosome (or any challenging genomic locus)
employing a targeting vector, a smallTVEC, or an LTVEC can be performed in any cell type,
and is not limited to an XY pluripotent and/or totipotent cell. Such cell types include, but are
not limited to, a human cell, a man cell, a mammalian cell, non-human mammalian
cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, a fibroblast cell or any other host
cell. Such cells include pluripotent cells, including, for example, d pluripotent stem
(iPS) cells, mouse embryonic stem (ES) cells, rat embryonic stem (ES) cells, human
embryonic (ES) cells, or developmentally restricted human progenitor cells.
Methods are further disclosed to te a large deletion on the Y chromosome
employing any of the various nuclease agents provided herein (e. g., CRISPR gRNAs in
ation with Cas9; ZFNs; or ). Such a deletion on the Y chromosome can be a
deletion of an endogenous nucleic acid sequence. The deletion can range from about 5 kb to
about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about
40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb,
from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to
about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from
about 500 kb to about 600 kb, from about 600 kb to about 700 kb, from about 700 kb to about
800 kb, from about 800 kb to about 900 kb, from about 900 kb to about 1 Mb, from about
500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,
from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb. In one embodiment,
the deletion is greater than 500 kb. In another embodiment, the deletion is from about 500 kb
to about 600 kb. In a specific ment, the deletion is about 500 kb. Such a deletion on
the Y some can be a deletion of any nucleic acid sequence. In one embodiment, the
deletion comprises a gene that is associated with fertility/infertility. The deletion on the Y
chromosome can comprise a deletion of multiple genes. In such methods, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more genes can be d. In specific embodiments, the Kdm5d gene e (K)-
specific demethylase 5d; for example, Entrez Gene ID 20592 (mus musculus)) and/or the
Usp9y gene (ubiquitin specific peptidase 9, y-linked; for e, Entrez Gene ID
107868(mus us)) is targeted for deletion. In other embodiments, the Sry gene is
targeted for deletion.
A. Nuclease Agents and Recognition Sites for Nuclease Agents
The term “recognition site for a nuclease agent” includes a DNA sequence at
which a nick or double-strand break is induced by a nuclease agent. The recognition site for
a nuclease agent can be endogenous (or native) to the cell or the recognition site can be
exogenous to the cell. In specific embodiments, the recognition site is exogenous to the cell
and thereby is not naturally occurring in the genome of the cell. In still further embodiments,
the recognition site is exogenous to the cell and to the polynucleotides of interest that one
desires to be positioned at the target locus. In further embodiments, the exogenous or
endogenous recognition site is present only once in the genome of the host cell. In specific
embodiments, an endogenous or native site that occurs only once within the genome is
identified. Such a site can then be used to design nuclease agents that will produce a nick or
double-strand break at the nous recognition site.
The length of the recognition site can vary, and includes, for example, recognition
sites that are about 30-36 bp for a zinc finger nuclease (ZFN) pair (i.e., about 15-18 bp for
each ZFN), about 36 bp for a ription tor-Like Effector Nuclease (TALEN), or
about 20bp for a /Cas9 guide RNA.
In one embodiment, each monomer of the nuclease agent recognizes a recognition
site of at least 9 nucleotides. In other ments, the recognition site is from about 9 to
about 12 nucleotides in length, from about 12 to about 15 nucleotides in length, from about
to about 18 tides in length, or from about 18 to about 21 nucleotides in length, and
any combination of such subranges (e. g., 9-18 nucleotides). It is recognized that a given
nuclease agent can bind the ition site and cleave that binding site or alternatively, the
nuclease agent can bind to a sequence that is different from the recognition site. Moreover,
the term recognition site comprises both the nuclease agent binding site and the nick/cleavage
site irrespective r the nick/cleavage site is within or outside the nuclease agent g
site. In another variation, the cleavage by the nuclease agent can occur at nucleotide
positions immediately opposite each other to produce a blunt end cut or, in other cases, the
incisions can be staggered to produce single-stranded overhangs, also called “sticky ends”,
which can be either 5' overhangs, or 3' ngs.
Any nuclease agent that induces a nick or double-strand break into a desired
recognition site can be used in the methods and compositions disclosed herein. A naturallyoccurring
or native nuclease agent can be employed so long as the nuclease agent induces a
nick or double-strand break in a desired recognition site. Alternatively, a modified or
engineered nuclease agent can be employed. An “engineered nuclease agent” includes a
nuclease that is engineered (modified or d) from its native form to specifically
recognize and induce a nick or double-strand break in the desired recognition site. Thus, an
engineered nuclease agent can be derived from a native, naturally-occurring nuclease agent or
it can be artificially created or synthesized. The modification of the se agent can be as
little as one amino acid in a protein cleavage agent or one tide in a nucleic acid
cleavage agent. In some embodiments, the engineered nuclease induces a nick or double-
strand break in a recognition site, wherein the recognition site was not a sequence that would
have been recognized by a native (non-engineered or non-modified) nuclease agent.
Producing a nick or double-strand break in a recognition site or other DNA can be ed to
herein as “cutting” or “cleaving” the recognition site or other DNA.
Active ts and fragments of the exemplified recognition sites are also
provided. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given
recognition site, wherein the active variants retain ical activity and hence are capable of
being recognized and cleaved by a nuclease agent in a sequence-specific manner. Assays to
e the double-strand break of a recognition site by a nuclease agent are known in the art
(e. g., TaqMan® qPCR assay, Frendewey D. et (11., Methods in logy, 2010, 476295-
307, which is incorporated by nce herein in its ty).
In specific embodiments, the recognition site is positioned within the
polynucleotide encoding the selection marker. Such a position can be located within the
coding region of the selection marker or within the tory regions, which influence the
sion of the selection marker. Thus, a recognition site of the nuclease agent can be
located in an intron of the selection marker, a promoter, an enhancer, a regulatory , or
any non-protein-coding region of the polynucleotide encoding the selection marker. In
specific embodiments, a nick or double-strand break at the recognition site disrupts the
activity of the selection marker. s to assay for the presence or absence of a functional
selection marker are known.
In one ment, the se agent is a Transcription Activator-Like Effector
Nuclease (TALEN). TAL effector nucleases are a class of sequence-specific ses that
can be used to make double-strand breaks at specific target sequences in the genome of a
prokaryotic or eukaryotic organism. TAL effector nucleases are created by fusing a native or
engineered transcription activator-like (TAL) effector, or functional part thereof, to the
catalytic domain of an endonuclease, such as, for example, Fold. The unique, modular TAL
effector DNA binding domain allows for the design of proteins with potentially any given
DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases
can be engineered to recognize specific DNA target sites and thus, used to make double-
strand breaks at desired target sequences. See, ; Morbitzer et a1. (2010)
PNAS 10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et
a1. Genetics (2010) 186:757-761; Li et a1. (2010) Nuc. Acids Res. (2010)
doi:10.1093/nar/gkq704; and Miller et a1. (2011) Nature Biotechnology 29:143—148; all of
which are herein incorporated by reference.
Examples of suitable TAL nucleases, and methods for preparing suitable TAL
nucleases, are disclosed, e. g., in US Patent Application No. 2011/0239315 A1, 2011/0269234
A1, 2011/0145940 A1, 232410 A1, 2005/0208489 A1, 2005/0026157 A1,
2005/0064474 A1, 2006/0188987 A1, and 063231 A1 (each hereby incorporated by
reference). In various embodiments, TAL effector nucleases are engineered that cut in or
near a target nucleic acid sequence in, e.g., a locus of interest or a genomic locus of interest,
wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting
vector. The TAL nucleases le for use with the various methods and compositions
ed herein include those that are specifically designed to bind at or near target c
acid sequences to be modified by targeting vectors as described herein.
In one embodiment, each r of the TALEN comprises 33-35 TAL repeats
that recognize a single base pair via two hypervariable residues. In one embodiment, the
nuclease agent is a chimeric protein comprising a TAL repeat-based DNA binding domain
operably linked to an independent nuclease. In one embodiment, the independent nuclease is
a FokI endonuclease. In one embodiment, the nuclease agent comprises a first TAL-repeat-
based DNA binding domain and a second TAL-repeat-based DNA binding domain, wherein
each of the first and the second TAL-repeat-based DNA binding domain is operably linked to
a FokI nuclease, wherein the first and the second TAL-repeat-based DNA binding domain
recognize two uous target DNA sequences in each strand of the target DNA sequence
separated by a spacer ce of varying length (12-20 bp), and wherein the FokI nuclease
ts dimerize to create an active nuclease that makes a double strand break at a target
sequence.
The nuclease agent employed in the various methods and compositions disclosed
herein can r comprise a zinc-finger nuclease (ZFN). In one embodiment, each
monomer of the ZFN comprises 3 or more zinc finger-based DNA binding domains, wherein
each zinc finger-based DNA binding domain binds to a 3bp subsite. In other embodiments,
the ZFN is a chimeric protein sing a zinc finger-based DNA binding domain operably
linked to an independent nuclease. In one embodiment, the ndent endonuclease is a
FokI clease. In one embodiment, the nuclease agent comprises a first ZFN and a
second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a FokI
nuclease t, wherein the first and the second ZFN recognize two contiguous target DNA
sequences in each strand of the target DNA sequence separated by about 5-7 bp spacer, and
wherein the Fokl nuclease subunits dimerize to create an active nuclease to make a double
strand break. See, for example, US20060246567; US20080182332; US20020081614;
US20030021776; WO/2002/057308A2; US20130123484; 0291048;
WO/2011/017293A2; and Gaj et al. (2013) Trends in hnology, 31(7):397-405 each of
which is herein incorporated by reference.
In still another ment, the nuclease agent is a meganuclease.
Meganucleases have been classified into four families based on conserved sequence motifs,
the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These
motifs participate in the coordination of metal ions and hydrolysis of odiester bonds.
Meganucleases are notable for their long ition sites, and for tolerating some sequence
rphisms in their DNA substrates. Meganuclease domains, structure and on are
known, see for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38: 199-
248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol
Life Sci 4-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat
Struct Biol 9:764. In some examples a naturally occurring variant, and/or engineered
derivative meganuclease is used. Methods for modifying the kinetics, cofactor interactions,
sion, optimal conditions, and/or recognition site specificity, and screening for activity
are known, see for example, Epinat et al., (2003) Nucleic Acids Res 31:2952-62; Chevalier et
al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et
al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) JMol Biol 342:31-41; Rosen
et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res
33:el78; Smith et al., (2006) c Acids Res 9; Gruen et al., (2002) Nucleic Acids
Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:el54; WO2005105989;
WO2003078619; WO2006097854; WO2006097853; WO2006097784; and WO2004031346.
Any meganuclease can be used herein, including, but not limited to, I-Scel, I-
SceII, I-SceIII, I—SceIV, I-SceV, I—SceVI, I-SceVII, I-Ceul, I-CeuAIIP, I—Crel, I-CrepsbIP, I-
CrepsbIIP, I—CrepsbIIIP, I-CrepsbIVP, I-Tlil, I-Ppol, PI-Pspl, F-SceI, F-SceII, , F-
TevI, F—TevII, I-Amal, I-Anil, I—Chul, I—Cmoel, I-Cpal, I-CpaII, I-Csml, I-Cvul, I-CvuAIP,
, I-DdiII, I—DirI, I—Dmol, I-HmuI, I—HmuII, I-HsNIP, I-LlaI, I—Msol, I-NaaI, , I-
NcIIP, I-NngP, I-NitI, I-NjaI, 36IP, I-Pakl, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, 1-
PngP, I-PobIP, I-Porl, I-PorIIP, I-PprP, I-SpBetaIP, I—Scal, P, I-SneIP, I-SpomI, I-
SpomCP, I-SpomIP, I-SpomlIP, P, I-Ssp6803l, I-SthPhiJP, hiST3P, I-
SthPhiSTe3bP, I-TdeIP, I-TevI, I-Tevll, I—TevIII, I-UarAP, I—UarHGPAIP, I-UarHGPAl3P,
I-VinIP, I-ZbiIP, PI-Mtul, PI—MtuHIP HIIP, PI-Pful, PI-PfuII, PI-Pkol, PI—Pkoll, PI-
Rma43812IP, PI—SpBetaIP, I, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, II, or any
active variants or fragments thereof.
In one ment, the meganuclease recognizes double-stranded DNA
ces of 12 to 40 base pairs. In one embodiment, the meganuclease recognizes one
perfectly matched target sequence in the genome. In one embodiment, the meganuclease is a
homing nuclease. In one embodiment, the homing nuclease is a LAGLIDADG family of
homing nuclease. In one embodiment, the LAGLIDADG family of homing nuclease is
selected from I-SceI, I-CreI, and I-Dmol.
Nuclease agents can further comprise restriction endonucleases, which include
Type I, Type II, Type III, and Type IV endonucleases. Type I and Type III restriction
endonucleases recognize specific recognition sites, but typically cleave at a variable position
from the nuclease binding site, which can be hundreds of base pairs away from the cleavage
site (recognition site). In Type II systems the restriction activity is independent of any
methylase activity, and ge typically occurs at specific sites within or near to the
binding site. Most Type II enzymes cut palindromic sequences, however Type IIa enzymes
recognize non-palindromic recognition sites and cleave outside of the recognition site, Type
IIb enzymes cut sequences twice with both sites outside of the recognition site, and Type IIs
enzymes recognize an tric recognition site and cleave on one side and at a defined
ce of about 1-20 nucleotides from the recognition site. Type IV restriction enzymes
target methylated DNA. Restriction enzymes are further described and classified, for example
in the REBASE database (webpage at rebase.neb.com; Roberts et al., (2003) Nucleic Acids
Res 31 0), s et al., (2003) Nucleic Acids Res 31 :1805-12, and Belfort et al.,
(2002) in Mobile DNA 11, pp. 761-783, Eds. Craigie et al., (ASM Press, gton, DC).
The nuclease agent employed in the various s and compositions can
also comprise a CRISPR/Cas system. Such systems can employ a Cass? nuclease, which in
some instances, is cation—Optimized fer the desired cell type in which it is to he expressed.
The system further s a fused chNA~trachNA construct that functions with the
coden—optimized Cas9. This single RNA is Often referred to as a guide RNA or gRNA,
Within a gRNA, the chNA perticn is identified as the ‘target sequence" for the given
recognition site and the A is often referred to as the ‘scaffold’. This system has been
shown to functien in a variety of euliar‘yotic and prokaryetic cells, Briefly, a short DNA
fragment ning the target sequence is inserted into a guide RNA expression plasmid.
The gRNA expression plasmid ises the target sequence (in some einbcdinients around
’20 nuclentides), a foiin of the A sequence (the scaffold) as well as a suitable
promoter that is active in the cell and necessary elements for proper processing in enharyotic
cells. Many of the s rely on custom, complementary oligos that are ed to form a
double stranded DNA and then cloned into the gRNA expression plasmid. The gRNA
expression cassette and the Case”) expression cassette are then introduced into the cell. See,
for example, Mali P et al. (2013) Science 2013 Feb 15; 339 (6121):823-6; Jinek M et al.
Science 2012 Aug 17;337(6096):816-21; Hwang WY et al. Nat Biotechnol 2013
Mar;31(3):227-9; Jiang W et al. Nat Biotechnol 2013 Mar;31(3):233-9; and, Cong L et al.
e 2013 Feb 15;339(6121):819-23, each of which is herein incorporated by reference.
The methods and compositions disclosed herein can utilize Clustered
Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)
systems or components of such systems to modify a genome within a cell. CRISPR/Cas
systems include transcripts and other ts involved in the expression of, or directing the
activity of, Cas genes. A CRISPR/Cas system can be a type I, a type II, or a type 111 system.
The methods and compositions disclosed herein employ CRISPR/Cas systems by utilizing
CRISPR complexes (comprising a guide RNA (gRNA) xed with a Cas protein) for
site-directed cleavage of nucleic acids.
Some CRISPR/Cas systems used in the methods disclosed herein are non-
naturally occurring. A “non-naturally occurring” system es anything indicating the
involvement of the hand of man, such as one or more components of the system being altered
or mutated from their naturally occurring state, being at least substantially free from at least
one other component with which they are naturally associated in nature, or being associated
with at least one other ent with which they are not naturally ated. For example,
some CRISPR/Cas systems employ turally occurring CRISPR complexes comprising
a gRNA and a Cas protein that do not naturally occur er.
(i) A. Cas RNA-Guided cleases
Cas proteins generally comprise at least one RNA recognition or binding
domain. Such domains can ct with guide RNAs (gRNAs, bed in more detail
below). Cas proteins can also comprise nuclease domains (e.g., DNase or RNase domains),
DNA binding domains, helicase domains, protein-protein interaction domains, dimerization
domains, and other domains. A nuclease domain possesses catalytic activity for nucleic acid
cleavage. Cleavage includes the breakage of the covalent bonds of a nucleic acid molecule.
Cleavage can produce blunt ends or staggered ends, and it can be single-stranded or double-
stranded.
Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5,
Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al or
, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl
Csx12), Cale, Caled, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB),
Cse3 (CasE), Cse4 , Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,
Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX,
Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions
Cas proteins can be from a type II CRISPR/Cas system. For example, the Cas
n can be a Cas9 protein or be derived from a Cas9 protein. Cas9 proteins typically
share four key motifs with a conserved architecture. Motifs l, 2, and 4 are Rqu-like motifs,
and motif 3 is an HNH motif. The Cas9 protein can be from, for example, Streptococcus
pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus,
Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, omyces viridochromogenes,
Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium ,
AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens,
Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla
marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.,
Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.,
Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus
Desulforudis, Clostridium botulinum, Clostridium diflicile, Finegoldia magna,
aerobius philus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus,
Acidithiobacillusferrooxidans, Allochromatium vinosum, bacter sp., Nitrosococcus
halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter
racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,
Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.,
Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, sipho africanus, or
Acaryochloris marina. Additional examples of the Cas9 family members are described in
WC 2014/131833, herein incorporated by nce in its ty. Cas9 protein from S.
es or derived therefrom is a preferred enzyme. Cas9 protein from S. pyogenes is
assigned SwissProt ion number Q99ZW2.
Cas proteins can be wild type ns (i.e., those that occur in nature),
modified Cas proteins (i.e., Cas protein variants), or nts of wild type or modified Cas
proteins. Cas proteins can also be active ts or fragments of wild type or modified Cas
proteins. Active variants or fragments can comprise at least 80%, 85%, 90%, 91%, 92%,
2015/038001
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the wild type or
modified Cas protein or a portion thereof, wherein the active variants retain the ability to cut
at a desired cleavage site and hence retain nick-inducing or double-strand-break-inducing
activity. Assays for nick-inducing or -strand-break-inducing activity are known and
generally measure the overall activity and specificity of the Cas protein on DNA substrates
ning the cleavage site.
Cas proteins can be modified to increase or decrease nucleic acid binding
affinity, nucleic acid binding specificity, and/or enzymatic ty. Cas proteins can also be
modified to change any other activity or property of the protein, such as stability. For
example, one or more se domains of the Cas n can be modified, deleted, or
inactivated, or a Cas protein can be truncated to remove domains that are not ial for the
on of the protein or to optimize (e. g., enhance or reduce) the activity of the Cas protein.
Some Cas proteins comprise at least two nuclease domains, such as DNase
domains. For example, a Cas9 protein can comprise a ke se domain and an
HNH-like nuclease domain. The Rqu and HNH domains can each cut a different strand of
double-stranded DNA to make a double-stranded break in the DNA. See, e. g., Jinek et al.
