U.S. patent application number 17/245405 was filed with the patent office on 2021-08-26 for methods and compositions for assessing crispr/cas-mediated disruption or excision and crispr/cas-induced recombination with an exogenous donor nucleic acid in vivo.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Susannah Brydges, David Frendewey, Guochun Gong, Suzanne Hartford, Charleen Hunt, Andrew J. Murphy, Brian Zambrowicz.
Application Number | 20210261985 17/245405 |
Document ID | / |
Family ID | 1000005583056 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261985 |
Kind Code |
A1 |
Gong; Guochun ; et
al. |
August 26, 2021 |
METHODS AND COMPOSITIONS FOR ASSESSING CRISPR/CAS-MEDIATED
DISRUPTION OR EXCISION AND CRISPR/CAS-INDUCED RECOMBINATION WITH AN
EXOGENOUS DONOR NUCLEIC ACID IN VIVO
Abstract
Methods and compositions are provided for assessing
CRISPR/Cas-mediated non-homologous end joining (NHEJ) activity
and/or CRISPR/Cas-induced recombination of a target genomic locus
with an exogenous donor nucleic acid in vivo or ex vivo. The
methods and compositions employ non-human animals comprising a
CRISPR reporter such as a genomically integrated CRISPR reporter
for detecting and measuring targeted excision of a sequence between
two CRISPR/Cas nuclease cleavage sites or disruption of a sequence
near a CRISPR/Cas nuclease cleavage site and/or measuring
CRISPR/Cas-induced recombination of the CRISPR reporter with an
exogenous donor nucleic acid to convert the coding sequence for a
first reporter protein to the coding sequence for a different
second reporter protein. Methods and compositions are also provided
for making and using these non-human animals.
Inventors: |
Gong; Guochun;
(Pleasantville, NY) ; Hunt; Charleen; (Montvale,
NJ) ; Brydges; Susannah; (Scarsdale, NY) ;
Hartford; Suzanne; (Putnam Valley, NY) ; Frendewey;
David; (New York, NY) ; Zambrowicz; Brian;
(Sleepy Hollow, NY) ; Murphy; Andrew J.;
(Croton-on-Hudson, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
1000005583056 |
Appl. No.: |
17/245405 |
Filed: |
April 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16050806 |
Jul 31, 2018 |
11021719 |
|
|
17245405 |
|
|
|
|
62539279 |
Jul 31, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/861 20130101;
C12N 2320/11 20130101; A01K 67/0275 20130101; A01K 2267/0393
20130101; C12N 9/2471 20130101; A01K 2227/105 20130101; C12N
15/8775 20130101; A01K 2217/07 20130101; C12N 15/907 20130101; C12N
2310/20 20170501; A01K 2217/206 20130101; C12Y 302/01023
20130101 |
International
Class: |
C12N 15/90 20060101
C12N015/90; C12N 15/861 20060101 C12N015/861; C12N 15/877 20060101
C12N015/877; A01K 67/027 20060101 A01K067/027; C12N 9/38 20060101
C12N009/38 |
Claims
1. A method of testing the ability of a CRISPR/Cas nuclease to
excise a genomic nucleic acid in vivo, comprising: (a) introducing
into a non-human animal comprising a CRISPR reporter for assessing
CRISPR/Cas-induced excision of a nucleic acid between first and
second guide RNA target sequences: (i) a first guide RNA or a DNA
encoding the first guide RNA, wherein the first guide RNA is
designed to hybridize to the first guide RNA target sequence in the
CRISPR reporter; (ii) a second guide RNA or a DNA encoding the
second guide RNA, wherein the second guide RNA is designed to
hybridize to the second guide RNA target sequence in the CRISPR
reporter; and (iii) a Cas protein or a nucleic acid encoding the
Cas protein; wherein the CRISPR reporter is integrated at a target
genomic locus and comprises a first polyadenylation signal flanked
by the first and second guide RNA target sequences followed by a
reporter cassette comprising a coding sequence for a first reporter
protein and a coding sequence for a second reporter protein in any
order, wherein the first reporter protein and the second reporter
protein are different; and (b) measuring the activity or expression
of at least one of the first and second reporter proteins.
2. The method of claim 1, wherein one of the first and second
reporter proteins comprises a fluorescent reporter protein.
3. The method of claim 2, wherein the fluorescent reporter protein
comprises an enhanced green fluorescent protein (eGFP) or an
enhanced blue fluorescent protein (eBFP).
4. The method of claim 2, wherein the first and second reporter
proteins comprise the fluorescent reporter protein and a
non-fluorescent reporter protein.
5. The method of claim 4, wherein the fluorescent reporter protein
can be detected in a flow cytometry assay, and the non-fluorescent
protein can be detected in a histochemical assay.
6. The method of claim 1, wherein one of the first and second
reporter proteins comprises a beta-galactosidase protein.
7. The method of claim 1, wherein the first polyadenylation signal
is flanked by recombinase recognition sites for a first
recombinase.
8. The method of claim 7, wherein the recombinase recognition sites
for the first recombinase are loxP sequences.
9. The method of claim 1, wherein the reporter cassette comprises a
multicistronic nucleic acid comprising the coding sequence for the
first reporter protein and the coding sequence for the second
reporter protein separated by an intervening internal ribosome
entry site (IRES) or an intervening 2A peptide coding sequence.
10. The method of claim 9, wherein the multicistronic nucleic acid
comprises a beta-galactosidase coding sequence and an enhanced blue
fluorescent protein (eBFP) coding sequence or an enhanced green
fluorescent protein (eGFP) coding sequence separated by an
intervening P2A peptide coding sequence.
11. The method of claim 1, wherein the CRISPR reporter is operably
linked to an endogenous promoter at the target genomic locus.
12. The method of claim 1, wherein the 5' end of the CRISPR
reporter comprises a 3' splicing sequence.
13. The method of claim 1, wherein the CRISPR reporter further
comprises a selection cassette.
14. The method of claim 13, wherein the selection cassette is
flanked by recombinase recognition sites for a second
recombinase.
15. The method of claim 13, wherein the selection cassette
comprises a drug resistance gene.
16. The method of claim 1, wherein the distance between the first
guide RNA target sequence and the second guide RNA target sequence
is less than 500 base pairs.
17. The method of claim 1, wherein the first guide RNA target
sequence and the second guide RNA target sequence are identical,
and each comprises SEQ ID NO: 41.
18. The method of claim 1, wherein the non-human animal is a mouse
or a rat.
19. The method of claim 1, wherein the non-human animal is a
rat.
20. The method of claim 1, wherein the non-human animal is a
mouse.
21. The method of claim 1, wherein the target genomic locus is a
safe harbor locus.
22. The method of claim 21, wherein the safe harbor locus is a
Rosa26 locus.
23. The method of claim 22, wherein the CRISPR reporter is inserted
into the first intron of the Rosa26 locus.
24. The method of claim 1, wherein the non-human animal is a mouse,
wherein the target genomic locus is a Rosa26 locus, and wherein the
CRISPR reporter is operably linked to an endogenous Rosa26
promoter, is inserted into the first intron of the Rosa26 locus,
and comprises from 5' to 3': (a) a 3' splicing sequence; (b) a
first polyadenylation signal flanked by: (i) first and second loxP
sites; and (ii) first and second guide RNA target sequences,
wherein the first guide RNA target sequence and the second guide
RNA target sequence are identical, and each comprises SEQ ID NO:
41; and (c) a reporter cassette, comprising from 5' to 3': (i) a
beta-galactosidase coding sequence; (ii) a P2A coding sequence;
(iii) an enhanced blue fluorescent protein (eBFP) coding sequence,
wherein the eBFP coding sequence comprises a third guide RNA target
sequence comprising SEQ ID NO: 42; and (iv) a second
polyadenylation signal, wherein the first polyadenylation signal
and the second polyadenylation signal are different.
25. The method of claim 24, wherein the CRISPR reporter further
comprises: (d) a selection cassette 3' of the reporter cassette,
wherein the selection cassette is flanked by FRT sites and
comprises from 5' to 3': (i) a neomycin phosphotransferase coding
sequence operably linked to a human ubiquitin promoter; and (ii) a
third polyadenylation signal.
