U.S. patent application number 16/531901 was filed with the patent office on 2019-12-19 for methods and compositions for generating or maintaining pluripotent cells.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Wojtek Auerbach, Junko Kuno, David M. Valenzuela.
Application Number | 20190382730 16/531901 |
Document ID | / |
Family ID | 54361190 |
Filed Date | 2019-12-19 |
United States Patent
Application |
20190382730 |
Kind Code |
A1 |
Kuno; Junko ; et
al. |
December 19, 2019 |
METHODS AND COMPOSITIONS FOR GENERATING OR MAINTAINING PLURIPOTENT
CELLS
Abstract
Methods and compositions are provided for generating or
maintaining human iPS cells in culture. Methods include the use of
a low osmolality medium to make human iPS cells, or use of a low
osmolality medium to maintain human iPS cells. Methods for making
targeted genetic modification to human iPS cells cultured in low
osmolality medium are also included. Compositions include human iPS
cells cultured and maintained using the low osmolality medium
defined herein.
Inventors: |
Kuno; Junko; (Holmes,
NY) ; Auerbach; Wojtek; (Ridgewood, NJ) ;
Valenzuela; David M.; (Yorktown Heights, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
54361190 |
Appl. No.: |
16/531901 |
Filed: |
August 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14884293 |
Oct 15, 2015 |
10428310 |
|
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16531901 |
|
|
|
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62064384 |
Oct 15, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/727 20130101;
C12N 2500/32 20130101; C12N 2501/235 20130101; C12N 2500/60
20130101; C12N 2500/38 20130101; C12N 5/0696 20130101; C12N 2509/00
20130101; C12N 2500/44 20130101 |
International
Class: |
C12N 5/074 20060101
C12N005/074 |
Claims
1. A method for modifying a target genomic locus in a human induced
pluripotent stem cell (hiPSC), comprising: (a) providing a
population of naive hiPSCs that display a morphology characterized
by compact, dome-shaped colonies, wherein the hiPSCs are cultured
in a low osmolality medium comprising a base medium and
supplements, wherein the low osmolality medium comprises: (i) a
leukemia inhibitory factor (LIF) polypeptide; (ii) a glycogen
synthase kinase 3 (GSK3) inhibitor; and (iii) a MEK inhibitor, and
wherein the low osmolality medium has an osmolality of about 200
mOsm/kg to about 250 mOsm/kg; (b) introducing into the population
of hiPSCs a targeting vector comprising an insert nucleic acid
flanked by 5' and 3' homology arms corresponding to 5' and 3'
target sites at the target genomic locus; and (c) identifying a
genetically modified hiPSC comprising in its genome the insert
nucleic acid integrated at the target genomic locus.
2.-31. (canceled)
32. A method for modifying a target genomic locus in a human
induced pluripotent stem cell (hiPSC), comprising: (a) providing a
population of naive hiPSCs that display a morphology characterized
by compact, dome-shaped colonies, wherein the hiPSCs are cultured
in a low osmolality medium comprising a base medium and
supplements, wherein the low osmolality medium comprises: (i) a
leukemia inhibitory factor (LIF) polypeptide; (ii) a glycogen
synthase kinase 3 (GSK3) inhibitor; and (iii) a MEK inhibitor, and
wherein the low osmolality medium has an osmolality of about 200
mOsm/kg to about 250 mOsm/kg; (b) introducing into the population
of hiPSCs a nuclease agent that induces one or more nicks or
double-strand breaks at a recognition site at the target genomic
locus; and (c) identifying a genetically modified hiPSC comprising
in its genome a modification at the target genomic locus.
33.-58. (canceled)
59. The method of claim 1, wherein the targeting vector is a large
targeting vector (LTVEC), wherein: (I) the LTVEC is at least 10 kb
in size; (II) the LTVEC is from about 50 kb to about 300 kb in
size; (III) the sum total of the 5' and 3' homology arms is at
least 10 kb; (IV) the sum total of the 5' and 3' homology arms is
from about 10 kb to about 200 kb; (V) the 5' homology arm is from
about 5 kb to about 100 kb and/or the 3' homology arm is from about
5 kb to about 100 kb; or (VI) the LTVEC comprises a nucleic acid
insert ranging from about 5 kb to about 200 kb.
60. The method of claim 1, wherein the targeted genetic
modification comprises: (a) deletion of an endogenous human nucleic
acid sequence; (b) insertion of an exogenous nucleic acid sequence;
or (c) replacement of the endogenous human nucleic acid sequence
with the exogenous nucleic acid sequence.
61. The method of claim 1, wherein the targeted genetic
modification comprises insertion of an exogenous nucleic acid
sequence, wherein the exogenous nucleic acid sequence comprises one
or more of the following: (a) a nucleic acid sequence that is
homologous or orthologous to the endogenous human nucleic acid
sequence; (b) a chimeric nucleic acid sequence; (c) a conditional
allele flanked by site-specific recombinase target sequences; and
(d) a reporter gene operably linked to a promoter active in the
hiPSC.
62. The method of claim 1, wherein introducing step (b) further
comprises introducing a nuclease agent that promotes homologous
recombination between the targeting vector and the target genomic
locus in the hiPSC.
63. The method of claim 62, wherein the nuclease agent comprises:
(a) a Zinc Finger Nuclease (ZFN); (b) a Transcription
Activator-Like Effector Nuclease (TALEN); (c) a meganuclease; or
(d) a Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) associated (Cas) protein and a guide RNA (gRNA) comprising
a CRISPR RNA (crRNA) that recognizes a genomic target sequence and
a trans-activating CRISPR RNA (tracrRNA).
64. The method of claim 63, wherein the nuclease agent comprises
the Cas protein and the guide RNA, wherein the Cas protein is
Cas9.
65. The method of claim 1, wherein prior to step (b), the hiPSCs
are enzymatically dissociated into a single-cell suspension and
subcultured.
66. The method of claim 65, wherein the enzymatic dissociation: (a)
is performed using trypsin; (b) is performed in the absence of a
ROCK inhibitor; or (c) a combination thereof, and wherein the
subcultured hiPSCs: (a) continue to express one or more
pluripotency markers; (b) maintain a naive state and display a
morphology characterized by compact dome-shaped colonies; or (c) a
combination thereof, and wherein the subcultured hiPSCs maintain a
normal karyotype.
67. The method of claim 1, wherein the hiPSCs have a normal
karyotype, and wherein the hiPSCs: (a) express one or more
pluripotency markers, wherein the pluripotency markers comprise
NANOG, alkaline phosphatase, or a combination thereof; (b) can
differentiate into cells of any one of the endoderm, ectoderm, or
mesoderm germ layers; (c) have a doubling time of between about 16
hours and about 24 hours; or (d) any combination of (a) to (c).
68. The method of claim 1, wherein the hiPSCs are derived from
non-pluripotent cells transformed to express a pluripotent state,
wherein the transformed cells express reprogramming genes
comprising Oct4, Sox2, Klf4, Myc, or any combination thereof.
69. The method of claim 1, wherein the base medium has an
osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
70. The method of claim 69, wherein the base medium comprises
sodium chloride at about 3 mg/mL, sodium bicarbonate at about 2.2
mg/mL, and glucose at about 4.5 mg/mL, and has an osmolality of
about 200 mOsm/kg.
71. The method of claim 1, wherein the low osmolality medium has an
osmolality of about 220 mOsm/kg to about 240 mOsm/kg.
72. The method of claim 71, wherein the low osmolality medium has
an osmolality of about 233 mOsm/kg.
73. The method of claim 1, wherein: (a) the supplements comprise:
(i) F-12 medium; (ii) N2 supplement; (iii) B-27 supplement; (iv)
L-glutamine; (v) 2-mercaptoethanol; or (vi) any combination of (i)
to (v); (b) the LIF polypeptide is a human LIF (hLIF) polypeptide;
(c) the GSK3 inhibitor comprises CHIR99021; (d) the MEK inhibitor
comprises PD0325901; (e) the hiPSCs are cultured on newborn human
foreskin fibroblast (NuFF) feeder cells; or (f) any combination of
(a) to (e).
74. The method of claim 1, wherein the low osmolality medium
comprises inhibitors consisting essentially of the glycogen
synthase kinase 3 (GSK3) inhibitor and the MEK inhibitor.
75. The method of claim 1, wherein the low osmolality medium
comprises base medium at about 24.75% (v/v), F-12 medium at about
24.75% (v/v), N2 supplement at about 0.5% (v/v), B-27 supplement at
about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at
about 0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 and
PD0325901 at about 0.5 optionally wherein the hiPSCs are cultured
on MATRIGEL, newborn human foreskin fibroblast (NuFF) feeder cells,
or GELTREX.
76. The method of claim 1, wherein the low osmolality medium does
not comprise one or more of the following: bFGF supplement;
TGF-.beta.1 supplement; JNK inhibitor; p38 inhibitor; ROCK
inhibitor; and PKC inhibitor.
77. The method of claim 76, wherein the low osmolality medium does
not comprise bFGF supplement.
78. The method of claim 32, wherein the nuclease agent comprises:
(a) a Zinc Finger Nuclease (ZFN); (b) a Transcription
Activator-Like Effector Nuclease (TALEN); (c) a meganuclease; or
(d) a Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) associated (Cas) protein and a guide RNA (gRNA) comprising
a CRISPR RNA (crRNA) that recognizes a genomic target sequence and
a trans-activating CRISPR RNA (tracrRNA).
79. The method of claim 78, wherein the nuclease agent comprises
the Cas protein and the guide RNA, wherein the Cas protein is
Cas9.
80. The method of claim 32, wherein prior to step (b), the hiPSCs
are enzymatically dissociated into a single-cell suspension and
subcultured.
81. The method of claim 80, wherein the enzymatic dissociation: (a)
is performed using trypsin; (b) is performed in the absence of a
ROCK inhibitor; or (c) a combination thereof, and wherein the
subcultured hiPSCs: (a) continue to express one or more
pluripotency markers; (b) maintain a naive state and display a
morphology characterized by compact dome-shaped colonies; or (c) a
combination thereof, and wherein the subcultured hiPSCs maintain a
normal karyotype.
82. The method of claim 32, wherein the hiPSCs have a normal
karyotype, and wherein the hiPSCs: (a) express one or more
pluripotency markers, wherein the pluripotency markers comprise
NANOG, alkaline phosphatase, or a combination thereof; (b) can
differentiate into cells of any one of the endoderm, ectoderm, or
mesoderm germ layers; (c) have a doubling time of between about 16
hours and about 24 hours; or (d) any combination of (a) to (c).
83. The method of claim 32, wherein the hiPSCs are derived from
non-pluripotent cells transformed to express a pluripotent state,
wherein the transformed cells express reprogramming genes
comprising Oct4, Sox2, Klf4, Myc, or any combination thereof.
84. The method of claim 32, wherein the base medium has an
osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
85. The method of claim 84, wherein the base medium comprises
sodium chloride at about 3 mg/mL, sodium bicarbonate at about 2.2
mg/mL, and glucose at about 4.5 mg/mL, and has an osmolality of
about 200 mOsm/kg.
86. The method of claim 32, wherein the low osmolality medium has
an osmolality of about 220 mOsm/kg to about 240 mOsm/kg.
87. The method of claim 86, wherein the low osmolality medium has
an osmolality of about 233 mOsm/kg.
88. The method of claim 32, wherein: (a) the supplements comprise:
(i) F-12 medium; (ii) N2 supplement; (iii) B-27 supplement; (iv)
L-glutamine; (v) 2-mercaptoethanol; or (vi) any combination of (i)
to (v); (b) the LIF polypeptide is a human LIF (hLIF) polypeptide;
(c) the GSK3 inhibitor comprises CHIR99021; (d) the MEK inhibitor
comprises PD0325901; (e) the hiPSCs are cultured on newborn human
foreskin fibroblast (NuFF) feeder cells; or (f) any combination of
(a) to (e).
89. The method of claim 32, wherein the low osmolality medium
comprises inhibitors consisting essentially of the glycogen
synthase kinase 3 (GSK3) inhibitor and the MEK inhibitor.
90. The method of claim 32, wherein the low osmolality medium
comprises base medium at about 24.75% (v/v), F-12 medium at about
24.75% (v/v), N2 supplement at about 0.5% (v/v), B-27 supplement at
about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at
about 0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 and
PD0325901 at about 0.5 optionally wherein the hiPSCs are cultured
on MATRIGEL, newborn human foreskin fibroblast (NuFF) feeder cells,
or GELTREX.
91. The method of claim 32, wherein the low osmolality medium does
not comprise one or more of the following: bFGF supplement;
TGF-.beta.1 supplement; JNK inhibitor; p38 inhibitor; ROCK
inhibitor; and PKC inhibitor.
92. The method of claim 91, wherein the low osmolality medium does
not comprise bFGF supplement.
93. A method for making a population of naive human induced
pluripotent stem cells (hiPSCs) that display a morphology
characterized by compact, dome-shaped colonies, comprising
culturing in vitro a population of non-pluripotent cells,
transformed to express a pluripotent state, in a low osmolality
medium comprising a base medium and supplements, wherein the low
osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)
polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(c) a MEK inhibitor; wherein the low osmolality medium has an
osmolality of about 200 mOsm/kg to about 250 mOsm/kg.
94. The method of claim 93, wherein the transformed cells are first
cultured in a high osmolality medium prior to culturing in the low
osmolality medium, wherein the high osmolality medium comprises
bFGF and has an osmolality of at least about 290 mOsm/kg, and
wherein: (a) the transformed cells are first cultured in the high
osmolality medium until they express characteristics of a naive
state; (b) the transformed cells are first cultured in the high
osmolality medium for a period of about two months; (c) the
transformed cells are first cultured in the high osmolality medium
until they display a morphology characterized by three-dimensional
cell clumps; or (d) a combination thereof.
95. A method for maintaining in an in vitro culture a population of
naive human induced pluripotent stem cells (hiPSCs) that display a
morphology characterized by compact, dome-shaped colonies,
comprising culturing the population of hiPSCs in a low osmolality
medium comprising a base medium and supplements, wherein the low
osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)
polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(c) a MEK inhibitor; wherein the low osmolality medium has an
osmolality of about 200 mOsm/kg to about 250 mOsm/kg.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/884,293, filed Oct. 15, 2015, which claims
the benefit of U.S. Patent Application No. 62/064,384, filed Oct.
15, 2014, all of which are herein incorporated by reference in
their entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS
WEB
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 532569SEQLIST.TXT, created on Aug. 5, 2019, and
having a size of 792 bytes, and is filed concurrently with the
specification. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety.
BACKGROUND
[0003] Human induced pluripotent stem (iPS) cells can display a
naive or primed state of pluripotency (Nichols and Smith, Cell Stem
Cell (2009) Vol. 4(6), pp. 487-492). Primed human iPS cells express
characteristics similar to those of post-implantation epiblast
cells, and are committed for lineage specification and
differentiation. By contrast, naive human iPS cells express
characteristics similar to those of embryonic stem (ES) cells of
the inner cell mass of a pre-implantation embryo. In some respects,
naive iPS cells are more pluripotent than primed cells, as they are
not committed for lineage specification. Various culture conditions
can be used to maintain human iPS in a naive state or in a primed
state.
SUMMARY
[0004] Methods are provided for making a population of human
induced pluripotent stem cells (hiPSCs). Such methods comprise
culturing in vitro a population of non-pluripotent cells,
transformed to express a pluripotent state, in a low osmolality
medium comprising a base medium and supplements, wherein the low
osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)
polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(c) a MEK inhibitor; wherein the medium has an osmolality of about
175 mOsm/kg to about 280 mOsm/kg. Such methods can also comprise
culturing in vitro a population of non-pluripotent cells,
transformed to express a pluripotent state, in a low osmolality
medium comprising a base medium and supplements, wherein the low
osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)
polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(c) a MEK inhibitor; wherein the base medium has an osmolality of
about 180 mOsm/kg to about 250 mOsm/kg.
[0005] Further provided are methods for maintaining a population of
hiPSCs in an in vitro culture, the methods comprising culturing the
population of hiPSCs in a low osmolality medium comprising a base
medium and supplements, wherein the low osmolality medium
comprises: (a) a leukemia inhibitory factor (LIF) polypeptide; (b)
a glycogen synthase kinase 3 (GSK3) inhibitor; and (c) a MEK
inhibitor; wherein the medium has an osmolality of about 175
mOsm/kg to about 280 mOsm/kg. Such methods can also comprise
culturing the population of hiPSCs in a low osmolality medium
comprising a base medium and supplements, wherein the low
osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)
polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(c) a MEK inhibitor; wherein the base medium has an osmolality of
about 180 mOsm/kg to about 250 mOsm/kg.