(2012) Science 337:816-821, hereby incorporated by reference in its entirety.
One or both of the nuclease domains can be deleted or mutated so that they are
no longer onal or have reduced nuclease activity. If one of the nuclease domains is
deleted or mutated, the resulting Cas protein (e. g., Cas9) can be referred to as a nickase and
can generate a single-strand break at a CRISPR RNA ition sequence within a -
stranded DNA but not a double-strand break (i.e., it can cleave the complementary strand or
the non-complementary , but not both). If both of the nuclease domains are deleted or
mutated, the resulting Cas protein (e. g., Cas9) will have a reduced ability to cleave both
strands of a double-stranded DNA. An example of a mutation that converts Cas9 into a
nickase is a DlOA (aspartate to alanine at position 10 of Cas9) mutation in the Rqu domain
of Cas9 from S. pyogenes. Likewise, H939A (histidine to alanine at amino acid position 839)
or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from
S. es can convert the Cas9 into a nickase. Other es of mutations that convert
Cas9 into a nickase include the corresponding mutations to Cas9 from S. thermophilus. See,
e. g., Sapranauskas et a1. (2011) Nucleic Acids Research 39:9275-9282 and WC 2013/141680,
each of which is herein incorporated by nce in its entirety. Such mutations can be
generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or
total gene synthesis. Examples of other mutations creating nickases can be found, for
e, in WO/2013/176772Al and WO/2013/142578Al, each of which is herein
incorporated by reference.
Cas proteins can also be fusion proteins. For example, a Cas protein can be
fused to a cleavage , an epigenetic cation domain, a transcriptional activation
domain, or a transcriptional sor domain. See , incorporated herein by
reference in its entirety. Cas proteins can also be fused to a heterologous polypeptide
providing increased or decreased stability. The fused domain or logous polypeptide
can be located at the N-terminus, the C-terminus, or internally within the Cas protein.
A Cas protein can be fused to a heterologous polypeptide that provides for
subcellular localization. Such heterologous peptides include, for example, a nuclear
localization signal (NLS) such as the SV40 NLS for targeting to the nucleus, a mitochondrial
localization signal for ing to the mitochondria, an ER retention signal, and the like.
See, e.g., Lange et a1. (2007) J. Biol. Chem. 282:5101-5105. Such subcellular localization
signals can be d at the N-terminus, the C-terminus, or anywhere within the Cas protein.
An NLS can se a stretch of basic amino acids, and can be a monopartite sequence or a
bipartite sequence.
Cas proteins can also be linked to a enetrating . For example, the
cell-penetrating domain can be derived from the HIV-1 TAT protein, the TLM cell-
penetrating motif from human hepatitis B virus, MPG, Pep-l, VP22, a cell penetrating
peptide from Herpes simplex virus, or a polyarginine peptide sequence. See, for e,
, herein incorporated by reference in its entirety. The cell-penetrating
domain can be located at the N-terminus, the C-terminus, or anywhere within the Cas protein.
Cas proteins can also comprise a heterologous polypeptide for ease of tracking
or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples
of fluorescent proteins include green fluorescent proteins (e. g., GFP, GFP-2, ,
turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, , AceGFP,
ZsGreenl), yellow fluorescent proteins (e. g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP,
ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFPZ, Azurite, mKalamal, GFPuv,
Sapphire, T-sapphire), cyan fluorescent proteins (e.g. eCFP, an, CyPet, AmCyanl,
ishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer,
mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, ,
AsRed2, eqFP6ll, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (mOrange,
mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, thomato), and any
other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST),
chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), p01y(NANP),
tandem affinity purification (TAP) tag, myc, ACV5, AU1
, AU5, E, ECS, E2, FLAG,
hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1 , T7,
V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.
Cas proteins can be ed in any form. For example, a Cas protein can be
provided in the form of a protein, such as a Cas n complexed with a gRNA.
Alternatively, a Cas protein can be ed in the form of a nucleic acid encoding the Cas
protein, such as an RNA (e. g., messenger RNA (mRNA)) or DNA. Optionally, the nucleic
acid encoding the Cas protein can be codon optimized for efficient translation into protein in
a particular cell or organism.
Nucleic acids encoding Cas proteins can be stably integrated in the genome of
the cell and operably linked to a promoter active in the cell. Alternatively, nucleic acids
encoding Cas proteins can be operably linked to a promoter in an expression uct.
Expression constructs e any nucleic acid constructs capable of directing expression of a
gene or other nucleic acid sequence of interest (e.g., a Cas gene) and which can transfer such
a nucleic acid sequence of interest to a target cell. ers that can be used in an
expression construct include, for example, promoters active in a pluripotent rat, eukaryotic,
mammalian, non-human mammalian, human, rodent, mouse, or hamster cell. Examples of
other promoters are described elsewhere herein.
(ii) B. Guide RNAs (gRNAs)
A “guide RNA” or “gRNA” includes an RNA molecule that binds to a Cas
protein and targets the Cas protein to a ic location within a target DNA. Guide RNAs
can se two segments: a argeting segment” and a “protein-binding segment.”
“Segment” includes a segment, section, or region of a molecule, such as a contiguous stretch
of nucleotides in an RNA. Some gRNAs comprise two separate RNA molecules: an
“activator-RNA” and a “targeter-RNA.” Other gRNAs are a single RNA molecule (single
RNA polynucleotide), which can also be called a e-molecule gRNA,” a “single-guide
RNA,” or an “ngNA.” See, e.g., WO/2013/176772A1, WO/2014/065596A1,
WO/2014/089290A1, WO/2014/093622A2, WO/2014/099750A2, WO/2013142578A1, and
WC 2014/131833A1, each of which is herein incorporated by reference. The terms “guide
RNA” and “gRNA” e both double-molecule gRNAs and single-molecule gRNAs.
An exemplary two-molecule gRNA comprises a chNA-like (“CRISPR RNA”
or “targeter-RNA” or “chNA” or “chNA ”) molecule and a ponding trachNA-
like (“trans-acting CRISPR RNA” or ator-RNA” or “trachNA” or “scaffold”)
molecule. A chNA comprises both the DNA-targeting segment (single-stranded) of the
gRNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-
binding segment of the gRNA.
A corresponding trachNA (activator-RNA) comprises a stretch of nucleotides
that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA.
A stretch of nucleotides of a chNA are complementary to and hybridize with a stretch of
nucleotides of a trachNA to form the dsRNA duplex of the protein-binding domain of the
gRNA. As such, each chNA can be said to have a ponding trachNA.
The chNA and the corresponding trachNA ize to form a gRNA. The
chNA additionally provides the single-stranded DNA-targeting segment that hybridizes to a
CRISPR RNA recognition sequence. If used for modification within a cell, the exact
sequence of a given chNA or trachNA molecule can be designed to be ic to the
species in which the RNA molecules will be used. See, for e, Mali et al. (2013)
Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat.
Biotechnol. -229; Jiang et al. (2013) Nat. Biotechnol. 31 :233-239; and Cong et al.
(2013) Science 339:819-823, each of which is herein incorporated by reference.
The DNA-targeting t (chNA) of a given gRNA comprises a
nucleotide sequence that is complementary to a sequence in a target DNA. The DNA-
targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via
hybridization (i.e., base g). As such, the nucleotide sequence of the rgeting
segment may vary and determines the location within the target DNA with which the gRNA
and the target DNA will interact. The DNA-targeting segment of a subject gRNA can be
modified to hybridize to any desired sequence within a target DNA. Naturally occurring
chNAs differ depending on the Cas9 system and organism but often contain a targeting
segment of n 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a
length of between 21 to 46 nucleotides (see, e. g., WO2014/131833). In the case of S.
pyogenes, the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long.
The 3’ located DR is complementary to and hybridizes with the ponding trachNA,
which in turn binds to the Cas9 protein.
The rgeting segment can have a length of from about 12 tides to
about 100 nucleotides. For example, the DNA-targeting segment can have a length of from
about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to
about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12
nt to about 20 nt, or from about 12 nt to about 19 nt. Alternatively, the DNA-targeting
t can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt,
from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40
nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about
60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to
about 90 nt, from about 19 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20
nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about
nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to about 60 nt, from
about 20 nt to about 70 nt, from about 20 nt to about 80 nt, from about 20 nt to about 90 nt, or
from about 20 nt to about 100 nt.
The nucleotide sequence of the DNA-targeting segment that is complementary
to a nucleotide sequence (CRISPR RNA recognition sequence) of the target DNA can have a
length at least about 12 nt. For example, the DNA-targeting sequence (i.e., the sequence
within the DNA-targeting segment that is complementary to a CRISPR RNA recognition
sequence within the target DNA) can have a length at least about 12 nt, at least about 15 nt, at
least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about
nt, at least about 35 nt, or at least about 40 nt. Alternatively, the rgeting sequence
can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about
50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to
about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12
nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about
19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from
about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt,
from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30
nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about
45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the
DNA-targeting sequence can have a length of at about 20 nt.
TrachNAs can be in any form (e.g., full-length trachNAs or active partial
trachNAs) and of varying lengths. They can include primary ripts or processed forms.
For example, trachNAs (as part of a -guide RNA or as a separate molecule as part of a
two-molecule gRNA) may comprise or t of all or a portion of a wild-type trachNA
sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more
nucleotides of a wild-type trachNA sequence). Examples of wild-type trachNA sequences
from S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and 65-nucleotide
versions. See, for example, Deltcheva et a1. (2011) Nature 471 :602-607; ,
each of which is incorporated herein by reference in their entirety. Examples of trachNAs
within single-guide RNAs ) include the trachNA segments found within +48, +54,
+67, and +85 ns of ngNAs, where “+n” indicates that up to the +n nucleotide of wild-
type trachNA is included in the ngNA. See US 8,697,359, incorporated herein by
reference in its entirety.
The percent mentarity between the DNA-targeting sequence and the
CRISPR RNA ition sequence within the target DNA can be at least 60% (e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 98%, at least 99%, or 100%). The percent complementarity between the DNA-
targeting sequence and the CRISPR RNA recognition sequence within the target DNA can be
at least 60% over about 20 contiguous nucleotides. As an example, the percent
complementarity between the DNA-targeting sequence and the CRISPR RNA recognition
sequence within the target DNA is 100% over the 14 contiguous nucleotides at the 5’ end of
the CRISPR RNA recognition sequence within the complementary strand of the target DNA
and as low as 0% over the remainder. In such a case, the DNA-targeting sequence can be
considered to be 14 nucleotides in length. As another e, the percent complementarity
between the DNA-targeting sequence and the CRISPR RNA recognition sequence within the
target DNA is 100% over the seven contiguous tides at the 5’ end of the CRISPR RNA
recognition sequence within the complementary strand of the target DNA and as low as 0%
over the remainder. In such a case, the DNA-targeting sequence can be considered to be 7
nucleotides in length.
The protein-binding segment of a gRNA can comprise two stretches of
nucleotides that are mentary to one another. The complementary nucleotides of the
protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The
protein-binding segment of a subject gRNA interacts with a Cas n, and the gRNA
s the bound Cas n to a specific nucleotide sequence within target DNA via the
DNA-targeting t.
Guide RNAs can include modifications or sequences that provide for
additional desirable features (e. g., modified or ted ity; subcellular targeting;
tracking with a fluorescent label; a binding site for a protein or protein complex; and the
like). Examples of such modifications include, for example, a 5' cap (e. g., a 7-
methylguanylate cap (m7G)); a 3' enylated tail (i.e., a 3' poly(A) tail); a riboswitch
sequence (e. g., to allow for regulated stability and/or regulated accessibility by proteins
and/or protein complexes); a stability control sequence; a sequence that forms a dsRNA
duplex (i.e., a hairpin)); a modification or sequence that targets the RNA to a subcellular
on (e. g., nucleus, mitochondria, chloroplasts, and the like); a cation or sequence
that provides for tracking (e. g., direct conjugation to a fluorescent molecule, conjugation to a
moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection,
and so forth); a modification or sequence that provides a binding site for proteins (e. g.,
ns that act on DNA, including transcriptional activators, transcriptional repressors,
DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone
deacetylases, and the like); and ations thereof.
Guide RNAs can be provided in any form. For example, the gRNA can be
provided in the form of RNA, either as two molecules (separate chNA and trachNA) or as
one molecule (ngNA), and optionally in the form of a x with a Cas protein. The
gRNA can also be provided in the form of DNA encoding the RNA. The DNA encoding the
gRNA can encode a single RNA molecule (ngNA) or separate RNA molecules (e.g.,
separate chNA and trachNA). In the latter case, the DNA encoding the gRNA can be
provided as separate DNA molecules encoding the chNA and trachNA, respectively.
DNAs encoding gRNAs can be stably integrated in the genome of the cell and
operably linked to a promoter active in the cell. Alternatively, DNAs encoding gRNAs can be
operably linked to a promoter in an expression construct. Such promoters can be , for
example, in a pluripotent rat, eukaryotic, mammalian, non-human mammalian, human,
rodent, mouse, or r cell. In some instances, the promoter is an RNA polymerase III
promoter, such as a human U6 promoter, a rat U6 polymerase III promoter, or a mouse U6
polymerase III promoter. Examples of other promoters are described elsewhere .
Alternatively, gRNAs can be prepared by various other s. For
example, gRNAs can be ed by in vitro transcription using, for example, T7 RNA
polymerase (see, for example, and ). Guide RNAs can
also be a synthetically produced molecule prepared by chemical synthesis.
(iii) C. CRISPR RNA Recognition ces
The term "CRISPR RNA recognition sequence" includes nucleic acid
sequences present in a target DNA to which a DNA-targeting segment of a gRNA will bind,
provided sufficient conditions for binding exist. For example, CRISPR RNA recognition
sequences e sequences to which a guide RNA is designed to have mentarity,
where hybridization between a CRISPR RNA recognition sequence and a DNA targeting
sequence promotes the formation of a CRISPR x. Full complementarity is not
necessarily ed, provided there is sufficient complementarity to cause hybridization and
promote formation of a CRISPR complex. CRISPR RNA recognition sequences also include
ge sites for Cas proteins, described in more detail below. A CRISPR RNA recognition
sequence can comprise any polynucleotide, which can be located, for e, in the nucleus
or cytoplasm of a cell or within an organelle of a cell, such as a mitochondrion or chloroplast.
The CRISPR RNA ition sequence within a target DNA can be targeted
by (i.e., be bound by, or hybridize with, or be mentary to) a Cas protein or a gRNA.
Suitable DNA/RNA binding conditions include physiological conditions normally present in
a cell. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free )
are known in the art (see, e. g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook
et al., Harbor tory Press 2001)). The strand of the target DNA that is complementary
to and hybridizes with the Cas protein or gRNA can be called the "complementary ,"
and the strand of the target DNA that is complementary to the "complementary strand" (and
is therefore not complementary to the Cas protein or gRNA) can be called
"noncomplementary strand" or "template strand.”
The Cas n can cleave the nucleic acid at a site within or outside of the
nucleic acid sequence present in the target DNA to which the DNA-targeting t of a
gRNA will bind. The “cleavage site” includes the position of a nucleic acid at which a Cas
protein produces a single-strand break or a double-strand break. For example, formation of a
CRISPR complex (comprising a gRNA hybridized to a CRISPR RNA recognition sequence
and xed with a Cas protein) can result in cleavage of one or both strands in or near
(e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid
sequence present in a target DNA to which a DNA-targeting t of a gRNA will bind.
If the cleavage site is outside of the nucleic acid sequence to which the DNA-targeting
segment of the gRNA will bind, the cleavage site is still considered to be within the “CRISPR
RNA recognition sequence.” The cleavage site can be on only one strand or on both strands
of a nucleic acid. Cleavage sites can be at the same position on both strands of the nucleic
acid (producing blunt ends) or can be at different sites on each strand (producing staggered
ends). Staggered ends can be ed, for example, by using two Cas proteins, each of
which produces a single-strand break at a different cleavage site on each strand, y
producing a -strand break. For example, a first nickase can create a single-strand
break on the first strand of double-stranded DNA (dsDNA), and a second nickase can create a
single-strand break on the second strand of dsDNA such that overhanging sequences are
created. In some cases, the CRISPR RNA recognition sequence of the e on the first
strand is separated from the CRISPR RNA recognition sequence of the nickase on the second
strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000
base pairs.
] Site-specific cleavage of target DNA by Cas9 can occur at locations
determined by both (i) base-pairing complementarity between the gRNA and the target DNA
and (ii) a short motif, called the protospacer adjacent motif (PAM), in the target DNA. The
PAM can flank the CRISPR RNA recognition sequence. Optionally, the CRISPR RNA
recognition sequence can be flanked by the PAM. For example, the cleavage site of Cas9 can
be about 1 to about 10 or about 2 to about 5 base pairs (e. g., 3 base pairs) am or
downstream of the PAM sequence. In some cases (e. g., when Cas9 from S. pyogenes or a
closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-
N1GG-3', where N1is any DNA nucleotide and is ately 3' of the CRISPR RNA
recognition ce of the non-complementary strand of the target DNA. As such, the PAM
sequence of the complementary strand would be 5'-CC N2-3', where N2 is any DNA
nucleotide and is immediately 5' of the CRISPR RNA recognition sequence of the
mentary strand of the target DNA. In some such cases, N1 and N2 can be
complementary and the N1- N2 base pair can be any base pair (e. g., N1=C and N2=G; N1=G
and N2=C; N1=A and N2=T, N1=T, and N2=A).
Examples of CRISPR RNA recognition sequences include a DNA sequence
complementary to the rgeting segment of a gRNA, or such a DNA sequence in
addition to a PAM sequence. For example, the target motif can be a 20-nucleotide DNA
ce immediately preceding an NGG motif recognized by a Cas protein (see, for
example, WC 2014/165825). The guanine at the 5’ end can facilitate ription by RNA
polymerase in cells. Other examples of CRISPR RNA recognition sequences can include two
guanine nucleotides at the 5’ end (e. g., GGNZONGG; SEQ ID NO: 9) to facilitate efficient
transcription by T7 polymerase in vitro. See, for example, .
The CRISPR RNA recognition sequence can be any nucleic acid sequence
nous or exogenous to a cell. The CRISPR RNA recognition sequence can be a
sequence coding a gene product (e. g., a n) or a non-coding sequence (e. g., a regulatory
sequence) or can include both.
In one ment, the target sequence is immediately flanked by a Protospacer
Adjacent Motif (PAM) sequence. In one embodiment, the locus of interest comprises the
nucleotide sequence of SEQ ID NO: 1. In one embodiment, the gRNA comprises a third
nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) RNA (chNA) and a trans-activating CRISPR RNA (trachNA). In another
embodiment, the genome of the otent rat cell comprises a target DNA region
complementary to the target sequence. In some such methods, the Cas protein is Cas9. In
some embodiments, the gRNA comprises (a) the chimeric RNA of the nucleic acid sequence
of SEQ ID NO: 2; or (b) the chimeric RNA of the nucleic acid sequence of SEQ ID NO: 3.
In some such methods, the chNA ses the sequence set forth in SEQ ID NO: 4, SEQ
ID NO: 5, or SEQ ID NO: 6. In some such s, the trachNA comprises the sequence
set forth in SEQ ID NO: 7 or SEQ ID NO: 8.