26. The method of claim 1, wherein the non-human animal is
heterozygous for the CRISPR reporter at the target genomic
locus.
27. The method of claim 1, wherein the non-human animal is
homozygous for the CRISPR reporter at the target genomic locus.
28. The method of claim 1, wherein the Cas protein is a Cas9
protein.
29. The method of claim 1, wherein step (a) comprises introducing
the Cas protein into the non-human animal.
30. The method of claim 1, wherein step (a) comprising introducing
the nucleic acid encoding the Cas protein into the non-human
animal, wherein the nucleic acid encoding the Cas protein is a
messenger RNA encoding the Cas protein.
31. The method of claim 1, wherein step (a) comprises introducing
the nucleic acid encoding the Cas protein into the non-human
animal, wherein the nucleic acid encoding the Cas protein is a DNA
encoding the Cas protein, wherein the DNA is operably linked to a
promoter active in one or more cell types in the non-human
animal.
32. The method of claim 1, wherein the first guide RNA and the
second guide RNA are identical, and each comprises the sequence set
forth in SEQ ID NO: 2.
33. The method of claim 1, wherein step (a) comprises introducing
the first guide RNA and the second guide RNA into the non-human
animal.
34. The method of claim 1, wherein step (a) comprises introducing
the DNA encoding first guide RNA and the DNA encoding the second
guide RNA into the non-human animal, wherein the DNA encoding the
first guide RNA and the DNA encoding the second guide RNA are each
operably linked to a promoter active in one or more cell types in
the non-human animal.
35. The method of claim 1, wherein the introducing in step (a)
comprises adeno-associated virus (AAV)-mediated delivery, lipid
nanoparticle-mediated delivery, or hydrodynamic delivery.
36. The method of claim 35, wherein the introducing in step (a)
comprises AAV-mediated delivery.
37. The method of claim 36, wherein the introducing in step (a)
comprises AAV8-mediated delivery, and step (b) comprises measuring
activity of the at least one of the first and second reporter
proteins in the liver of the non-human animal.
38. The method of claim 1, wherein the at least one of the first
and second reporter proteins measured in step (b) is a fluorescent
reporter protein, and step (b) comprises a flow cytometry
assay.
39. The method of claim 1, wherein the at least one of the first
and second reporter proteins measured in step (b) is a
beta-galactosidase protein, and step (b) comprises a histochemical
staining assay.
40. A method of optimizing the ability of a CRISPR/Cas nuclease to
excise a genomic nucleic acid in vivo, comprising: (I) performing
the method of claim 1 a first time in a first non-human animal;
(II) changing a variable and performing the method of step (I) a
second time with the changed variable in a second non-human animal;
and (III) comparing the activity or expression of the at least one
of the first and second reporter proteins in step (I) with the
activity or expression of the at least one of the first and second
reporter proteins in step (II), and selecting the method resulting
in the higher activity or expression of the at least one of the
first and second reporter proteins.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 16/050,806, filed Jul. 31, 2018, which claims the benefit of
U.S. Application No. 62/539,279, filed Jul. 31, 2017, each of which
is herein incorporated by reference in its entirety for all
purposes.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS
WEB
[0002] The Sequence Listing written in file 558388SEQLIST.txt is
72.7 kilobytes, was created on Apr. 29, 2021, and is hereby
incorporated by reference.
BACKGROUND
[0003] CRISPR/Cas technology is a promising new therapeutic
modality. However, there is a need for better means of assessing
the efficiency of mutation generation or targeted gene modification
by an introduced CRISPR/Cas agent in vivo. Assessing such activity
in vivo currently relies on difficult molecular assays, such as
single-strand DNase sensitivity assays, digital PCR, or next
generation sequencing. Better methods and tools are needed to more
effectively assess the activity of introduced CRISPR/Cas agents and
to assess different delivery methods and parameters for targeting
specific tissues or cell types in vivo.
SUMMARY
[0004] Methods and compositions are provided for assessing
CRISPR/Cas-mediated non-homologous end joining activity and/or
CRISPR/Cas-induced recombination activity in vivo. In one aspect,
provided are non-human animals comprising a CRISPR reporter for
assessing CRISPR/Cas-induced excision of a nucleic acid between
first and second guide RNA target sequences, wherein the CRISPR
reporter is integrated at a target genomic locus and comprises a
first polyadenylation signal flanked by the first and second guide
RNA target sequences followed by a reporter cassette comprising a
coding sequence for a first reporter protein and a coding sequence
for a second reporter protein in any order, wherein the first
reporter protein and the second reporter protein are different.
[0005] In some such non-human animals, the CRISPR reporter is also
for assessing CRISPR/Cas-induced recombination of the CRISPR
reporter with an exogenous donor nucleic acid. Optionally, the
first reporter protein comprises a third guide RNA target sequence,
wherein recombination of the CRISPR reporter with the exogenous
donor nucleic acid changes the coding sequence for the first
reporter protein into a coding sequence for a third reporter
protein. Optionally, the coding sequence for the first reporter
protein is changed into the coding sequence for the third reporter
protein by changing a single codon. Optionally, the third guide RNA
target sequence overlaps with the portion of the coding sequence
for the first reporter protein modified by the exogenous donor
nucleic acid.
[0006] In some such non-human animals, one of the first and second
reporter proteins comprises a fluorescent reporter protein.
Optionally, the fluorescent reporter protein comprises an enhanced
green fluorescent protein (eGFP) or an enhanced blue fluorescent
protein (eBFP). Optionally, the first and second reporter proteins
comprise a fluorescent reporter protein and a non-fluorescent
reporter protein. Optionally, the fluorescent reporter protein can
be detected in a flow cytometry assay, and the non-fluorescent
protein can be detected in a histochemical assay. In some such
non-human animals, one of the first and second reporter proteins
comprises a beta-galactosidase protein.
[0007] In some such non-human animals, the first polyadenylation
signal is also flanked by recombinase recognition sites for a first
recombinase. Optionally, the recombinase recognition sites for the
first recombinase are loxP sequences.
[0008] In some such non-human animals, the reporter cassette
comprises a multicistronic nucleic acid comprising the coding
sequence for the first reporter protein and the coding sequence for
the second reporter protein separated by an intervening internal
ribosome entry site (IRES) or an intervening 2A peptide coding
sequence. Optionally, the multicistronic nucleic acid comprises a
beta-galactosidase coding sequence and an enhanced blue fluorescent
protein (eBFP) coding sequence or an enhanced green fluorescent
protein (eGFP) coding sequence separated by an intervening P2A
peptide coding sequence.
[0009] In some such non-human animals, the CRISPR reporter is
operably linked to an endogenous promoter at the target genomic
locus.
[0010] In some such non-human animals, the 5' end of the CRISPR
reporter further comprises a 3' splicing sequence.
[0011] In some such non-human animals, the CRISPR reporter does not
comprise a selection cassette. In some such non-human animals, the
CRISPR reporter further comprises a selection cassette. Optionally,
the selection cassette is flanked by recombinase recognition sites
for a second recombinase. Optionally, the selection cassette
comprises a drug resistance gene.
[0012] In some such non-human animals, the distance between the
first guide RNA target sequence and the second guide RNA target
sequence is less than about 500 base pairs.
[0013] In some such non-human animals, the first guide RNA target
sequence and the second guide RNA target sequence are identical,
and each comprises SEQ ID NO: 41.
[0014] In some such non-human animals, the non-human animal is a
rat or mouse. Optionally, the non-human animal is a mouse.
[0015] In some such non-human animals, the target genomic locus is
a safe harbor locus. Optionally, the safe harbor locus is a Rosa26
locus. Optionally, the CRISPR reporter is inserted into the first
intron of the Rosa26 locus.