[0006] In some methods, the hiPSCs comprise naive or naive-looking
hiPSCs. In some methods, the hiPSCs comprise naive-like hiPSCs.
[0007] In some methods, the method enriches for a population of
naive or naive-looking hiPSCs. In some methods, the method enriches
for a population of naive-like hiPSCs.
[0008] In some methods, the transformed cells express reprogramming
genes comprising Oct4, Sox2, Klf4, Myc, or any combination thereof.
In some methods, the transformed cells comprise primed hiPSCs.
[0009] In some methods, the base medium has an osmolality of about
200 mOsm/kg. In some methods, the base medium comprises NaCl at
about 3 mg/ml, sodium bicarbonate at about 2.2 mg/mL, and has an
osmolality of about 200 mOsm/kg.
[0010] In some methods, the base medium comprises glucose at about
4.5 mg/mL.
[0011] In some methods, the low osmolality medium has an osmolality
of about 200 mOsm/kg to about 250 mOsm/kg. In some methods, the low
osmolality medium has an osmolality of about 233 mOsm/kg.
[0012] In some methods, the supplements comprise: (a) F-12 medium;
(b) N2 supplement; (c) NEUROBASAL medium; (d) B-27 supplement; (e)
L-glutamine; (f) 2-mercaptoethanol; or (g) any combination of (a)
to (f).
[0013] In some methods, the LIF polypeptide is a human LIF (hLIF)
polypeptide. In some methods, the GSK3 inhibitor comprises
CHIR99021. In some methods, the MEK inhibitor comprises PD0325901.
In some methods, the low osmolality medium comprises inhibitors
consisting essentially of a GSK3 inhibitor and a MEK inhibitor.
[0014] In some methods, the low osmolality medium comprises base
medium at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2
supplement at about 0.5% (v/v), NEUROBASAL medium at about 49%
(v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2
mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL,
CHIR99021 at about 3 .mu.M, and PD0325901 at about 0.5 .mu.M.
[0015] In some methods, the low osmolality medium does not comprise
one or more of the following: bFGF supplement, TGF-.beta.1
supplement, JNK inhibitor, p38 inhibitor, ROCK inhibitor, and PKC
inhibitor. In some methods, the low osmolality medium does not
comprise basic fibroblast growth factor (bFGF).
[0016] In some methods, the hiPSCs or the transformed cells are
cultured on MATRIGEL.TM., newborn human foreskin fibroblast (NuFF)
feeder cells, or GELTREX.TM..
[0017] In some methods, the hiPSCs express one or more pluripotency
markers. In some methods, the one or more pluripotency markers
comprises NANOG, alkaline phosphatase, or a combination thereof. In
some methods, the hiPSCs have a normal karyotype.
[0018] In some methods, the hiPSCs display a morphology
characterized by compact dome-shaped colonies.
[0019] In some methods, the hiPSCs can be enzymatically dissociated
into a single-cell suspension and subcultured. In some methods, the
enzymatic dissociation is performed using trypsin. In some methods,
the enzymatic dissociation can be performed in the absence of a
Rho-associated protein kinase (ROCK) inhibitor. In some methods,
the subcultured hiPSCs continue to express the one or more
pluripotency markers. In some methods, the subcultured hiPSCs
maintain a naive or naive-looking state and display a morphology
characterized by compact dome-shaped colonies. In some methods, the
subcultured hiPSCs maintain a normal karyotype.
[0020] In some methods, the hiPSCs can differentiate into cells of
any one of the endoderm, ectoderm, or mesoderm germ layers.
[0021] In some methods, the hiPSCs have a doubling time of between
about 16 hours and about 24 hours.
[0022] In some methods, the transformed cells are first cultured in
a high osmolality medium prior to culturing in the low osmolality
medium, wherein the high osmolality medium comprises bFGF.
Optionally, the high osmolality medium has an osmolality of at
least about 290 mOsm/kg.
[0023] In some methods, the transformed cells are first cultured in
the high osmolality medium until they express characteristics of a
naive or naive-looking state. In some methods, the transformed
cells are first cultured in the high osmolality medium for a period
of about two months. In some methods, the transformed cells are
first cultured in the high osmolality medium until they display a
morphology characterized by three-dimensional cell clumps.
[0024] Further provided are hiPSCs made by any of the above
methods.
[0025] Further provided are methods for modifying a target genomic
locus in a hiPSC, comprising: (a) introducing into the hiPSC a
targeting vector comprising an insert nucleic acid flanked by 5'
and 3' homology arms corresponding to 5' and 3' target sites at the
target genomic locus; and (b) identifying a genetically modified
hiPSC comprising in its genome the insert nucleic acid integrated
at the target genomic locus; wherein the hiPSC is cultured in a low
osmolality medium comprising a base medium and supplements, wherein
the low osmolality medium comprises: (a) a leukemia inhibitory
factor (LIF) polypeptide; (b) a glycogen synthase kinase 3 (GSK3)
inhibitor; and (c) a MEK inhibitor; wherein the medium has an
osmolality of about 175 mOsm/kg to about 280 mOsm/kg. Such methods
can also comprise: (a) introducing into the hiPSC a targeting
vector comprising an insert nucleic acid flanked by 5' and 3'
homology arms corresponding to 5' and 3' target sites at the target
genomic locus; and (b) identifying a genetically modified hiPSC
comprising in its genome the insert nucleic acid integrated at the
target genomic locus; wherein the hiPSC is cultured in a low
osmolality medium comprising a base medium and supplements, wherein
the low osmolality medium comprises: (a) a leukemia inhibitory
factor (LIF) polypeptide; (b) a glycogen synthase kinase 3 (GSK3)
inhibitor; and (c) a MEK inhibitor; wherein the base medium has an
osmolality of about 180 mOsm/kg to about 250 mOsm/kg. In some
methods, the targeting vector is a large targeting vector (LTVEC),
wherein the sum total of the 5' and 3' homology arms is at least 10
kb. In some methods, introducing step (a) further comprises
introducing a nuclease agent that promotes homologous recombination
between the targeting vector and the target genomic locus in the
hiPSC. In some methods, the targeted genetic modification
comprises: (a) deletion of an endogenous human nucleic acid
sequence; (b) insertion of an exogenous nucleic acid sequence; or
(c) replacement of the endogenous human nucleic acid sequence with
the exogenous nucleic acid sequence. In some methods, the exogenous
nucleic acid sequence comprises one or more of the following: (a) a
nucleic acid sequence that is homologous or orthologous to the
endogenous human nucleic acid sequence; (b) a chimeric nucleic acid
sequence; (c) a conditional allele flanked by site-specific
recombinase target sequences; and (d) a reporter gene operably
linked to a promoter active in the hiPSC.
[0026] Such methods for modifying a target genomic locus in a
hiPSC, can also comprise: (a) introducing into the hiPSC one or
more nuclease agents that induces one or more nicks or
double-strand breaks at a recognition site at the target genomic
locus; and (b) identifying at least one cell comprising in its
genome a modification at the target genomic locus; wherein the
hiPSC is cultured in a low osmolality medium comprising a base
medium and supplements, wherein the low osmolality medium
comprises: (i) a leukemia inhibitory factor (LIF) polypeptide; (ii)
a glycogen synthase kinase 3 (GSK3) inhibitor; and (iii) a MEK
inhibitor; wherein the medium has an osmolality of about 175
mOsm/kg to about 280 mOsm/kg. Such methods can also comprise: (a)
introducing into the hiPSC one or more nuclease agents that induces
one or more nicks or double-strand breaks at a recognition site at
the target genomic locus; and (b) identifying at least one cell
comprising in its genome a modification at the target genomic
locus; wherein the hiPSC is cultured in a low osmolality medium
comprising a base medium and supplements, wherein the low
osmolality medium comprises: (i) a leukemia inhibitory factor (LIF)
polypeptide; (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;
and (iii) a MEK inhibitor; wherein the base medium has an
osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
[0027] In any such methods for modifying a target genomic locus in
a hiPSC, the hiPSCs can be enzymatically dissociated into a
single-cell suspension and subcultured prior to step (a).
Optionally, the enzymatic dissociation is performed using trypsin.
Optionally, the enzymatic dissociation is performed in the absence
of a ROCK inhibitor. In some methods, the subcultured hiPSCs
continue to express one or more pluripotency markers. In some
methods, the subcultured hiPSCs maintain a naive or naive-looking
state and display a morphology characterized by compact dome-shaped
colonies. In some methods, the subcultured hiPSCs maintain a normal
karyotype.
[0028] In some methods, the nuclease agent comprises a zinc finger
nuclease (ZFN). In some methods, the nuclease agent comprises a
Transcription Activator-Like Effector Nuclease (TALEN). In some
methods, the nuclease agent comprises a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) associated (Cas)
protein and a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) that
recognizes a genomic target sequence and a trans-activating CRISPR
RNA (tracrRNA). Optionally, the Cas protein is Cas9.
[0029] In some methods, the targeted genetic modification is
biallelic.
[0030] In some methods, the hiPSCs comprise naive or naive-looking
hiPSCs. In some methods, the hiPSCs comprise naive-like hiPSCs. In
some methods, the hiPSCs express one or more pluripotency markers.
Optionally, the pluripotency markers comprise NANOG, alkaline
phosphatase, or a combination thereof. In some methods, the hiPSCs
display a morphology characterized by compact dome-shaped colonies.
In some methods, the hiPSCs can differentiate into cells of any one
of the endoderm, ectoderm, or mesoderm germ layers. In some
methods, the hiPSCs have a doubling time of between about 16 hours
and about 24 hours. In some methods, the hiPSCs have a normal
karyotype.
[0031] In some methods, the hiPSCs are derived from non-pluripotent
cells transformed to express a pluripotent state. Optionally, the
transformed cells express reprogramming genes comprising Oct4,
Sox2, Klf4, Myc, or any combination thereof. Optionally, the
transformed cells comprise primed hiPSCs. In some methods, the
transformed cells are first cultured in a high osmolality medium
prior to culturing in the low osmolality medium, wherein the high
osmolality medium comprises bFGF. Optionally, the high osmolality
medium has an osmolality of at least 290 mOsm/kg. In some methods,
the transformed cells are first cultured in the high osmolality
medium until they express characteristics of a naive or
naive-looking state. In some methods, the transformed cells are
first cultured in the high osmolality medium for a period of about
two months. In some methods, the transformed cells are first
cultured in the high osmolality medium until they display a
morphology characterized by three-dimensional cell clumps.
[0032] In some methods, the base medium has an osmolality of about
200 mOsm/kg. In some methods, the base medium comprises NaCl at
about 3 mg/ml, sodium bicarbonate at about 2.2 mg/mL, and has an
osmolality of about 200 mOsm/kg.
[0033] In some methods, the base medium comprises glucose at about
4.5 mg/mL.
[0034] In some methods, the low osmolality medium has an osmolality
of about 200 mOsm/kg to about 250 mOsm/kg. In some methods, the low
osmolality medium has an osmolality of about 233 mOsm/kg.
[0035] In some methods, the supplements comprise: (i) F-12 medium;
(ii) N2 supplement; (iii) NEUROBASAL medium; (iv) B-27 supplement;
(v) L-glutamine; (vi) 2-mercaptoethanol; or (vii) any combination
of (i) to (vi). In some methods, the LIF polypeptide is a human LIF
(hLIF) polypeptide. In some methods, the GSK3 inhibitor comprises
CHIR99021. In some methods, the MEK inhibitor comprises
PD0325901.
[0036] In some methods, the low osmolality medium comprises
inhibitors consisting essentially of a glycogen synthase kinase 3
(GSK3) inhibitor and a MEK inhibitor.
[0037] In some methods, the low osmolality medium comprises base
medium at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2
supplement at about 0.5% (v/v), NEUROBASAL medium at about 49%
(v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2
mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL,
CHIR99021 at about 3 .mu.M, and PD0325901 at about 0.5 .mu.M.
[0038] In some methods, the low osmolality medium does not comprise
one or more of the following: bFGF supplement; TGF-.beta.1
supplement; JNK inhibitor; p38 inhibitor; ROCK inhibitor; and PKC
inhibitor. In some methods, the low osmolality medium does not
comprise bFGF supplement.
[0039] In some methods, the hiPSCs are cultured on MATRIGEL, NuFF
feeder cells, or GELTREX.
[0040] Further provided are modified hiPSCs made by any of the
above methods.
[0041] Further provided are in vitro cultures comprising: (a) a
population of hiPSCs; and (b) a low osmolality medium comprising a
base medium and supplements, wherein the low osmolality medium
comprises: (i) a leukemia inhibitory factor (LIF) polypeptide; (ii)
a glycogen synthase kinase 3 (GSK3) inhibitor; and (iii) a MEK
inhibitor; wherein the medium has an osmolality of about 175
mOsm/kg to about 280 mOsm/kg. Such in vitro cultures can also
comprise (a) a population of hiPSCs; and (b) a low osmolality
medium comprising a base medium and supplements, wherein the low
osmolality medium comprises: (i) a leukemia inhibitory factor (LIF)
polypeptide; (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;
and (iii) a MEK inhibitor; wherein the base medium has an
osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
[0042] Further provided are populations of hiPSCs made or
maintained in a low osmolality medium comprising a base medium and
supplements, wherein the low osmolality medium comprises: (a) a
leukemia inhibitory factor (LIF) polypeptide; (b) a glycogen
synthase kinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor;
wherein the medium has an osmolality of about 175 mOsm/kg to about
280 mOsm/kg. Such populations of hiPSCs can also be made or
maintained in a low osmolality medium comprising a base medium and
supplements, wherein the low osmolality medium comprises: (a) a
leukemia inhibitory factor (LIF) polypeptide; (b) a glycogen
synthase kinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor;
wherein the base medium has an osmolality of about 180 mOsm/kg to
about 250 mOsm/kg.
[0043] In some populations or in vitro cultures, the hiPSCs
comprise naive or naive-looking hiPSCs. In some populations or in
vitro cultures, the hiPSCs comprise naive-like hiPSCs.
[0044] In some populations or in vitro cultures, the hiPSCs are
derived from non-pluripotent cells transformed to express a
pluripotent state. In some populations or in vitro cultures, the
transformed cells express reprogramming genes comprising Oct4,
Sox2, Klf4, Myc, or any combination thereof. In some populations or
in vitro cultures, the transformed cells comprise primed
hiPSCs.
[0045] In some populations or in vitro cultures, the base medium
has an osmolality of about 200 mOsm/kg. In some populations or in
vitro cultures, the base medium comprises NaCl at about 3 mg/ml,
sodium bicarbonate at about 2.2 mg/mL, and has an osmolality of
about 200 mOsm/kg.
[0046] In some populations or in vitro cultures, the base medium
comprises glucose at about 4.5 mg/mL.
[0047] In some populations or in vitro cultures, the low osmolality
medium comprising the base medium and supplements has an osmolality
of about 200 mOsm/kg to about 250 mOsm/kg. In some populations or
in vitro cultures, the low osmolality medium has an osmolality of
about 233 mOsm/kg.
[0048] In some populations or in vitro cultures, the supplements
comprise: (a) F-12 medium; (b) N2 supplement; (c) NEUROBASAL
medium; (d) B-27 supplement; (e) L-glutamine; (f)
2-mercaptoethanol; or (g) any combination of (a) to (f).
[0049] In some populations or in vitro cultures, the LIF
polypeptide is a human LIF (hLIF) polypeptide. In some populations
or in vitro cultures, the GSK3 inhibitor comprises CHIR99021. In
some populations or in vitro cultures, the MEK inhibitor comprises
PD0325901. In some populations or in vitro cultures, the low
osmolality medium comprises inhibitors consisting essentially of a
GSK3 inhibitor and a MEK inhibitor.
[0050] In some populations or in vitro cultures, the low osmolality
medium comprises base medium at about 24.75% (v/v), F-12 medium at
about 24.75% (v/v), N2 supplement at about 0.5% (v/v), NEUROBASAL
medium at about 49% (v/v), B-27 supplement at about 1% (v/v),
L-glutamine at about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF
at about 100 units/mL, CHIR99021 at about 3 .mu.M, and PD0325901 at
about 0.5 .mu.M.
[0051] In some populations or in vitro cultures, the low osmolality
medium does not comprise one or more of the following: bFGF
supplement, TGF-.beta.1 supplement, JNK inhibitor, p38 inhibitor,
ROCK inhibitor, and PKC inhibitor. In some populations or in vitro
cultures, the low osmolality medium does not comprise basic
fibroblast growth factor (bFGF).