] Active variants and fragments of nuclease agents (i.e. an engineered nuclease
agent) are also provided. Such active variants can comprise at least 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
the native nuclease agent, wherein the active variants retain the ability to cut at a desired
recognition site and hence retain nick or double-strand-break-inducing activity. For example,
any of the nuclease agents described herein can be modified from a native endonuclease
sequence and designed to recognize and induce a nick or -strand break at a recognition
site that was not recognized by the native nuclease agent. Thus, in some embodiments, the
engineered nuclease has a specificity to induce a nick or double-strand break at a recognition
site that is ent from the corresponding native nuclease agent recognition site. Assays for
nick or double-strand-break-inducing activity are known and generally e the l
ty and specificity of the endonuclease on DNA substrates containing the recognition
site.
The nuclease agent may be introduced into the cell by any means known in the art.
The polypeptide encoding the nuclease agent may be directly introduced into the cell.
Alternatively, a polynucleotide ng the se agent can be introduced into the cell.
When a polynucleotide encoding the nuclease agent is introduced into the cell, the nuclease
agent can be transiently, conditionally or constitutive expressed within the cell. Thus, the
polynucleotide encoding the nuclease agent can be contained in an sion cassette and be
operably linked to a conditional promoter, an inducible promoter, a constitutive promoter, or
a tissue-specific promoter. Such promoters of interest are discussed in further detail
elsewhere herein. Alternatively, the nuclease agent is introduced into the cell as an mRNA
encoding a nuclease agent.
] In specific embodiments, the polynucleotide encoding the se agent is stably
integrated in the genome of the cell and operably linked to a promoter active in the cell. In
other embodiments, the polynucleotide encoding the nuclease agent is in the same targeting
vector comprising the insert polynucleotide, while in other instances the polynucleotide
WO 00805
encoding the nuclease agent is in a vector or a d that is separate from the ing
vector comprising the insert polynucleotide.
When the se agent is provided to the cell through the introduction of a
polynucleotide encoding the nuclease agent, such a polynucleotide encoding a nuclease agent
can be modified to substitute codons having a higher frequency of usage in the cell of
interest, as compared to the naturally occurring polynucleotide sequence encoding the
nuclease agent. For e the polynucleotide encoding the nuclease agent can be modified
to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic
cell of interest, including a ial cell, a yeast cell, a human cell, a non-human cell, a
mammalian cell, a rodent cell, a mouse cell, a rat cell or any other host cell of interest, as
compared to the naturally occurring polynucleotide sequence.
B. ing the CRISPR/Cas System in Combination with a Large Targeting
Vector (LTVEC) or a Small Targeting Vector (SmallTVEC) to Modify a Challenging
Genomic Loci or a Y Chromosome Locus
Non-limiting methods for modifying a challenging genomic locus or a locus of the
Y some comprise exposing the chromosome (i.e., the Y chromosome) to a Cas protein
and a CRISPR RNA in the presence of a large targeting vector (LTVEC) comprising a
nucleic acid sequence of at least 10 kb, n following exposure to the Cas protein, the
CRISPR RNA, and the LTVEC, the chromosome (i.e., the Y chromosome) is modified to
n at least 10 kb nucleic acid sequence.
The method can employ any of the LTVECs or smallTVECs described herein. In
non-limiting embodiments, the LTVEC or smallTVEC comprises a nucleic acid sequence of
at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at
least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, or at least 200 kb. In other
embodiments, the sum total of 5’ and 3’ homology arms of the LTVEC is from about 10 kb
to about 150 kb, about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40
kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from
about 100 kb to about 120 kb, or from about 120 kb to 150 kb. In another embodiment, the
sum total of 5’ and 3’ homology arms of the smallTVEC is about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3
kb, 4 kb, 5kb, 6kb, 7kb, 8kb, 9kb, about 0.5 kb to about 1 kb, about 1 kb to about 1.5 kb,
about 1.5 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to
about 5kb, about 5kb to about 6 kb, about 6 kb to about 7 kb, about 8 kb to about 9 kb, or is at
least 10 kb.
Further provided is a method for modifying a challenging target locus or a target
genomic locus on the Y chromosome, comprising: (a) providing a mammalian cell
comprising the challenging target locus or a target genomic locus on the Y some,
wherein the target c locus comprises a guide RNA (gRNA) target sequence; (b)
introducing into the mammalian cell: (i) a large targeting vector (LTVEC) comprising a first
nucleic acid flanked with targeting arms homologous to the target c locus, wherein the
LTVEC is at least 10 kb; (ii) a first expression construct comprising a first promoter operably
linked to a second nucleic acid encoding a Cas protein, and (iii) a second expression construct
comprising a second promoter operably linked to a third nucleic acid encoding a guide RNA
(gRNA) sing a tide sequence that hybridizes to the gRNA target sequence and a
trans-activating CRISPR RNA (trachNA), wherein the first and the second promoters are
active in the mammalian cell; and, (c) identifying a modified mammalian cell comprising a
targeted genetic modification at the challenging target c locus or at the target genomic
locus on the Y chromosome. In specific embodiments, the first and the second expression
constructs are on a single nucleic acid molecule. In other embodiments, the target genomic
locus of the Y chromosomes is the Sry locus.
As ed above, in one ment, the Cas protein can comprise a Cas9
protein. In another embodiment, the gRNA target sequence is immediately flanked by a
Protospacer Adjacent Motif (PAM) sequence.
The method can employ any of the LTVECs or smallTVECs described herein. In
non-limiting ments, the LTVEC or smallTVEC is at least 0.5 kb, at least 1 kb, at least
kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30kb, at least 40 kb, at least 50 kb, at
least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, or at
least 200 kb. In other embodiments, the sum total of 5’ and 3’ homology arms of the LTVEC
is from about 10 kb to about 150 kb, about 10 kb to about 20 kb, from about 20 kb to about
40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to
about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb.
The various methods employing the CRISPR/Cas system (or any method
disclosed herein) can be performed on, for example, mammalian cells, non-human
mammalian cells, fibroblast cells, rodent cells, rat cells, mouse cells, or hamster cells. The
cell can be a otent cell, an induced pluripotent stem (iPS) cell, a mouse embryonic stem
(ES) cell, a rat nic stem (ES) cell, a human embryonic stem (ES) cell or a
developmentally restricted human progenitor cell.
As discussed in detail below, following the modification a challenging genomic
locus or a genomic locus of interest on the Y chromosome (i.e., the Sry locus) of a non-
human pluripotent cell employing, for e, using the /CAS system outline
above, the cally modified non-human pluripotent cell that is produced can be
introduced into a man host embryo; and the non-human host embryo comprising the
modified pluripotent cell in a surrogate mother is gestated. The surrogate mother es
F0 progeny comprising the ed genetic modification. In ic embodiments, the
targeted genetic modification is e of being transmitted through the germline.
C. Selection Markers
] Various selection markers can be used in the s and compositions disclosed
herein which provide for modifying a target c locus on the Y chromosome or a
challenging target genomic locus. Such markers are disclosed elsewhere herein and include,
but are not limited to, selection markers that impart resistance to an antibiotic such as G418,
hygromycin, blastocidin, neomycin, or puromycin. The polynucleotide encoding the
selection s are operably linked to a promoter active in the cell. Such expression
cassettes and their various regulatory components are discussed in further detailed elsewhere
herein.
D. Target Genomic Locus
Various s and compositions are provided which allow for the integration of
at least one insert polynucleotide at a target genomic locus on the Y chromosome or a
challenging target genomic locus. As used herein, a “target genomic locus on the Y
chromosome” comprises any segment or region of DNA on the Y chromosome that one
desires to ate an insert polynucleotide.
The genomic locus on the Y chromosome or a challenging target genomic locus
being targeted can be native to the cell, or alternatively can comprise a heterologous or
exogenous segment of DNA that was ated into the chromosome of the cell. Such
logous or exogenous segments of DNA can include transgenes, expression cassettes,
polynucleotide encoding selection makers, or heterologous or exogenous regions of genomic
DNA. The target genomic locus on the Y chromosome or the challenging target genomic
locus can comprise any of the targeted genomic integration system including, for example,
the recognition site, the selection marker, previously integrated insert polynucleotides,
polynucleotides encoding nuclease agents, promoters, etc. Alternatively, the target genomic
locus on the Y chromosome or the challenging target genomic locus can be located within a
yeast artificial some (YAC), bacterial artificial some (BAC), a human
artificial chromosome, or any other engineered genomic region contained in an appropriate
host cell. Thus, in specific embodiments, the targeted genomic locus on the Y chromosome
or the challenging target genomic locus can comprise native, heterologous or exogenous
genomic c acid ce from a non-human mammal, a non-human cell, a rodent, a
human, a rat, a mouse, a r, a rabbit, a pig, a bovine, a deer, a sheep, a goat, a chicken, a
cat, a dog, a ferret, a primate (e. g., marmoset, rhesus monkey), domesticated mammal or an
agricultural mammal or any other organism of interest or a combination thereof.
Non-limiting examples of the target genomic locus on the Y chromosome include,
the Sry gene, the Uty gene, the Eif2s3y gene, the Ddx3y gene, the gene, the Ubely gene, the
Tspy gene, the Usp9y gene, the ny1 gene, and the ny2 gene and the region on the Y
chromosome assing the Kdm5d, y, Tspy, Uty, Ddx3y, and Usp9y genes. Such
a locus on the Y chromosome can be from a non-human mammal, a mammal, a rodent, a
human, a rat, a mouse, a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a goat, a chicken, a
cat, a dog, a ferret, a primate (e. g., marmoset, rhesus monkey), domesticated mammal or an
agricultural mammal or any other organism of interest or a combination f. Such cells
include pluripotent cells, including, for example, induced pluripotent stem (iPS) cells, mouse
embryonic stem (ES) cells, rat embryonic stem (ES) cells, human embryonic stem (ES) cell,
or developmentally restricted human progenitor cells.
] As described elsewhere , various s and compositions are provided
which comprise XY pluripotent and/or totipotent cells (such as XY ES cells or iPS cells)
having a decreased activity or level of the Sry protein. The various methods described herein
to modify genomic locus on the Y chromosome can also be used to introduce targeted c
modifications to polynucleotides of interest that are not located on the Y chromosome.
E. Targeting Vectors and Insert Polynucleotides
As outlined above, methods and compositions provided herein employ targeting
vectors alone or in combination with a nuclease agent. “Homologous recombination” is used
conventionally to refer to the exchange of DNA fragments between two DNA molecules at
cross-over sites within the regions of homology.
1'. Insert Polynucleotide
As used herein, the “insert polynucleotide” comprises a segment of DNA that one
desires to integrate at the target genomic locus. In ic embodiments, the target genomic
locus is on the Y chromosome. In other ments, the target genomic locus is a
nging genomic locus. In one embodiment, the insert polynucleotide comprises one or
more cleotides of interest. In other embodiments, the insert polynucleotide can
comprise one or more expression cassettes. A given expression cassette can comprise a
polynucleotide of interest, a polynucleotide encoding a selection marker and/or a reporter
gene along with the various regulatory components that influence expression. Non-limiting
es of polynucleotides of interest, selection markers, and reporter genes that can be
included within the insert polynucleotide are discussed in detail elsewhere herein.
In specific embodiments, the insert polynucleotide can comprise a genomic
nucleic acid. In one embodiment, the genomic nucleic acid is derived from an animal, a
mouse, a human, a non-human, a rodent, a non-human, a rat, a r, a rabbit, a pig, a
, a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret, a e (e.g., marmoset,
rhesus monkey), domesticated mammal or an agricultural , an avian, or any other
organism of interest or a combination thereof.
In further embodiments, the insert cleotide comprises a ional allele.
In one embodiment, the conditional allele is a multifunctional allele, as described in US
2011/0104799, which is incorporated by reference in its entirety. In specific embodiments,
the conditional allele comprises: (a) an actuating sequence in sense orientation with respect to
transcription of a target gene, and a drug selection cassette in sense or antisense orientation;
(b) in antisense orientation a tide sequence of interest (NSI) and a conditional by
inversion module (COIN, which utilizes an exon-splitting intron and an invertible genetrap-
like module; see, for example, US 2011/0104799, which is incorporated by reference in its
entirety); and (c) recombinable units that recombine upon exposure to a first recombinase to
form a conditional allele that (i) lacks the actuating sequence and the DSC, and (ii) contains
the NSI in sense orientation and the COIN in antisense ation.
The insert polynucleotide can be from about 5kb to about 200kb, from about 5kb
to about 10kb, from about 10kb to about 20kb, from about 20kb to about 30kb, from about
30kb to about 40kb, from about 40kb to about 50kb, from about 60kb to about 70kb, from
about 80kb to about 90kb, from about 90kb to about 100kb, from about 100kb to about
110kb, from about 120kb to about 130kb, from about 130kb to about 140kb, from about
140kb to about 150kb, from about 150kb to about 160kb, from about 160kb to about 170kb,
from about 170kb to about 180kb, from about 180kb to about 190kb, from about 190kb to
about 200kb, from about 200kb to about 250kb, from about 250kb to about 300kb, from
about 300kb to about 350kb, or from about 350kb to about 400kb.
] In specific embodiments, the insert polynucleotide comprises a nucleic acid
flanked with site-specific ination target ces. It is recognized that while the
entire insert polynucleotide can be flanked by such pecific recombination target
sequence, any region or individual polynucleotide of interest within the insert polynucleotide
can also be flanked by such sites. The term "recombination site" as used herein includes a
nucleotide sequence that is ized by a site-specific recombinase and that can serve as a
substrate for a recombination event. The term "site-specific inase" as used herein
es a group of enzymes that can facilitate recombination between recombination sites
where the two recombination sites are physically separated within a single nucleic acid
molecule or on separate nucleic acid molecules. Examples of site-specific recombinases
include, but are not limited to, Cre, Flp, and Dre recombinases. The site-specific
recombinase can be introduced into the cell by any means, including by introducing the
recombinase polypeptide into the cell or by introducing a polynucleotide ng the site-
specific recombinase into the host cell. The polynucleotide ng the site-specific
recombinase can be located within the insert polynucleotide or within a separate
polynucleotide. The site-specific recombinase can be operably linked to a promoter active in
the cell including, for example, an inducible er, a promoter that is endogenous to the
cell, a promoter that is heterologous to the cell, a cell-specific promoter, a tissue-specific
promoter, or a developmental stage-specific promoter. Site-specific recombination target
sequences which can flank the insert polynucleotide or any polynucleotide of interest in the
insert polynucleotide can include, but are not limited to, loxP, lox511, lox2272, lox66, lox71,
loxM2, lox5171, FRT, FRTl 1, FRT71, attp, att, FRT, rox, and a combination thereof.
In other embodiments, the site-specific recombination sites flank a polynucleotide
encoding a selection marker and/or a reporter gene contained within the insert
polynucleotide. In such ces following ation of the insert polynucleotide at the
targeted genomic locus the sequences between the site-specific recombination sites can be
In one embodiment, the insert polynucleotide comprises a polynucleotide
encoding a selection marker. Such selection markers include, but are not limited, to in
phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-
acetyltransferase (puror), blasticidin S deaminase (bsrr), xanthine/guanine phosphoribosyl
erase (gpt), or herpes simplex virus ine kinase (HSV-k), or a ation
thereof. In one embodiment, the polynucleotide encoding the ion marker is operably
linked to a promoter active in the cell. When serially tiling polynucleotides of interest into a
targeted genomic locus, the selection marker can comprise a recognition site for a nuclease
agent, as outlined above. In one embodiment, the polynucleotide encoding the ion
marker is flanked with a pecific recombination target sequences.
The insert polynucleotide can further comprise a reporter gene operably linked to
a promoter, wherein the er gene encodes a reporter protein selected from the group
consisting of LacZ, mPlum, mCherry, thomato, mStrawberry, J-Red, DsRed, mOrange,
mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald,
enhanced green fluorescent protein (EGFP), CyPet, cyan fluorescent protein (CFP), Cerulean,
T-Sapphire, luciferase, alkaline atase, and a combination thereof. Such reporter genes
can be operably linked to a promoter active in the cell. Such promoters can be an inducible
promoter, a promoter that is endogenous to the er gene or the cell, a promoter that is
heterologous to the er gene or to the cell, a cell-specific promoter, a -specific
promoter manner or a developmental stage-specific promoter.
ii. Targeting Vectors
Targeting vectors are employed to introduce the insert polynucleotide into the
targeted genomic locus on the Y chromosome or into a challenging target locus or on another
chromosome of interest. The targeting vector ses the insert polynucleotide and further
comprises an upstream and a downstream homology arm that flank the insert polynucleotide.
The homology arms that flank the insert polynucleotide pond to genomic regions
within the targeted genomic locus. For ease of reference, the corresponding genomic s
within the targeted genomic locus are referred to herein as “target sites”. Thus, in one
example, a targeting vector can comprise a first insert polynucleotide flanked by a first and a
second homology arm corresponding to a first and a second target site located in sufficient
proximity to the first recognition site within the polynucleotide encoding the selection
marker. As such, the targeting vector thereby aids in the integration of the insert
polynucleotide into the targeted genomic locus through a gous recombination event
that occurs between the gy arms and the corresponding target sites within the genome
of the cell.
A homology arm of the targeting vector can be of any length that is sufficient to
promote a homologous recombination event with a corresponding target site, including for
example, from about 400 bp to about 500 bp, from about 500 bp to about 600 bp, from about
600 bp to about 700 bp, from about 700 bp to about 800 bp, from about 800 bp to about 900
bp, or from about 900 bp to about 1000 bp; or at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-
40, 5-45, 5- 50, 5-55, 5-60, 5-65, 5- 70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 100-200, or 200-
300 kilobases in length or greater. In specific embodiments, the sum total of the targeting
arms is at least 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb or at least 10kb.
In other embodiments, the sum total of the gy arms is between about 0.5 kb to about 1
kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 3 kb, about 3
kb to about 4 kb, about 4kb to about 5kb, about 5kb to about 6kb, about 6kb to about 7kb,
about 7kb to about 8kb, about 8kb to about 9kb, or about 10kb to about 150kb. As outlined
in further detail below, large targeting vectors can employ targeting arms of greater length.
The target sites within the targeted c locus that correspond to the upstream
and downstream homology arms of the targeting vector are located in “sufficient ity to
the recognition site” located in the polynucleotide encoding the selection marker. As used
herein, the upstream and downstream homology arms of a targeting vector are “located in
sufficient ity” to a recognition site when the distance is such as to promote the
occurrence of a homologous recombination event between the target sites and the homology
arms upon a nick or double-strand break at the recognition site. Thus, in specific
embodiments, the target sites corresponding to the upstream and/or ream homology
arm of the targeting vector are within at least 10 nucleotide to about 14 kb of a given
recognition site. In specific embodiments, the recognition site is ately nt to at
least one or both of the target sites.
The spatial relationship of the target sites that correspond to the homology arms of
the targeting vector to the recognition site within the polynucleotide ng the selection
marker can vary. For example, both target sites can be located 5’ to the ition site, both
target sites can be located 3’ to the recognition site, or the target sites can flank the
recognition site.