[0016] In some such non-human animals, the non-human animal is a
mouse, the target genomic locus is the Rosa26 locus, and the CRISPR
reporter is operably linked to the endogenous Rosa26 promoter, is
inserted into the first intron of the Rosa26 locus, and comprises
from 5' to 3': (a) a 3' splicing sequence; (b) a first
polyadenylation signal flanked by: (i) first and second loxP sites;
and (ii) first and second guide RNA target sequences, wherein the
first guide RNA target sequence and the second guide RNA target
sequence are identical, and each comprises SEQ ID NO: 41, 43, 44,
45, 46, or 47; and (c) a reporter cassette, comprising from 5' to
3': (i) a beta-galactosidase coding sequence; (ii) a P2A coding
sequence; (iii) an enhanced blue fluorescent protein (eBFP) coding
sequence, wherein the eBFP coding sequence comprises a third guide
RNA target sequence comprising SEQ ID NO: 42; and (iv) a second
polyadenylation signal, wherein the first polyadenylation signal
and the second polyadenylation signal are different. Optionally,
the CRISPR reporter further comprises: (d) a selection cassette 3'
of the reporter cassette, wherein the selection cassette is flanked
by FRT sites and comprises from 5' to 3': (i) a neomycin
phosphotransferase coding sequence operably linked to a human
ubiquitin promoter; and (ii) a third polyadenylation signal.
[0017] In some such non-human animals, the non-human animal is
heterozygous for the CRISPR reporter at the target genomic locus.
In some such non-human animals, the non-human animal is homozygous
for the CRISPR reporter at the target genomic locus.
[0018] In another aspect, provided are methods of testing the
ability of a CRISPR/Cas nuclease to excise a genomic nucleic acid
in vivo. Some such methods comprise: (a) introducing into any of
the above non-human animals: (i) a first guide RNA designed to
hybridize to the first guide RNA target sequence in the CRISPR
reporter; (ii) a second guide RNA designed to hybridize to the
second guide RNA target sequence in the CRISPR reporter; and (iii)
a Cas protein; and (b) measuring the activity or expression of at
least one of the first and second reporter proteins.
[0019] In some such methods, the Cas protein is a Cas9 protein. In
some such methods, the Cas protein is introduced into the non-human
animal in the form of a protein. In some such methods, the Cas
protein is introduced into the non-human animal in the form of a
messenger RNA encoding the Cas protein. In some such methods, the
Cas protein is introduced into the non-human animal in the form of
a DNA encoding the Cas protein, wherein the DNA is operably linked
to a promoter active in one or more cell types in the non-human
animal.
[0020] In some such methods, the first guide RNA and the second
guide RNA are identical, and each comprises the sequence set forth
in SEQ ID NO: 2.
[0021] In some such methods, the reporter protein measured in step
(b) is a fluorescent reporter protein, and step (b) comprises a
flow cytometry assay. In some such methods, the reporter protein
measured in step (b) is a beta-galactosidase protein, and step (b)
comprises a histochemical staining assay.
[0022] In some such methods, the guide RNAs in step (a) are
introduced in the form of RNA. In some such methods, the guide RNAs
in step (a) are each introduced into the non-human animal in the
form of a DNA encoding the guide RNA, wherein the DNA is operably
linked to a promoter active in one or more cell types in the
non-human animal.
[0023] In some such methods, the introducing comprises
adeno-associated virus (AAV)-mediated delivery, lipid
nanoparticle-mediated delivery, or hydrodynamic delivery.
Optionally, the introducing comprises AAV-mediated delivery.
Optionally, the introducing comprises AAV8-mediated delivery, and
step (b) comprises measuring activity of the reporter protein in
the liver of the non-human animal.
[0024] In another aspect, provided are methods of optimizing the
ability of a CRISPR/Cas nuclease to excise a genomic nucleic acid
in vivo. Some such methods comprise: (I) performing any of the
above methods of testing the ability of a CRISPR/Cas nuclease to
excise a genomic nucleic acid in vivo a first time in a first
non-human animal; (II) changing a variable and performing the
method of step (I) a second time with the changed variable in a
second non-human animal; and (III) comparing the activity or
expression of the reporter protein in step (I) with the activity or
expression of the at least one of the reporter protein in step
(II), and selecting the method resulting in the higher activity or
expression of the reporter protein.
[0025] In some such methods, the changed variable in step (II) is
the delivery method. In some such methods, the changed variable in
step (II) is the delivery method of introducing the guide RNAs
and/or the Cas protein into the non-human animal. In some such
methods, the changed variable in step (II) is the route of
administration. In some such methods, the changed variable in step
(II) is the route of administration of introducing the guide RNAs
and/or the Cas protein into the non-human animal. In some such
methods, the changed variable in step (II) is the concentration or
amount of the guide RNAs introduced into the non-human animal. In
some such methods, the changed variable in step (II) is the
concentration or amount of the guide RNAs and/or the Cas protein
introduced into the non-human animal. In some such methods, the
changed variable in step (II) is the concentration or amount of the
guide RNAs introduced into the non-human animal relative to the
concentration or amount of Cas protein introduced into the
non-human animal. In some such methods, the changed variable in
step (II) is the guide RNAs (e.g., the form of guide RNAs or the
sequence of the guide RNAs) introduced into the non-human animal.
In some such methods, the changed variable in step (II) is Cas
protein (e.g., the form of Cas protein) introduced into the
non-human animal.
[0026] In another aspect, provided are methods of testing
CRISPR/Cas-induced recombination of a genomic nucleic acid with an
exogenous donor nucleic acid in vivo. Some such methods comprise:
(a) providing any of the above non-human animals, wherein the
CRISPR reporter is also for assessing CRISPR/Cas-induced
recombination of the CRISPR reporter with an exogenous donor
nucleic acid, wherein the first polyadenylation signal has been
removed from the CRISPR reporter, and wherein the coding sequence
for the first reporter protein comprises a third guide RNA target
sequence, and introducing into the non-human animal: (i) a guide
RNA designed to hybridize to the third guide RNA target sequence in
the CRISPR reporter; (ii) a Cas protein; and (iii) an exogenous
donor nucleic acid capable of recombining with the CRISPR reporter
and changing the coding sequence for the first reporter protein
into a coding sequence for a third reporter protein; and (b)
measuring the activity or expression of the third reporter
protein.
[0027] In some such methods, the Cas protein is a Cas9 protein. In
some such methods, the Cas protein is introduced into the non-human
animal in the form of a protein. In some such methods, the Cas
protein is introduced into the non-human animal in the form of a
messenger RNA encoding the Cas protein. In some such methods, the
Cas protein is introduced into the non-human animal in the form of
a DNA encoding the Cas protein, wherein the DNA is operably linked
to a promoter active in one or more cell types in the non-human
animal.
[0028] In some such methods, the third reporter protein measured in
step (b) is a fluorescent reporter protein, and step (b) comprises
a flow cytometry assay. In some such methods, the first reporter
protein is an enhanced blue fluorescent protein (eBFP), and the
third guide RNA comprises the sequence set forth in SEQ ID NO: 14.
In some such methods, the first reporter protein is an enhanced
blue fluorescent protein (eBFP), and the third reporter protein is
an enhanced green fluorescent protein (eGFP).
[0029] In some such methods, the exogenous donor nucleic acid
comprises the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.
In some such methods, the exogenous donor nucleic acid is a
single-stranded deoxynucleotide.
[0030] In some such methods, the guide RNA in step (a) is
introduced in the form of RNA. In some such methods, the guide RNA
in step (a) is introduced into the non-human animal in the form of
a DNA encoding the guide RNA, wherein the DNA is operably linked to
a promoter active in one or more cell types in the non-human
animal.
[0031] In some such methods, the introducing comprises
adeno-associated virus (AAV)-mediated delivery, lipid
nanoparticle-mediated delivery, or hydrodynamic delivery.
Optionally, the introducing comprises AAV-mediated delivery.
Optionally, the introducing comprises AAV8-mediated delivery, and
step (b) comprises measuring activity of the reporter protein in
the liver of the non-human animal.
[0032] In another aspect, provided are methods of optimizing the
ability of CRISPR/Cas to induce recombination of a genomic nucleic
acid with an exogenous donor nucleic acid in vivo. Some such
methods comprise: (I) performing any of the above methods of
testing CRISPR/Cas-induced recombination of a genomic nucleic acid
with an exogenous donor nucleic acid in vivo a first time in a
first non-human animal; (II) changing a variable and performing the
method of step (I) a second time with the changed variable in a
second non-human animal; and (III) comparing the activity or
expression of the third reporter protein in step (I) with the
activity or expression of the third reporter protein in step (II),
and selecting the method resulting in the higher activity or
expression of the third reporter protein.