[0052] In some populations or in vitro cultures, the hiPSCs or the
transformed cells are cultured on MATRIGEL.TM., newborn human
foreskin fibroblast (NuFF) feeder cells, or GELTREX.TM..
[0053] In some populations or in vitro cultures, the hiPSCs express
one or more pluripotency markers. In some populations or in vitro
cultures, the one or more pluripotency markers comprises NANOG,
alkaline phosphatase, or a combination thereof. In some populations
or in vitro cultures, the hiPSCs have a normal karyotype.
[0054] In some populations or in vitro cultures, the hiPSCs display
a morphology characterized by compact dome-shaped colonies.
[0055] In some populations or in vitro cultures, the hiPSCs can be
enzymatically dissociated into a single-cell suspension and
subcultured. In some populations or in vitro cultures, the
enzymatic dissociation is performed using trypsin. In some
populations or in vitro cultures, the enzymatic dissociation can be
performed in the absence of a Rho-associated protein kinase (ROCK)
inhibitor. In some populations or in vitro cultures, the
subcultured hiPSCs continue to express the one or more pluripotency
markers. In some populations or in vitro cultures, the subcultured
hiPSCs maintain a naive or naive-looking state and display a
morphology characterized by compact dome-shaped colonies. In some
populations or in vitro cultures, the subcultured hiPSCs maintain a
normal karyotype.
[0056] In some populations or in vitro cultures, the hiPSCs can
differentiate into cells of any one of the endoderm, ectoderm, or
mesoderm germ layers.
[0057] In some populations or in vitro cultures, the hiPSCs have a
doubling time of between about 16 hours and about 24 hours.
[0058] In some populations or in vitro cultures, the transformed
cells are first cultured in a high osmolality medium prior to
culturing in the low osmolality medium, wherein the high osmolality
medium comprises bFGF. Optionally, the high osmolality medium has
an osmolality of at least about 290 mOsm/kg.
[0059] In some populations or in vitro cultures, the transformed
cells are first cultured in the high osmolality medium until they
express characteristics of a naive or naive-looking state. In some
populations or in vitro cultures, the transformed cells are first
cultured in the high osmolality medium for a period of about two
months. In some populations or in vitro cultures, the transformed
cells are first cultured in the high osmolality medium until they
display a morphology characterized by three-dimensional cell
clumps.
BRIEF DESCRIPTION OF THE FIGURES
[0060] FIG. 1 depicts a schematic for replacement of a portion of
the human ADAM6 locus with a nucleic acid comprising the mouse
Adam6a and mouse Adam6b loci using an LTVEC and a guide RNA in
human iPS cells. The target site for the guide RNA is indicated by
the arrow.
[0061] FIG. 2A depicts the morphology displayed by human iPS cells
cultured for 8 days in 2i medium.
[0062] FIG. 2B depicts the morphology displayed by human iPS cells
cultured for 12 days in 2i medium.
[0063] FIGS. 3A-3D depict the morphology of human iPS cells
cultured in mTeSR.TM.-hLIF medium or low osmolality VG2i medium for
6 days. FIGS. 3A and 3B depict the morphology of human iPS cells
cultured in mTeSR.TM.-hLIF medium (FIG. 3A) or VG2i medium (FIG.
3B) for 6 days. FIGS. 3C and 3D depict the morphology of human iPS
cells cultured on newborn human foreskin fibroblast (NuFF) feeder
cells in mTeSR.TM.-hLIF medium (FIG. 3C) or VG2i medium (FIG. 3D)
for 6 days.
[0064] FIG. 4A depicts reprogrammed human iPS cells cultured in
VG2i medium that have been stained for alkaline phosphatase. FIGS.
4B and 4C depict reprogrammed human iPS cells cultured in VG2i
medium that have been immunostained for the expression of
NANOG.
[0065] FIGS. 5A-5C illustrate enzymatic dissociation and subculture
of reprogrammed human iPS cells cultured in VG2i medium. FIG. 5A
depicts reprogrammed human iPS cells cultured in VG2i medium prior
to enzymatic dissociation with trypsin in the absence of a ROCK
inhibitor. FIG. 5B depicts human iPS cells cultured in VG2i medium
for 1 day after subculture. FIG. 5C depicts human iPS cells
cultured in VG2i medium for 4 days after subculture.
[0066] FIGS. 6A and 6B depict the karyotypes of cells from two
different human iPS cell clones at passage 10 following
dissociation with trypsin to create a single-cell suspension.
BRIEF DESCRIPTION OF THE SEQUENCES
[0067] SEQ ID NO: 1 sets forth a nucleic acid sequence comprised by
ADAM6 gRNA.
[0068] SEQ ID NO: 2 sets forth the nucleic acid sequence of a
target sequence for a CRISPR/Cas complex.
Definitions
[0069] 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 polymers that have been modified, such as
polypeptides having modified peptide backbones.
[0070] The terms "nucleic acid" and "polynucleotide," used
interchangeably herein, include polymeric forms of nucleotides of
any length, including ribonucleotides, deoxyribonucleotides, or
analogs or modified versions thereof. They include single-,
double-, 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.
[0071] "Codon optimization" generally includes a process of
modifying a nucleic acid sequence for enhanced 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
protein can be modified to substitute codons having a higher
frequency of usage in a human cell. 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 al. (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).
[0072] "Operable linkage" or being "operably linked" includes
juxtaposition of two or more components (e.g., a promoter and
another sequence element) such that both components function
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.
[0073] "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 another 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
Watson-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 conditions
(e.g., salt concentration 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 complexes
formed between two nucleic acid strands 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
separation 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 1 M NaCl solution by using, e.g., Tm=81.5+0.41(%
G+C), although other known Tm computations take into account
nucleic acid structural characteristics.
[0074] "Hybridization 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 incubation time or
reaction chamber dimensions may contribute to the environment. See,
e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual,
2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 1 1.47-11.57 (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0075] 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 temperature (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, 11.7-11.8). Typically, the length for a
hybridizable nucleic acid is at least about 10 nucleotides.
Illustrative minimum lengths for a hybridizable nucleic acid
include 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.
[0076] 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
involved 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 nucleotides may be clustered or interspersed with
complementary nucleotides and need not be contiguous to each other
or to complementary nucleotides.
[0077] Percent complementarity between 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 al. (1990) J
Mol. Biol. 215:403-410; Zhang and Madden (1997) Genome Res.
7:649-656) or by using the Gap program (Wisconsin 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).
[0078] "Sequence identity" or "identity" in the context of two
polynucleotides or polypeptide 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 positions which are not identical often
differ by conservative amino acid substitutions, where amino acid
residues are substituted for other amino acid residues with similar
chemical 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 correct for the
conservative nature of the substitution. Sequences that differ by
such conservative substitutions are said to have "sequence
similarity" or "similarity." Means for making this adjustment 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 PC/GENE (Intelligenetics, Mountain View,
Calif.).
[0079] "Percentage of sequence identity" includes the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein 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 deletions) 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 residue 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.
[0080] 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; % identity 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
identical nucleotide or amino acid residue matches and an identical
percent sequence identity when compared to the corresponding
alignment generated by GAP Version 10.
[0081] Compositions or methods "comprising" or "including" one or
more recited elements may include other elements not specifically
recited. For example, a composition that "comprises" or "includes"
a protein may contain the protein alone or in combination with
other ingredients.
[0082] Designation of a range of values includes all integers
within or defining the range, and all subranges defined by integers
within the range.
[0083] The term "about" means that the specified value can vary by
some percentage. In some examples, the percentage can be 1, 2, 3,
4, 8, or 10% of the specified value.
[0084] The singular forms of the articles "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a cell" or "at least one cell"
can include a plurality of cells, including mixtures thereof.
DETAILED DESCRIPTION
A. Low Osmolality Medium for Making and Maintaining Human Induced
Pluripotent Stem Cells.
[0085] A cell culture medium is provided for use in the methods and
compositions of the invention. In one embodiment, the medium is
suitable for making a population of human iPS cells. In another
embodiment, the medium is suitable for maintaining human iPS cells
in culture. In some embodiments, the human iPS cells are naive or
naive-looking.
[0086] The medium provided herein comprises at least a base medium,
supplements, a leukemia inhibitory factor (LIF) polypeptide, a
glycogen synthase kinase 3 (GSK3) inhibitor, and a
mitogen-activated protein kinase kinase (MEK) inhibitor. A "base
medium" or "base media" includes, for example, a base medium known
in the art (e.g., Dulbecco's Modified Eagle's Medium (DMEM)) that
is suitable for use (with added supplements) in growing or
maintaining pluripotent cells (e.g., iPS cells) in culture. Base
medium is typically supplemented with a number of supplements known
in the art when used to maintain cells in culture.
[0087] The present medium is a low osmolality medium. In one
example, the osmolality is between about 175-280 mOsm/kg. In
further examples, the osmolality of the medium is about 180-270
mOsm/kg, about 200-250 mOsm/kg, about 220-240 mOsm/kg, or about
225-235 mOsm. In a particular embodiment, the osmolality of the
medium is about 233 mOsm/kg.
[0088] The base medium provided for the invention is a low
osmolality base medium to which supplements are added. The present
base medium differs from base media typically used to maintain
human iPS cells in culture, which include Dulbecco's Modified
Eagle's Medium (DMEM), in various forms (e.g., Invitrogen DMEM,
Cat. No. 1 1971-025), and a low salt DMEM available commercially as
KO-DMEM.TM. (Invitrogen Cat. No. 10829-018).
[0089] The base medium provided herein is a low osmolality medium
but exhibits characteristics that are not limited to low
osmolality. For example, the DMEM formulation shown in Table 1 can
be made suitable for the purposes of the invention by altering the
sodium chloride and/or sodium bicarbonate concentrations as
provided herein, which will result in a different osmolality as
compared with the standard DMEM base medium or low-salt DMEM base
medium (KO-DMEM) shown in Table 1.
TABLE-US-00001 TABLE 1 DMEM base medium formulation. Component Mg/L
mM Glycine 30 0.4 L-Arginine.cndot.HCl 84 0.398
L-Cystine.cndot.2HCl 63 0.201 L-Glutamine 584 4
L-Histidine.cndot.HCl.cndot.H2O 42 0.2 L-Isoleucine 105 0.802
L-Leucine 105 0.802 L-Lysine.cndot.HCl 146 0.798 L-Methionine 30
0.201 L-Phenylalanine 66 0.4 L-Serine 42 0.4 L-Threonine 95 0.798
L-Tryptophan 16 0.0784 L-Tyrosine disodium salt dihydrate 104 0.398
L-Valine 94 0.803 Choline chloride 4 0.0286 D-Calcium pantothenate
4 8.39 .times. 10.sup.-3 Folic Acid 4 9.07 .times. 10.sup.-3
Niacinamide 4 0.0328 Pyridoxine.cndot.HCl 4 0.0196 Riboflavin 0.4
1.06 .times. 10.sup.-3 Thiamine.cndot.HCl 4 0.0119 i-Inositol 7.2
0.04 Calcium Chloride (CaCl.sub.2) (anhydrous) 200 1.8 Ferric
Nitrate (Fe(NO.sub.3).sub.3.cndot.9H.sub.2O) 0.1 2.48 .times.
10.sup.-4 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 97.67 0.814
Potassium Chloride (KCl) 400 5.33 D-Glucose (Dextrose) 4500 25
Phenol Red 15 0.0399 NaCl/NaHCO.sub.3 Content of DMEM Sodium
Bicarbonate (NaHCO.sub.3) 3700 44.05 Sodium Chloride (NaCl) 6400
110.34 Osmolality 340 mOsm/kg NaCl/NaHCO.sub.3 Content of Low Salt
DMEM (KO-DMEM) Sodium Bicarbonate (NaHCO.sub.3) 2200 26 Sodium
Chloride (NaCl) 5100 87.7 Osmolality 275 mOsm/kg NaCl/NaHCO.sub.3
Content of Low Osmolality DMEM (VG-DMEM) Sodium Bicarbonate
(NaHCO.sub.3) 2200 26 Sodium Chloride (NaCl) 3000 50 Osmolality 200
mOsm/kg
[0090] The present base medium can include a salt of an alkaline
metal and a halide, such as sodium chloride (NaCl). Exemplary
concentrations of NaCl in the base medium include 50.+-.5 mM or
about 3 mg/mL. The concentration of a salt of an alkaline metal and
a halide in the base medium or a medium comprising the base medium
and supplements can be, for example, no more than about 100, 90,
80, 70, 60, or 50 mM. For example, the base medium or a medium
comprising the base medium and supplements can comprise a
concentration of a salt of an alkaline metal and halide of about
50-110, 60-105, 70-95, 80-90, 90 mM, or 85 mM. Alternatively, the
concentration of a salt of an alkaline metal and halide can be, for
example, 50.+-.5 mM, 87.+-.5 mM, 110.+-.5 mM, about 3 mg/mL, about
5.1 mg/mL, or about 6.4 mg/mL.
[0091] In another embodiment, the base medium exhibits a
concentration of a salt of carbonic acid. The salt of carbonic acid
can be a sodium salt. In such an example, the sodium salt can be
sodium bicarbonate. In a particular embodiment, sodium bicarbonate
is present in the base medium at a concentration of about 26.+-.5
mM or about 2.2 mg/mL. The concentration of a salt of carbonic acid
in the base medium or a medium comprising the base medium and
supplements can be, for example, no more than 45, 40, 35, 30, 25,
or 20 mM. For example, the base medium or a medium comprising the
base medium and supplements can comprise a concentration of
carbonic acid salt in the base medium of about 10-40, 18-44, 17-30,
18-26, 13-25, 20-30, 25-26, 18, or 26 mM. Alternatively, the
concentration of carbonic acid salt can be, for example, 18.+-.5
mM, 26.+-.5 mM, about 1.5 mg/mL, or about 2.2 mg/mL.
[0092] The sum of the concentration of the salt of the alkaline
metal and halide and the salt of carbonic acid in the base medium
or a medium comprising the base medium and supplements can be, for
example, no more than 140, 130, 120, 110, 100, 90, or 80 mM. For
example, the base medium or a medium comprising the base medium and
supplements can comprise a sum concentration of a salt of an
alkaline metal and halide and a salt of carbonic acid of about
80-140, 85-130, 90-120, 95-120, 100-120, or 115 mM.
[0093] The molar ratio of the salt of the alkaline metal and halide
and the salt of carbonic acid in the base medium or a medium
comprising the base medium and supplements can be, for example,
higher than 2.5. For example, the base medium or a medium
comprising the base medium and supplements can comprise a molar
ratio of a salt of an alkaline metal and halide and a salt of
carbonic acid of about 2.6-4.0, 2.8-3.8, 3.0-3.6, 3.2-3.4, 3.3-3.5,
or 3.4.
[0094] In yet another embodiment, the base medium is a low
osmolality base medium. The osmolality of the base medium can be
within a range of about 175-280 mOsm/kg, about 180-250 mOsm/kg,
about 190-225 mOsm/kg, or about 195-205 mOsm/kg. An exemplary
osmolality of the base medium can be 200, 214, 216, or 218 mOsm/kg.
In a particular example, the osmolality of the base medium is 200
mOsm/kg. The osmolality can be determined when cells are cultured
in different concentrations of CO.sub.2. In some examples, cells
are cultured at 3% CO.sub.2 or 5% CO.sub.2. The osmolality of the
base medium or a medium comprising the base medium and supplements
can be, for example, no more than about 330, 320, 310, 300, 290,
280, 275, 270, 260, 250, 240, 230, 220, 210, or 200 mOsm/kg. For
example, the base medium or the medium comprising the base medium
and supplements can comprise an osmolality of about 200-329,
218-322, 240-320, 250-310, 275-295, or 260-300 mOsm/kg. For
example, the base medium or the medium comprising the base medium
and the supplements can comprise an osmolality of about 270
mOsm/kg, about 261 mOsm/kg, or about 218 mOsm/kg. Alternatively,
the osmolality can be 218.+-.22 mOsm/kg, 261.+-.26 mOsm/kg,
294.+-.29 mOsm/kg, or 322.+-.32 mOsm/kg.
[0095] The osmolality of the base medium can be, for example, about
130-270, 140-260, 150-250, 160-240, 170-230, 180-220, 190-210,
195-205, or 200 mOsm/kg. Alternatively, the osmolality of the base
medium can be, for example, about 200.+-.70, 200.+-.60, 200.+-.50,
200.+-.40, 200.+-.35, 200.+-.30, 200.+-.25, 200.+-.20, 200.+-.15,
200.+-.10, 200.+-.5, or 200 mOsm/kg. Alternatively, the osmolality
of the base medium can be, for example, about 130-140, about
140-150, about 150-160, about 160-170, about 170-180, about
180-190, about 190-200, about 200-210, about 210-220, about
220-230, about 230-240, about 240-250, about 250-260, about
260-270, about 270-280, about 280-290, about 290-300, about
300-310, about 310-320, or about 320-330 mOsm/kg. Alternatively,
the osmolality of the base medium can be, for example, less than
about 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220,
210, 200, 190, 180, 170, 160, 150, 140, or 130 mOsm/kg.