In specific embodiments, the target genomic locus comprises (i) a 5’ target
sequence that is gous to a 5’ homology arm; and (ii) a 3’ target sequence that is
homologous to a 3’ homology arm. In specific embodiments, the 5’ target sequence and the
3’ target sequence is separated by at least 5 kb but less than 3 Mb, at least 5 kb but less than
kb, at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb, at least 40 kb but
less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb, at
least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb, at least about 200 kb
but less than about 300 kb, at least about 300 kb but less than about 400 kb, at least about 400
kb but less than about 500 kb, at least about 500 kb but less than about 1Mb, at least about 1
Mb but less than about 1.5 Mb, at least about 1.5 Mb but less than about 2 Mb, at least about
2 Mb but less than about 2.5 Mb, or at least about 2.5 Mb but less than about 3 Mb.
As used herein, a homology arm and a target site “correspond” or are
“corresponding” to one r when the two regions share a ient level of sequence
identity to one another to act as substrates for a homologous recombination reaction. By
“homology” is meant DNA sequences that are either identical or share sequence identity to a
corresponding sequence. The sequence identity between a given target site and the
corresponding homology arm found on the targeting vector can be any degree of sequence
identity that allows for homologous recombination to occur. For e, the amount of
sequence identity shared by the homology arm of the targeting vector (or a fragment thereof)
and the target site (or a fragment thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo
homologous recombination. Moreover, a corresponding region of homology between the
gy arm and the corresponding target site can be of any length that is sufficient to
promote homologous recombination at the cleaved recognition site. For example, a given
homology arm and/or ponding target site can comprise corresponding regions of
gy that are from about 400 bp to about 500 bp, from about 500 bp to about 600 bp,
from about 600 bp to about 700 bp, from about 700 bp to about 800 bp, from about 800 bp to
about 900 bp, or from about 900 bp to about 1000 bp (such as described for the smallTVEC
vectors described ere herein); or at least about 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40,
-45, 5- 50, 5-55, 5-60, 5-65, 5- 70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 0, or 200-300
kilobases in length or more (such as described in the LTVEC vectors described elsewhere
herein) such that the homology arm has sufficient homology to undergo gous
recombination with the ponding target sites within the genome of the cell.
For ease of reference the homology arms are referred to herein an upstream and a
downstream gy arm. This terminology relates to the relative position of the homology
arms to the insert polynucleotide within the targeting vector.
The gy arms of the targeting vector are therefore designed to correspond to
a target site with the targeted genomic locus on the Y chromosome or within a challenging
target locus. Thus, the homology arms can correspond to a genomic locus that is native to the
cell, or alternatively they can correspond to a region of a heterologous or exogenous segment
of DNA that was integrated into the Y chromosome, including, but not d to, transgenes,
expression cassettes, or heterologous or exogenous regions of c DNA. atively,
the homology arms of the targeting vector can correspond to a region of a yeast artificial
chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial
chromosome, or any other engineered c region contained in an riate host cell.
Still further the homology arms of the targeting vector can correspond to or be d from a
region of a BAC library, a cosmid library, or a P1 phage library. Thus, in specific
embodiments, the homology arms of the targeting vector correspond to a genomic locus on
the Y chromosome or to a challenging target locus that is native, heterologous or exogenous
to a non-human mammal, a rodent, a human, a rat, a mouse, a hamster a rabbit, a pig, a
bovine, a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret, a primate (e.g., marmoset,
rhesus ), domesticated mammal or an agricultural mammal, an avian, or any other
sm of interest. In further embodiments, the homology arms correspond to a genomic
locus of the cell that is not targetable using a conventional method or can be targeted only
incorrectly or only with significantly low efficiency, in the absence of a nick or double-strand
break induced by a nuclease agent. In one embodiment, the homology arms are derived from
a synthetic DNA.
In still other embodiments, the upstream and downstream homology arms
correspond to the same genome as the targeted genome. In one embodiment, the homology
arms are from a related , e. g., the ed genome is a mouse genome of a first strain,
and the targeting arms are from a mouse genome of a second strain, wherein the first strain
and the second strain are different. In other embodiments, the homology arms are from the
genome of the same animal or are from the genome of the same strain, e.g., the targeted
genome is a mouse genome of a first strain, and the targeting arms are from a mouse genome
from the same mouse or from the same strain.
] The targeting vector (such as a large targeting ) can also comprise a
selection cassette or a reporter gene as discussed elsewhere herein. The selection cassette can
comprise a nucleic acid sequence encoding a selection marker, wherein the nucleic acid
sequence is operably linked to a promoter. Such promoters can be an inducible promoter, a
promoter that is endogenous to the report gene or the cell, a promoter that is heterologous to
the reporter gene or to the cell, a cell-specific promoter, a tissue-specific promoter manner or
a developmental stage-specific promoter. In one embodiment, the selection marker is
selected from neomycin phosphotransferase (neor), ycin B phosphotransferase (hygr),
cin-N-acetyltransferase (puror), blasticidin S ase (bsrr), xanthine/guanine
phosphoribosyl transferase (gpt), and herpes simplex virus ine kinase (HSV-k), and a
combination thereof. The selection marker of the targeting vector can be flanked by the
upstream and downstream homology arms or found either 5’ or 3’ to the homology arms.
In one embodiment, the targeting vector (such as a large targeting vector)
comprises a reporter gene operably linked to a er, wherein the reporter gene encodes a
reporter protein selected from the group consisting of LacZ, mPlum, mCherry, thomato,
mStrawberry, J-Red, DsRed, mOrange, mKO, ne, Venus, YPet, enhanced yellow
fluorescent protein (EYFP), Emerald, enhanced green fluorescent protein (EGFP), CyPet,
cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, and a
combination f. Such reporter genes can be operably linked to a promoter active in the
cell. Such promoters can be an ble promoter, a er that is endogenous to the
report gene or the cell, a promoter that is heterologous to the reporter gene or to the cell, a
cell-specific promoter, a -specific promoter manner or a developmental stage-specific
promoter.
In one non-limiting embodiment, the combined use of the targeting vector
(including, for example, a large targeting vector) with the nuclease agent results in an
increased ing efficiency compared to the use of the targeting vector alone. In one
embodiment, when the ing vector is used in conjunction with the nuclease agent,
targeting efficiency of the targeting vector is increased at least by two-fold, at least three-fold,
at least 4-fold, or at least d when compared to when the targeting vector is used alone.
iii. Large Targeting Vectors
The term “large targeting vector” or “LTVEC” as used herein includes large
targeting vectors that comprise homology arms that pond to and are derived from
nucleic acid sequences larger than those typically used by other ches ed to
perform homologous ing in cells and/or sing insert polynucleotides comprising
nucleic acid sequences larger than those typically used by other approaches intended to
perform homologous recombination targeting in cells. In specific embodiments, the
homology arms and/or the insert polynucleotide of the LTVEC comprises a genomic
sequence of a eukaryotic cell. The size of the LTVEC is too large to enable screening of
targeting events by conventional assays, e. g., southern blotting and long-range (e. g., lkb-5kb)
PCR. Examples of the LTVEC, include, but are not limited to, s derived from a
bacterial artificial chromosome (BAC), a human artificial chromosome or a yeast artificial
chromosome (YAC). Non-limiting examples of LTVECs and methods for making them are
described, e.g., in US Pat. No. 6,586,251, 6,596,541, 7,105,348, and
(PCT/USOl/45375), each of which is herein incorporated by reference.
The LTVEC can be of any length, including, but not limited to, at least about
10kb, about 15kb, about 20kb, about 30kb, about 40kb, about 50kb, about 60kb, about 70kb,
about 80kb, about 90kb, about 100kb, about 150kb, about 200kb, from about 10kb to about
15kb, about 15 kb to about 20kb, about 20kb to about 30kb, from about 30kb to about 50kb,
from about 50kb to about 300kb, from about 50kb to about 75kb, from about 75kb to about
100kb, from about 100kb to 125kb, from about 125kb to about 150kb, from about 150kb to
about 175kb, about 175kb to about 200kb, from about 200kb to about 225kb, from about
225kb to about 250kb, from about 250kb to about 275kb or from about 275kb to about
300kb.
In one embodiment, the LTVEC ses an insert polynucleotide ranging from
about 5kb to about 200kb, from about 5kb to about 10kb, from about 10kb to about 20kb,
from about 20kb to about 30kb, from about 30kb to about 40kb, from about 40kb to about
50kb, from about 60kb to about 70kb, from about 80kb to about 90kb, from about 90kb to
about 100kb, from about 100kb to about 110kb, from about 120kb to about 130kb, from
about 130kb to about 140kb, from about 140kb to about 150kb, from about 150kb to about
160kb, from about 160kb to about 170kb, from about 170kb to about 180kb, from about
180kb to about 190kb, or from about 190kb to about 200kb, from about 200kb to about
250kb, from about 250kb to about 300kb, from about 300kb to about 350kb, or from about
350kb to about 400kb.
In other instances, the LTVEC design can be such as to allow for the replacement
of a given ce that is from about 5kb to about 200kb or from about 5kb to about 3.0Mb
as described herein. In one embodiment, the replacement is from about 5kb to about 10kb,
from about 10kb to about 20kb, from about 20kb to about 30kb, from about 30kb to about
40kb, from about 40kb to about 50kb, from about 50kb to about 60kb, from about 60kb to
about 70kb, from about 80kb to about 90kb, from about 90kb to about 100kb, from about
100kb to about 110kb, from about 110kb to about 120kb, from about 120kb to about 130kb,
from about 130kb to about 140kb, from about 140kb to about 150kb, from about 150kb to
about 160kb, from about 160kb to about 170kb, from about 170kb to about 180kb, from
about 180kb to about 190kb, from about 190kb to about 200kb, from about 5kb to about
10kb, from about 10kb to about 20kb, from about 20kb to about 40kb, from about 40kb to
about 60kb, from about 60kb to about 80kb, from about 80kb to about 100kb, from about
100kb to about 150kb, or from about 150kb to about 200kb, from about 200kb to about
WO 00805
300kb, from about 300kb to about 400kb, from about 400kb to about 500kb, from about
500kb to about 1Mb, from about 1Mb to about 1.5Mb, from about 1.5Mb to about 2Mb, from
about 2Mb to about 2.5Mb, or from about 2.5Mb to about 3Mb.
In one embodiment, the homology arms of the LTVEC are derived from a BAC
library, a cosmid library, or a P1 phage library. In other embodiments, the homology arms
are derived from the targeted genomic locus of the cell and in some instances the target
genomic locus that the LTVEC is designed to target is not targetable using a conventional
method. In still other embodiments, the homology arms are derived from a synthetic DNA.
In one embodiment, a sum total of the upstream homology arm and the
downstream gy arm in the LTVEC is at least 10kb. In other embodiments, the
upstream homology arm ranges from about 5kb to about 100kb. In one ment, the
downstream homology arm ranges from about 5kb to about 100kb. In other ments,
the sum total of the upstream and downstream homology arms are from about 5kb to about
10kb, from about 10kb to about 20kb, from about 20kb to about 30kb, from about 30kb to
about 40kb, from about 40kb to about 50kb, from about 50kb to about 60kb, from about 60kb
to about 70kb, from about 70kb to about 80kb, from about 80kb to about 90kb, from about
90kb to about 100kb, from about 100kb to about 110kb, from about 110kb to about 120kb,
from about 120kb to about 130kb, from about 130kb to about 140kb, from about 140kb to
about 150kb, from about 150kb to about 160kb, from about 160kb to about 170kb, from
about 170kb to about 180kb, from about 180kb to about 190kb, or from about 190kb to about
200kb. In one embodiment, the size of the deletion is the same or similar to the size of the
sum total of the 5' and 3' gy arms of the LTVEC.
In one embodiment, the LTVEC comprises a selection cassette or a reporter gene
as sed elsewhere herein.
iv. Methods ofIntegrating an Insert Polynucleotide Near the Recognition Site
on the Y Chromosome by Homologous Recombination
Methods are provided for modifying a target genomic locus on the Y chromosome
in a cell comprising: (a) providing a cell comprising a target genomic locus on the Y
chromosome, (b) introducing into the cell a first targeting vector sing a first insert
polynucleotide flanked by a first and a second homology arm corresponding to a first and a
second target site; and (c) identifying at least one cell sing in its genome the first insert
polynucleotide integrated at the target genomic locus on the Y chromosome. Similar
methods can be performed to target a challenging chromosomal locus. As discussed in detail
elsewhere herein, in specific embodiments, the sum total of the first homology arm and the
second homology arm of the targeting vector is about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb,
5kb, 6kb, 7kb, 8kb, 9kb, about 0.5 kb to about 1 kb, about 1 kb to about 1.5 kb, about 1.5 kb
to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5kb,
about 5kb to about 6 kb, about 6 kb to about 7 kb, about 8 kb to about 9 kb, or is at least 10
kb or at least 10 kb and less than 150 kb. In ic embodiments, an LTVEC is employed.
In other specific embodiments, a smallTVEC is employed. In one miting embodiment,
such methods are performed employing the culture media that promotes the development of
XY F0 fertile females disclosed herein and thereby generating XY F0 fertile female animals.
In other instance, the s described herein are employed to produce a targeted genetic
modification in the Sry gene, as discussed ere herein.
Further ed are methods for modifying a target genomic locus on the Y
chromosome in a cell comprising: (a) providing a cell comprising a target genomic locus on
the Y chromosome comprising a recognition site for a nuclease agent, (b) introducing into the
cell (i) the nuclease agent, wherein the se agent induces a nick or double-strand break
at the first recognition site; and, (ii) a first targeting vector comprising a first insert
polynucleotide flanked by a first and a second homology arm corresponding to a first and a
second target site located in sufficient proximity to the first recognition site; and (c)
fying at least one cell comprising in its genome the first insert polynucleotide integrated
at the target c locus on the Y chromosome. Similar methods can be performed to
target a challenging target locus. As sed in detail elsewhere herein, in specific
embodiments, the sum total of the first homology arm and the second homology arm of the
targeting vector is about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 7kb, 8kb, 9kb, about
0.5 kb to about 1 kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to
about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5kb, about 5kb to about 6 kb, about
6 kb to about 7 kb, about 8 kb to about 9 kb, or is at least 10 kb or at least 10 kb and less than
150 kb. In specific embodiments, an LTVEC is employed. In other specific embodiments, a
smallTVEC is employed. In one non-limiting embodiment, such methods are performed
employing the culture media that promotes the development of XY F0 fertile females
sed herein and thereby generating XY F0 fertile female animals. In other instance, the
methods described herein are employed to e a ed genetic modification in the Sry
gene, as sed elsewhere herein.
Various methods can also be employed to identify cells having the insert
polynucleotide integrated at the genomic target locus. Insertion of the insert polynucleotide
at the genomic target locus s in a “modification of allele”. The term ication of
allele” or “MOA” includes the modification of the exact DNA sequence of one allele of a
gene(s) or chromosomal locus (loci) in a genome. es of “modification of allele
(MOA)” include, but are not limited to, deletions, substitutions, or insertions of as little as a
single nucleotide or deletions of many kilobases spanning a gene(s) or chromosomal locus
(loci) of interest, as well as any and all possible modifications between these two extremes.
In various ments, to facilitate identification of the targeted cation, a
high-throughput quantitative assay, , modification of allele (MOA) assay, is
employed. The MOA assay described herein allows a large-scale screening of a modified
(s) in a parental chromosome following a genetic modification. The MOA assay can be
carried out via various analytical techniques, ing, but not limited to, a quantitative
PCR, e.g., a real-time PCR (qPCR). For example, the real-time PCR comprises a first
primer-probe set that recognizes the target locus and a second primer-probe set that
recognizes a non-targeted reference locus. In addition, the primer-probe set comprises a
fluorescent probe that recognizes the amplified sequence. The quantitative assay can also be
carried out via a variety of analytical techniques, including, but not limited to, cence-
mediated in situ hybridization , comparative genomic hybridization, isothermic DNA
amplification, quantitative hybridization to an immobilized probe(s), Invader Probes®, MMP
assays®, TaqMan® Molecular Beacon, and EclipseTM probe technology. (See, for example,
U82005/0144655, incorporated by reference herein in its entirety).
In various embodiments, in the ce of the nick or double strand bread,
targeting efficiency of a targeting vector (such as a LTVEC or a smallTVEC) at the target
genomic locus is at least about 2-fold higher, at least about 3-fold , at least about 4-fold
higher than in the absence of the nick or double-strand break (using, e. g., the same targeting
vector and the same homology arms and corresponding target sites at the c locus of
interest but in the absence of an added nuclease agent that makes the nick or double strand
break).
The various methods set forth above can be tially repeated to allow for the
targeted integration of any number of insert polynucleotides into a given targeted genomic
locus on the Y chromosome or into a challenging target locus. Thus, the various methods
provide for the insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12,13, 14,15, 16,17, 18,
19, 20 or more insert polynucleotides into the target c locus on the Y chromosome or
into a challenging target locus. In particular embodiments, such sequential tiling methods
allow for the reconstruction of large genomic regions from an animal cell or from a
mammalian cell (i.e., a human, a man, a rodent, a mouse, a monkey, a rat, a hamster, a
domesticated mammal or an agricultural animal) into a ed genomic locus on a Y
chromosome. In such instances, the transfer and reconstruction of genomic regions that
include both coding and non-coding s allow for the complexity of a given region to be
preserved by retaining, at least in part, the coding regions, the ding regions and the
copy number variations found within the native genomic region. Thus, the various methods
provide, for example, methods to generate “heterologous” or “exogenous” genomic s
within any mammalian cell or animal of interest. In one miting example, a
“humanized” genomic region within a non-human animal is generated.
It is further recognized that along with modifying the target genomic locus on the
Y chromosome, the various methods and compositions disclosed herein can be ed to
also generate at targeted genetic modification on another chromosome.
v. Polynucleotides ofInterest
] Any polynucleotide of interest may be contained in the various insert
polynucleotides and thereby integrated at the target genomic locus on the Y chromosome or
into a challenging target locus. The methods disclosed herein, provide for at least 1, 2, 3, 4,
, 6 or more polynucleotides of interest to be integrated into the targeted genomic locus.
The cleotide of interest within the insert polynucleotide when integrated at
the target genomic locus on the Y chromosome or at a challenging target locus can uce
one or more genetic modifications into the cell. The genetic modification can comprise a
deletion of an endogenous nucleic acid sequence and/or the addition of an exogenous or
heterologous or orthologous polynucleotide into the target genomic locus. In one
embodiment, the genetic modification comprises a replacement of an endogenous c
acid sequence with an exogenous polynucleotide of interest at the target genomic locus.
Thus, methods ed herein allow for the generation of a genetic modification comprising
a knockout, a deletion, an insertion, a replacement (“knock-in”), a point mutation, a domain
swap, an exon swap, an intron swap, a tory sequence swap, a gene swap, or a
combination thereof in a target genomic locus on the Y chromosome. Such cations
may occur upon integration of the first, second, third, fourth, fifth, six, seventh, or any
subsequent insert polynucleotides into the target genomic locus.
The polynucleotide of interest within the insert polynucleotide and/or integrated at
the target genomic locus can comprise a ce that is native or gous to the cell it is
introduced into; the polynucleotide of interest can be heterologous to the cell it is introduced
to; the polynucleotide of interest can be exogenous to the cell it is introduced into; the
polynucleotide of interest can be orthologous to the cell it is introduced into; or the
polynucleotide of interest can be from a different species than the cell it is introduced into.
As used herein “homologous” in reference to a sequence is a sequence that is native to the
cell. As used herein, “heterologous” in reference to a sequence is a sequence that originates
from a foreign species, or, if from the same species, is substantially modified from its native
form in composition and/or c locus by deliberate human intervention. As used herein,
“exogenous” in reference to a sequence is a sequence that originates from a foreign species.