[0033] In some such methods, the changed variable in step (II) is
the delivery method. In some such methods, the changed variable in
step (II) is the delivery method of introducing one or more of the
guide RNA, the Cas protein, and the exogenous donor nucleic acid
into the non-human animal. In some such methods, the changed
variable in step (II) is the route of administration. In some such
methods, the changed variable in step (II) is the route of
administration of introducing one or more of the guide RNA, the Cas
protein, and the exogenous donor nucleic acid into the non-human
animal. In some such methods, the changed variable in step (II) is
the concentration or amount of one or more of the guide RNA, the
Cas protein, and the exogenous donor nucleic acid introduced into
the non-human animal. In some such methods, the changed variable in
step (II) is exogenous donor nucleic acid (e.g., the form or
sequence of the exogenous donor nucleic acid) introduced into the
non-human animal. In some such methods, the changed variable in
step (II) is the concentration or amount of the guide RNA
introduced into the non-human animal relative to the concentration
or amount of Cas protein introduced into the non-human animal. In
some such methods, the changed variable in step (II) is the guide
RNA (e.g., the form or sequence of the guide RNA) introduced into
the non-human animal. In some such methods, the changed variable in
step (II) is the Cas protein (e.g., the form or sequence of the Cas
protein) introduced into the non-human animal.
BRIEF DESCRIPTION OF THE FIGURES
[0034] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0035] FIG. 1A shows a LSL-LacZ:eBFP CRISPR reporter allele
(MAID2634; not to scale), comprising from 5' to 3': a 3' splicing
sequence; a first loxP site; a first guide RNA target sequence; a
first Pgk polyadenylation signal; a second guide RNA target
sequence; a second loxP site; a lacZ gene; a P2A coding sequence;
an enhanced blue fluorescent protein (eBFP) coding sequence; an
SV40 polyadenylation signal; a first Frt site; human ubiquitin and
Em7 promoters operably linked to a neomycin resistance gene coding
sequence; a second Pgk polyadenylation signal; and a second Frt
site.
[0036] FIG. 1B shows the positions of various guide RNA target
sequences in a schematic of the region of the LSL-LacZ:eBFP CRISPR
reporter allele comprising the first loxP site, the first Pgk
polyadenylation signal, and the second loxP site (not to
scale).
[0037] FIG. 2 shows a general schematic for targeting a transgene
into the first intron of the Rosa26 locus.
[0038] FIG. 3 shows a LSL-eBFP CRISPR reporter allele (MAID2652;
not to scale) comprising from 5' to 3': a 3' splicing sequence; a
first loxP site; an Em7 promoter; a neomycin resistance gene coding
sequence; a triple polyadenylation signal; a second loxP site; an
enhanced blue fluorescent protein (eBFP) coding sequence; and an
SV40 polyadenylation signal.
[0039] FIGS. 4A-4E show lacZ-stained mouse embryonic stem cells
(mESCs) comprising the LSL-LacZ:eBFP CRISPR reporter allele. The
cells in FIG. 4A are untreated, the cells in FIG. 4B were
electroporated with a Cre recombinase plasmid to excise the first
Pgk polyadenylation signal, the cells in FIG. 4C were
electroporated with a ribonucleoprotein complex comprising Cas9
protein complexed together with the gU3 and gD1 synthetic sgRNAs to
target the first Pgk polyadenylation signal for excision, the cells
in FIG. 4D were electroporated with a ribonucleoprotein complex
comprising Cas9 protein complexed together with the gU3 and gD2
synthetic sgRNAs to target the first Pgk polyadenylation signal for
excision, and the cells in FIG. 4E were electroporated with a
ribonucleoprotein complex comprising Cas9 protein complexed
together with the gU2 and gD1 synthetic sgRNAs to target the first
Pgk polyadenylation signal for excision.
[0040] FIG. 5 shows lacZ-stained mouse embryonic stem cells (mESCs)
comprising the LSL-LacZ:eBFP CRISPR reporter allele three days
post-electroporation. The cells were electroporated with a
ribonucleoprotein complex comprising Cas9 protein complexed
together with synthetic sgRNAs cGM4 and cGM5, a ribonucleoprotein
complex comprising Cas9 protein complexed together with synthetic
sgRNAs cGM4 and cGM, a ribonucleoprotein complex comprising Cas9
protein complexed together with synthetic sgRNAs cGM4 and cGM3, a
ribonucleoprotein complex comprising Cas9 protein complexed
together with synthetic sgRNA 9172 (non-cutting control guide RNA),
Cas9 protein alone, or a Cre recombinase plasmid.
[0041] FIG. 6 shows lacZ-stained mouse embryonic stem cells (mESCs)
comprising the LSL-LacZ:eBFP CRISPR reporter allele three days
post-electroporation. The cells were either untreated, were
electroporated with a Cas9 plasmid and sgRNA #16 plasmid, or were
electroporated with a Cre recombinase plasmid.
[0042] FIG. 7 shows lacZ-stained livers isolated from LSL-LacZ:eBFP
mice 1 week post-injection with lipid nanoparticles comprising
guide RNA, Cas9 mRNA and guide RNA, or Cre recombinase mRNA. The
treatment conditions included Cas9 plus pA removal sgRNA (sgRNA
#16), Cas9 together with sgRNAs gU2 and gD1, Cas9 together with a
control non-cutting sgRNA, and Cre recombinase.
[0043] FIG. 8 shows lacZ immunohistochemistry of liver samples from
LSL-LacZ:eBFP CRISPR reporter mice treated with Cas9 plus pA
removal sgRNA (sgRNA #16), Cas9 together with sgRNAs gU2 and gD1,
or LNP-Cre recombinase. Liver samples from untreated mice were used
as a control. Brown-stained cells indicate expression of the LACZ
protein.
[0044] FIG. 9 shows brightfield and fluorescence microscopy (eGFP)
images of mouse embryonic stem cells (mESCs) comprising the eBFP
CRISPR reporter allele (LSL-eBFP CRISPR reporter allele after
treatment with Cre recombinase to generate MAID20090) following
treatment with CRISPR/Cas9 and an ssODN repair template to convert
eBFP to eGFP. The top row shows brightfield images, and the bottom
row shows fluorescence microscopy images (eGFP).
[0045] FIG. 10A shows brightfield and fluorescence microscopy
(eGFP) images of hematopoietic stem and progenitor cell (HSPC)
isolated from mice comprising the eBFP CRISPR reporter allele
(LSL-eBFP CRISPR reporter allele after treatment with Cre
recombinase to generate MAID20090) genomically integrated at the
Rosa26 locus following treatment with CRISPR/Cas9 ribonucleoprotein
complexes and an ssODN repair template (FW or REV) via
electroporation to convert eBFP to eGFP. The top row shows
brightfield images, and the bottom row shows fluorescence
microscopy (eGFP) 48 hours after electroporation.
[0046] FIG. 10B shows brightfield and fluorescence microscopy
(eGFP) images of hematopoietic stem and progenitor cell (HSPC)
isolated from mice comprising eBFP CRISPR reporter allele (LSL-eBFP
CRISPR reporter allele after treatment with Cre recombinase to
generate MAID20090) genomically integrated at the Rosa26 locus
following treatment with CRISPR/Cas9 ribonucleoprotein complexes
and an ssODN FW repair template via electroporation to convert eBFP
to eGFP. The top row shows untreated control cells, and the bottom
row shows treated cells. The first column shows brightfield images,
and the second and third columns show fluorescence microscopy
images (eGFP) 7 days after electroporation.
[0047] FIG. 11 shows non-homologous end joining efficiency (number
of reads) as determined by next-generating sequencing in cells
isolated from livers harvested from mice comprising the
LSL-LacZ:eBFP CRISPR reporter allele integrated at the Rosa26 locus
one week following injection with the indicated CRISPR/Cas9 or Cre
recombinase components.
DEFINITIONS
[0048] 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.
[0049] Proteins are said to have an "N-terminus" and a
"C-terminus." The term "N-terminus" relates to the start of a
protein or polypeptide, terminated by an amino acid with a free
amine group (--NH2). The term "C-terminus" relates to the end of an
amino acid chain (protein or polypeptide), terminated by a free
carboxyl group (--COOH).
[0050] 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.