[0096] The osmolality of the medium comprising the base medium and
supplements can be, for example, about 205-260, 215-250, 225-240,
230-235, or 233 mOsm/kg. Alternatively, the osmolality of the
medium comprising the base medium and supplements can be, for
example, about 233.+-.27, 233.+-.25, 233.+-.20, 233.+-.15,
233.+-.10, 233.+-.5, or 233 mOsm/kg. Alternatively, the osmolality
of the medium comprising the base medium and supplements can be,
for example, about 200-205, 205-210, 210-215, 215-220, 220-225,
225-230, 230-235, 235-240, 240-245, 245-250, 250-255, or 255-260
mOsm/kg. Alternatively, the osmolality of the medium comprising the
base medium and supplements can be, for example, less than 260,
255, 250, 245, 240, 235, 230, 225, 220, 215, 210, 205, or 200
mOsm/kg.
[0097] In some low osmolality media, the base medium comprises
about 87.+-.5 mM NaCl and about 26.+-.5 mM carbonate. For example
the base media can comprise about 5.1 mg/mL NaCl, about 2.2 mg/mL
sodium bicarbonate, and an osmolality of about 275 mOsm/kg.
[0098] In some low osmolality media, the base medium comprises
about 50.+-.5 mM NaCl and about 26.+-.5 mM carbonate. For example,
the base medium can comprise about 3.0 mg/mL NaCl, about 2.2 mg/mL
sodium bicarbonate, and an osmolality of about 200 mOsm/kg. In a
preferred embodiment, the base medium comprises NaCl at a
concentration of 3.0 mg/mL, sodium bicarbonate at a concentration
of about 2.2 mg/mL, and has an osmolality of 200 mOsm/kg.
[0099] Other examples of low osmolality media are described in WO
2011/156723, US 2011/0307968, and US 2015/0067901, each of which is
herein incorporated by reference in its entirety.
[0100] Supplements formulated with the base medium of the invention
are suitable for making, maintaining, or enriching populations of
human iPS cells disclosed herein. Such supplements are indicated as
"supplements" or "+supplements" in this disclosure. The term
"supplements" or the phrase "+supplements," includes one or more
additional elements added to the components of the base medium
described in Table 1. For example, supplements can include, without
limitation, F-12.RTM. medium (Gibco), N2.RTM. supplement (Gibco;
100.times. solution), NEUROBASAL.RTM. medium (Gibco), B-27.RTM.
supplement (Gibco; 50.times. solution), L-glutamine, glucose,
2-mercaptoethanol, a Leukemia Inhibitory Factor (LIF) polypeptide,
a glycogen synthase kinase 3 inhibitor, a MEK inhibitor, or any
combination thereof. Supplements can also include, for example,
fetal bovine serum (FBS), antibiotic(s), penicillin and
streptomycin (i.e., penstrep), pyruvate salts (e.g., sodium
pyruvate), and nonessential amino acids (e.g., MEM NEAA).
[0101] In a particular embodiment, the LIF polypeptide is a human
LIF (hLIF) polypeptide. In some examples, a hLIF polypeptide is
used at a concentration of about 1-1000 units/mL, about 20-800
units/mL, about 50-500 units/mL, about 75-250 units/mL, or about
100 units/mL.
[0102] The media can comprise inhibitors, for example, consisting
essentially of a GSK3 inhibitor and a MEK inhibitor. For example,
the medium can comprise inhibitors consisting of a GSK3 inhibitor
and a MEK inhibitor.
[0103] In another particular embodiment, the GSK3 inhibitor
comprises CHIR99021. In some examples, CHIR99021 is used at a
concentration of about 0.1 to 10 .mu.M, about 1-5 .mu.M, about 2-4
.mu.M, or about 3 .mu.M.
[0104] In another particular embodiment, the MEK inhibitor
comprises PD0325901. In some examples, PD0325901 is used at a
concentration of about 0.1-5 .mu.M, about 0.2-1 .mu.M, about
0.3-0.7 .mu.M, or about 0.5 .mu.M.
[0105] An exemplary medium comprises a low osmolality base medium
described herein at about 24.75% (v/v), F-12 medium at about 24.75%
(v/v), N2 supplement at about 0.5% (v/v), NEUROBASAL medium at
about 49% (v/v), B-27 supplement at about 1% (v/v), L-glutamine at
about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100
units/mL, CHIR99021 at about 3 .mu.M, and PD0325901 at about 0.5
.mu.M.
[0106] In another particular embodiment, the medium may or may not
comprise basic fibroblast growth factor (bFGF, also known as FGF2
or FGF-.beta.). Preferably the present medium does not comprise
bFGF.
[0107] The medium may or may not comprise one or more of
transforming growth factor beta 1 (TGF-.beta.1) supplement, bFGF
supplement, c-Jun N-terminal kinase (JNK) inhibitor (e.g.,
SP600125), p38 mitogen-activated protein kinase (p38) inhibitor
(e.g., SB203580), rho-associated protein kinase (ROCK) inhibitor
(e.g., Y-27632), and protein kinase C (PKC) inhibitor (e.g.,
Go6983). The medium may or may not comprise forskolin. For example,
some media do not comprise one or more of TGF-.beta.1 supplement,
bFGF supplement, JNK inhibitor (e.g., SP600125), p38 inhibitor
(e.g., SB203580), ROCK inhibitor (e.g., Y-27632), and PKC inhibitor
(e.g., Go6983). Some media do not comprise one or more of p38
inhibitor and INK inhibitor. Some media do not comprise bFGF
supplement or TGF-.beta.1 supplement. Some media do not comprise
TGF-.beta.1 supplement. Some media do not comprise any one of
TGF-.beta.1 supplement, bFGF supplement, INK inhibitor (e.g.,
SP600125), p38 inhibitor (e.g., SB203580), ROCK inhibitor (e.g.,
Y-27632), and PKC inhibitor (e.g., Go6983). Some media do not
comprise forskolin.
B. Human Induced Pluripotent Stem Cells
[0108] Methods and compositions are provided herein for making a
population of human iPS cells. Methods and compositions are further
provided for maintaining human iPS cells in culture. Human iPS
cells that are produced or maintained in culture are also
provided.
[0109] The term "pluripotent cell" or "pluripotent stem cell"
includes an undifferentiated cell that possesses the ability to
develop into more than one differentiated cell type. Such
pluripotent cells can be, for example, a mammalian embryonic stem
(ES cell) cell or a mammalian induced pluripotent stem cell (iPS
cell). Examples of pluripotent cells include human iPS cells.
[0110] The term "embryonic stem cell" or "ES cell" means an
embryo-derived totipotent or pluripotent stem cell, derived from
the inner cell mass of a blastocyst, that can be maintained in an
in vitro culture under suitable conditions. ES cells are capable of
differentiating into cells of any of the three vertebrate germ
layers, e.g., the endoderm, the ectoderm, or the mesoderm. ES cells
are also characterized by their ability propagate indefinitely
under suitable in vitro culture conditions. See, for example,
Thomson et al. (Science (1998) Vol. 282(5391), pp. 1145-1147).
[0111] The term "induced pluripotent stem cell" or "iPS cell"
includes a pluripotent stem cell that can be derived directly from
a differentiated adult cell. Human iPS cells can be generated by
introducing specific sets of reprogramming factors into a
non-pluripotent cell which can include, for example, Oct3/4, Sox
family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc
family transcription factors (e.g., c-Myc, 1-Myc, n-Myc),
Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2,
KLF4, KLF5), and/or related transcription factors, such as NANOG,
LIN28, and/or Glis1. Human iPS cells can also be generated, for
example, by the use of miRNAs, small molecules that mimic the
actions of transcription factors, or lineage specifiers. Human iPS
cells are characterized by their ability to differentiate into any
cell of the three vertebrate germ layers, e.g., the endoderm, the
ectoderm, or the mesoderm. Human iPS cells are also characterized
by their ability propagate indefinitely under suitable in vitro
culture conditions. See, for example, Takahashi and Yamanaka (Cell
(2006) Vol. 126(4), pp. 663-676).
[0112] The terms "naive" and "primed" identify different
pluripotency states of human iPS cells. The term "naive-looking"
identifies a cell expressing a pluripotent state that exhibits one
or more characteristics of a naive pluripotent cell. Naive-looking
human iPS cells can also be referred to as "naive-like" human iPS
cells. The terms "naive-looking" and "naive-like" are intended to
be equivalent. In some embodiments, naive-looking human iPS cells
exhibit one or more morphological characteristics of naive human
iPS cells, such as a morphology characterized by compact
dome-shaped colonies. In some embodiments, naive-looking human iPS
cells express one or more of the pluripotency markers described
herein. In some embodiments, naive or naive-looking human iPS cells
are naive human iPS cells. In other embodiments, naive or
naive-looking human iPS cells are naive-looking iPS cells.
[0113] Characteristics of naive and primed iPS cells are described
in the art. See, for example, Nichols and Smith (Cell Stem Cell
(2009) Vol. 4(6), pp. 487-492). Naive human iPS cells exhibit a
pluripotency state similar to that of ES cells of the inner cell
mass of a pre-implantation embryo. Such naive cells are not primed
for lineage specification and commitment. Female naive iPS cells
are characterized by two active X chromosomes. In culture,
self-renewal of naive human iPS cells is dependent on leukemia
inhibitory factor (LIF) and other inhibitors. Cultured naive human
iPS cells display a clonal morphology characterized by rounded
dome-shaped colonies and a lack of apico-basal polarity. Cultured
naive cells can further display one or more pluripotency makers as
described elsewhere herein. Under appropriate conditions, the
doubling time of naive human iPS cells in culture can be between 16
and 24 hours.
[0114] Primed human iPS cells express a pluripotency state similar
to that of post-implantation epiblast cells. Such cells are primed
for lineage specification and commitment. Female primed iPS cells
are characterized by one active X chromosome and one inactive X
chromosome. In culture, self-renewal of primed human iPS cells is
dependent on fibroblast growth factor (FGF) and activin. Cultured
primed human iPS cells display a clonal morphology characterized by
an epithelial monolayer and display apico-basal polarity. Under
appropriate conditions, the doubling time of primed human iPS cells
in culture can be 24 hours or more.
[0115] In one embodiment, human iPS cells can be derived from
non-pluripotent cells transformed to express a pluripotent state.
Such transformed cells include, for example, cells that have been
transformed to express reprogramming genes that induce
pluripotency. A pluripotent state can include, for example,
expression of one or more of the pluripotency markers described
herein. Such cells (such as human foreskin fibroblasts) can be
transformed to express reprogramming genes, or any additional genes
of interest, by any means known in the art. See, for example,
Takahashi and Yamanaka (Cell (2006) Vol. 126(4), pp. 663-676). For
example, they can be introduced into the cells using one or more
plasmids, lentiviral vectors, or retroviral vectors. In some cases,
the vectors integrate into the genome and can be removed after
reprogramming is complete. In particular embodiments, the
non-pluripotent cells are transformed with reprogramming genes
comprising Oct4, Sox2, Klf4, Myc, or any combination thereof. In
some examples, the transformed cells comprise primed human iPS
cells.
[0116] In some embodiments, the human iPS cells cultured in the low
osmolality medium described herein express one or more phenotypes,
gene expression profiles, or markers characteristic of a naive
state. In one example, the human iPS cells express one or more
pluripotency markers whose expression is indicative of a naive
state. Such pluripotency markers can include alkaline phosphatase,
NANOG, 5T4, ABCG2, Activin RIB/ALK-4, Activin RIM, E-Cadherin,
Cbx2, CD9, CD30/TNFRSF8, CD117/c-kit, CDX2, CHD1, Cripto, DNMT3B,
DPPA2, DPPA4, DPPA5/ESG1, EpCAM/TROP1, ERR beta/NR3B2, ESGP, F-box
protein 15/FBXO15, FGF-4, FGF-5, FoxD3, GBX2, GCNF/NR6A1, GDF-3,
Gi24/VISTA/B7-H5, integrin alpha 6/CD49f, integrin alpha 6 beta 1,
integrin alpha 6 beta 4, integrin beta 1/CD29, KLF4, KLF5, L1 TD1,
Lefty, Lefty-1, Lefty-A, LIN-28A, LIN-28B, LIN-41, cMaf, cMyc,
Oct-3/4, Oct-4A, Podocalyxin, Rex-1/ZFP42, Smad2, Smad2/3, SOX2,
SSEA-1, SSEA-3, SSEA-4, STAT3, Stella/Dppa3, SUZ12, TBX2, TBX3,
TBX5, TERT, TEX19, TEX19.1, THAP11, TRA-1-60(R), TROP-2, UTF1,
and/or ZIC3. In a specific example, the expressed pluripotency
marker is alkaline phosphatase, NANOG, or both.
[0117] In another embodiment, human iPS cells cultured in the low
osmolality medium described herein display morphological
characteristics indicative of a naive state. An exemplary
morphology is characterized by cells having compact dome-shaped
colonies in culture.
[0118] The human iPS cells cultured in the low osmolality medium
described herein can have a normal karyotype. A normal karyotype
includes a karyotype in which all chromosomes normally
characteristic of the species are present and have not been
noticeably altered or a state of cells lacking any visible
numerical or structural chromosomal abnormality detectable with
chromosome banding analysis. The human iPS cells cultured in the
low osmolality medium described herein can have a normal karyotype,
for example, after about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, or 100 passages in the low
osmolality medium described herein.
[0119] In another embodiment, human iPS cells cultured in the low
osmolality medium described herein can be mechanically or
enzymatically dissociated into a single-cell suspension, passaged,
and/or subcultured. Such human iPS cells cultured in the low
osmolality medium described herein can have a normal karyotype and
can maintain the normal karyotype after being mechanically or
enzymatically dissociated into a single-cell suspension, passaged,
and/or subcultured. For example, such human iPS cells cultured in
the low osmolality medium described herein can have a normal
karyotype and can maintain the normal karyotype after being
mechanically or enzymatically dissociated into a single-cell
suspension, modified at a target genomic locus using the methods
described elsewhere herein, and subcultured. In one example,
enzymatic dissociation can be performed using trypsin.
[0120] When cultured in the present low osmolality medium, human
iPS cells can provide greater transformation efficiency due to
enhanced dissociation into a single-cell suspension. With other
types of medium (e.g., mTeSR.TM. medium or 2i medium) typically
used to maintain human iPS cells in culture, dissociation of human
iPS cells must be performed mechanically or with enzymes such as
collagenase that are less harsh than trypsin. It is generally not
recommended to passage human iPS cells as single cells, as this
practice has been demonstrated to place unwanted selective
pressures on cell populations that can lead to, for example,
genetic aberrations in culture. Human iPS cells are vulnerable to
apoptosis upon cellular detachment and dissociation, and typically
undergo massive cell death after complete dissociation. See
Watanabe et al. (2007) Nature 25(6):681-686, herein incorporated by
reference in its entirety for all purposes. Thus, dissociation of
human iPS cells is typically performed with reagents or methods
that minimize the breakup of colonies when passaging and do not
create single-cell suspensions. Consequently, the cells are not
dissociated as effectively or as completely. However, complete
dissociation can be important for procedures such as clonal
isolation following gene transfer or generation of a targeted
genetic modification, particularly when attempting to isolate
relatively rare clones such as those undergoing homologous
recombination to produce a desired targeted modification. In
contrast, with the present low osmolality medium, trypsin can be
used to dissociate the cells, and the enhanced dissociation results
in increased transformation efficiency. For example, such
dissociation can create single-cell suspensions that result in
greater targeting efficiencies when targeting, for example, via
electroporation or using the methods for making targeted genetic
modifications described elsewhere herein. Furthermore, unlike with
other types of medium typically used to maintain human iPS cells in
culture (e.g., mTeSR.TM. medium or 2i medium), enzymatic
dissociation of human iPS cells cultured with the present low
osmolality medium (preferably a low osmolality medium not
comprising bFGF) can be performed in the absence of one or more
inhibitors that are generally necessary for the passage of such
cells. An exemplary inhibitor that can be omitted is a
Rho-associated protein kinase (ROCK) inhibitor. A ROCK inhibitor is
generally necessary when passaging human iPS cells to inhibit the
activation of pro-apoptotic pathways. In particular, addition of a
ROCK inhibitor is generally recommended when plating single-cell
suspensions of human iPS cells, as this has been reported to
increase cell survival. See Watanabe et al. (2007) Nature
25(6):681-686. When using the low osmolality medium disclosed
herein, however, such ROCK inhibitors are not needed, even when
passaging as single-cell suspensions. Such single-cell suspensions
can maintain pluripotency and a normal karyotype following
trypsinization and replating when the low osmolality medium
disclosed herein is used.