As used herein, “orthologous” is a polynucleotide from one species that is functionally
equivalent to a known reference sequence in another species (i.e., a species t). The
polynucleotide of interest can be from any organism of interest including, but not d to,
non-human, a rodent, a hamster, a mouse, a rat, a human, a monkey, an avian, an agricultural
mammal or a ricultural mammal. The polynucleotide of interest can further comprise
a coding region, a ding , a regulatory region, or a genomic DNA. Thus, the 1“,
2nd, 3rd, 4th, 5th, 6th, 7m, and/or any of the subsequent insert cleotides can comprise such
sequences.
In one embodiment, the polynucleotide of interest within the insert polynucleotide
and/or integrated at the target genomic locus on the Y chromosome is homologous to a
mouse nucleic acid sequence, a human nucleic acid, a non-human nucleic acid, a rodent
nucleic acid, a rat nucleic acid, a hamster nucleic acid, a monkey nucleic acid, an agricultural
mammal nucleic acid, or a non-agricultural mammal nucleic acid. In still further
embodiments, the polynucleotide of interest integrated at the target locus is a fragment of a
genomic nucleic acid. In one embodiment, the genomic c acid is a mouse genomic
nucleic acid, a human genomic nucleic acid, a man nucleic acid, a rodent c acid,
a rat c acid, a hamster nucleic acid, a monkey c acid, an agricultural mammal
c acid or a non-agricultural mammal nucleic acid or a combination thereof.
In one embodiment, the polynucleotide of interest can range from about 500
nucleotides to about 200kb as described above. The polynucleotide of interest can be from
about 500 nucleotides to about 5kb, from about 5kb to about 200kb, from about 5kb to about
10kb, from about 10kb to about 20kb, from about 20kb to about 30kb, from about 30kb to
about 40kb, from about 40kb to about 50kb, from about 60kb to about 70kb, from about 80kb
to about 90kb, from about 90kb to about 100kb, from about 100kb to about 110kb, from
about 120kb to about 130kb, from about 130kb to about 140kb, from about 140kb to about
150kb, from about 150kb to about 160kb, from about 160kb to about 170kb, from about
l70kb to about 180kb, from about 180kb to about 190kb, or from about l90kb to about
200kb.
The polynucleotide of interest within the insert polynucleotide and/or inserted at
the target c locus on the Y chromosome or into a nging target locus can encode
a polypeptide, can encode an miRNA, can encode a long non-coding RNA, or it can comprise
any regulatory regions or non-coding regions of interest including, for example, a regulatory
sequence, a promoter sequence, an enhancer sequence, a transcriptional repressor-binding
sequence, or a deletion of a non-protein-coding sequence, but does not comprise a deletion of
a protein-coding sequence. In addition, the polynucleotide of interest within the insert
polynucleotide and/or inserted at the target c locus on the Y chromosome or at a
challenging target locus can encode a protein expressed in the s system, the skeletal
system, the digestive system, the circulatory system, the ar system, the respiratory
system, the cardiovascular system, the lymphatic system, the endocrine system, the urinary
system, the reproductive system, or a combination thereof.
The polynucleotide of interest within the insert polynucleotide and/or integrated at
the target genomic locus on the Y chromosome or at a challenging target locus can comprises
a genetic modification in a coding sequence. Such genetic modifications include, but are not
limited to, a deletion mutation of a coding sequence or the fusion of two coding sequences.
The polynucleotide of interest within the insert polynucleotide and/or integrated at
the target c locus on the Y some or at a challenging target locus can comprise
a polynucleotide encoding a mutant protein. In one embodiment, the mutant n is
characterized by an altered binding characteristic, altered localization, altered expression,
and/or altered expression pattern. In one embodiment, the polynucleotide of interest within
the insert cleotide and/or integrated at the genomic target locus on the Y chromosome
or at a challenging target locus comprises at least one disease allele. In such ces, the
disease allele can be a dominant allele or the disease allele is a recessive . Moreover,
the disease allele can se a single nucleotide polymorphism (SNP) allele. The
polynucleotide of st encoding the mutant protein can be from any organism, including,
but not limited to, a , a non-human mammal, rodent, mouse, rat, a human, a monkey,
an agricultural mammal or a domestic mammal polynucleotide encoding a mutant protein.
The cleotide of interest within the insert polynucleotide and/or integrated at
the target genomic locus on the Y chromosome or at a challenging target locus can also
comprise a regulatory sequence, including for example, a promoter sequence, an enhancer
sequence, a transcriptional repressor-binding ce, or a transcriptional terminator
sequence. In specific embodiments, the polynucleotide of interest within the insert
polynucleotide and/or integrated at the target genomic locus on the Y chromosome or at a
challenging target locus comprises a polynucleotide having a on of a non-protein-
coding sequence, but does not comprise a deletion of a protein-coding sequence. In one
embodiment, the deletion of the otein-coding ce comprises a deletion of a
regulatory sequence. In another embodiment, the deletion of the regulatory element
comprises a deletion of a er sequence. In one embodiment, the deletion of the
regulatory element comprises a deletion of an enhancer sequence. Such a polynucleotide of
interest can be from any organism, including, but not limited to, a mammal, a non-human
mammal, , mouse, rat, a human, a monkey, an agricultural mammal or a domestic
mammal polynucleotide encoding a mutant protein.
The various methods disclosed herein can be employed to generate a variety of
modifications in a nging genomic locus or in the Y chromosome locus (such as Sry).
Such modifications include, for example, a replacement of an endogenous nucleic acid
sequence with a homologous or an orthologous c acid sequence; a on of an
endogenous nucleic acid sequence; a deletion of an endogenous nucleic acid sequence,
wherein the deletion ranges from about 5 kb to about 10 kb, from about 10 kb to about 20 kb,
from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about
80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150
kb to about 200 kb, from about 200 kb to about 300 kb, from about 300 kb to about 400 kb,
from about 400 kb to about 500 kb, from about 500 kb to about 600 kb, from about 600 kb to
about 700 kb, from about 700 kb to about 800 kb, from about 800 kb to about 900 kb, from
about 900 kb to about 1 Mb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5
Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5
Mb to about 3 Mb; an insertion of an exogenous nucleic acid sequence; an insertion of an
exogenous nucleic acid sequence ranging from about 5kb to about 10kb, from about 10 kb to
about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about
60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to
about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb; an
insertion of an exogenous nucleic acid sequence sing a homologous or an orthologous
nucleic acid sequence; an insertion of a chimeric c acid sequence comprising a human
and a non-human nucleic acid sequence; an insertion of a ional allele flanked with site-
2015/038001
specific recombinase target sequences; an insertion of a selectable marker or a er gene
operably linked to a third er active in the mammalian cell; or a combination thereof.
III. s ofIntroducing ces and Generation of Transgenic Animals
As outlined above, methods and compositions are provided herein to allow for the
ed genetic modification of one or more polynucleotides of interest located on the Y
chromosome, at a challenging target locus, or a se in the level and/or activity of the Sry
protein. It is further recognized that in addition to a targeted genetic modification to a
sequence on the Y chromosome or on a challenging target chromosomal locus, additional
targeted genetic modification can be made on other chromosomes. Such systems that allow
for these targeted genetic modifications can employ a variety of components and for ease of
nce, herein the term “targeted genomic integration system” generically includes all the
components required for an integration event (i.e. the various nuclease agents, recognition
sites, insert DNA polynucleotides, targeting vectors, target genomic locus, and
polynucleotides of interest).
The methods provided herein comprise introducing into a cell one or more
polynucleotides or polypeptide constructs comprising the various components of the ed
genomic integration system. "Introducing" means presenting to the cell the sequence
(polypeptide or polynucleotide) in such a manner that the sequence gains access to the
interior of the cell. The methods provided herein do not depend on a particular method for
ucing any ent of the targeted genomic integration system into the cell, only that
the polynucleotide gains access to the interior of a least one cell. Methods for introducing
polynucleotides into various cell types are known in the art and include, but are not limited
to, stable transfection methods, ent transfection methods, and virus-mediated methods.
In some embodiments, the cells employed in the methods and compositions have a
DNA construct stably incorporated into their genome. "Stably orated" or "stably
introduced" means the introduction of a polynucleotide into the cell such that the nucleotide
sequence integrates into the genome of the cell and is capable of being inherited by progeny
thereof. Any protocol may be used for the stable incorporation of the DNA constructs or the
various ents of the ed genomic integration system.
ection protocols as well as protocols for introducing polypeptides or
polynucleotide sequences into cells may vary. Non-limiting transfection methods include
chemical-based transfection s include the use of liposomes; nanoparticles; calcium
phosphate (Graham et al. (1973). Virology 52 (2): 456—67, Bacchetti et al. (1977) Proc Natl
2015/038001
Acad Sci USA 74 (4): 1590—4 and, Kriegler, M (1991). Transfer and Expression: A
Laboratory Manual. New York: W. H. Freeman and Company. pp. ; dendrimers; or
cationic polymers such as DEAE-dextran or polyethylenimine. Non chemical s
include oporation; Sono-poration; and optical transfection . Particle-based transfection
include the use of a gene gun, magnet assisted transfection ( Bertram, J. (2006) Current
Pharmaceutical Biotechnology 7, 277—28). Viral methods can also be used for transfection.
In one embodiment, the nuclease agent is introduced into the cell simultaneously
with the targeting vector, the smallTVEC, or the large targeting vector (LTVEC).
Alternatively, the nuclease agent is introduced separately from the targeting vector,
smallTVEC, or the LTVEC over a period of time. In one embodiment, the nuclease agent is
introduced prior to the introduction of the targeting vector, smallTVEC, or the LTVEC, while
in other embodiments, the se agent is introduced following uction of the
ing vector, smallTVEC, or the LTVEC.
man animals can be generated employing the various methods disclosed
herein. Such methods comprises (1) integrating one or more polynucleotide of interest at the
target genomic locus of the Y some of a otent cell of the non-human animal to
generate a genetically modified pluripotent cell comprising the insert polynucleotide in the
targeted genomic locus of the Y chromosome employing the methods disclosed herein; (2)
ing the cally modified pluripotent cell having the one or more polynucleotides of
interest at the target genomic locus of the Y chromosome; (3) introducing the genetically
modified pluripotent cell into a host embryo of the non-human animal at a pre-morula stage;
and (4) implanting the host embryo comprising the genetically ed pluripotent cell into
a surrogate mother to generate an F0 generation derived from the cally modified
pluripotent cell. Similar methods can be employed to target a challenging target
chromosomal locus. The non-human animal can be a non-human , a rodent, a
mouse, a rat, a hamster, a monkey, an agricultural mammal or a domestic mammal, or a fish
or a bird.
The pluripotent cell can be a human ES cell, a non-human ES cell, a rodent ES
cell, a mouse ES cell, a rat ES cell, a hamster ES cell, a monkey ES cell, an agricultural
mammal ES cell or a domesticated mammal ES cell. In other embodiments, the pluripotent
cell is a mammalian cell, human cell, a non-human mammalian cell, a human pluripotent cell,
a human ES cell, a human adult stem cell, a developmentally-restricted human progenitor
cell, a human iPS cell, a human cell, a rodent cell, a rat cell, a mouse cell, a hamster cell. In
one embodiment, the targeted genetic modification decreases the level and/or activity of the
2015/038001
Sry protein. In such instances, the pluripotent cell can comprise an XY ES cell or an XY iPS
cell. Methods of culturing such cells to e the development of F0 fertile XY female
s are described in detail elsewhere herein.
Nuclear transfer techniques can also be used to generate the non-human
mammalian animals. Briefly, methods for nuclear transfer include the steps of: (l)
ating an oocyte; (2) ing a donor cell or nucleus to be combined with the
enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a
reconstituted cell; (4) implanting the reconstituted cell into the womb of an animal to form an
embryo; and (5) allowing the embryo to develop. In such methods oocytes are generally
retrieved from deceased animals, although they may be isolated also from either oviducts
and/or ovaries of live animals. s can be d in a variety of medium known to
those of ordinary skill in the art prior to enucleation. Enucleation of the oocyte can be
performed in a number of manners well known to those of ry skill in the art. Insertion
of the donor cell or nucleus into the enucleated oocyte to form a reconstituted cell is usually
by microinjection of a donor cell under the zona pellucida prior to fusion. Fusion may be
induced by application of a DC electrical pulse across the contact/fusion plane
(electrofusion), by exposure of the cells to fusion-promoting chemicals, such as polyethylene
glycol, or by way of an vated virus, such as the Sendai virus. A reconstituted cell is
typically activated by ical and/or non-electrical means before, during, and/or after
fusion of the nuclear donor and recipient oocyte. tion methods include electric pulses,
chemically induced shock, penetration by sperm, increasing levels of nt cations in the
, and reducing phosphorylation of cellular proteins (as by way of kinase inhibitors) in
the oocyte. The activated reconstituted cells, or embryos, are typically cultured in medium
well known to those of ordinary skill in the art and then transferred to the womb of an animal.
See, for example, US20080092249, WO/l999/005266A2, US20040177390,
WO/2008/017234Al, and US Patent No. 7,612,250, each of which is herein incorporated by
reference.
Other methods for making a non-human animal comprising in its germline one or
more genetic modifications as bed herein is provided, comprising: (a) modifying a
targeted genomic locus on the Y chromosome of a non-human animal in a prokaryotic cell
employing the various methods described ; (b) selecting a modified prokaryotic cell
comprising the genetic modification at the targeted genomic locus; (c) isolating the
genetically modified targeting vector from the genome of the modified yotic cell; (d)
introducing the genetically modified targeting vector into a pluripotent cell of the non-human
animal to generate a genetically modified pluripotent cell comprising the insert nucleic acid
at the targeted genomic locus of the Y some; (e) selecting the genetically modified
pluripotent cell; (f) introducing the genetically modified pluripotent cell into a host embryo of
the man animal at a pre-morula stage; and (g) implanting the host embryo comprising
the genetically modified pluripotent cell into a surrogate mother to generate an F0 generation
derived from the genetically modified otent cell. In such methods the targeting vector
can comprise a large ing vector or a smallTVEC. Similar methods can be employed to
target a challenging target locus. The man animal can be a man mammal, a
, a mouse, a rat, a hamster, a , an agricultural mammal or a domestic mammal.
The pluripotent cell can be a human ES cell, a non-human ES cell, a rodent ES cell, a mouse
ES cell, a rat ES cell, a hamster ES cell, a monkey ES cell, an agricultural mammal ES cell or
a domestic mammal ES cell. In other embodiments, the pluripotent cell is a mammalian cell,
human cell, a man mammalian cell, a human pluripotent cell, a human ES cell, a
human adult stem cell, a developmentally-restricted human itor cell, a human iPS cell,
a human cell, a rodent cell, a rat cell, a mouse cell, a hamster cell. In one embodiment, the
targeted genetic modification decreases the level and/or activity of the Sry protein. In such
instances, the pluripotent cell can comprise an XY ES cell or an XY iPS cell. Methods of
culturing such cells to promote the development of F0 fertile XY female animals are
described in detail elsewhere herein.
In further methods, the isolating step (c) further comprises (cl) linearizing the
cally modified targeting vector (i.e., the genetically modified LTVEC). In still r
embodiments, the introducing step (d) further comprises (dl) introducing a nuclease agent as
described herein into the pluripotent cell. In one ment, selecting steps (b) and/or (e)
are carried out by applying a selectable agent as described herein to the prokaryotic cell or the
pluripotent cell. In one embodiment, selecting steps (b) and/or (e) are carried out via a
modification of allele (MOA) assay as described herein.
r methods for modifying a target genomic locus of an animal cell via
bacterial homologous recombination (BHR) in a prokaryotic cell are provided and comprise:
(a) providing a prokaryotic cell sing a target genomic locus of the Y chromosome; (b)
introducing into the prokaryotic cell a targeting vector (as described above) comprising an
insert polynucleotide flanked with a first upstream homology arm and a first downstream
gy arm, wherein the insert polynucleotide comprises a mammalian genomic region,
and introducing into the prokaryotic cell a nuclease agent that makes a nick or double-strand
break at or near the first recognition site, and (c) selecting a targeted prokaryotic cell
comprising the insert polynucleotide at the target genomic locus of the chromosome, wherein
the prokaryotic cell is capable of sing a recombinase that mediates the BHR. Similar
methods can be employed to target a challenging target locus. Steps (a)-(c) can be serially
repeated as disclosed herein to allow the introduction of multiple insert polynucleotides at the
ed genomic locus in the prokaryotic cell. Once the targeted genomic locus is “built”
with the prokaryotic cell, a targeting vector sing the ed target genomic locus of
the Y chromosome can be isolated from the prokaryotic cell and introduced into a target
genomic locus of the Y chromosome within a mammalian cell. Mammalian cells comprising
the modified genomic locus of the Y chromosome can then be made into non-human
transgenic animals.
Further methods for modifying a target genomic locus of an animal cell via
bacterial homologous recombination (BHR) in a prokaryotic cell are provided and comprise:
(a) providing a prokaryotic cell comprising a target genomic locus of the Y chromosome; (b)
introducing into the prokaryotic cell a targeting vector (as described above) comprising an
insert polynucleotide flanked with a first upstream homology arm and a first downstream
homology arm, n the insert polynucleotide comprises a mammalian genomic region,
and (c) ing a targeted prokaryotic cell comprising the insert cleotide at the target
genomic locus of the chromosome, wherein the prokaryotic cell is capable of expressing a
recombinase that mediates the BHR. Similar methods can be employed to target a
challenging target locus. Steps (a)-(c) can be serially repeated as disclosed herein to allow
the introduction of le insert polynucleotides at the targeted genomic locus in the
prokaryotic cell. Once the ed genomic locus is “built” with the prokaryotic cell, a
targeting vector comprising the ed target genomic locus of the Y chromosome can be
isolated from the prokaryotic cell and introduced into a target genomic locus of the Y
chromosome within a mammalian cell. Mammalian cells comprising the modified genomic
locus of the Y chromosome can then be made into man transgenic animals
In some embodiments, various genetic modifications of the target genomic loci
described herein can be carried out by a series of homologous recombination ons (BHR)
in bacterial cells using an LTVEC derived from Bacterial Artificial some (BAC)
DNA using VELOCIGENE® genetic engineering logy (see, e.g., US Pat. No.
6,586,251 and Valenzuela, D. M. et a1. (2003), High-throughput engineering of the mouse
genome d with high-resolution expression analysis, Nature Biotechnology 21(6): 652-
659, which is incorporated herein by nce in their entireties).
In some embodiments, targeted XY pluripotent and/or tent cells (i.e., X YES
cells or XY iPS cells) comprising various genetic cations as described herein are used
as insert donor cells and uced into a pre-morula stage embryo from a corresponding
organism, e. g., an 8-cell stage mouse embryo, via the VELOCIMOUSE® method (see, e. g.,
US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 Al, all of which are
incorporated by reference herein in their ties). The non-human animal embryo
comprising the genetically modified XY pluripotent and/or totipotent cells (i.e., XY ES cells
or XY iPS cells) is incubated until the blastocyst stage and then implanted into a surrogate
mother to produce an F0 generation. In some embodiments, targeted mammalian ES cells
sing various c modifications as described herein are introduced into a blastocyst
stage embryo. Non-human s bearing the genetically modified genomic locus of the Y
chromosome can be identified via modification of allele (MOA) assay as described herein.