[0051] Nucleic acids are said to have "5' ends" and "3' ends"
because mononucleotides are reacted to make oligonucleotides in a
manner such that the 5' phosphate of one mononucleotide pentose
ring is attached to the 3' oxygen of its neighbor in one direction
via a phosphodiester linkage. An end of an oligonucleotide is
referred to as the "5' end" if its 5' phosphate is not linked to
the 3' oxygen of a mononucleotide pentose ring. An end of an
oligonucleotide is referred to as the "3' end" if its 3' oxygen is
not linked to a 5' phosphate of another mononucleotide pentose
ring. A nucleic acid sequence, even if internal to a larger
oligonucleotide, also may be said to have 5' and 3' ends. In either
a linear or circular DNA molecule, discrete elements are referred
to as being "upstream" or 5' of the "downstream" or 3'
elements.
[0052] The term "genomically integrated" refers to a nucleic acid
that has been introduced into a cell such that the nucleotide
sequence integrates into the genome of the cell. Any protocol may
be used for the stable incorporation of a nucleic acid into the
genome of a cell.
[0053] The term "expression vector" or "expression construct"
refers to a recombinant nucleic acid containing a desired coding
sequence operably linked to appropriate nucleic acid sequences
necessary for the expression of the operably linked coding sequence
in a particular host cell or organism. Nucleic acid sequences
necessary for expression in prokaryotes usually include a promoter,
an operator (optional), and a ribosome binding site, as well as
other sequences. Eukaryotic cells are generally known to utilize
promoters, enhancers, and termination and polyadenylation signals,
although some elements may be deleted and other elements added
without sacrificing the necessary expression.
[0054] The term "targeting vector" refers to a recombinant nucleic
acid that can be introduced by homologous recombination,
non-homologous-end-joining-mediated ligation, or any other means of
recombination to a target position in the genome of a cell.
[0055] The term "viral vector" refers to a recombinant nucleic acid
that includes at least one element of viral origin and includes
elements sufficient for or permissive of packaging into a viral
vector particle. The vector and/or particle can be utilized for the
purpose of transferring DNA, RNA, or other nucleic acids into cells
either ex vivo or in vivo. Numerous forms of viral vectors are
known.
[0056] The term "isolated" with respect to proteins, nucleic acids,
and cells includes proteins, nucleic acids, and cells that are
relatively purified with respect to other cellular or organism
components that may normally be present in situ, up to and
including a substantially pure preparation of the protein, nucleic
acid, or cell. The term "isolated" also includes proteins and
nucleic acids that have no naturally occurring counterpart or
proteins or nucleic acids that have been chemically synthesized and
are thus substantially uncontaminated by other proteins or nucleic
acids. The term "isolated" also includes proteins, nucleic acids,
or cells that have been separated or purified from most other
cellular components or organism components with which they are
naturally accompanied (e.g., other cellular proteins, nucleic
acids, or cellular or extracellular components).
[0057] The term "wild type" includes entities having a structure
and/or activity as found in a normal (as contrasted with mutant,
diseased, altered, or so forth) state or context. Wild type genes
and polypeptides often exist in multiple different forms (e.g.,
alleles).
[0058] The term "endogenous sequence" refers to a nucleic acid
sequence that occurs naturally within a cell or non-human animal.
For example, an endogenous Rosa26 sequence of a non-human animal
refers to a native Rosa26 sequence that naturally occurs at the
Rosa26 locus in the non-human animal.
[0059] "Exogenous" molecules or sequences include molecules or
sequences that are not normally present in a cell in that form.
Normal presence includes presence with respect to the particular
developmental stage and environmental conditions of the cell. An
exogenous molecule or sequence, for example, can include a mutated
version of a corresponding endogenous sequence within the cell,
such as a humanized version of the endogenous sequence, or can
include a sequence corresponding to an endogenous sequence within
the cell but in a different form (i.e., not within a chromosome).
In contrast, endogenous molecules or sequences include molecules or
sequences that are normally present in that form in a particular
cell at a particular developmental stage under particular
environmental conditions.
[0060] The term "heterologous" when used in the context of a
nucleic acid or a protein indicates that the nucleic acid or
protein comprises at least two segments that do not naturally occur
together in the same molecule. For example, the term
"heterologous," when used with reference to segments of a nucleic
acid or segments of a protein, indicates that the nucleic acid or
protein comprises two or more sub-sequences that are not found in
the same relationship to each other (e.g., joined together) in
nature. As one example, a "heterologous" region of a nucleic acid
vector is a segment of nucleic acid within or attached to another
nucleic acid molecule that is not found in association with the
other molecule in nature. For example, a heterologous region of a
nucleic acid vector could include a coding sequence flanked by
sequences not found in association with the coding sequence in
nature. Likewise, a "heterologous" region of a protein is a segment
of amino acids within or attached to another peptide molecule that
is not found in association with the other peptide molecule in
nature (e.g., a fusion protein, or a protein with a tag).
Similarly, a nucleic acid or protein can comprise a heterologous
label or a heterologous secretion or localization sequence.
[0061] "Codon optimization" takes advantage of the degeneracy of
codons, as exhibited by the multiplicity of three-base pair codon
combinations that specify an amino acid, and 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
Cas9 protein can be modified to substitute codons having a higher
frequency of usage in a given prokaryotic or eukaryotic cell,
including a bacterial cell, a yeast cell, a human cell, a non-human
cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a
hamster cell, or any other host cell, as compared to the naturally
occurring nucleic acid sequence. Codon usage tables are readily
available, for example, at the "Codon Usage Database." These tables
can be adapted in a number of ways. See Nakamura et al. (2000)
Nucleic Acids Research 28:292, herein incorporated by reference in
its entirety for all purposes. Computer algorithms for codon
optimization of a particular sequence for expression in a
particular host are also available (see, e.g., Gene Forge).
[0062] A "promoter" is a regulatory region of DNA usually
comprising a TATA box capable of directing RNA polymerase II to
initiate RNA synthesis at the appropriate transcription initiation
site for a particular polynucleotide sequence. A promoter may
additionally comprise other regions which influence the
transcription initiation rate. The promoter sequences disclosed
herein modulate transcription of an operably linked polynucleotide.
A promoter can be active in one or more of the cell types disclosed
herein (e.g., a eukaryotic cell, a non-human mammalian cell, a
human cell, a rodent cell, a pluripotent cell, a one-cell stage
embryo, a differentiated cell, or a combination thereof). A
promoter can be, for example, a constitutively active promoter, a
conditional promoter, an inducible promoter, a temporally
restricted promoter (e.g., a developmentally regulated promoter),
or a spatially restricted promoter (e.g., a cell-specific or
tissue-specific promoter). Examples of promoters can be found, for
example, in WO 2013/176772, herein incorporated by reference in its
entirety for all purposes.
[0063] A constitutive promoter is one that is active in all tissues
or particular tissues at all developing stages. Examples of
constitutive promoters include the human cytomegalovirus immediate
early (hCMV), mouse cytomegalovirus immediate early (mCMV), human
elongation factor 1 alpha (hEF1.alpha.), mouse elongation factor 1
alpha (mEF1.alpha.), mouse phosphoglycerate kinase (PGK), chicken
beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin
promoters.
[0064] Examples of inducible promoters include, for example,
chemically regulated promoters and physically-regulated promoters.
Chemically regulated promoters include, for example,
alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA)
gene promoter), tetracycline-regulated promoters (e.g., a
tetracycline-responsive promoter, a tetracycline operator sequence
(tetO), a tet-On promoter, or a tet-Off promoter), steroid
regulated promoters (e.g., a rat glucocorticoid receptor, a
promoter of an estrogen receptor, or a promoter of an ecdysone
receptor), or metal-regulated promoters (e.g., a metalloprotein
promoter). Physically regulated promoters include, for example
temperature-regulated promoters (e.g., a heat shock promoter) and
light-regulated promoters (e.g., a light-inducible promoter or a
light-repressible promoter).
[0065] Tissue-specific promoters can be, for example,
neuron-specific promoters, glia-specific promoters, muscle
cell-specific promoters, heart cell-specific promoters, kidney
cell-specific promoters, bone cell-specific promoters, endothelial
cell-specific promoters, or immune cell-specific promoters (e.g., a
B cell promoter or a T cell promoter).