[0121] In a further embodiment, subcultured human iPS cells
cultured in the low osmolality medium described herein can maintain
a naive or naive-looking state following enzymatic dissociation and
subculture. Subcultured human iPS cells cultured in the low
osmolality medium described herein can maintain a naive or
naive-looking state following enzymatic dissociation and subculture
even when passaged as single-cell suspensions and/or when modified
at a target genomic locus using the methods described elsewhere
herein. In some examples, subcultured human iPS cells can continue
to display a morphology characterized by compact dome-shaped
colonies. Subcultured human iPS cells can also continue to express
one or pluripotency markers as described herein.
C. Methods of Making and Maintaining a Population of Human Induced
Pluripotent Stem Cells
[0122] Methods and compositions are provided for making human iPS
cells in an in vitro culture. Methods and compositions are further
provided for maintaining human iPS cells in an in vitro
culture.
[0123] The term "making" includes culturing non-pluripotent cells
transformed to express one or more reprogramming factors as
described herein, under suitable conditions to induce a change in
cell phenotype, gene expression, or both, such that the cells
display a naive or naive-looking state, i.e., express one or more
characteristics of naive human iPS cells. A naive or naive-looking
state can be expressed in response to particular culture
conditions, e.g., culture in a low osmolality medium as described
herein. In some examples, the proportion of cells expressing a
naive or naive-looking state is at least about 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, and up to 100% of the cells in culture.
[0124] In one embodiment, the method enriches an in vitro culture
for a population of naive or naive-looking human iPS cells. In such
an embodiment, naive or naive-looking human iPS cells can be
propagated in culture preferentially over cells that do not express
a naive or naive-looking state. In another embodiment, naive or
naive-looking human iPS cells can be selected from a culture, be
enzymatically dissociated, and subcultured to produce an enriched
population of naive or naive-looking human iPS cells.
[0125] In one embodiment, non-pluripotent cells transformed to
express a pluripotent state, are cultured in vitro in a medium
provided herein that is suitable for inducing expression of a naive
or naive-looking state for a period of at least 1, 2, 5, 7, 10, 14,
21, or 28 days, or any period of time sufficient to induce
expression of a naive or naive-looking state in culture.
Transformed cells can be cultured in the present medium for at
least 1, 2, 3, or 4 weeks. Sometimes transformed cells are cultured
for 1-4 weeks. Expression of a naive or naive-looking state can be
determined by observing morphological characteristics or the
expression of pluripotency markers, characteristic of a naive or
naive-looking state, that are described elsewhere herein.
[0126] In one embodiment, non-pluripotent cells transformed to
express a pluripotent state, are cultured in the present low
osmolality medium until they express characteristics of a naive or
naive-looking state. Cells can then be cultured in the present
medium to maintain a naive or naive-looking state. In another
embodiment, non-pluripotent cells transformed to express a
pluripotent state, are first cultured in a high osmolality medium
prior to culturing in the present low osmolality medium. Such high
osmolality medium exhibits an osmolality higher than the present
low osmolality medium and can comprise bFGF. The osmolality of the
high osmolality medium can be, for example, about 300-380, 310-370,
320-360, 330-350, or 340 mOsm/kg. Alternatively, the osmolality of
the high osmolality medium can be, for example, 340.+-.70,
340.+-.60, 340.+-.50, 340.+-.40, 340.+-.30, 340.+-.20, or 340.+-.10
mOsm/kg. For example, the osmolality of the high osmolality medium
can be about 270-280, 280-290, 290-300, 300-310, 310-320, 320-330,
330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, or
400-410 mOsm/kg. Alternatively, the osmolality of the high
osmolality medium can be at least about 270, 280, 290, 300, 310,
320, 330, 340, 350, 360, 370, 380, 390, 400, or 410 mOsm/kg. Some
high osmolality medium comprises one or more of bovine serum
albumin, bFGF, transforming growth factor .beta. (TGF.beta.),
lithium chloride, pipecolic acid, and gamma-aminobutyric acid
(GABA). Examples of a high osmolality medium include mTeSR.TM.
medium (Stemcell Technologies).
[0127] In some embodiments, non-pluripotent cells transformed to
express a pluripotent state, can first be cultured in high
osmolality medium comprising bFGF until they begin to express
characteristics of a naive or naive-looking state, at which time
the cells are cultured in the present low osmolality medium. In one
example, cells can be cultured in high osmolality medium comprising
bFGF for a period of at least 1, 2, 5, 10, 30, 60, or 90 days, a
period of 1, 2, 4, 8, or 12 weeks, or a period between 1 day to 3
months. An exemplary time period for culture in a high osmolality
medium comprising bFGF is 2 months.
[0128] In other embodiments, non-pluripotent cells transformed to
express a pluripotent state, can first be cultured in high
osmolality medium comprising bFGF until they begin to display a
morphology characterized by three-dimensional cell clumps, at which
time cells are cultured in the present low osmolality medium. In
such embodiments, cells displaying three-dimensional clumps can be
selected, dissociated (e.g., with trypsin), and transferred to a
new culture in the low osmolality medium described herein.
[0129] The terms "maintain," "maintaining," and "maintenance"
include the preservation of at least one or more of the
characteristics or phenotypes of the human iPS cells described
herein. Such characteristics can include maintaining pluripotency,
cell morphology, gene expression profiles, and/or other functional
characteristics of naive cells. The terms "maintain,"
"maintaining," and "maintenance" can also encompass the propagation
of cells and/or an increase in the number of naive cells being
cultured. The terms include culture conditions that prevent cells
from converting to a primed or non-pluripotent state. The terms
further include culture conditions that permit the cells to remain
pluripotent and/or naive, while the cells may or may not continue
to divide and increase in number.
[0130] In one embodiment, human iPS cells are cultured in vitro in
a medium provided herein that is suitable for maintaining such
cells in a naive or naive-looking state. In a particular example,
human iPS cells can be cultured in a suitable medium for a period
of 1, 2, 5, 7, 10, 14, 21, or 28 days, or for a period of about 2
weeks, about 3 weeks, about 4 weeks, or more, so long as the
cultured cells are maintained in a naive or naive-looking state.
Cells can be cultured for at least 1, 2, 3 or 4 weeks. Sometimes
cells are cultured for 1-4 weeks. Human iPS cells can be
maintained, for example, for any period of time sufficient for
propagation of the cells in culture, genetic modification of the
cells, and/or subculture of the cells.
[0131] In another embodiment, human iPS cells or non-pluripotent
cells transformed to express a pluripotent state, can be cultured
on a substrate or feeder cell layer suitable for in vitro culture.
In a particular example, cells are cultured on MATRIGEL.TM. (BD
Biosciences). In another example, cells are cultured on newborn
human foreskin fibroblast (NuFF) feeder cells. In another example,
cells are cultured on GELTREX.TM. (Life Technologies). In another
example, the cells are cultured on vitronectin (e.g., VITRONECTIN
XF.TM. (STEMCELL Technologies).
[0132] In a further embodiment, the doubling time of human iPS
cells cultured in the present low osmolality medium is reduced as
compared to primed human iPS cells or non-pluripotent cells
transformed to express a pluripotent state. In a particular
example, the doubling time of the present human iPS cells is
between about 16-24 hours.
D. Genetic Modifications and Methods for Making Targeted Genetic
Modifications
[0133] In some embodiments, the methods for making and maintaining
human iPS cells comprise introducing a genetic modification into
the human iPS cells. Likewise, the invention provides human iPS
cells that comprise a genetic modification.
[0134] In particular embodiments, the genetic modification
comprises a modification of one or more endogenous nucleic acids, a
substitution of one or more endogenous nucleic acids, a replacement
of an endogenous nucleic acid with a heterologous nucleic acid, a
knockout, or a knock-in. In specific examples, the genetic
modification is introduced by introducing a large targeting vector
(LTVEC) into the human iPS cells or the non-pluripotent cells
transformed to express a pluripotent state. In such an example, the
LTVEC can comprise DNA to be inserted into the genome of the
cells.
[0135] Various methods for making targeted genetic modifications in
human iPS cells can be used. For example, various methods for
making targeted genetic modifications that modify the level and/or
the activity of proteins in human iPS cells can be used. For
example, in one instance, the targeted genetic modification employs
a system that will generate a targeted genetic modification via a
homologous recombination (HR) event. Homology-directed repair (HDR)
or HR includes a form of nucleic acid repair that can require
nucleotide sequence homology, uses a "donor" molecule to template
repair of a "target" molecule (i.e., the one that experienced the
double-strand break), and leads to transfer of genetic information
from the donor to target. Without wishing to be bound by any
particular theory, such transfer can involve mismatch correction of
heteroduplex DNA that forms between the broken target and the
donor, and/or synthesis-dependent strand annealing, in which the
donor is used to resynthesize genetic information that will become
part of the target, and/or related processes. In some cases, the
donor polynucleotide, a portion of the donor polynucleotide, a copy
of the donor polynucleotide, or a portion of a copy of the donor
polynucleotide integrates into the target DNA. In other instances,
a cell can be modified using nuclease agents that generate 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). NHEJ includes the repair of double-strand
breaks in a nucleic acid by direct ligation of the break ends to
one another without the need for a homologous template. Ligation of
non-contiguous sequences by NHEJ can often result in deletions,
insertions, or translocations near the site of the double-strand
break. Such systems find use, for example, in generating targeted
loss of function genetic modifications. Non-limiting methods for
generating such targeted genetic modification are discussed in
detail elsewhere herein, including, for example, the use of
targeting plasmids or large targeting vectors. 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.
[0136] It is recognized that in specific embodiments, the targeted
genetic modification of any polynucleotide of interest can occur
while the pluripotent cell (i.e., human iPS cell) is being
maintained in the culture medium described herein. Alternatively,
the targeted genetic modification of any polynucleotide of interest
can occur while the pluripotent cell (i.e., human iPS cell) is
being maintained in different culture medium, and subsequently
transferred to the low osmolality culture medium disclosed
herein.
[0137] In general, the level and/or activity of a protein is
modified if the protein level and/or the activity level of the
protein is statistically higher or lower than the protein level in
an appropriate control cell that has not been genetically modified
or mutagenized to alter the expression and/or activity of the
protein. In specific embodiments, the concentration and/or activity
of the protein is altered by at least 1%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% relative to a control cell which has not
been modified to have a modified level and/or activity of the
protein.
[0138] A "subject cell" is one in which a genetic alteration, such
as a genetic 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
reference point for measuring changes in phenotype of the subject
cell. In one embodiment, a control cell is as closely matched as
possible with the cell with reduced protein activity except it
lacks the genetic modification or mutation resulting in the
modified activity (for example, the respective cells can originate
from the same cell line). In other instances, 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 alteration 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 control cell and the subject 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 interest; or (e) the subject
cell itself, under conditions in which the genetic modification
does not result in an alteration in expression of the
polynucleotide of interest.
[0139] The expression level of the polypeptide may be measured
directly, for example, by assaying for the level of the polypeptide
in the cell or organism, or indirectly, for example, by measuring
the activity of the polypeptide.
[0140] In other instances, cells having the targeted genetic
modification are selected using methods that include, but are not
limited to, Southern blot analysis, DNA sequencing, PCR analysis,
or phenotypic analysis. Such cells are then employed in the various
methods and compositions described herein.
[0141] A targeted genetic modification can comprise a targeted
alteration to a polynucleotide of interest. 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 portion thereof, a knock-in of the
polynucleotide of interest 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, 100, 500 or more nucleotides
or at least 10 kb to 500 kb or more are changed to form the
targeted genomic modification.
[0142] In other embodiments, the activity and/or level of a
polypeptide is modified by introducing into the cell a
polynucleotide that alters the level or activity of the
polypeptide. The polynucleotide may modify the expression of the
polypeptide directly, by altering translation of the messenger RNA,
or indirectly, by encoding a polypeptide that alters the
transcription or translation of the gene encoding a protein. In
other embodiments, the activity of a polypeptide is modified by
introducing into the cell a sequence encoding a polypeptide that
alters the activity of the target polypeptide.
[0143] In one embodiment, human iPS cells can comprise a
conditional allele that modifies the activity and/or level of a
protein. A "conditional allele" includes a modified gene designed
to have the modified level and/or activity of the protein at a
desired developmental time and/or within a desired tissue of
interest. The modified level and/or activity can be compared with a
control cell lacking the modification giving rise to the
conditional allele, or in the case of modified 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 allele comprises a
conditional null allele of the gene that can be switched off or on
at a desired developmental time point and/or in specific
tissues.
[0144] In a non-limiting 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 (DSC) in sense or antisense
orientation; (b) in antisense 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 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 orientation.
[0145] The present invention allows for modifying a target genomic
locus on a chromosome in a cell. In particular embodiments, the
methods provided herein allow for the targeting of a genomic locus
on a chromosome by employing a targeting vector in the absence of,
or in combination with, a nuclease agent.
[0146] Methods for making targeted genetic modifications can
comprise, for example, the use of a targeting vector (e.g., an
LTVEC), either alone or in combination with one or more nucleases
as described elsewhere herein. See, e.g., US 2015/0159175, US
2015/0159174, US 2014/0310828, US 2014/0309487, and US
2013/0309670, each of which is herein incorporated by reference in
its entirety for all purposes. Likewise, methods for making
targeted genetic modifications can comprise the use of one or more
nucleases either alone or in combination with a targeting
vector.
[0147] For example, methods are provided for modifying a target
genomic locus in a human iPS cell, comprising: (a) introducing into
the cell one or more nuclease agents that induces one or more nicks
or double-strand breaks at a recognition site at or near the target
genomic locus; and (b) identifying at least one cell comprising in
its genome a modification at the target genomic locus. Such methods
can result in disruption of the target genomic locus. Disruption of
the endogenous nucleic acid sequence can result, for example, when
a double-strand break created by a nuclease is repaired by
non-homologous end joining (NHEJ)-mediated DNA repair, which
generates a mutant allele comprising an insertion or a deletion of
a nucleic acid sequence and thereby causes disruption of that
genomic locus. Examples of disruption include alteration of a
regulatory element (e.g., promoter or enhancer), a missense
mutation, a nonsense mutation, a frame-shift mutation, a truncation
mutation, a null mutation, or an insertion or deletion of small
number of nucleotides (e.g., causing a frameshift mutation).
Disruption can result in inactivation (i.e., loss of function) or
loss of the allele.
[0148] Other methods for modifying a target genomic locus in a
human iPS cell comprise: (a) introducing into the cell a targeting
vector comprising an insert nucleic acid flanked by 5' and 3'
homology arms corresponding to 5' and 3' target sites; and (b)
identifying at least one cell comprising in its genome the insert
nucleic acid integrated at the target genomic locus.
[0149] Other methods for modifying a target genomic locus in a
human iPS cell comprise: (a) introducing into the cell: (i) a
nuclease agent, wherein the nuclease agent induces a nick or
double-strand break at a recognition site within the target genomic
locus; and (ii) a targeting vector comprising an insert nucleic
acid flanked by 5' and 3' homology arms corresponding to 5' and 3'
target sites located in sufficient proximity to the recognition
site; and (c) identifying at least one cell comprising a
modification (e.g., integration of the insert nucleic acid) at the
target genomic locus. Such methods can result in various types of
targeted genetic modifications. Such targeted modifications can
include, for example, additions of one or more nucleotides,
deletions of one or more nucleotides, substitutions of one or more
nucleotides, a point mutation, a knockout of a polynucleotide of
interest or a portion thereof, a knock-in of a polynucleotide of
interest or a portion thereof, a replacement of an endogenous
nucleic acid sequence with a heterologous, exogenous, or
orthologous nucleic acid sequence, a domain swap, an exon swap, an
intron swap, a regulatory sequence swap, a gene swap, or a
combination thereof. For example, at least 1, 2, 3, 4, 5, 7, 8, 9,
10 or more nucleotides can be changed to form the targeted genomic
modification. The deletions, insertions, or replacements can be of
any size, as disclosed elsewhere herein.
[0150] a. Nuclease Agents and Recognition Sites for Nuclease
Agents
[0151] 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 desired 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 endogenous recognition site.
[0152] 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 Transcription Activator-Like Effector Nuclease
(TALEN), or about 20 bp for a CRISPR/Cas9 guide RNA.