The resulting F0 generation non-human animal derived from the genetically modified XY
pluripotent and/or totipotent cells (i.e., X YES cells or XY iPS cells) is crossed to a wild-type
non-human animal to obtain F1 generation offspring. Following ping with specific
primers and/or probes, Fl non-human animals that are heterozygous for the genetically
modified genomic locus are crossed to each other to produce F2 generation non-human
animal offspring that are homozygous for the genetically modified genomic locus of the Y
chromosome or for the cally modified challenging target locus.
IV. Cells and Expression Cassettes
The various methods described herein employ a c locus targeting system
for the Y chromosome or for a challenging target locus in a cell. Such cells include
prokaryotic cells such as bacterial cells including E. coli, or eukaryotic cells such as yeast,
insect, amphibian, plant, or mammalian cells, including, but not limited to a mouse cell, a rat
cell, a rabbit cell, a pig cell, a bovine cell, a deer cell, a sheep cell, a goat cell, a n cell,
a cat cell, a dog cell, a ferret cell, a primate (e. g., et, rhesus monkey) cell, and the like
and cells from domesticated mammals or cells from ltural mammals. Some cells are
non-human, particularly non-human mammalian cells. In some embodiments, for those
mammals for which suitable genetically modifiable pluripotent cells are not readily ble,
other methods are employed to reprogram somatic cells into pluripotent cells, e. g., via
introduction into somatic cells of a combination of pluripotency-inducing factors, including,
but not limited to, Oct3/4, Sox2, KLF4, Myc, Nanog, LIN28, and Glisl. In such methods,
the cell can also be a mammalian cell, human cell, a non-human mammalian cell, a non-
human cell, a cell from a rodent, a rat, a mouse, a hamster, a last cell or any other host
cell. In other embodiments, the cell is a pluripotent cell, an induced otent stem (iPS)
cell, a non-human embryonic stem (ES) cell. Such cells include pluripotent cells, including,
for example, d pluripotent stem (iPS) cells, mouse embryonic stem (ES) cells, rat
embryonic stem (ES) cells, human embryonic (ES) cells, or developmentally restricted
human progenitor cells, a rodent nic stem (ES) cell, a mouse nic stem (ES)
cell or a rat embryonic stem (ES) cell.
The terms “polynucleotide,” “polynucleotide sequence,77 (Cnucleic acid sequence,”
and “nucleic acid fragment” are used interchangeably herein. These terms encompass
nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that
is single- or double-stranded, that optionally contains synthetic, non-natural or d
nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of
one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
Polynucleotides can comprise deoxyribonucleotides and ribonucleotides include both
lly occurring molecules and synthetic analogues, and any combination these. The
polynucleotides provided herein also encompass all forms of ces including, but not
limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures,
and the like.
Further provided are recombinant polynucleotides. The terms “recombinant
polynucleotide” and “recombinant DNA construct” are used interchangeably herein. A
recombinant construct comprises an artificial or heterologous combination of nucleic acid
sequences, e. g., regulatory and coding sequences that are not found together in nature. In
other embodiments, a inant construct may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory sequences and coding
ces derived from the same source, but arranged in a manner different than that found
in nature. Such a uct may be used by itself or may be used in conjunction with a
vector. If a vector is used, then the choice of vector is dependent upon the method that is
used to transform the host cells as is well known to those skilled in the art. For example, a
plasmid vector can be used. Screening may be accomplished by Southern analysis of DNA,
Northern analysis of mRNA expression, blotting analysis of protein expression, or
phenotypic is, among others.
In specific embodiments, one or more of the components bed herein can be
provided in an expression cassette for expression in the pluripotent and/or totipotent cell.
The cassette can include 5' and 3' tory sequences operably linked to a polynucleotide
provided . bly linked” means a functional linkage between two or more
elements. For example, an operable linkage between a polynucleotide of interest and a
regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the
polynucleotide of interest. ly linked ts may be contiguous or non-contiguous.
When used to refer to the joining of two protein coding regions, operably linked means that
the coding regions are in the same reading frame. In another instance, a nucleic acid sequence
encoding a protein may be operably linked to regulatory sequences (e. g., promoter, enhancer,
silencer sequence, etc.) so as to retain proper riptional regulation. The cassette may
additionally contain at least one additional polynucleotide of interest to be co-introduced into
the ES cell. Alternatively, the additional polynucleotide of st can be provided on
multiple expression cassettes. Such an expression cassette is provided with a plurality of
restriction sites and/or recombination sites for ion of a recombinant polynucleotide to be
under the riptional regulation of the regulatory regions. The sion cassette may
additionally contain selection marker genes.
The sion cassette can e in the 5'-3' direction of transcription, a
transcriptional and translational initiation region (i.e., a promoter), a recombinant
cleotide provided herein, and a transcriptional and translational termination region
(i.e., ation region) functional in mammalian cell or a host cell of interest. The
regulatory regions (i.e., promoters, transcriptional regulatory regions, and transcriptional and
translational termination regions) and/or a polynucleotide provided herein may be
native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or
a cleotide provided herein may be heterologous to the host cell or to each other. For
example, a promoter operably linked to a heterologous polynucleotide is from a species
different from the species from which the cleotide was derived, or, if from the
same/analogous species, one or both are substantially modified from their original form
and/or genomic locus, or the er is not the native promoter for the operably linked
polynucleotide. Alternatively, the regulatory regions and/or a recombinant polynucleotide
provided herein may be entirely synthetic.
The termination region may be native with the transcriptional tion region,
may be native with the operably linked recombinant polynucleotide, may be native with the
host cell, or may be derived from another source (i.e., foreign or heterologous) to the
promoter, the recombinant polynucleotide, the host cell, or any combination thereof.
In preparing the expression cassette, the various DNA fragments may be
manipulated, so as to provide for the DNA sequences in the proper orientation. Toward this
end, rs or linkers may be ed to join the DNA fragments or other manipulations
may be involved to provide for convenient restriction sites, l of superfluous DNA,
removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair,
restriction, annealing, resubstitutions, e. g., transitions and transversions, may be involved.
A number of promoters can be used in the expression cassettes provided herein.
The ers can be selected based on the desired outcome. It is recognized that different
applications can be enhanced by the use of different promoters in the expression tes to
modulate the timing, location and/or level of sion of the polynucleotide of interest.
Such expression constructs may also contain, if desired, a er regulatory region (e. g.,
one conferring inducible, constitutive, environmentally- or developmentally-regulated, or
cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome
binding site, an RNA processing signal, a transcription termination site, and/or a
polyadenylation signal.
Non-limiting embodiments include:
1. An in vitro culture comprising
(a) a non-human mammalian XY embryonic stem (ES) cell having a modification
that decreases the level and/or activity of an Sry protein; and,
(b) a medium comprising a base medium and supplements suitable for
maintaining the non-human mammalian ES cell in culture, n the medium exhibits one
or more of the following characteristic: an osmolality from about 200 mOsm/kg to less than
about 329 g; a tivity of about 11 mS/cm to about 13 mS/cm; a salt of an
alkaline metal and a halide in a tration of about 50 mM to about 110 mM; a carbonic
acid salt concentration of about l7mM to about 30 mM; a total alkaline metal halide salt and
carbonic acid salt concentration of about 85mM to about 130 mM; and/or a combination of
any two or more thereof.
2. The in vitro culture of claim 1, wherein the non-human ian XY ES
cell is from a rodent.
3. The in vitro culture of claim 2, n the rodent is a mouse.
4. The in vitro culture of embodiment 3, wherein the mouse XY ES cell is a
VGFl mouse ES cell.
. The in vitro culture of embodiment 2, wherein the rodent is a rat or a hamster.
6. The in vitro culture of any one of embodiments 1-5, wherein the decreased
level and/or activity of the Sry protein is from a genetic modification in the Sry gene.
7. The in vitro culture of embodiment 6, wherein the genetic modification in the
Sry gene comprises an insertion of one or more nucleotides, a deletion of one or more
nucleotides, a substitution of one or more nucleotides, a knockout, a n, a replacement
of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence or a
combination thereof.
8. The in vitro culture of any one of embodiments 1-7, n the non-human
mammalian ES cell comprises one, two, three or more targeted genetic modifications.
9. The in vitro culture of embodiment 8, wherein the targeted genetic
modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a
combination thereof.
. The in vitro culture of embodiment 8, wherein the targeted genetic
modification comprises at least one insertion of a heterologous polynucleotide into the
genome of the XY ES cell.
11. The in vitro culture of any one of embodiments 8-10, wherein the targeted
genetic cation is on an autosome.
12. The in vitro culture of any one of embodiments 1-11, wherein the base
medium exhibits 50 i 5 mM NaCl, 26 i 5 mM carbonate, and 218 i 22 mOsm/kg.
13. The in vitro e of any one of embodiments 1-11, n the base
medium exhibits about 3 mg/mL NaCl, 2.2 mg/mL sodium onate, and 218 mOsm/kg.
14. The in vitro culture of any one of embodiments 1-11, wherein the base
medium exhibits 87 i 5 mM NaCl, 18 i 5 mM carbonate, and 261 i 26 mOsm/kg.
. The in vitro culture of any one of embodiments 1-11, wherein the base
medium ts about 5.1 mg/mL NaCl, 1.5 mg/mL sodium onate, and 261 mOsm/kg.
16. The in vitro e of any one of embodiments 1-11, wherein the base
medium exhibits 110 i 5 mM NaCl, 18 i 5 mM carbonate, and 294 i 29 mOsm/kg.
17. The in vitro culture of any one of embodiments 1-11, wherein the base
medium exhibits about 6.4 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 294 mOsm/kg.
18. The in vitro culture of any one of embodiments 1-11, wherein the base
medium exhibits 87 i 5 mM NaCl, 26 i 5 mM carbonate, and 270 i 27 g.
19. The in vitro culture of any one of embodiments 1-11, wherein the base
medium ts about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 270 mOsm/kg.
. The in vitro e of any one of embodiments 1-11, wherein the base
medium exhibits 87 i 5 mM NaCl, 26 i 5 mM carbonate, 86 i 5 mM glucose, and 322 i 32
mOsm/kg.
21. The in vitro culture of any one of ments 1-11, wherein the base
medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, 15.5 mg/mL
glucose, and 322 mOsm/kg.
22. The in vitro culture of any one of embodiments 1-21, wherein upon
introduction of the non-human mammalian XY ES cells into a host embryo and following
ion of the host embryo, at least 80% of the F0 non-human mammals are XY females
which upon attaining sexual maturity the F0 XY female non-human mammal is fertile.
23. A method for making a fertile female XY non-human mammal in an F0
generation, comprising:
(a) culturing a donor non-human ian XY nic stem (ES) cell
having a modification that decreases the level and/or activity of an Sry protein in a medium
comprising a base medium and supplements suitable for maintaining the non-human
mammalian ES cell in culture, wherein the medium exhibits a characteristic comprising one
or more of the following: an osmolality from about 200 mOsm/kg to less than about 329
mOsm/kg; a conductivity of about 11 mS/cm to about 13 mS/cm; a salt of an alkaline metal
and a halide in a concentration of about 50mM to about 110 mM; a carbonic acid salt
concentration of about 17 mM to about 30 mM; a total alkaline metal halide salt and carbonic
acid salt tration of about 85 mM to about 130 mM; and/or a combination of any two or
more thereof;
(b) introducing the donor XY non-human mammalian ES cell into a host embryo;
(c) gestating the host embryo; and,
(d) obtaining an F0 XY female non-human mammal, wherein upon attaining
sexual maturity the F0 XY female non-human mammal is fertile.
24. The method of embodiment 23, wherein the man mammalian XY ES
cell is from a rodent.
. The method of embodiment 24, wherein the rodent is a mouse.
26. The method of embodiment 25, n the mouse XY ES cell is a VGFl
mouse ES cell.
27. The method of embodiment 24, wherein the rodent is a rat or a hamster.
28. The method of any one of embodiments 23-27, wherein the decreased level
and/or activity of the Sry protein is from a c modification in the Sry gene.
29. The method of ment 28, wherein the genetic modification in the Sry
gene comprises an ion of one or more nucleotides, a deletion of one or more
nucleotides, a substitution of one or more nucleotides, a knockout, a knockin, a replacement
WO 00805
of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence or a
combination f.
. The method of any one of embodiments 23-29, wherein the non-human
mammalian ES cell comprises one, two, three or more ed genetic modifications.
31. The method of embodiment 30, wherein the targeted genetic modification
comprises an insertion, a deletion, a knockout, a n, a point mutation, or a combination
thereof.
32. The method of embodiment 30, wherein the targeted genetic modification
comprises at least one insertion of a heterologous polynucleotide into a genome of the XY ES
cell.
33. The method of any one of embodiments 30-32, wherein the targeted genetic
modification is on an autosome.
34. The method of any one of embodiments 23-33, wherein the base medium
exhibits 50 i 5 mM NaCl, 26 i 5 mM carbonate, and 218 i 22 g.
. The method of any one of embodiments 23-33, wherein the base medium
exhibits about 3 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 218 mOsm/kg.
36. The method of any one of embodiments 23-33, wherein the base medium
exhibits 87 i 5 mM NaCl, 18 i 5 mM carbonate, and 261 i 26 mOsm/kg.
37. The method of any one of embodiments 23-33, n the base medium
exhibits about 5.1 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 261 mOsm/kg.
38. The method of any one of embodiments 23-33, wherein the base medium
exhibits 110 i 5 mM NaCl, 18 i 5 mM carbonate, and 294 i 29 mOsm/kg.
39. The method of any one of embodiments 23-33, wherein the base medium
exhibits about 6.4 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 294 mOsm/kg.
40. The method of any one of embodiments 23-33, wherein the base medium
exhibits 87 i 5 mM NaCl, 26 i 5 mM carbonate, and 270 i 27 mOsm/kg.
41. The method of any one of embodiments 23-33, wherein the base medium
exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 270 mOsm/kg.
42. The method of any one of embodiments 23-33, n the base medium
ts 87 i 5 mM NaCl, 26 i 5 mM carbonate, 86 i 5 mM glucose, and 322 i 32
43. The method of any one of embodiments 23-33, wherein the base medium
exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, 15.5 mg/mL glucose, and
322 mOsm/kg.
44. A method of producing a transgenic non-human mammal homozygous for a
targeted genetic mutation in the F1 generation comprising: (a) crossing an F0 XY fertile
female having a decreased level and/or ty of the Sry protein with a cohort clonal sibling,
derived from the same ES cell clone, F0 XY male non-human mammal, wherein the F0 XY
fertile female non-human mammal and the F0 XY male non-human mammal each is
heterozygous for the genetic mutation; and, (b) obtaining an F1 progeny mouse that is
homozygous for the genetic modification.
45. A method for modifying a target genomic locus on the Y chromosome in a cell
comprising: (a) providing a cell comprising a target c locus on the Y some
comprising a recognition site for a nuclease agent, (b) introducing into the cell (i) the
nuclease agent, wherein the nuclease agent induces a nick or double-strand break at the first
recognition site; and, (ii) a first targeting vector comprising a first insert polynucleotide
flanked by a first and a second homology arm corresponding to a first and a second target site
d in sufficient proximity to the first recognition site, wherein a sum total of the first
homology arm and the second homology arm is at least 4kb but less than 150kb; and, (c)
fying at least one cell sing in its genome the first insert polynucleotide integrated
at the target genomic locus.
46. A method for modifying a target genomic locus on the Y chromosome in a cell
comprising:
(a) providing a cell comprising a target genomic locus on the Y chromosome
comprising a recognition site for a nuclease agent,
(b) ucing into the cell a first targeting vector sing a first insert
polynucleotide flanked by a first and a second homology arm corresponding to a first and a
second target site, wherein a sum total of the first homology arm and the second homology
arm is at least 4kb but less than 150kb; and,
(c) identifying at least one cell comprising in its genome the first insert
polynucleotide ated at the target c locus.
47. The method of embodiment 45 or 46, wherein the cell is a mammalian cell.
48. The method of embodiment 47, wherein the mammalian cell is a non-human
cell.
49. The method of embodiment 47, wherein the mammalian cell is from a rodent.
50. The method of embodiment 49, wherein the rodent is a rat, a mouse or a
hamster.
51. The method of any one of embodiments 45-50, wherein the cell is a
pluripotent cell.
52. The method of any one of embodiments 45-50, wherein the mammalian cell is
an induced pluripotent stem (iPS) cell.
53. The method of embodiment 51, wherein the pluripotent cell is a man
embryonic stem (ES) cell.
54. The method of embodiment 51, wherein the pluripotent cell is a rodent
embryonic stem (ES) cell, a mouse embryonic stem (ES) cell or a rat embryonic stem (ES)
cell.
55. The method of any one of embodiments 45 and 47-54, wherein the nuclease
agent is an mRNA encoding a nuclease.
56. The method of any one of embodiments 45 and 47-54, wherein the nuclease
agent is a zinc finger nuclease (ZFN).
57. The method of any one of embodiments 45 and 47-54, wherein the nuclease
agent is a ription Activator-Like Effector Nuclease (TALEN).
58. The method of any one of embodiments 45 and 47-54, wherein the nuclease
agent is a meganuclease.
59. The method any one of embodiments 45 and 47-54, wherein the nuclease
agent is a CRISPR RNA guided Cas9 endonuclease.
60. A method for modifying the Y chromosome comprising exposing the Y
chromosome to a Cas protein and a CRISPR RNA in the presence of a large targeting vector
(LTVEC) comprising a c acid ce of at least 10 kb, wherein following exposure
to the Cas protein, the CRISPR RNA, and the LTVEC, the Y chromosome is modified to
contain at least 10 kb nucleic acid sequence.
61. The method of ment 60, wherein the LTVEC comprises a nucleic acid
sequence of at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least
70 kb, at least 80 kb, or at least 90 kb.
62. The method of embodiment 60, n the LTVEC comprises a nucleic acid
ce of at least 100 kb, at least 150 kb, or at least 200 kb.
63. A method for modifying a target genomic locus on the Y chromosome,
comprising: (a) providing a mammalian cell comprising the target genomic locus on the Y
chromosome, n the target genomic locus comprises a guide RNA (gRNA) target
ce; (b) introducing into the mammalian cell: (i) a large targeting vector (LTVEC)
comprising a first nucleic acid flanked with ing arms homologous to the target genomic
locus, wherein the LTVEC is at least 10 kb; (ii) a first expression construct comprising a first
er operably linked to a second nucleic acid encoding a Cas protein, and (iii) a second
expression construct comprising a second er ly linked to a third nucleic acid
encoding a guide RNA (gRNA) comprising a nucleotide sequence that hybridizes to the
gRNA target ce and a trans-activating CRISPR RNA (trachNA), wherein the first and
the second ers are active in the ian cell; and (c) identifying a modified
mammalian cell comprising a targeted genetic modification at the target genomic locus on the
Y some.
64. The method of embodiment 63, wherein the LTVEC is at least 15 kb, at least
kb, at least 30kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb,
or at least 90 kb.
65. The method of embodiment 63, wherein the LTVEC is at least 100 kb, at least
150 kb, or at least 200 kb.
66. The method of embodiment 63, wherein the mammalian cell is a non-human
mammalian cell.
67. The method of embodiment 63, wherein the mammalian cell is a last
cell.
68. The method of embodiment 63, wherein the mammalian cell is from a rodent.
69. The method of embodiment 68, wherein the rodent is a rat, a mouse, or a
hamster.