[0066] Developmentally regulated promoters include, for example,
promoters active only during an embryonic stage of development, or
only in an adult cell.
[0067] "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. Operable
linkage can include such sequences being contiguous with each other
or acting in trans (e.g., a regulatory sequence can act at a
distance to control transcription of the coding sequence).
[0068] "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 (melting temperature) 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 (i.e., a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands). 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.
[0069] "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), herein
incorporated by reference in its entirety for all purposes.
[0070] 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 which are well known. 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.
[0071] 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.
[0072] 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 (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).
[0073] The methods and compositions provided herein employ a
variety of different components. Some components throughout the
description can have active variants and fragments. Such components
include, for example, Cas proteins, CRISPR RNAs, tracrRNAs, and
guide RNAs. Biological activity for each of these components is
described elsewhere herein. The term "functional" refers to the
innate ability of a protein or nucleic acid (or a fragment or
variant thereof) to exhibit a biological activity or function. Such
biological activities or functions can include, for example, the
ability of a Cas protein to bind to a guide RNA and to a target DNA
sequence. The biological functions of functional fragments or
variants may be the same or may in fact be changed (e.g., with
respect to their specificity or selectivity or efficacy) in
comparison to the original, but with retention of the basic
biological function.
[0074] The term "variant" refers to a nucleotide sequence differing
from the sequence most prevalent in a population (e.g., by one
nucleotide) or a protein sequence different from the sequence most
prevalent in a population (e.g., by one amino acid).
[0075] The term "fragment" when referring to a protein means a
protein that is shorter or has fewer amino acids than the
full-length protein. The term "fragment" when referring to a
nucleic acid means a nucleic acid that is shorter or has fewer
nucleotides than the full-length nucleic acid. A fragment can be,
for example, an N-terminal fragment (i.e., removal of a portion of
the C-terminal end of the protein), a C-terminal fragment (i.e.,
removal of a portion of the N-terminal end of the protein), or an
internal fragment.
[0076] "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,
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.
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.).
[0077] "Percentage of sequence identity" includes the value
determined by comparing two optimally aligned sequences (greatest
number of perfectly matched residues) 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. Unless
otherwise specified (e.g., the shorter sequence includes a linked
heterologous sequence), the comparison window is the full length of
the shorter of the two sequences being compared.
[0078] 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.
[0079] The term "conservative amino acid substitution" refers to
the substitution of an amino acid that is normally present in the
sequence with a different amino acid of similar size, charge, or
polarity. Examples of conservative substitutions include the
substitution of a non-polar (hydrophobic) residue such as
isoleucine, valine, or leucine for another non-polar residue.
Likewise, examples of conservative substitutions include the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine, or
between glycine and serine. Additionally, the substitution of a
basic residue such as lysine, arginine, or histidine for another,
or the substitution of one acidic residue such as aspartic acid or
glutamic acid for another acidic residue are additional examples of
conservative substitutions. Examples of non-conservative
substitutions include the substitution of a non-polar (hydrophobic)
amino acid residue such as isoleucine, valine, leucine, alanine, or
methionine for a polar (hydrophilic) residue such as cysteine,
glutamine, glutamic acid or lysine and/or a polar residue for a
non-polar residue. Typical amino acid categorizations are
summarized in Table 1 below.
TABLE-US-00001 TABLE 1 Amino Acid Categorizations. Alanine Ala A
Nonpolar Neutral 1.8 Arginine Arg R Polar Positive -4.5 Asparagine
Asn N Polar Neutral -3.5 Aspartic acid Asp D Polar Negative -3.5
Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu E Polar
Negative -3.5 Glutamine Gln Q Polar Neutral -3.5 Glycine Gly G
Nonpolar Neutral -0.4 Histidine His H Polar Positive -3.2
Isoleucine Ile I Nonpolar Neutral 4.5 Leucine Leu L Nonpolar
Neutral 3.8 Lysine Lys K Polar Positive -3.9 Methionine Met M
Nonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8
Proline Pro P Nonpolar Neutral -1.6 Serine Ser S Polar Neutral -0.8
Threonine Thr T Polar Neutral -0.7 Tryptophan Trp W Nonpolar
Neutral -0.9 Tyrosine Tyr Y Polar Neutral -1.3 Valine Val V
Nonpolar Neutral 4.2
[0080] The term "in vitro" includes artificial environments and to
processes or reactions that occur within an artificial environment
(e.g., a test tube). The term "in vivo" includes natural
environments (e.g., a cell or organism or body) and to processes or
reactions that occur within a natural environment. The term "ex
vivo" includes cells that have been removed from the body of an
individual and to processes or reactions that occur within such
cells.
[0081] The term "reporter gene" refers to a nucleic acid having a
sequence encoding a gene product (typically an enzyme) that is
easily and quantifiably assayed when a construct comprising the
reporter gene sequence operably linked to an endogenous or
heterologous promoter and/or enhancer element is introduced into
cells containing (or which can be made to contain) the factors
necessary for the activation of the promoter and/or enhancer
elements. Examples of reporter genes include, but are not limited,
to genes encoding beta-galactosidase (lacZ), the bacterial
chloramphenicol acetyltransferase (cat) genes, firefly luciferase
genes, genes encoding beta-glucuronidase (GUS), and genes encoding
fluorescent proteins. A "reporter protein" refers to a protein
encoded by a reporter gene.
[0082] The term "fluorescent reporter protein" as used herein means
a reporter protein that is detectable based on fluorescence wherein
the fluorescence may be either from the reporter protein directly,
activity of the reporter protein on a fluorogenic substrate, or a
protein with affinity for binding to a fluorescent tagged compound.
Examples of fluorescent proteins include green fluorescent proteins
(e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green,
Monomeric Azami Green, CopGFP, AceGFP, and ZsGreen1), yellow
fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet,
PhiYFP, and ZsYellow1), blue fluorescent proteins (e.g., BFP, eBFP,
eBFP2, Azurite, mKalama1, GFPuv, Sapphire, and T-sapphire), cyan
fluorescent proteins (e.g., CFP, eCFP, Cerulean, CyPet, AmCyan1,
and Midoriishi-Cyan), red fluorescent proteins (e.g., RFP, mKate,
mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express,
DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611,
mRaspberry, mStrawberry, and Jred), orange fluorescent proteins
(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,
mTangerine, and tdTomato), and any other suitable fluorescent
protein whose presence in cells can be detected by flow cytometry
methods.
[0083] Repair in response to double-strand breaks (DSBs) occurs
principally through two conserved DNA repair pathways: homologous
recombination (HR) and non-homologous end joining (NHEJ). See
Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol.
22:886-897, herein incorporated by reference in its entirety for
all purposes. Likewise, repair of a target nucleic acid mediated by
an exogenous donor nucleic acid can include any process of exchange
of genetic information between the two polynucleotides.
[0084] The term "recombination" includes any process of exchange of
genetic information between two polynucleotides and can occur by
any mechanism. Recombination can occur via homology directed repair
(HDR) or homologous recombination (HR). HDR or HR includes a form
of nucleic acid repair that can require nucleotide sequence
homology, uses a "donor" molecule as a template for 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. See 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 in its entirety
for all purposes.
[0085] NHEJ includes the repair of double-strand breaks in a
nucleic acid by direct ligation of the break ends to one another or
to an exogenous sequence 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. For example, NHEJ can also result in the
targeted integration of an exogenous donor nucleic acid through
direct ligation of the break ends with the ends of the exogenous
donor nucleic acid (i.e., NHEJ-based capture). Such NHEJ-mediated
targeted integration can be preferred for insertion of an exogenous
donor nucleic acid when homology directed repair (HDR) pathways are
not readily usable (e.g., in non-dividing cells, primary cells, and
cells which perform homology-based DNA repair poorly). In addition,
in contrast to homology-directed repair, knowledge concerning large
regions of sequence identity flanking the cleavage site (beyond the
overhangs created by Cas-mediated cleavage) is not needed, which
can be beneficial when attempting targeted insertion into organisms
that have genomes for which there is limited knowledge of the
genomic sequence. The integration can proceed via ligation of blunt
ends between the exogenous donor nucleic acid and the cleaved
genomic sequence, or via ligation of sticky ends (i.e., having 5'
or 3' overhangs) using an exogenous donor nucleic acid that is
flanked by overhangs that are compatible with those generated by
the Cas protein in the cleaved genomic sequence. See, e.g., US
2011/020722, WO 2014/033644, WO 2014/089290, and Maresca et al.