[0153] In one embodiment, each monomer of the nuclease agent
recognizes a recognition site of at least 9 nucleotides. In other
embodiments, the recognition site is from about 9 to about 12
nucleotides in length, from about 12 to about 15 nucleotides in
length, from about 15 to about 18 nucleotides 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 recognition 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 whether the
nick/cleavage site is within or outside the nuclease agent binding
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' overhangs.
[0154] 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 naturally-occurring 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 derived) 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 nuclease agent can be as little as one amino acid in a protein
cleavage agent or one nucleotide 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
referred to herein as "cutting" or "cleaving" the recognition site
or other DNA.
[0155] Active variants 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 biological activity and hence
are capable of being recognized and cleaved by a nuclease agent in
a sequence-specific manner. Assays to measure the double-strand
break of a recognition site by a nuclease agent are known in the
art (e.g., TaqMang qPCR assay, Frendewey D. et al., Methods in
Enzymology, 2010, 476:295-307, which is incorporated by reference
herein in its entirety).
[0156] The recognition site of the nuclease agent can be positioned
anywhere in or near the target locus. The recognition site can be
located within a coding region of a gene, or within regulatory
regions that influence expression of the gene. A recognition site
of the nuclease agent can be located in an intron, an exon, a
promoter, an enhancer, a regulatory region, or any non-protein
coding region. 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 regulatory regions, which influence
the expression 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 region, 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. Methods to assay for the presence or absence of a
functional selection marker are known.
[0157] In one embodiment, the nuclease agent is a Transcription
Activator-Like Effector Nuclease (TALEN). TAL effector nucleases
are a class of sequence-specific nucleases 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, Fokl.
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, WO 2010/079430; Morbitzer et al. (2010) PNAS
10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence
1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al.
(2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et
al. (2011) Nature Biotechnology 29:143-148; all of which are herein
incorporated by reference.
[0158] 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,
2003/0232410 A1, 2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1,
2006/0188987 A1, and 2006/0063231 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 suitable for use
with the various methods and compositions provided herein include
those that are specifically designed to bind at or near target
nucleic acid sequences to be modified by targeting vectors as
described herein.
[0159] In one embodiment, each monomer of the TALEN comprises 12-25
TAL repeats, wherein each TAL repeat binds a 1 bp subsite. In some
TALENs, each monomer 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 Fokl 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 domains is operably linked
to a Fokl nuclease, wherein the first and the second
TAL-repeat-based DNA binding domain recognize two contiguous target
DNA sequences in each strand of the target DNA sequence separated
by about 6 bp to about 40 bp cleavage site, and wherein the Fokl
nucleases dimerize and make a double strand break at a target
sequence. For example, the nuclease agent can comprise 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 domains is operably linked to a Fokl
nuclease, wherein the first and the second TAL-repeat-based DNA
binding domain recognize two contiguous target DNA sequences in
each strand of the target DNA sequence separated by a spacer
sequence of varying length (12-20 bp), and wherein the Fokl
nuclease subunits dimerize to create an active nuclease that makes
a double strand break at a target sequence.
[0160] The nuclease agent employed in the various methods and
compositions disclosed herein can further 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 3 bp subsite.
In other embodiments, the ZFN is a chimeric protein comprising a
zinc finger-based DNA binding domain operably linked to an
independent nuclease. In one embodiment, the independent
endonuclease is a Fokl endonuclease. 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 Fokl
nuclease, wherein the first and the second ZFN recognize two
contiguous target DNA sequences in each strand of the target DNA
sequence separated by about 6 bp to about 40 bp cleavage site, and
wherein the Fokl nucleases dimerize and make a double strand break.
For example, the nuclease agent can comprise a first ZFN and a
second ZFN, wherein each of the first ZFN and the second ZFN is
operably linked to a Fokl nuclease subunit, 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 that makes a double strand break. See, for
example, US20060246567; US20080182332; US20020081614;
US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and,
WO/2011/017293A2, each of which is herein incorporated by
reference.
[0161] In still another embodiment, 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 phosphodiester
bonds. Meganucleases are notable for their long recognition sites,
and for tolerating some sequence polymorphisms in their DNA
substrates. Meganuclease domains, structure and function 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 55:1304-26; 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, expression, 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) J Mol
Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800;
Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al.,
(2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic
Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154;
WO2005105989; WO2003078619; WO2006097854; WO2006097853;
WO2006097784; and WO2004031346.
[0162] Any meganuclease can be used herein, including, but not
limited to, I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI,
I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP,
I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI,
F-SceII, F-SuvI, F-TeVI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI,
I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI,
I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI,
I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP,
I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP,
I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP,
I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P,
I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP,
I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP
PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP,
PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI,
PI-TliII, or any active variants or fragments thereof.
[0163] In one embodiment, the meganuclease recognizes
double-stranded DNA sequences 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-DmoI.
[0164] 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 cleavage 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 asymmetric recognition site and cleave on one side and
at a defined distance 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 31:418-20), Roberts et al., (2003)
Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) in Mobile
DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press, Washington,
D.C.).
[0165] The nuclease agent employed in the various methods and
compositions can also comprise a Clustered Regularly Interspersed
Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system
or components of such a system. CRISPR/Cas systems include
transcripts and other elements 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 III system. The methods and
compositions disclosed herein employ CRISPR/Cas systems by
utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexed
with a Cas protein) for site-directed cleavage of nucleic
acids.
[0166] Some CRISPR/Cas systems used in the methods disclosed herein
are non-naturally occurring. A "non-naturally occurring" system
includes 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 component with which they are not naturally
associated. For example, some CRISPR/Cas systems employ
non-naturally occurring CRISPR complexes comprising a gRNA and a
Cas protein that do not naturally occur together.
[0167] Cas proteins generally comprise at least one RNA recognition
or binding domain. Such domains can interact with guide RNAs
(gRNAs, described 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.
[0168] Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,
Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2,
Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG,
CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4
(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,
Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10,
Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966,
and homologs or modified versions thereof.
[0169] In some instances, a Cas protein is from a type II
CRISPR/Cas system. For example, the Cas protein can be a Cas9
protein or be derived from a Cas9 protein. Cas9 proteins typically
share four key motifs with a conserved architecture. Motifs 1, 2,
and 4 are RuvC-like motifs, and motif 3 is an HNH motif. The Cas9
protein can be from, for example, Streptococcus pyogenes,
Streptococcus thermophilus, Streptococcus sp Nocardiopsis
dassonvillei, Streptomyces pristinaespiralis, Streptomyces
viridochromogenes, Streptomyces viridochromogenes,
Streptosporangium roseum, Streptosporangium roseum, 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 difficile, Finegoldia magna, Natranaerobius
thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus
caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum,
Marinobacter 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, Thermosipho africanus, or Acaryochloris
marina. The Cas9 protein can be from Staphylococcus aureus.
Additional examples of the Cas9 family members include those
described in WO 2014/131833, herein incorporated by reference in
its entirety. In a specific example, the Cas9 protein is a Cas9
protein from S. pyogenes or is derived therefrom. The amino acid
sequence of a Cas9 protein from S. pyogenes can be found, for
example, in the SwissProt database under accession number
Q99ZW2.
[0170] Cas proteins can be wild type proteins (i.e., those that
occur in nature), modified Cas proteins (i.e., Cas protein
variants), or fragments of wild type or modified Cas proteins. Cas
proteins can also be active variants or fragments of wild type or
modified Cas proteins. Active variants or fragments can comprise at
least 80%, 85%, 90%, 91%, 92%, 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
double-strand-break-inducing activity are known and generally
measure the overall activity and specificity of the Cas protein on
DNA substrates containing the cleavage site.
[0171] Cas proteins can be modified to increase or decrease nucleic
acid binding affinity, nucleic acid binding specificity, and/or
enzymatic activity. Cas proteins can also be modified to change any
other activity or property of the protein, such as stability. For
example, one or more nuclease domains of the Cas protein can be
modified, deleted, or inactivated, or a Cas protein can be
truncated to remove domains that are not essential for the function
of the protein or to optimize (e.g., enhance or reduce) the
activity of the Cas protein.
[0172] Some Cas proteins comprise at least two nuclease domains,
such as DNase domains. For example, a Cas9 protein can comprise a
RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC
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.
[0173] One or both of the nuclease domains can be deleted or
mutated so that they are no longer functional 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 target
sequence within a double-stranded DNA but not a double strand break
(i.e., it can cleave the complementary strand or the
non-complementary strand, 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 D10A (aspartate to alanine at position 10 of
Cas9) mutation in the RuvC 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. pyogenes can convert the Cas9 into a
nickase. Other examples of mutations that convert Cas9 into a
nickase include the corresponding mutations to Cas9 from S.
thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic Acids
Research 39:9275-9282 and WO 2013/141680, each of which is herein
incorporated by reference in its entirety. Such mutations can be
generated using well-known methods such as site-directed
mutagenesis, PCR-mediated mutagenesis, or total gene synthesis.
Examples of other mutations creating nickases can be found, for
example, in WO/2013/176772A1 and WO/2013/142578A1, each of which is
herein incorporated by reference.
[0174] Cas proteins can also be fusion proteins. For example, a Cas
protein can be fused to a cleavage domain, an epigenetic
modification domain, a transcriptional activation domain, or a
transcriptional repressor domain. See WO 2014/089290, 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 heterologous polypeptide can be
located at the N-terminus, the C-terminus, or internally within the
Cas protein.
[0175] One example of a Cas fusion protein is a Cas protein fused
to a heterologous polypeptide that provides for subcellular
localization. Such sequences can include, for example, a nuclear
localization signal (NLS) such as the SV40 NLS for targeting to the
nucleus, a mitochondrial localization signal for targeting to the
mitochondria, an ER retention signal, and the like. See, e.g.,
Lange et al. (2007) J. Biol. Chem. 282:5101-5105. Such subcellular
localization signals can be located at the N-terminus, the
C-terminus, or anywhere within the Cas protein. An NLS can comprise
a stretch of basic amino acids, and can be a monopartite sequence
or a bipartite sequence.
[0176] Cas proteins can also comprise a cell-penetrating domain.
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-1, VP22, a cell penetrating peptide
from Herpes simplex virus, or a polyarginine peptide sequence. See,
for example, WO 2014/089290, 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.
[0177] 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, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric
Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins
(e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue
fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv,
Sapphire, T-sapphire), cyan fluorescent proteins (e.g. eCFP,
Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent
proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,
DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,
eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent
proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,
mTangerine, tdTomato), and any other suitable fluorescent protein.
Examples of tags include glutathione-S-transferase (GST), chitin
binding protein (CBP), maltose binding protein, thioredoxin (TRX),
poly(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.
[0178] Cas proteins can be provided in any form. For example, a Cas
protein can be provided in the form of a protein, such as a Cas
protein complexed with a gRNA. Alternatively, a Cas protein can be
provided 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
(i.e., a human cell). When a nucleic acid encoding the Cas protein
is introduced into the cell, the Cas protein can be transiently,
conditionally, or constitutively expressed in the cell.
[0179] 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 construct.
Expression constructs include 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. For example, the
nucleic acid encoding the Cas protein can be in a targeting vector
comprising a nucleic acid insert and/or a vector comprising a DNA
encoding the gRNA. Alternatively, it can be in a vector or a
plasmid that is separate from the targeting vector comprising the
nucleic acid insert and/or separate from the vector comprising the
DNA encoding the gRNA. Promoters that can be used in an expression
construct include, for example, promoters active in a human iPS
cell or a non-pluripotent cell transformed to express a naive
state. Such promoters can be, for example, conditional promoters,
inducible promoters, constitutive promoters, or tissue-specific
promoters.
[0180] A "guide RNA" or "gRNA" includes an RNA molecule that binds
to a Cas protein and targets the Cas protein to a specific location
within a target DNA. Guide RNAs can comprise two segments: a
"DNA-targeting 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 "single-molecule gRNA,"
a "single-guide RNA," or an "sgRNA." See, e.g., WO/2013/176772A1,
WO/2014/065596A1, WO/2014/089290A1, WO/2014/093622A2,
WO/2014/099750A2, WO/2013142578A1, and WO 2014/131833A1, each of
which is herein incorporated by reference. The terms "guide RNA"
and "gRNA" are inclusive, including both double-molecule gRNAs and
single-molecule gRNAs.
[0181] An exemplary two-molecule gRNA comprises a crRNA-like
("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA repeat")
molecule and a corresponding tracrRNA-like ("trans-acting CRISPR
RNA" or "activator-RNA" or "tracrRNA" or "scaffold") molecule. A
crRNA 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.
[0182] A corresponding tracrRNA (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
crRNA are complementary to and hybridize with a stretch of
nucleotides of a tracrRNA to form the dsRNA duplex of the
protein-binding domain of the gRNA. As such, each crRNA can be said
to have a corresponding tracrRNA.
[0183] The crRNA and the corresponding tracrRNA hybridize to form a
gRNA. The crRNA additionally provides the single stranded
DNA-targeting segment that hybridizes to a target sequence. If used
for modification within a cell, the exact sequence of a given crRNA
or tracrRNA molecule can be designed to be specific to the species
in which the RNA molecules will be used. See, for example, Mali et
al. (2013) Science 339:823-826; Jinek et al. (2012) Science
337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; 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.
[0184] The DNA-targeting segment (crRNA) 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 pairing). As such, the nucleotide sequence of the
DNA-targeting 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 crRNAs differ depending on the Cas9 system and
organism but often contain a targeting segment of between 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 corresponding tracrRNA,
which in turn binds to the Cas9 protein.
[0185] The DNA-targeting segment can have a length of from about 12
nucleotides 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 segment 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 20 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.
[0186] The nucleotide sequence of the DNA-targeting segment that is
complementary to a nucleotide sequence (target 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 target 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 30 nt, at least about
35 nt, or at least about 40 nt. Alternatively, the DNA-targeting
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.
[0187] TracrRNAs can be in any form (e.g., full-length tracrRNAs or
active partial tracrRNAs) and of varying lengths. They can include
primary transcripts or processed forms. For example, tracrRNAs (as
part of a single-guide RNA or as a separate molecule as part of a
two-molecule gRNA) may comprise or consist of all or a portion of a
wild-type tracrRNA sequence (e.g., about or more than about 20, 26,
32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type
tracrRNA sequence). Examples of wild-type tracrRNA sequences from
S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide,
and 65-nucleotide versions. See, for example, Deltcheva et al.
(2011) Nature 471:602-607; WO 2014/093661, each of which is
incorporated herein by reference in their entirety. Examples of
tracrRNAs within single-guide RNAs (sgRNAs) include the tracrRNA
segments found within +48, +54, +67, and +85 versions of sgRNAs,
where "+n" indicates that up to the +n nucleotide of wild-type
tracrRNA is included in the sgRNA. See U.S. Pat. No. 8,697,359,
incorporated herein by reference in its entirety.
[0188] The percent complementarity between the DNA-targeting
sequence and the target 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%). In some cases, the percent
complementarity between the DNA-targeting sequence and the target
sequence within the target DNA is at least 60% over about 20
contiguous nucleotides. In one example, the percent complementarity
between the DNA-targeting sequence and the target sequence within
the target DNA is 100% over the 14 contiguous nucleotides at the 5'
end of the target 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. In another example, the percent complementarity between the
DNA-targeting sequence and the target sequence within the target
DNA is 100% over the seven contiguous nucleotides at the 5' end of
the target 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.
[0189] The protein-binding segment of a gRNA can comprise two
stretches of nucleotides that are complementary 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 protein, and the
gRNA directs the bound Cas protein to a specific nucleotide
sequence within target DNA via the DNA-targeting segment.
[0190] Guide RNAs can include modifications or sequences that
provide for additional desirable features (e.g., modified or
regulated stability; 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' polyadenylated
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
location (e.g., nucleus, mitochondria, chloroplasts, and the like);
a modification 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, etc.); a modification or sequence that
provides a binding site for proteins (e.g., proteins that act on
DNA, including transcriptional activators, transcriptional
repressors, DNA methyltransferases, DNA demethylases, histone
acetyltransferases, histone deacetylases, and the like); and
combinations thereof.
[0191] 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 crRNA and tracrRNA) or as one molecule (sgRNA), and
optionally in the form of a complex 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 (sgRNA) or
separate RNA molecules (e.g., separate crRNA and tracrRNA). In the
latter case, the DNA encoding the gRNA can be provided as separate
DNA molecules encoding the crRNA and tracrRNA, respectively.