70. The method of embodiment 63, wherein the mammalian cell is a pluripotent
cell.
71. The method of embodiment 70, wherein the pluripotent cell is an induced
pluripotent stem (iPS) cell.
72. The method of embodiment 70, wherein the pluripotent cell is a mouse
embryonic stem (ES) cell or a rat embryonic stem (ES) cell.
73. The method of embodiment 70, wherein the pluripotent cell is a
developmentally restricted human progenitor cell.
74. The method of embodiment 63, wherein the Cas protein is a Cas9 protein.
75. The method of embodiment 74, wherein the gRNA target ce is
immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.
76. The method of embodiment 63, wherein the sum total of 5’ and 3’ homology
arms of the LTVEC is from about 10 kb to about 150 kb.
77. The method of embodiment 76, wherein the sum total of the 5’ and the 3’
2015/038001
homology arms of the LTVEC is from about 10 kb to about 20 kb, from about 20 kb to about
40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to
about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb.
78. The method of embodiment 63, wherein the targeted genetic modification
comprises: (a) a replacement of an endogenous nucleic acid sequence with a homologous or
an orthologous nucleic acid sequence; (b) a deletion of an nous nucleic acid
sequence; (c) a on of an nous nucleic acid sequence, wherein the deletion ranges
from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about
40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to
about 100 kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200 kb, from
about 200 kb to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about
500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5
Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb;
(d) insertion of an exogenous nucleic acid sequence; (e) insertion of an exogenous
nucleic acid sequence ranging from about 5kb to about 10kb, from about 10 kb to about 20
kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to
about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from
about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about
300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb; (f)
insertion of an exogenous nucleic acid sequence comprising a homologous or an orthologous
nucleic acid sequence; (g) insertion of a chimeric nucleic acid sequence comprising a
human and a non-human nucleic acid ce; (h) insertion of a conditional allele flanked
with site-specific recombinase target sequences; (i) insertion of a selectable marker or a
reporter gene operably linked to a third promoter active in the mammalian cell; or (1') a
combination thereof.
79. The method of embodiment 63, wherein the target genomic locus comprises
(i) a 5’ target sequence that is homologous to a 5’ homology arm; and (ii) a 3’ target sequence
that is homologous to a 3’ homology arm.
80. The method of ment 79, n the 5’ target sequence and the 3’
target sequence is separated by at least 5 kb but less than 3 Mb.
81. The method of embodiment 79, wherein the 5’ target sequence and the 3’
target sequence is separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20
kb, at least 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but less
than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb, or at
least 150 kb but less than 200 kb, at least about 200 kb but less than about 300 kb, at least
about 300 kb but less than about 400 kb, at least about 400 kb but less than about 500 kb, at
least about 500 kb but less than about le, at least about 1 Mb but less than about 1.5 Mb, at
least about 1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than about 2.5 Mb,
or at least about 2.5 Mb but less than about 3 Mb.
82. The method of embodiment 63, n the first and the second expression
constructs are on a single nucleic acid molecule.
83. The method of embodiment 63, wherein the target genomic locus comprises
the Sry locus.
84. A method for targeted genetic modification on the Y chromosome of a non-
human animal, comprising: (a) modifying a genomic locus of interest on the Y chromosome
of a non-human pluripotent cell according to the method of embodiment 4, thereby producing
a genetically modified non-human pluripotent cell comprising a targeted genetic modification
on the Y chromosome; (b) introducing the modified non-human pluripotent cell of (a) into a
non-human host embryo; and (c) gestating the man host embryo comprising the
ed pluripotent cell in a surrogate mother, wherein the surrogate mother produces F0
progeny comprising the targeted genetic modification, wherein the targeted genetic
modification is capable of being transmitted h the germline.
85. The method of embodiment 84, wherein the c locus of interest
comprises the Sry locus.
86. A method for modifying a target c locus on the Y chromosome in a cell
comprising: (a) providing a cell comprising a target genomic locus on the Y chromosome
comprising a ition site for a nuclease agent, (b) ucing into the cell (i) the
nuclease agent, wherein the nuclease agent induces a nick or double-strand break at the first
recognition site; and, (ii) a first targeting vector comprising a first insert polynucleotide
flanked by a first and a second homology arm corresponding to a first and a second target site
located in sufficient proximity to the first recognition site, wherein the length of the first
gy arm and/or the second homology arm is at least 400 bp but less than 1000 bp; and,
(c) identifying at least one cell comprising in its genome the first insert polynucleotide
integrated at the target genomic locus.
87. The method of embodiment 86, wherein the length of the first homology arm
and/or the second homology arm is from about 700 bp to about 800 bp.
88. The method of embodiment 86, n the modification comprises a deletion
of an endogenous nucleic acid sequence.
2015/038001
89. The method of embodiment 88, wherein the deletion ranges from about 5 kb to
about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about
40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb,
from about 100 kb to about 150 kb, or from about 150 kb to about 200 kb, from about 200 kb
to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from
about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2
Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb.
90. The method of embodiment 88, wherein the deletion is at least 500 kb.
The present methods and compositions may be embodied in many different forms
and should not be ued as limited to the embodiments set forth herein; rather, these
embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like
numbers refer to like elements throughout.
Many modifications and other embodiments of the s and compositions set
forth herein will come to mind to one skilled in the art to which this methods and compositions
pertains having the benefit of the teachings presented in the ing ptions and the
associated drawings. Therefore, it is to be understood that the methods and compositions are not
to be limited to the specific embodiments disclosed and that modifications and other
embodiments are included within the scope of the appended . Although specific terms are
employed herein, they are used in a generic and ptive sense only and not for purposes of
limitation.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Example 1. Targeting of the Y Chromosome Gene S[y Assisted by TALENs or CRISPR
A targeted deletion comprising a lacZ replacement allele for Sry was created with
a targeting vector comprising, in order, an upstream homology arm of approximately 700 bp,
a beta-galactosidase coding sequence (lacZ) followed by a polyadenylation signal, a
neomycin resistance cassette flanked by loxP sites sing a human ubiquitin C promoter,
ing the first exon, first intron, and part of the second exon, a in
phosphotransferase coding sequence, and a polyadenylation , and a downstream
homology arm of approximately 650 bp. The allele created by correct targeting of the Sry
gene with the targeting vector comprises a deletion of the approximately 1 kb Sry open
[Link]
http://www.velocigene.com/komp/detail/12778
[Link]
http://www.velocigene.com/komp/detail/12778
reading frame and replacement with the eo cassette such that the beta-galactosidase
coding sequence is fused in-frame at the Sry start codon. The targeting vector was used to
target the Sry gene in both the VGB6 (a.k.a. B6A6) C57BL/6 and the VGF1 (a.k.a.
F1H4) C57BL6/129 F1 hybrid ES cell lines. VGF1 (F1H4) mouse ES cells were derived
from hybrid embryos produced by crossing a female C57BL/6NTac mouse to a male
SvEvTac mouse. Therefore, VGF1 ES cells contain a Y chromosome from
129S6/SvEvTac mouse. The female XY mice produced from the VGF1 cell line contain a Y
chromosome derived from 129S6/SvEvTac mouse.
Example 2. TALEN- or CRISPR-induced Mutations in the Y Chromosome Gene Sry
Deletion mutations, presumably the result of non-homologous end joining (NHEJ)
repair of double strand DNA , ranging from 3 bp to 1.2 kb and larger were created in
the Sry gene by the action of a TALEN or of CRISPR guide RNAs, in combination with Cas9
DNA endonuclease (see, . ES cells sing the TALEN- and CRISPR-induced
mutations in the Sry gene also carried random transgenic insertions of the NIH KOMP project
VG12778 LTVEC ( available via internet on the world wide web (www) at the URL
igene.com/komp/detail/12778”), which comprises a deletion of the Sry coding
sequence and ement with an insertion cassette comprising lacZ fused in-frame with the
Sry start codon and a neomycin resistance gene flanked by gy arms of 38 and 37 kb
and based on a BAC from the bMQ library (129S7/SvEv Brd-Hprt b-m2). The LTVEC
comprises in its homology arms all the known control ts for the expression of Sry. Its
lacZ-encoded beta-galactosidase serves as a reporter for the tissue-specific and
developmental stage-specific expression of the Sry gene. TALEN- and CRISPR-induced
mutations accompanied by LTVEC insertions were created in both the VGB6 (a.k.a. B6A6)
and VGF1 (a.k.a. F1H4) ES cell lines.
] We obtained a TALEN (TALEN-1) designed to target part of the HMG box DNA
binding motif coding sequence (upstream recognition sequence: 5´-
TCCCGTGGTGAGAGGCAC-3´ (SEQ ID NO. 72); downstream ition sequence: 5´-
TATTTTGCATGCTGGGAT-3´ (SEQ ID NO. 73) in the Sry gene. TALEN-1 was active in
creating NHEJ mutations at the Sry locus in multiple experiments.
Both VGB6 and VGF1 mouse ES cells were created with TALEN-induced
mutations. Table 1 contains a list of all the clones and the sizes of the deletion mutations
they carry. (ND in Table 1 indicates that a mutation was detected by a qPCR assay, but the
exact molecular nature of the mutation was not determined.) All clones also carry at least one
copy of the NIH KOMP project VG12778 LTVEC.
Table 1
TALEN- and CRISPR-induced mutations in the Sry gene
ES cell Clone Mutation inducing agent Deletion (bp)
VGB6 DE7 TALEN- 1 9
DEl 1 TALEN- 1 303*
DG5 TALEN- 1 627
DH1 TALEN- 1 ND
EA2 TALEN- 1 ND
ED4 TALEN- 1 > 1200
EF4 TALEN- 1 16
EG7 TALEN-l >1200
VGB6 RD3 TALEN- 1 >1200
RE9 TALEN- 1 ND
RF3 TALEN- 1 9
RG7 TALEN- 1 15
SF7 TALEN- 1 3
SGl 1 TALEN- 1 6
SH2 TALEN- 1 >200
SHl 1 TALEN- 1 2
VGFl TB1 TALEN- 1 1 1
TC2 TALEN-l 5
UA5 TALEN- 1 15
UB5 TALEN- 1 1201
UE12 TALEN- 1 9
WEI 1 TALEN- 1 >1200
VGB6 0G6 CRISPR-2 9
QE8 CRISPR-3 5
VGFl AU-B6 CRISPR-4 5
AU-C12 CRISPR-4 8
AW-H5 CRISPR-S 22
*Also contained a 50 bp insertion
The results of njections of the Sry mutant clones are set forth in Table 2 and
the breeding results of sex-reversed females are set forth in Table 3.
2015/038001
Table 2
F0 generation VelociMice produced by microinjection of Sry mutant ES cell clones into 8—cell embryos
ES Cell Clone Sry mutation Female VM Male VM
VGB6 ED4 >1 kb deletion 2 O
EG7 >1 kb deletion 19 O
GB4 None 3
GGl None 5
DEll 303 bp deletion; 50 bp insertion O
DG5 627 bp deletion ll 0
VGFl TA3 None 0 5
TA4 None 0 l l
TBl 11 bp deletion 2 0
TC2 5 bp deletion 8 O
TH4 None 2 6
UB5 1,201 bp deletion 6 0
WEll >1.2 kb deletion 7 0
UA5 15 bp deletion 4 O
UE12 9 bp deletion 8 O
Table 3
Breeding results of XY female VelociMice with mutations in the Sry gene
2:11 SW Elgéetion
Clone XYIlSeglale 1:351:86 Pups born
VGB6 EG7 >1,200 1460403 0
1460404 0
1460405 0
1460406 1 0*
1460408 0
1460409 0
1460410 0
VGB6 DG5 627 1460428 0
1460429 0
1460430 0
1460431 0
1460432 0
1460436 0
1460437 0
1460438 0
1460410 0
VGF1 UB5 1,201 1525585 5 33
1525586 5 25
1525587 5 32
1525588 3 35
1525589 3 19
VGF1 WE11 >1,200 1525573 3 4
1525574 5 21
1525575 4 11
1525576 4 14
1525577 4 16
1525578 2 4
1525579 2 6
VGF1 TB1 11 1525700 5 30
1525701 4 28
VGF1 TC2 5 6 1 2
1525707 5 10
1525708 1 6
9 4 17
0 1 3
1525711 3 9
2 4 12
1525713 2 7
VGF1 UA5 15 1594102 2 5
1594103 2 7
1594104 1 3
1594105 2 15
VGF1 UE12 9 1594117 1 6
1594118 2 12
9 1 11
1594120 2 8
1594121 2 21
1594122 2 15
1594123 2 10
*XY Female ID# 1460406 had to be euthanized before birth because she had a erm crisis and could not
deliver. Her dead pups (4 male, 5 females) were recovered by dissection and none carried the Sry mutation.
All of the VelociMice with Sry mutations derived from VGB6 ES cells were
female, as expected for inactivation of Sry (Table 2). Those without Sry mutations but
carrying at least one copy of the NIH KOMP VG12778 LTVEC produced only male
VelociMice (Table 2, clones GB4 and GGl). When 17 Sry mutant female B6 VelociMice
were test bred, only one became pregnant after about four months of breeding set-up (Table
3), and that female had to be euthanized before birth because she had a near-term crisis and
could not deliver. Her dead pups (4 male, 5 females) recovered by dissection were all WT;
none carried the Sry on. It was concluded that nearly all Sry mutant mice made from
VGB6 ES cells are sterile, which is in agreement with the literature on Sry mutations.
However, our data demonstrated very different result with the VGF1 clones.
First, the VGF1 ES cells were maintained, as usual, in our M-like low
osmotic strength growth medium that is zing: some of the microinj ected XY clones
grown in this medium will produce fertile XY females, i.e. an XY female enon, even
though they do not carry mutations. An example is clone TH4, which has no Sry mutation
but carries at least one copy of the NIH KOMP VG12778 LTVEC. This clone produced 2
female and 6 male VelociMice (Table 2). Two other VGF1 clones with no Sry mutations
(TA3 and TA4, Table 2) produced only male VelociMice. We wanted to determine if VGF1
XY ES cells with ons in Sry might also be feminized by the medium. In other words,
would they, unlike the VGB6 Sry mutant ES cells, produce some fertile XY Sry mutant
females? (Note that VGB6 ES cells cannot be maintained in KO-DMEM-like low c
strength media and retain the ability to produce mice.) The answer is yes as shown in Table
Six VGF1 ES cell clones with induced small deletions ranging from 5 bp
to over 1 kb were microinjected. All produced female VelociMice, 32 of which were bred.
Remarkably, all of the Sry mutant XY female VelociMice were fertile; each produced at least
one litter (Table 3). Many of the Sry mutant XY females produced multiple litters with
normal litter sizes, while some of the XY females produced only one or two small litters. Out
of 299 F1 mice from these breedings that have been genotyped, approximately half (146,
49%) are normal XY males or normal XX females. 174 (58%) of the F1 mice were
ypic females, while 125 (42%) were phenotypic males. 26 of the s (15% of
females, 8.7% of the total F1 generation) were XY females that inherited a mutant Sry allele.
Because of meiotic sjunction events associated with XY oocytes, a number of aberrant
genoytpes — XXY, XYY, XO, XXYY — some of which included mutant Sry alleles were
observed in the F1 progeny of Sry mutant XY female Mice.
A method for the efficient creation of fertile XY female VelociMice from XY ES
cells has been discovered. If inactivating mutations in the Sry gene in ES cells are created
that have been ined in the feminizing growth medium, a high proportion of fertile XY
female mice are obtained that when bred to males produce mostly male and female mice with
normal X and Y chromosomes.
Example 3. Embryo ry in KO-DMEM or DMEM after TALEN-induced Mutations in
the Y Chromosome Gene Sry
Correct targeting of mouse Sry by LTVEC was confirmed or negated by
genotyping of F1 offspring derived from F0 females, which were XY and carried Sry
mutation. Co-segregation in F1 mice of the LacZ/Neo cassette with the Sry mutation (as
assessed by Sry LOA assays) strongly suggests correct ing. Failure of LacZ/Neo to co-
segregate with the mutation indicates that the original clone contained an Sry deletion
mutation (induced by TALEN) coupled with a LacZ/Neo transgenic insertion elsewhere in
the genome.
Offspring from XY females with Sry ons exhibited a variety of abnormal
karyotypes at a high frequency (including XXY, XYY, and X0). Sex chromosome count was
ed by using unrelated loss of allele (LOA) assays for genes on X and Y chromosomes.
The copy number of Sry was then determined using LOA assays. The presence of mutant Sry
allele was inferred in mice in which the Y chromosome copy number exceeded the Sry copy
number (for instance, 1 copy of Y and 0 copies of Sry, or 2 copies of Y and 1 copy of Sry).
Lastly the ce of LacZ and Neo were determined using TaqMan assays.
In the original set of clones, which were created by Sry LTVEC together with
TALEN nuclease and grown in M, it was evident that LacZ/Neo cassette was not
co-segregating with the Sry mutation. A sample litter from these clones is shown in Table 4.
2015/038001
Table 4: Screening of clones generated by Sry LTVEC together with TALEN nuclease
Mouse SeX X Chr Y Chr Sry LacZ Neo Genotype ts
Copy # Copy # Copy
1656721 M 1 1 1 X+Y+
1656722 M 1 1 1 X+Y+ LacZ/Neo
present by Sry
mutation absent
1656723 M 1 1 1 0 0 X+Y+
1656724 M 1 2 1 0 0 X+Y+YA Sry mutation
present but
LacZ/Neo
absent
1656725 F 2 0 0 X+X+ LacZ/Neo
present but Sry
mutation absent
1656726 F 2 1 0 X+X+ Sry on
YA present but
LacZ/Neo
absent
1656727 F 2 1 0 X+X+
1656728 F 1 0 0 X+ LacZ/Neo
present but Sry
mutation absent
1656729 F 1 0 0 X+
In the subsequent set of clones, which were created by Sry LTVEC together with
TALEN nuclease and grown in DMEM, the eo cassette was completely co-
segregating with the Sry mutation, indicating correct targeting. A typical litter from these
clones is shown in Table 5.
Table 5: Screening s for clones created by Sry LTVEC together with TALEN nuclease
Mouse SeX X Chr Y Chr Sry LacZ Neo Genotype
Copy # Copy # Copy
1848360 M 1 1 1 0 0 X+Y+
1848361 M 1 1 1 0 0 X+Y+
1848362 M 1 1 1 0 0 X+Y+
1848363 1 1 0 0
1848364 1 1
1848365
1848366
1848367
Example 4: TALEN and -Assisted Targeting of Sry by SmallTVECs or LTVECs
As depicted in a targeted deletion comprising a lacZ replacement allele for
Sry was created with either a LTVEC or a small ing vector (smallTVEC) together with
either TALEN nuclease or CRISPR guide RNAs, in combination with Cas9 DNA
endonuclease. The smallTVEC comprised, in order, an upstream homology arm of
approximately 700-800 bp, a beta-galactosidase coding sequence (lacZ) followed by a
polyadenylation signal, a neomycin resistance cassette flanked by loxP sites comprising a
human ubiquitin C promoter, including the first exon, first , and part of the second
exon, a neomycin phosphotransferase coding sequence, and a polyadenylation signal, and a
downstream homology arm of approximately 0 bp. The allele created by correct
targeting of the Sry gene with the targeting vector comprises a deletion of the imately
1 kb Sry open reading frame and replacement with the lacZ-neo cassette such that the beta-
galactosidase coding sequence is fused in-frame at the Sry start codon. The targeting vector
was used to target the Sry gene in the VGFl (a.k.a. FlH4) /129 F1 hybrid ES cell
line and in the VGB6 ES cell line (a.k.a. B6A6).