(2013) Genome Res. 23(3):539-546, each of which is herein
incorporated by reference in its entirety for all purposes. If
blunt ends are ligated, target and/or donor resection may be needed
to generation regions of microhomology needed for fragment joining,
which may create unwanted alterations in the target sequence.
[0086] 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. The transitional phrase "consisting essentially
of" means that the scope of a claim is to be interpreted to
encompass the specified elements recited in the claim and those
that do not materially affect the basic and novel characteristic(s)
of the claimed invention. Thus, the term "consisting essentially
of" when used in a claim of this invention is not intended to be
interpreted to be equivalent to "comprising."
[0087] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur and that the
description includes instances in which the event or circumstance
occurs and instances in which it does not.
[0088] Designation of a range of values includes all integers
within or defining the range, and all subranges defined by integers
within the range.
[0089] Unless otherwise apparent from the context, the term "about"
encompasses values within a standard margin of error of measurement
(e.g., SEM) of a stated value.
[0090] The term "and/or" refers to and encompasses any and all
possible combinations of one or more of the associated listed
items, as well as the lack of combinations when interpreted in the
alternative ("or").
[0091] The term "or" refers to any one member of a particular list
and also includes any combination of members of that list.
[0092] The singular forms of the articles "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a Cas protein" or "at least one
Cas protein" can include a plurality of Cas proteins, including
mixtures thereof.
[0093] Statistically significant means p.ltoreq.0.05.
DETAILED DESCRIPTION
I. Overview
[0094] Assessing the efficiency of delivery and the efficiency of
mutation generation or targeted gene modification by an introduced
CRISPR/Cas agent in vivo currently relies on difficult molecular
assays, such as single-strand DNase sensitivity assays, digital
PCR, or next generation sequencing. Better methods and tools are
needed to more effectively assess the activity of CRISPR/Cas agents
and to assess different delivery methods and parameters for
targeting specific tissues or cell types in vivo.
[0095] Methods and compositions are provided for assessing
CRISPR/Cas-mediated non-homologous end joining (NHEJ) activity
and/or CRISPR/Cas-induced recombination of a target genomic nucleic
acid with an exogenous donor nucleic acid in vivo and ex vivo. The
methods and compositions employ cells and non-human animals
comprising a CRISPR reporter (e.g., a genomically integrated CRISPR
reporter) for detecting and measuring targeted excision of a
sequence between two CRISPR/Cas nuclease cleavage sites or
disruption of a sequence near a CRISPR/Cas nuclease cleavage site
and/or detecting and measuring CRISPR/Cas-induced recombination of
the CRISPR reporter with an exogenous donor nucleic acid to convert
the coding sequence for a first reporter protein into a coding
sequence for a different second reporter protein. Some such CRISPR
reporters can be multifunctional reporters comprising two or more
different types of reporter genes. These reporters are an
improvement over CRISPR reporters with a single type of reporter
gene because they enable the use of different methods for detecting
and measuring CRISPR/Cas activity in vivo and ex vivo, such as in
vivo imaging of the non-human animal, detection of fluorescence in
cells isolated from the animal via flow cytometry or other methods,
or histochemical staining of tissues isolated from the non-human
animal. In this way, the limitations of one reporter protein can be
offset by the advantages of another reporter protein. For example,
beta-galactosidase (encoded by the lacZ gene) and fluorescent
proteins are used in combination in some reporters. LacZ allows for
the ability to take sections of tissue to visualize the precise
boundaries of CRISPR/Cas-induced repair. LacZ staining is permanent
and can be visualized with the naked eye or standard brightfield
magnification. Fluorescent reporter proteins such as eBFP or eGFP
allow for a precise count of correctly edited, unedited, and
alternatively edited cells. Fluorescent reporter proteins allow
tissues to be analyzed on a single cell basis (e.g., via FACS
analysis) for exact ratios of edited cells.
[0096] The CRISPR reporters described herein that have a lacZ gene
have additional advantages over other reporters, such as "traffic
light" reporters that report a change from red fluorescent protein
to green fluorescent protein upon CRISPR/Cas action. The lacZ gene
through its encoded beta-galactosidase is the most thoroughly
established and reliable of all reporters. The reporters described
herein that comprise a lacZ gene are designed to produce a
histological color read-out upon the action of CRISPR/Cas. Because
beta-galactosidase is a multiple turnover enzyme that converts a
substrate into a visible blue dye, it has the potential to be more
sensitive than fluorescent reporter proteins, which require tens of
thousands of proteins per cell for a detectable fluorescent signal.
Compared with fluorescent proteins, beta-galactosidase produces a
higher definition signal that can reveal fine cell-type specific
expression patterns. Traffic light fluorescent reporters require
CRISPR/Cas-induced mutations that produce a fortuitous reading
frame change. In contrast, lacZ reporters described herein do not
depend on reading frames; rather, they require only a simple
deletion between to CRIPSR/Cas cleavage sites to delete or
inactivate a transcriptional polyadenylation termination signal,
thereby permitting expression of the downstream beta-galactosidase
coding sequence. Unlike the traffic light and similar dual
fluorescent protein reporter systems that require analysis and
interpretation of the ratio of two different fluorescent signals,
the lacZ reporter systems go from no signal in the unmodified state
to a strong reporter signal after CRISPR/Cas activation of the
allele.
[0097] In addition, some of the CRISPR reporters disclosed herein
are multifunctional reporters in that they enable testing of not
only CRISPR/Cas NHEJ activity in vivo and ex vivo but also enable
testing of CRISPR/Cas-induced HDR activity in vivo and ex vivo via
different readouts. Because some such reporters for testing
CRISPR-induced recombination require changing only a single codon
in the gene encoding a reporter protein to convert that reporter
protein into a different reporter protein, smaller exogenous donor
nucleic acids can be used than if an entire coding sequence for a
reporter protein needed to be deleted and replaced with the
sequence for a different reporter protein. In some such reporters,
a fluorescent reporter protein is converted into a different
fluorescent reporter protein (e.g., eBFP to eGFP, or vice versa).
Such conversion allows for a precise count of correctly edited,
unedited, and alternatively edited cells and allows tissue to be
analyzed on a single cell basis via FACs analysis for exact ratios
of edited cells.
[0098] Methods and compositions are also provided for making and
using these non-human animals to test and measure the ability of a
CRISPR/Cas nuclease to excise or disrupt a genomically integrated
nucleic acid and/or to facilitate recombination of a target genomic
nucleic acid with an exogenous donor in vivo and to optimize the
ability of a CRISPR/Cas nuclease to excise or disrupt a genomically
integrated nucleic acid and/or to facilitate recombination of a
target genomic locus with an exogenous donor in vivo.
II. Non-Human Animals Comprising CRISPR Reporters
[0099] The methods and compositions disclosed herein utilize a
CRISPR reporter to assess the ability of introduced Clustered
Regularly Interspersed Short Palindromic Repeats
(CRISPR)/CRISPR-associated (Cas) systems or components of such
systems to modify the CRISPR reporter in vivo or ex vivo.
[0100] The methods and compositions disclosed herein employ the
CRISPR/Cas systems by testing the ability of CRISPR complexes
(comprising a guide RNA (gRNA) complexed with a Cas protein) to
induce two site-directed cleavage events within a CRISPR reporter
in vivo or ex vivo and excise the intervening sequence between the
two cleavage sites via non-homologous end joining (NHEJ) or to
induce a site-directed cleavage event within a CRISPR reporter in
vivo or ex vivo and disrupt a nearby sequence via NHEJ-mediated
small insertions and deletions (indels). The methods and
compositions disclosed herein also employ the CRISPR/Cas systems by
testing the ability of CRISPR complexes (comprising a guide RNA
(gRNA) complexed with a Cas protein) to induce recombination
between a CRISPR reporter and an exogenous donor nucleic acid in
vivo or ex vivo to repair a coding sequence for a first reporter
protein in order to convert it into a coding sequence for a
different second reporter protein.