[0192] When a DNA encoding a gRNA is introduced into the cell, the
gRNA can be transiently, conditionally, or constitutively expressed
in the cell. 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. For example, the DNA
encoding the gRNA can be in a targeting vector comprising a nucleic
acid insert and/or a vector comprising a nucleic acid encoding a
Cas protein. Alternatively, it can be in a vector or a plasmid that
is separate from the targeting vector comprising the nucleic acid
insert and/or separate from the vector comprising the nucleic acid
encoding the Cas protein. Such promoters can be active, for
example, in a human iPS cell or a non-pluripotent cell transformed
to express a pluripotent state. Such promoters can be, for example,
conditional promoters, inducible promoters, constitutive promoters,
or tissue-specific promoters. In some instances, the promoter is an
RNA polymerase III promoter, such as a human U6 promoter.
[0193] Alternatively, gRNAs can be prepared by various other
methods. For example, gRNAs can be prepared by in vitro
transcription using, for example, T7 RNA polymerase (see, for
example, WO 2014/089290 and WO 2014/065596). Guide RNAs can also be
a synthetically produced molecule prepared by chemical
synthesis.
[0194] A target sequence for a CRISPR/Cas system 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, target sequences include sequences to
which a guide RNA is designed to have complementarity, where
hybridization between a target sequence and a DNA targeting
sequence promotes the formation of a CRISPR complex. Full
complementarity is not necessarily required, provided there is
sufficient complementarity to cause hybridization and promote
formation of a CRISPR complex. Target sequences also include
cleavage sites for Cas proteins, described in more detail below. A
target sequence can comprise any polynucleotide, which can be
located, for example, in the nucleus or cytoplasm of a cell or
within an organelle of a cell, such as a mitochondrion or
chloroplast.
[0195] The target sequence within a target DNA can be targeted by
(i.e., be bound by, or hybridize with, or be complementary 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 system)
are known in the art (see, e.g., Molecular Cloning: A Laboratory
Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory 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 strand," 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."
[0196] The Cas protein can cleave the nucleic acid at a site within
or outside of a nucleic acid sequence present in a target DNA to
which a DNA-targeting segment 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 target sequence and complexed 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 segment of a gRNA will bind. If the cleavage site is
outside of the nucleic acid sequence present in a target DNA to
which a DNA-targeting segment of a gRNA will bind, the cleavage
site is still considered to be within the "target 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 produced, for example, by using two Cas
proteins which produce a single-strand break at different cleavage
sites on each strand. For example, a first nickase can create a
single strand break on the first strand of double stranded DNA
(dsDNA), while 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 target sequence of the nickase on the
first strand is separated from the target 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.
[0197] 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 target sequence. Optionally, the target sequence can
be flanked on the 3' end 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) upstream 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'-XGG-3', where X is any DNA nucleotide and is immediately 3'
of the target sequence of the non-complementary strand of the
target DNA. As such, the PAM sequence of the complementary strand
would be 5'-CCY-3', where Y is any DNA nucleotide and is
immediately 5' of the target sequence of the complementary strand
of the target DNA. In some such cases, X and Y can be complementary
and the X-Y base pair can be any base pair (e.g., X=C and Y=G; X=G
and Y=C; X=A and Y=T, X=T, and Y=A).
[0198] Examples of target sequences include a DNA sequence
complementary to the DNA-targeting segment of a gRNA, or such a DNA
sequence in addition to a PAM sequence. One example of a target
sequence comprises the nucleotide sequence of
GNNNNNNNNNNNNNNNNNNNNGG (GN.sub.1-20 GG; SEQ ID NO: 2). Other
target sequences can have between 4-22 nucleotides in length of SEQ
ID NO: 2, including the 5' G and the 3' GG. Yet other target
sequences can have between 14 and 20 nucleotides in length of SEQ
ID NO: 2.
[0199] The target sequence can be any nucleic acid sequence
endogenous or exogenous to a cell. The target sequence can be a
sequence coding a gene product (e.g., a protein) or a non-coding
sequence (e.g., a regulatory sequence or junk DNA) or can include
both.
[0200] 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 double-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 different from
the corresponding native nuclease agent recognition site. Assays
for nick or double-strand-break-inducing activity are known and
generally measure the overall activity and specificity of the
endonuclease on DNA substrates containing the recognition site.
[0201] The nuclease agent may be introduced into the pluripotent
cell by any means known in the art. The polypeptide encoding the
nuclease agent may be directly introduced into the cell.
Alternatively, a polynucleotide encoding the nuclease 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 constitutively expressed within
the cell. Thus, the polynucleotide encoding the nuclease agent can
be contained in an expression cassette and be operably linked to a
conditional promoter, an inducible promoter, a constitutive
promoter, or a tissue-specific promoter. Alternatively, the
nuclease agent is introduced into the cell as an mRNA encoding a
nuclease agent.
[0202] In specific embodiments, the polynucleotide encoding the
nuclease agent is stably integrated in the genome of the
pluripotent 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
nucleic acid insert, while in other instances the polynucleotide
encoding the nuclease agent is in a vector or a plasmid that is
separate from the targeting vector comprising the nucleic acid
insert.
[0203] When the nuclease agent is provided to the pluripotent 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 example
the polynucleotide encoding the nuclease agent can be modified to
substitute codons having a higher frequency of usage in a human
cell, as compared to the naturally occurring polynucleotide
sequence.
[0204] b. Selection Markers
[0205] Various selection markers can be used in the methods and
compositions disclosed herein which provide for modifying a target
genomic locus on a chromosome. 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,
blasticidin, neomycin, or puromycin. The polynucleotide encoding
the selection markers are operably linked to a promoter active in a
human iPS cell or a non-pluripotent cell transformed to express a
naive state.
[0206] c. Target Genomic Locus
[0207] Various methods and compositions are provided which allow
for the integration of at least one nucleic acid insert at a target
genomic locus on a chromosome. A "target genomic locus on a
chromosome" comprises any segment or region of DNA on a chromosome
that one desires to integrate a nucleic acid insert. The genomic
locus on a chromosome being targeted can be native to human iPS
cell or a non-pluripotent cell transformed to express a pluripotent
state, or alternatively can comprise a heterologous or exogenous
segment of DNA that was integrated into a chromosome of the cell.
Such heterologous 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 chromosome can comprise any of the
targeted genomic integration system including, for example, the
recognition site, the selection marker, previously integrated
nucleic acid inserts, polynucleotides encoding nuclease agents,
promoters, etc. Alternatively, the target genomic locus on the
chromosome can be located within a yeast artificial chromosome
(YAC), bacterial artificial chromosome (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 chromosome can comprise native genomic
sequence from a human cell or heterologous or exogenous genomic
nucleic acid sequence from a non-human mammal, a non-human cell, 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 thereof.
[0208] Targeting Vectors and Nucleic Acid Inserts
[0209] 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.
[0210] i. Nucleic Acid Insert
[0211] One or more separate nucleic acid inserts can be employed in
the methods disclosed herein, and they can be introduced into a
cell via separate targeting vectors or on the same targeting
vector. Nucleic acid inserts include segments of DNA to be
integrated at genomic target loci. Integration of a nucleic acid
insert at a target locus can result in addition of a nucleic acid
sequence of interest to the target locus, deletion of a nucleic
acid sequence of interest at the target locus, and/or replacement
of a nucleic acid sequence of interest at the target locus.
[0212] The nucleic acid insert or the corresponding nucleic acid at
the target locus being replaced can be a coding region, an intron,
an exon, an untranslated region, a regulatory region, a promoter,
an enhancer, or any combination thereof. Moreover, the nucleic acid
insert or the corresponding nucleic acid at the target locus being
replaced can be of any desired length, including, for example,
between 10-100 nucleotides in length, 100-500 nucleotides in
length, 500 nucleotides-1 kb in length, 1 kb to 1.5 kb nucleotide
in length, 1.5 kb to 2 kb nucleotides in length, 2 kb to 2.5 kb
nucleotides in length, 2.5 kb to 3 kb nucleotides in length, 3 kb
to 5 kb nucleotides in length, 5 kb to 8 kb nucleotides in length,
8 kb to 10 kb nucleotides in length or more. In other cases, the
length can be 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 250 kb, from about 250
kb to about 300 kb, from about 300 kb to about 350 kb, from about
350 kb to about 400 kb, from about 400 kb to about 800 kb, from
about 800 kb to 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, from about
2.5 Mb to about 2.8 Mb, from about 2.8 Mb to about 3 Mb. In yet
other cases, the length can be at least 100, 200, 300, 400, 500,
600, 700, 800, or 900 nucleotides or at least 1 kb, 2 kb, 3 kb, 4
kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14
kb, 15 kb, 16 kb or greater.
[0213] In some targeting vectors, the nucleic acid insert can be
from about 5 kb to about 200 kb, from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 30 kb,
from about 30 kb to about 40 kb, from about 40 kb to about 50 kb,
from about 60 kb to about 70 kb, from about 80 kb to about 90 kb,
from about 90 kb to about 100 kb, from about 100 kb to about 110
kb, from about 120 kb to about 130 kb, from about 130 kb to about
140 kb, from about 140 kb to about 150 kb, from about 150 kb to
about 160 kb, from about 160 kb to about 170 kb, from about 170 kb
to about 180 kb, from about 180 kb to about 190 kb, from about 190
kb to about 200 kb. Alternatively, the nucleic acid insert can be
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 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.
[0214] In some cases, the replacement of the nucleic acid at the
target locus results in the deletion of a target sequence ranging
from about 1 kb to about 200 kb, from about 2 kb to about 20 kb, or
from about 0.5 kb to about 3 Mb. In some cases, the extent of the
deletion is greater than a total length of the 5' homology arm and
the 3' homology arm.
[0215] In some cases, the extent of the deletion of the target
sequence 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 20 kb to about 30 kb, from about 30 kb
to about 40 kb, from about 40 kb to about 50 kb, from about 50 kb
to about 60 kb, from about 60 kb to about 70 kb, from about 70 kb
to about 80 kb, from about 80 kb to about 90 kb, from about 90 kb
to about 100 kb, from about 100 kb to about 110 kb, from about 110
kb to about 120 kb, from about 120 kb to about 130 kb, from about
130 kb to about 140 kb, from about 140 kb to about 150 kb, from
about 150 kb to about 160 kb, from about 160 kb to about 170 kb,
from about 170 kb to about 180 kb, from about 180 kb to about 190
kb, from about 190 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, from about 350 kb to about 400 kb, from about 400 kb
to about 800 kb, from about 800 kb to 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, from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb
to about 3 Mb, 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.
[0216] In other cases, the nucleic acid insert or the corresponding
nucleic acid at the target locus being replaced can be at least 10
kb, 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, at least 200 kb, at least 250 kb, at
least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or
at least 500 kb or greater.
[0217] The nucleic acid insert can comprise genomic DNA or any
other type of DNA. For example, the nucleic acid insert can be from
a prokaryote, a eukaryote, a yeast, a bird (e.g., chicken), 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 cat, a dog, a
ferret, a primate (e.g., marmoset, rhesus monkey), a domesticated
mammal, an agricultural mammal, or any other organism of interest.
In one example, the insert polynucleotide can comprise any human or
non-human genomic locus.
[0218] The nucleic acid insert and/or the nucleic acid at the
target locus can comprise a coding sequence or a non-coding
sequence, such as a regulatory element (e.g., a promoter, an
enhancer, or a transcriptional repressor-binding element). For
example, the nucleic acid insert can comprise a knock-in allele of
at least one exon of an endogenous gene, or a knock-in allele of
the entire endogenous gene (i.e., "gene-swap knock-in").
[0219] The nucleic acid insert can also comprise a conditional
allele. The conditional allele can be a multifunctional allele, as
described in US 2011/0104799, which is incorporated by reference in
its entirety. For example, the conditional allele can comprise: (a)
an actuating sequence in sense orientation with respect to
transcription of a target gene; (b) a drug selection cassette (DSC)
in sense or antisense orientation; (c) a nucleotide sequence of
interest (NSI) in antisense orientation; and (d) a conditional by
inversion module (COIN, which utilizes an exon-splitting intron and
an invertible gene-trap-like module) in reverse orientation. See,
for example, US 2011/0104799, which is incorporated by reference in
its entirety. The conditional allele can further comprise
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 orientation. See US
2011/0104799.
[0220] Some nucleic acid inserts comprise a polynucleotide encoding
a selection marker. The selection marker can be contained in a
selection cassette. Such selection markers include, but are not
limited, to neomycin phosphotransferase (neo.sup.r), hygromycin B
phosphotransferase (hyg.sup.r), puromycin-N-acetyltransferase
(puro.sup.r), blasticidin S deaminase (bsr.sup.r), xanthine/guanine
phosphoribosyl transferase (gpt), or herpes simplex virus thymidine
kinase (HSV-k), or a combination thereof. The polynucleotide
encoding the selection marker can be operably linked to a promoter
active in a cell being targeted.
[0221] In some targeting vectors, the nucleic acid insert comprises
a reporter gene. Examples of reporter genes are genes encoding
luciferase, .beta.-galactosidase, green fluorescent protein (GFP),
enhanced green fluorescent protein (eGFP), cyan fluorescent protein
(CFP), yellow fluorescent protein (YFP), enhanced yellow
fluorescent protein (eYFP), blue fluorescent protein (BFP),
enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP,
mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO,
mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire,
alkaline phosphatase, and a combination thereof. Such reporter
genes can be operably linked to a promoter active in a cell being
targeted.
[0222] In some targeting vectors, the nucleic acid insert comprises
one or more expression cassettes or deletion cassettes. A given
cassette can comprise a nucleotide sequence of interest, a nucleic
acid encoding a selection marker, and/or a reporter gene, along
with various regulatory components that influence expression.
Examples of selectable markers and reporter genes that can be
included are discussed in detail elsewhere herein.
[0223] In some targeting vectors, the insert nucleic acid comprises
a nucleic acid flanked with site-specific recombination target
sequences. Although the entire insert nucleic acid can be flanked
by such site-specific recombination target sequences, any region or
individual polynucleotide of interest within the insert nucleic
acid can also be flanked by such sites. Site-specific recombination
target sequences, which can flank the insert nucleic acid or any
polynucleotide of interest in the insert nucleic acid can include,
for example, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171,
FRT, FRT11, FRT71, attp, att, FRT, rox, and a combination thereof.
In one example, the site-specific recombination sites flank a
polynucleotide encoding a selection marker and/or a reporter gene
contained within the insert nucleic acid. Following integration of
the insert nucleic acid at a targeted locus, the sequences between
the site-specific recombination sites can be removed.
[0224] ii. Targeting Vectors
[0225] Targeting vectors can be employed to introduce the nucleic
acid insert into a target genomic locus and comprise the nucleic
acid insert and homology arms that flank the nucleic acid insert.
Targeting vectors can be in linear form or in circular form, and
can be single-stranded or double-stranded. For ease of reference,
the homology arms are referred to herein as 5' and 3' (i.e.,
upstream and downstream) homology arms. This terminology relates to
the relative position of the homology arms to the nucleic acid
insert within the targeting vector. The 5' and 3' homology arms
correspond to regions within the targeted locus, which are referred
to herein as "5' target sequence" and "3' target sequence,"
respectively.
[0226] A homology arm and a target sequence "correspond" or are
"corresponding" to one another when the two regions share a
sufficient level of sequence identity to one another to act as
substrates for a homologous recombination reaction. The term
"homology" includes DNA sequences that are either identical or
share sequence identity to a corresponding sequence. The sequence
identity between a given target sequence 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 example, the amount of sequence identity shared by the
homology arm of the targeting vector (or a fragment thereof) and
the target sequence (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 homology arm and the corresponding target sequence 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 corresponding target sequence can comprise corresponding
regions of homology that are at least about 5-10 kb, 5-15 kb, 5-20
kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb,
5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95
kb, 5-100 kb, 100-200 kb, or 200-300 kb in length or more (such as
described in the LTVEC vectors described elsewhere herein) such
that the homology arm has sufficient homology to undergo homologous
recombination with the corresponding target sequences within the
genome of the cell.
[0227] The homology arms can correspond to a locus that is native
to a cell (e.g., the targeted locus), or alternatively they can
correspond to a region of a heterologous or exogenous segment of
DNA that was integrated into the genome of the cell, including, for
example, transgenes, expression cassettes, or heterologous or
exogenous regions of DNA. Alternatively, 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 region contained in
an appropriate host cell. Still further, the homology arms of the
targeting vector can correspond to or be derived from a region of a
BAC library, a cosmid library, or a P1 phage library. In certain
instances, the homology arms of the targeting vector correspond to
a locus that is native, heterologous, or exogenous to a human iPS
cell or a non-pluripotent cell transformed to express a naive
state. In some cases, the homology arms correspond to a locus of
the cell that is not targetable using a conventional method or that
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 (e.g., a Cas protein). In some cases, the
homology arms are derived from synthetic DNA.