As illustrated in Table 6, clones produced using four different gRNAs and one TALEN pair
were produced and screened for cleavage and loss of allele by TaqMan assays.
Table 6: ing results for cleavage and loss of allele
Small TVEC
Targeting
Location Clones Total targ. Eff.
Screened (%)
HMG box 192 2.1
HMG box 192 2.6
3’ end 192 1.6
3’ end gRNA 5 192 2.6
HMG box TALEN pair 1 384 0.3
The LTVEC transgenic clones produced embryos with the same lacZ n. illustrates LacZ expression in the embryos.
Table 7 reports the fertility results of XY Females derived from ES cells grown in
conventional ased medium that had TALEN-assisted LTVEC targeted deletion-
replacement ons of Sry. Unexpectedly compared with the results for a similar
experiment with ES cells grown in KO-DEMEM-based medium (Table 3), LTVEC targeting
in DMEM-based medium produced clones with tly targeted Sry ons and lacZ—neo
insertions. Forty out of 41 XYS'WQCZ) females derived from four targeted clones produced live
born pups upon mating — a 98% fertility rate. Thus, we have devised two new ways to
produce highly e XY females from mutant ES cells: (1) induced inactivating
mutations in Sry in ES cells grown in a KO-DMEM-based medium; and (2) TALEN-assisted
LTVEC targeted precise deletion-replacement mutations in ES cells grown in ased
Table 7: Production of Sry TALEN Mutant XY Females
Clone ES Allele XY female XY Fertile Fertility
cell description VelocMice females XY rates
line bred females (%)
X-C4 VGFl lacZ—neo
2 2 100
targeted
X-El 0 VGFl lacZ—neo
1 1 1 100
targeted
XOF3 VGFl lacZ—neo
2 5 3 3 100
m targeted
2 X-G3 VGFl lacZ—neo
Q 9 3 2 67
targeted
VGFl Total 53 41 40 98
Example 5: Large Deletion on the Y Chromosome ed by ZFNs
As illustrated in large deletions, 500 kb or greater, were made on the Y
chromosome using ZFNs targeting the Kdm5d and the Usp9y genes. Table 8 provides
examples of zinc finger sequences on the Y chromosome.
Table 8: Zinc Finger Sequence on the Y Chromosome
Target SEQ ID
Y CHR Plate ch Fmger Sequence_ _ ZFN#
Name N0:
ZFN1 42
NMOll419—r43102al ttAGGTAGGTAGACAGGGATgttttctg
KDMSD NMOll419—43108al atCCAGTCtCTGAAGGAAGCTctgacta
NMOll419—rl9880al caAAAGCTTCAGGGGGActcttacactc
NMOll419—19887al ttTGAGCAgGCTACACAGGAGtatactt
ZFNS 46
Oll4l9—rl7347al aaGCGGTGgCAATAGGCAaaagatgtgg
011419—17353al TCCCCAAGGGAGTAtggagatg
Oll4lg—rl7350al agAAAGCGGTGGCAaTAGGCAaaagatg
011419—17356al aaGTCCCCAAGGGAGTAtggagatgccc
012008—r8130al acTCCAACGACTATGACcactccgttca
012008—8136al acAGATCAGATGAAGATgactggtcaaa
—r7l72al ctTTCAAGGAAAAAAAGaacaaaaccca
DDX3Y 012008—7178al ggTCTGTGATAAGGACAGTTcaggatgg
OlZOO8—r20472al taAATCTGACTGAGAATGGGtagtagaa
012008—20479al GTCCAGGAGAGGCTttgaaggc
012008—r7267al atTGGGCTTCCCTCTGGAatcacgagat
OlZOO8—7274al ttTCAGTGATCGTGGAAGTGgatccagg
l48943—r9256lal ctGGTTTGGAAATCGTActgtaaaagac
l48943—92567al gcAAAGAGGTTGAGGATttggacatatt
l48943—r11830al gaGGAGTTGTTGGAGAAGTthattgga
[JSPQY l48943—ll836al atATGAACAAGGCCAAthgatgctcca
l48943—r108581al aCTCAGAAGAAGGATTAGGAatgctttg
148943—108588al atGCTTAGaAATGTATCAGTTcatcttg
l48943—rl6244al tcCATAAGGATTTTGGAaaaagacacag
ZFN8 65
—l6251al agGCTGTGAGTGGATGGAAGtttgaaat
In one experiment, 3.3 million ES cells from VGB6 clones D-G5 and E-G7
(Table 3) were electroporated with the ZFN mRNA pairs Kdm5d-ZFN5(NM01 1419-
al)/ZFN6(NM01l4l9-l7353al) and Usp9y-ZFN3(NM148943-
rl1830al)/ZFN4(NM148943-l1836al) (10 ug each) and with an LTVEC targeting the Ch25h
gene (0.67 ug), to e selection for puromycin resistance. Puromycin resistant colonies
were picked and screened for the deletion. The results are shown in Table 9.
Table 9: Screening Results for large Y chromosome deletion in 12778D-G5 and 12778E-G7
Parental Clone # of Puromycin- # of Colonies # Confirmed Deleted
resistant Colonies Screened Clones
12778E-G7 638 384 8
Table 10 shows the exact sizes of the greater than 500 kb deletions that were
precisely determined for one deletion clone (4306A-D5) derived from the E-G7 parental
clone (Table 3) and two deletion clones (4306E-C4 and 4306F-A12) derived from the D-G5
parental clone (Table 3).
Table 10: ZEN-mediated deletions of Kdm5d and Usp9y
Deletion Coordinates
Clone Size (bp)
on Y Chromosome
4306A-D5 250569-785404 534835
4306E-C4 520363-785402 535039
4306F—A12 250373-785404 535031
Deletion of the Kdm5d, Eif2s3y, Uty, Ddx3y, and Usp9y genes ( was
confirmed in the deletion loss-of-allele assays and DNA sequencing as shown in
Clone 4306A-D5 produced nine XY female fully ES cell-derived VelociMice upon
microinjection into 8-cell stage embryos and er to ate mothers. None of the XY
females from clone 4306A-D5 were fertile.
Example 6: Large Deletion on the Y Chromosome Mediated by CRISPR/Cas
A large deletion of the on the Y chromosome targeting the region n the
Kdm5d and the Usp9y genes was made utilizing CRISPR guide RNAs in combination with
Cas9 DNA endonuclease. gRNAs were designed to target the Kdm5d gene and the Usp9y
gene. The ing gRNAs were designed to target Kdm5d: A (Guide #1)
UUUGCCGAAUAUGCUCUCGU (SEQ ID ; Kdm5ng (Guide #2)
UUGCCGAAUAUGCUCUCGUG (SEQ ID NO:67); and Kdm5dgC (Guide #5)
CGGGCAUCUCCAUACUCCCU (SEQ ID NO:68). The following gRNAs were designed to
target Usp9y: A (Guide #1) UAGCUCGUUGUGUAGCACCU (SEQ ID NO:69);
B (Guide #1) UAUAGUUUCUUCGGGGUAAC (SEQ ID NO:70); and Usp9ng
(Guide #2) GGAUACCCUUCUAUAGGCCC (SEQ ID NO:7l).
VGFl mouse ES cells were electroporated with 5 ug of a plasmid that expressed
Cas9 and 10 ug each of plasmids that expressed the Kdm5d gRNA B and Usp9y gRNA C
and with an LTVEC targeting the Ch25h gene (0.67 ug), to e selection for puromycin
resistance. .
As illustrated in Kdm5ng (gRNA B) and Usp9ng (gRNA C) were used
to target the deletion of the Kdm5d and Usp9y genes. The resulting clones were screened for
deletion by loss-of-allele assays for sequences at the Kdm5d and Usp9y genes and the genes
in between (Eif2s3y, Uly, and Ddx3y) and for genes outside the ed deletion (ny2 and
Sry). As shown in Table 11, four clones comprising the large deletion were obtained. Clone
R-AS produced seven XY male and 3 XY female fully ES cell-derived VelociMice upon
microinjection into 8-cell stage embryos and transfer to surrogate mothers.
Table 11: TaqMan assay confirming large deletion mediated by CRISPR guide
RNAs and Cas9
Loss-of—allele Copy Number Determination
Clone 19178TD 16697TD DdX3yZF12 Note
Zjfi/Z Sry
(1317253y)
Large
Q_F1 0 1
deletion
R-A8 o 1 Large
deletion
Large
R-C2 0 1 deletion,
partial loss Y
clone add as
R4511 1 1
WT control
All publications and patent applications ned in the specification are
indicative of the level of those skilled in the art to which this ion pertains. All
publications and patent applications are herein incorporated by reference to the same extent
as if each individual publication or patent application was specifically and individually
indicated to be incorporated by nce. If the information associated with a on, such
as a deposit number changes with time, the version of the ation in effect at the
effective filing date of the application is intended, the effective filing date meaning the actual
filing date or date of a priority application first providing the citation. Unless otherwise
apparent from the context of any embodiment, aspect, step or feature of the invention can be
used in combination with any other. Reference to a range includes any integers within the
range, any subrange within the range. Reference to le ranges includes ites of
such ranges.
THE
Claims (22)
1. A method for making a mouse XY embryonic stem (ES) cell line capable of producing a fertile XY female mouse in an F0 generation, comprising: (a) modifying a mouse XY ES cell to comprise a genetic modification comprising a deletion that vates an nous Sry gene, wherein the mouse XY ES cell comprises a Y chromosome from a 129S6 strain; and (b) culturing the modified mouse XY ES cell under conditions to produce the mouse XY ES cell line capable of producing the fertile XY female non-human mammal in the F0 generation.
2. The method of claim 1, wherein the genetic modification is generated by introducing into the mouse XY ES cell a nuclease agent that induces a nick or a double-strand break at a recognition site on the Y chromosome or a cleotide encoding the nuclease agent.
3. The method of claim 2, wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription tor-Like Effector Nuclease (TALEN), a meganuclease, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA).
4. The method of claim 3, n the nuclease agent is the Cas protein and the gRNA, wherein the Cas n is a Cas9 protein, and wherein the gRNA comprises: (a) a CRISPR RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately d by a Protospacer Adjacent Motif (PAM) sequence; and (b) a trans-activating CRISPR RNA (tracrRNA), optionally wherein the gRNA targets a sequence comprising SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
5. The method of claim 3, wherein the nuclease agent is the TALEN, optionally wherein the TALEN targets a sequence comprising SEQ ID NO: 72 and a sequence comprising SEQ ID NO: 73.
6. The method of any one of claims 1-5, wherein the genetic cation is generated by introducing into the mouse XY ES cell a ing vector comprising an insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites at a target c locus on the Y chromosome, wherein the mouse XY ES cell is modified to se the insert polynucleotide at the target genomic locus.
7. The method of claim 6, wherein the first homology arm is from about 400- 1000 base pairs, and the second homology arm is from about 400-1000 base pairs.
8. The method of claim 6, wherein the sum total of the first homology arm and the second homology arm is at least 10 kb.
9. The method of any one of claims 6-8, wherein the genetic modification is generated by introducing into the mouse XY ES cell: (i) a nuclease agent that induces the nick or a double-strand break at the recognition site on the Y chromosome or a polynucleotide encoding the nuclease agent; and (ii) the targeting vector comprising the insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites at the target genomic locus on the Y chromosome, wherein the mouse XY ES cell is modified to comprise the insert polynucleotide at the target c locus.
10. The method of any one of claims 1-9, wherein the mouse XY ES cell is not cultured in a feminizing medium.
11. The method of any one of claims 1-9, wherein the culturing step comprises ing the ed mouse XY ES cell in a medium comprising: (1) a base medium comprising about 3 mg/mL sodium chloride and about 2.2 mg/mL sodium bicarbonate and having an osmolality of about 218 mOsm/kg; and (2) supplements that maintain the modified mouse XY ES cell in culture.
12. The method of any one of claims 1-11, wherein upon uction of the modified mouse XY ES cell into a mouse host embryo and following gestation of the mouse host embryo to produce F0 mice, at least 60% of the F0 mice are XY females which upon attaining sexual maturity are fertile.
13. A method for making a fertile XY female mouse in an F0 generation, comprising: (a) ucing a mouse XY nic stem (ES) cell into a mouse host embryo, wherein the mouse XY ES cell comprises a Y chromosome from a 129S6 strain and a genetic modification comprising a deletion that inactivates an endogenous mouse Sry gene, and optionally n the mouse host embryo is a pre-morula stage embryo; (b) gestating the mouse host embryo; and (c) ing F0 mice following gestation of the mouse host embryo, wherein at least 60% of the F0 mice are XY females which upon attaining sexual ty are fertile.
14. The method of claim 13, wherein the F0 XY female mouse is fertile when crossed to a wild-type mouse, optionally wherein the wild-type mouse is a C57BL/6 mouse.
15. The method of any one of claims 1-14, wherein the mouse XY ES cell is ed from a mouse that is a cross between a 129S6 strain and a C57BL/6 strain.
16. The method of claim 15, wherein the mouse XY ES cell is isolated from a hybrid embryo produced by crossing a female C57BL/6NTac mouse to a male 129S6/SvEvTac mouse.
17. The method of claim 16, wherein the mouse XY ES cell is a VGF1 mouse ES cell.
18. The method of any one of claims 1-17, wherein the genetic modification further comprises an insertion of one or more nucleotides, a substitution of one or more nucleotides, or a combination thereof, optionally wherein: (I) the genetic modification r comprises a knockout; a knockin; a replacement of an nous nucleic acid sequence with a homologous, orthologous, or heterologous sequence; or a combination f; (II) the genetic modification further comprises an insertion of a nucleic acid encoding a selectable marker and/or a nucleic acid encoding a reporter gene operably linked to a promoter active in the mouse XY ES cell; or (III) the genetic modification further comprises an insertion of a nucleic acid encoding a reporter gene ly linked to an endogenous Sry promoter.
19. The method of any one of claims 1-18, further comprising modifying the mouse XY ES cell to comprise at least one additional targeted genetic cation of a polynucleotide of interest.
20. The method of any one of claims 12-19, wherein upon introduction of the modified mouse XY ES cell into the mouse host embryo and following gestation of the mouse host embryo, at least 80% of the F0 mice are XY females which upon attaining sexual maturity are fertile, optionally wherein at least 90% of the F0 mice are XY females which upon attaining sexual maturity are fertile, and optionally wherein at least 95% of the F0 mice are XY females which upon ing sexual maturity are e.
21. A cell produced by the method of any one of claims 1-12.
22. A mouse produced by the method of any one of claims 13-20. B * E F C E !LX" - + / Z` D DZ`EC, DSaO WP @96<$ SVN]MON NOTO\SWV B^]TT!((+" DZ`EC,!?83" 7 E F QC@2*!G8$*" 4K[0 MTOK^ON MTWVO[ - / + , DZ`EC+ E 7 DZ`EC( DXRT!0'" Z` D >EG64 \KZQO\ON MTWVO[ ' ' V%K% ' ( 0 DZ`EB( W_ B + L 8 MO PZWU 2 WP 2E8 !LX" )* +/ ('. ' ( , *,+! " E Z` 14 ? D % 14 % ( 9 7 DZ`EC) E D Z` * E B + QC@2)!G8$)" Z ` D D Z`E7 DZ`E7* " $( 8 DZ`EB) ( !G 2 @ C Q EKZQO\ DOY]OVMO !B2?" DZ`E7) ,c$442E822E842EEE2E88E8!E88"$* b ,c$448E88E828288424228E!E88"$* b ,c$842284284E8882E84288!E88"$* c QC@2 G8$( G8$) G8$* QC@2+ QC@2, $ * 6 @ > E 2 =A?B ?2;5 ()../ >EG64 " .03$ !2 O X M W Z O O V ] V d Y [O X ZW Q 4 L S V F MW N R *,+! # D Z ` EKY?KV K[[K`[ \W [MZOOV PWZ QO KVN >A2 [\KZ\ \W [\WX MWNWV NOTO\SWV " .03$ Z X!2 [UKTTEG64 .' & ZO ) $ ) @ 6 4C;DBC QC@2[ $ ( 6 @ > W _ QC@2) QC@2* * E 2 L 8 ) ? 9 C < ( 1 )0,.- */41- (4736 *,+! $ .')& [\KSVSVQ 8&##"% $# 6+01 54206-5 IY6 (#,''#''' JP`) IY5 IY4 * ) IY4 ''' J7@*&+ QC@2 4$F[X0` IY4 ( #"!! -( *+.+2,0/ *,+! % F[X0` 5N_*` IY3 IY2 ) F\` ,''#''' 6SP)[*` E[X`$X[ ; IY2 ( J7@,&- QC@2 3$=NU,N FLK(` =NU,N MRZI !Y2(" MRZI1 JP`( #"#"%#!%%%%$##%"%%$!##"!##$#%%$"$$"$%!%$%%$$"$""$%#%#"%%$$""!$""%#"$"%$" $"$$"$%%$%%$$"$""$%#%#"%%$$ !!!!!!!!!%!$$"$""$%#%#"%%$$ !!!!!!!!!%!$$"$""$%#%#"%$$$ !!!!!!!!!%!$##$""$%#%#"%%$# !!!!!!!!!%$$$"$""$%#%#"%%$$ *,+! &' *,+! &( %#"$"%"$$"%"%%%%"$"$%%%%!#"%"%%$%"%$$%%"%"$ %#"$"%"$$"%"%%%%"$"$%%%%!#"%"%%$%!!!!!!!!!! %#"$"%"$$$%"%%%%"$"$%%%%!#"%"%%$%!!!!!!!!!! %###"%"$$"%"%%%%"$"$%%#%!#"%"%%$#!!!!!!!!!! %%%%"$"$%%%%%#"%"%%$%!!!!!!!!!! *,+! &) =NU, FB KVN F[X`0 5AH@ ""%%"##"#"%#%%%%%$##%"%%$%##""!!!!!!!!!!!!!"%"#%$$"$""$%#%#"%%$#""#$""%#"$"%$" ""%%"##"#"%#!%%%%$##%"%%$"#"""!!!!!!!!!!!!!"%$%%$$"$""$%#%#"%$$$""!$""%#"""%%$ #%"%#"$"%"$$"%"%%%%"$"$%%!%%#"%"%%$%"%$$"$$"$%%$%%$$"$""$%#%#"%%$$""$""%#"$"%$"%$"$"%#%%""% #%"%#"$"%"$$"%"%%%%"$"$%%$%%!!!!!!!!!!!!!!!!!!!!!!$$"$""$%#%#"%%$$""$""%#"$"%$"%$"$"%#%%""% #%"%#"$"%"$$"%"%%%%"$"$%%$%%!!!!!!!!!!!!!!!!!!!!!!$$"$""$%#%#"%%$$""$""%#"$"%$"%$"$"%#%%""% #%"%#"#"%"$$"%"%%%%"$"$%%$%%!!!!!!!!!!!!!!!!!!!!!!$$#$""$%#%#"%%$$""$""%#"$"%$"%$"$"%#%%""% =NU, FB KVN FX[` 5AH@ (,''7 ($5, (,''7 ($5, ('''C ('''C ('''7 (,''C ('''7 =NU, FB KVN F[X` 5AH@ (,''7 ('''C
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