[0228] In some targeting vectors, the 5' and 3' homology arms
correspond to a safe harbor locus. Interactions between integrated
exogenous DNA and a host genome can limit the reliability and
safety of integration and can lead to overt phenotypic effects that
are not due to the targeted genetic modification but are instead
due to unintended effects of the integration on surrounding
endogenous genes. For example, randomly inserted transgenes can be
subject to position effects and silencing, making their expression
unreliable and unpredictable. Likewise, integration of exogenous
DNA into a chromosomal locus can affect surrounding endogenous
genes and chromatin, thereby altering cell behavior and phenotypes.
Safe harbor loci include chromosomal loci where transgenes or other
exogenous nucleic acid inserts can be stably and reliably expressed
in all tissues of interest without overtly altering cell behavior
or phenotype. See, e.g., Sadelain et al. (2012) Nat. Rev. Cancer
12:51-58. For example, safe harbor loci can include chromosomal
loci where exogenous DNA can integrate and function in a
predictable manner without adversely affecting endogenous gene
structure or expression. Safe harbor loci can include extragenic
regions or intragenic regions such as, for example, loci within
genes that are non-essential, dispensable, or able to be disrupted
without overt phenotypic consequences.
[0229] For example, the Rosa26 locus and its equivalent in humans
offer an open chromatin configuration in all tissues and is
ubiquitously expressed during embryonic development and in adults.
See Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. USA
94:3789-3794. In addition, the Rosa26 locus can be targeted with
high efficiency, and disruption of the Rosa26 gene produces no
overt phenotype. Another example of a suitable locus is the Ch25h
locus.
[0230] A homology arm of a targeting vector can be of any length
that is sufficient to promote a homologous recombination event with
a corresponding target sequence, including, for example, at least
5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45
kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb,
5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb in
length or greater. As described in further detail below, large
targeting vectors can employ targeting arms of greater length.
[0231] Nuclease agents (e.g., CRISPR/Cas systems) can be employed
in combination with targeting vectors to aid in the modification of
a target locus. Such nuclease agents may promote homologous
recombination between the targeting vector and the target locus.
When nuclease agents are employed in combination with a targeting
vector, the targeting vector can comprise 5' and 3' homology arms
corresponding to 5' and 3' target sequences located in sufficient
proximity to a nuclease cleavage site so as to promote the
occurrence of a homologous recombination event between the target
sequences and the homology arms upon a nick or double-strand break
at the nuclease cleavage site. The term "nuclease cleavage site"
includes a DNA sequence at which a nick or double-strand break is
created by a nuclease agent (e.g., a Cas9 cleavage site). The
target sequences within the targeted locus that correspond to the
5' and 3' homology arms of the targeting vector are "located in
sufficient proximity" to a nuclease cleavage site if the distance
is such as to promote the occurrence of a homologous recombination
event between the 5' and 3' target sequences and the homology arms
upon a nick or double-strand break at the recognition site. Thus,
in specific instances, the target sequences corresponding to the 5'
and/or 3' homology arms of the targeting vector are within at least
1 nucleotide of a given recognition site or are within at least 10
nucleotides to about 14 kb of a given recognition site. In some
cases, the nuclease cleavage site is immediately adjacent to at
least one or both of the target sequences.
[0232] The spatial relationship of the target sequences that
correspond to the homology arms of the targeting vector and the
nuclease cleavage site can vary. For example, target sequences can
be located 5' to the nuclease cleavage site, target sequences can
be located 3' to the recognition site, or the target sequences can
flank the nuclease cleavage site.
[0233] Combined use of the targeting vector (including, for
example, a large targeting vector) with a nuclease agent can result
in an increased targeting efficiency compared to use of the
targeting vector alone. For example, when a targeting vector is
used in conjunction with a nuclease agent, targeting efficiency of
the targeting vector can be increased by at least two-fold, at
least three-fold, at least 4-fold, or at least 10-fold when
compared to use of the targeting vector alone.
[0234] iii. Large Targeting Vectors
[0235] Some targeting vectors are "large targeting vectors" or
"LTVECs," which includes targeting vectors that comprise homology
arms that correspond to and are derived from nucleic acid sequences
larger than those typically used by other approaches intended to
perform homologous recombination in cells. Examples of generating
targeted genetic modifications using LTVECs are disclosed, for
example, in WO 2015/088643, US 2015/0159175, US 2015/0159174, US
2014/0310828, US 2014/0309487, and US 2013-0309670, each of which
is herein incorporated by reference in its entirety for all
purposes. LTVECs also include targeting vectors comprising nucleic
acid inserts having nucleic acid sequences larger than those
typically used by other approaches intended to perform homologous
recombination in cells. For example, LTVECs make possible the
modification of large loci that cannot be accommodated by
traditional plasmid-based targeting vectors because of their size
limitations. For example, the targeted locus can be (i.e., the 5'
and 3' homology arms can correspond to) a locus of the cell that is
not targetable using a conventional method or that 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 (e.g., a Cas protein).
[0236] Examples of LTVECs include vectors 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 U.S. Pat. Nos.
6,586,251; 6,596,541; 7,105,348; and WO 2002/036789
(PCT/US01/45375), each of which is herein incorporated by
reference. LTVECs can be in linear form or in circular form.
[0237] LTVECs can be of any length, including, for example, from
about 50 kb to about 300 kb, from about 50 kb to about 75 kb, from
about 75 kb to about 100 kb, from about 100 kb to 125 kb, from
about 125 kb to about 150 kb, from about 150 kb to about 175 kb,
about 175 kb to about 200 kb, from about 200 kb to about 225 kb,
from about 225 kb to about 250 kb, from about 250 kb to about 275
kb or from about 275 kb to about 300 kb. Alternatively, an LTVEC
can be at least 10 kb, at least 15 kb, 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,
at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb,
at least 400 kb, at least 450 kb, or at least 500 kb or greater.
The size of an LTVEC can be too large to enable screening of
targeting events by conventional assays, e.g., southern blotting
and long-range (e.g., 1 kb to 5 kb) PCR.
[0238] In some cases, an LTVEC comprises a nucleic acid insert
ranging from about 5 kb to about 200 kb, from about 5 kb to about
10 kb, from about 10 kb to about 20 kb, from about 20 kb to about
30 kb, from about 30 kb to about 40 kb, from about 40 kb to about
50 kb, from about 60 kb to about 70 kb, from about 80 kb to about
90 kb, from about 90 kb to about 100 kb, from about 100 kb to about
110 kb, from about 120 kb to about 130 kb, from about 130 kb to
about 140 kb, from about 140 kb to about 150 kb, from about 150 kb
to about 160 kb, from about 160 kb to about 170 kb, from about 170
kb to about 180 kb, from about 180 kb to about 190 kb, or from
about 190 kb to about 200 kb. In other cases, the insert nucleic
acid 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 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.
[0239] In some LTVECs, the sum total of the upstream homology arm
and the downstream homology arm is at least 10 kb. In other LTVECs,
the upstream homology arm ranges from about 5 kb to about 100 kb
and/or the downstream homology arm ranges from about 5 kb to about
100 kb. The sum total of the upstream and downstream homology arms
can be, for example, from about 5 kb to about 10 kb, from about 10
kb to about 20 kb, from about 20 kb to about 30 kb, from about 30
kb to about 40 kb, from about 40 kb to about 50 kb, from about 50
kb to about 60 kb, from about 60 kb to about 70 kb, from about 70
kb to about 80 kb, from about 80 kb to about 90 kb, from about 90
kb to about 100 kb, from about 100 kb to about 110 kb, from about
110 kb to about 120 kb, from about 120 kb to about 130 kb, from
about 130 kb to about 140 kb, from about 140 kb to about 150 kb,
from about 150 kb to about 160 kb, from about 160 kb to about 170
kb, from about 170 kb to about 180 kb, from about 180 kb to about
190 kb, or from about 190 kb to about 200 kb.
[0240] In some cases, the LTVEC and nucleic acid insert are
designed to allow for a deletion at the target locus 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. Alternatively, the deletion can be at least 10 kb, 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, at least 200 kb, at least 250 kb, at least 300 kb, at
least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb
or greater.
[0241] In other cases, the LTVEC and nucleic acid insert are
designed to allow for an insertion into the target locus of an
exogenous nucleic acid sequence ranging 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 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.
Alternatively, the insertion can be at least 10 kb, 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, at least 200 kb, at least 250 kb, at least 300 kb, at
least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb
or greater.
[0242] In yet other cases, the nucleic acid insert and/or the
region of the endogenous locus being deleted is at least 100, 200,
300, 400, 500, 600, 700, 800, or 900 nucleotides or at least 1 kb,
2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12
kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.
[0243] iv. Methods of Integrating a Nucleic Acid Insert Near the
Recognition Site on a Chromosome by Homologous Recombination
[0244] In some examples, methods for modifying a target genomic
locus on a chromosome in a pluripotent cell can comprise: (a)
providing a cell comprising a target genomic locus on a chromosome,
(b) introducing into the cell a first targeting vector comprising a
first nucleic acid insert flanked by 5' and 3' homology arms
corresponding to 5' and 3' target sequences; and (c) identifying at
least one cell comprising in its genome the first nucleic acid
insert integrated at the target genomic locus on the chromosome. 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 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb,
about 4 kb to about 5 kb, about 5 kb 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 one non-limiting embodiment, such methods are
performed employing the culture medium provided herein.
[0245] In other examples, methods for modifying a target genomic
locus on a chromosome in a pluripotent cell can comprise: (a)
providing a cell comprising a target genomic locus on a chromosome
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 nucleic
acid insert flanked by 5' and 3' homology arms corresponding to 5'
and 3' target sequences located in sufficient proximity to the
first recognition site; and (c) identifying at least one cell
comprising in its genome the first nucleic acid insert integrated
at the target genomic locus on the chromosome. 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 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, about 4 kb to
about 5 kb, about 5 kb 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 one non-limiting embodiment, such methods are
performed employing the culture medium provided herein.
[0246] Various methods can also be employed to identify pluripotent
cells having the nucleic acid insert integrated at the genomic
target locus. Insertion of the nucleic acid insert at the genomic
target locus results in a "modification of allele." The term
"modification 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. Examples 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.
[0247] In various embodiments, to facilitate identification of the
targeted modification, a high-throughput quantitative assay,
namely, modification of allele (MOA) assay, is employed. The MOA
assay described herein allows a large-scale screening of a modified
allele(s) in a parental chromosome following a genetic
modification. The MOA assay can be carried out via various
analytical techniques, including, 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,
fluorescence-mediated in situ hybridization (FISH), comparative
genomic hybridization, isothermic DNA amplification, quantitative
hybridization to an immobilized probe(s), Invader Probes.RTM., MMP
Assays.RTM., TaqMan.RTM. Molecular Beacon, and Eclipse.TM. probe
technology. See, for example, US2005/0144655, incorporated by
reference herein in its entirety.
[0248] In various embodiments, in the presence of the nick or
double strand break, targeting efficiency of a targeting vector
(such as a LTVEC) at the target genomic locus is at least about
2-fold higher, at least about 3-fold higher, 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 genomic locus of interest but
in the absence of an added nuclease agent that makes the nick or
double strand break).
[0249] The various methods set forth above can be sequentially
repeated to allow for the targeted integration of any number of
nucleic acid inserts into a given targeted genomic locus on a
chromosome. 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 nucleic acid inserts into the target genomic
locus on a chromosome. 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 non-human, a rodent, a mouse, a monkey, a rat, a hamster,
a domesticated mammal or an agricultural animal) into a targeted
genomic locus on a chromosome. In such instances, the transfer and
reconstruction of genomic regions that include both coding and
non-coding regions allow for the complexity of a given region to be
preserved by retaining, at least in part, the coding regions, the
non-coding 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
regions within a human iPS cell or a non-pluripotent cell
transformed to express a pluripotent state.
[0250] v. Polynucleotides of Interest
[0251] Any polynucleotide of interest may be contained in the
various nucleic acid inserts and thereby integrated at the target
genomic locus on a chromosome. The methods disclosed herein,
provide for at least 1, 2, 3, 4, 5, 6 or more polynucleotides of
interest to be integrated into the targeted genomic locus.
[0252] The polynucleotide of interest within the nucleic acid
insert when integrated at the target genomic locus on a chromosome
can introduce one or more genetic modifications into the
pluripotent 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 nucleic acid sequence with
an exogenous polynucleotide of interest at the target genomic
locus. Thus, methods provided 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 regulatory sequence swap, a
gene swap, or a combination thereof in a target genomic locus on a
chromosome. Such modifications may occur upon integration of the
first, second, third, fourth, fifth, six, seventh, or any
subsequent nucleic acid inserts into the target genomic locus.
[0253] The polynucleotide of interest within the nucleic acid
insert and/or integrated at the target genomic locus can comprise a
sequence that is native or homologous to the pluripotent 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. "Homologous" in reference to a
sequence includes a sequence that is native to the cell.
"Heterologous" in reference to a sequence includes 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
genomic locus by deliberate human intervention. "Exogenous" in
reference to a sequence includes a sequence that originates from a
foreign species. "Orthologous" includes a polynucleotide from one
species that is functionally equivalent to a known reference
sequence in another species (i.e., a species variant). The
polynucleotide of interest can be from any organism of interest
including, but not limited to, non-human, a rodent, a hamster, a
mouse, a rat, a human, a monkey, an avian, an agricultural mammal
or a non-agricultural mammal. The polynucleotide of interest can
further comprise a coding region, a non-coding region, a regulatory
region, or a genomic DNA. Thus, the 1.sup.st, 2.sup.nd, 3.sup.rd,
4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th, and/or any of the
subsequent nucleic acid inserts can comprise such sequences.
[0254] In one embodiment, the polynucleotide of interest within the
nucleic acid insert and/or integrated at the target genomic locus
on a chromosome is homologous to a human 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 nucleic acid is a mouse genomic nucleic
acid, a human genomic 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 or a combination thereof.
[0255] In one embodiment, the polynucleotide of interest can range
from about 500 nucleotides to about 200 kb as described above. The
polynucleotide of interest can be from about 500 nucleotides to
about 5 kb, from about 5 kb to about 200 kb, from about 5 kb to
about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to
about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to
about 50 kb, from about 60 kb to about 70 kb, from about 80 kb to
about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to
about 110 kb, from about 120 kb to about 130 kb, from about 130 kb
to about 140 kb, from about 140 kb to about 150 kb, from about 150
kb to about 160 kb, from about 160 kb to about 170 kb, from about
170 kb to about 180 kb, from about 180 kb to about 190 kb, or from
about 190 kb to about 200 kb.
[0256] The polynucleotide of interest within the nucleic acid
insert and/or inserted at the target genomic locus on a chromosome
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 nucleic acid insert and/or inserted at the
target genomic locus on a chromosome can encode a protein expressed
in the nervous system, the skeletal system, the digestive system,
the circulatory system, the muscular system, the respiratory
system, the cardiovascular system, the lymphatic system, the
endocrine system, the urinary system, the reproductive system, or a
combination thereof.
[0257] The polynucleotide of interest within the nucleic acid
insert and/or integrated at the target genomic locus on a
chromosome 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.
[0258] The polynucleotide of interest within the nucleic acid
insert and/or integrated at the target genomic locus on a
chromosome can comprise a polynucleotide encoding a mutant protein.
In one embodiment, the mutant protein 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 nucleic acid insert
and/or integrated at the genomic target locus on a chromosome
comprises at least one disease allele. In such instances, the
disease allele can be a dominant allele or the disease allele is a
recessive allele. Moreover, the disease allele can comprise a
single nucleotide polymorphism (SNP) allele. The polynucleotide of
interest encoding the mutant protein can be from any organism,
including, but not limited to, a mammal, a non-human mammal,
rodent, mouse, rat, a human, a monkey, an agricultural mammal or a
domestic mammal polynucleotide encoding a mutant protein.
[0259] The polynucleotide of interest within the nucleic acid
insert and/or integrated at the target genomic locus on a
chromosome can also comprise a regulatory sequence, including for
example, a promoter sequence, an enhancer sequence, a
transcriptional repressor-binding sequence, or a transcriptional
terminator sequence. In specific embodiments, the polynucleotide of
interest within the nucleic acid insert and/or integrated at the
target genomic locus on a chromosome comprises a polynucleotide
having a deletion of a non-protein-coding sequence, but does not
comprise a deletion of a protein-coding sequence. In one
embodiment, the deletion of the non-protein-coding sequence
comprises a deletion of a regulatory sequence. In another
embodiment, the deletion of the regulatory element comprises a
deletion of a promoter sequence. In one embodiment, the deletion of
the regul