[0101] A. CRISPR Reporters for Measuring CRISPR/Cas-Mediated
Disruption or CRISPR/Cas-Mediated Excision Using Paired gRNAs
and/or for Measuring CRISPR/Cas-Induced Recombination of a Target
Genomic Nucleic Acid with an Exogenous Donor Nucleic Acid
[0102] Provided herein are CRISPR reporters for detecting and
measuring targeted excision of a sequence between two CRISPR/Cas
nuclease cleavage sites or targeted disruption of a sequence near a
CRISPR/Cas nuclease cleavage site and/or for detecting and
measuring CRISPR/Cas-induced recombination of a target nucleic acid
with an exogenous donor nucleic acid. The CRISPR reporters provided
herein can comprise a polyadenylation signal or transcription
terminator flanked by first and second guide RNA target sequences
followed by a reporter cassette comprising a coding sequence for
one or more reporter proteins. Alternatively or additionally, the
polyadenylation signal or transcription terminator can comprise a
third guide RNA target sequence at or near the canonical
polyadenylation hexamer (AATAAA, referred to as a poly A
recognition motif or poly A recognition sequence). Examples of
guide RNA target sequences near the canonical polyadenylation
hexamer in a Pgk polyadenylation signal include SEQ ID NOS: 48-52.
However, any desired guide RNA target sequence can be included in
or engineered into the CRISPR reporter so that any guide RNA or
combination of guide RNAs can be tested. Alternatively or
additionally, the polyadenylation signal or transcription
terminator can be flanked by first and second recombinase
recognition sites. The polyadenylation signal or transcription
terminator prevents transcription and expression of the one or more
reporter proteins. However, upon cleavage of the first and second
guide RNA target sequences by a CRISPR/Cas nuclease and excision of
the intervening sequence including the transcription terminator or
polyadenylation signal, transcription can proceed through the
coding sequence for the one or more reporter proteins, enabling
their expression. Alternatively, upon cleavage of a guide RNA
target sequence at or near the poly A recognition motif (canonical
polyadenylation hexamer AATAAA) and disruption of the poly A
recognition motif via NHEJ-mediated small insertions and deletions
(indels), transcription can proceed through the coding sequence for
the one or more reporter proteins, enabling their expression.
[0103] Any transcription terminator or polyadenylation signal can
be used. A "transcription terminator" as used herein refers to a
DNA sequence that causes termination of transcription. In
eukaryotes, transcription terminators are recognized by protein
factors, and termination is followed by polyadenylation, a process
of adding a poly(A) tail to the mRNA transcripts in presence of the
poly(A) polymerase. The mammalian poly(A) signal typically consists
of a core sequence, about 45 nucleotides long, that may be flanked
by diverse auxiliary sequences that serve to enhance cleavage and
polyadenylation efficiency. The core sequence consists of a highly
conserved and required upstream element (the poly A recognition
motif AATAAA or AAUAAA in the mRNA), recognized by cleavage and
polyadenylation-specificity factor (CPSF), and a poorly defined
downstream region (rich in Us or Gs and Us), bound by cleavage
stimulation factor (CstF). Examples of transcription terminators
that can be used include, for example, the human growth hormone
(HGH) polyadenylation signal, the simian virus 40 (SV40) late
polyadenylation signal, the rabbit beta-globin polyadenylation
signal, the bovine growth hormone (BGH) polyadenylation signal, the
phosphoglycerate kinase (PGK) polyadenylation signal, an AOX1
transcription termination sequence, a CYC1 transcription
termination sequence, or any transcription termination sequence
known to be suitable for regulating gene expression in eukaryotic
cells.
[0104] Optionally, the polyadenylation signal can also be flanked
by recombinase recognition sites for a site-specific recombinase.
The recombinase can be used as a positive control for excision of
the polyadenylation signal. Site-specific recombinases include
enzymes that can facilitate recombination between recombinase
recognition sites, where the two recombination sites are physically
separated within a single nucleic acid or on separate nucleic
acids. Examples of recombinases include Cre, Flp, and Dre
recombinases. One example of a Cre recombinase gene is Crei, in
which two exons encoding the Cre recombinase are separated by an
intron to prevent its expression in a prokaryotic cell. Such
recombinases can further comprise a nuclear localization signal to
facilitate localization to the nucleus (e.g., NLS-Crei).
Recombinase recognition sites include nucleotide sequences that are
recognized by a site-specific recombinase and can serve as a
substrate for a recombination event. Examples of recombinase
recognition sites include FRT, FRT11, FRT71, attp, att, rox, and
lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2, and
lox5171.
[0105] The first and second guide RNA target sequences can be the
same or different, and any suitable guide RNA target sequence can
be used. Any desired guide RNA target sequence can be included in
or engineered into the CRISPR reporter so that any guide RNA or
combination of guide RNAs can be tested. Guide RNA target sequences
are described in more detail elsewhere herein. As one example, the
first guide RNA target sequence can comprise the sequence set forth
in SEQ ID NO: 41, 43, 44, or 45, and the second guide RNA target
sequence can comprise the sequence set forth in SEQ ID NO: 41, 46,
or 47. The first and second guide RNA target sequences (or the
first and second Cas cleavage sites within the first and second
guide RNA target sequences, respectively) can be separated by any
desired distance to be tested. For example, they can be separated
by at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
500, 600, or 1000 base pairs (bp), no more than 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 100, 200, 300, 400, 500, 600, or 1000 bp, or
between about 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-1000,
6-100, 6-200, 6-300, 6-400, 6-500, 6-600, 6-1000, 10-100, 10-200,
10-300, 10-400, 10-500, 10-600, 10-1000, 50-100, 50-200, 50-300,
50-400, 50-500, 50-600, 50-1000, 100-1000, 200-1000, 300-1000,
400-1000, or 500-1000 bp. In a specific example, the guide RNA
target sequences or Cas cleavage sites can separated by less than
about 1000, less than about 600, less than about 500, less than
about 200, or less than about 100 base pairs. Alternatively, they
can be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 kb or more, or can be separated by between about
1-10, 1-20, 1-30, 1-40, 1-50, 1-100, 10-20, 10-30, 10-40, 10-50,
10-100, 20-30, 20-40, 20-50, 20-100, 30-40, 30-50, 30-100, 40-50,
40-100, or 50-100 kb. For example, the first and second guide RNA
target sequences or first and second Cas cleavage sites can be
separated by between about 5 bp to 10 kb, 6 bp to 10 kb, 10 bp to
10 kb, 50 bp to 10 kb, 100 bp to 10 kb, 200 bp to 10 kb, 300 bp to
10 kb, 400 bp to 10 kb, or 500 bp to 10 kb.
[0106] The first and second guide RNA target sequences (or the
first and second Cas cleavage sites) are optionally at (i.e.,
overlapping) or near the poly A recognition motif (canonical
polyadenylation signal hexamer AATAAA). For example, one or both of
the first and second guide RNA target sequences or the first and
second Cas cleavage sites can be within about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, or 1000 bp,
or between about 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-1000,
5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-1000, 10-100, 10-200,
10-300, 10-400, 10-500, 10-600, 10-1000, 50-100, 50-200, 50-300,
50-400, 50-500, 50-600, 50-1000, 100-1000, 200-1000, 300-1000,
400-1000, or 500-1000 bp from the canonical polyadenylation signal
hexamer. Optionally, a third guide RNA target sequence (or Cas
cleavage site) is at (i.e., overlapping) or near the poly A
recognition motif (canonical polyadenylation signal hexamer
AATAAA). For example, the third guide RNA target sequences or the
third Cas cleavage site can be within about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,
600, or 1000 bp, or between about 1-10, 1-20, 1-30, 1-40, 1-50,
1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600,
1-1000, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90,
5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-1000, 10-10, 10-20,
10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200,
10-300, 10-400, 10-500, 10-600, 10-1000, 50-100, 50-200, 50-300,
50-400, 50-500, 50-600, 50-1000, 100-1000, 200-1000, 300-1000,
400-1000, or 500-1000 bp from the canonical polyadenylation signal
hexamer. For example, the third guide RNA target sequence or third
Cas cleavage site can be within about 10-220, 20-200, 20-40, 29,
65-85, 76, 185-205, or 195 bp from the canonical polyadenylation
signal hexamer.
[0107] The first and second guide RNA target sequences can be
anywhere with respect to the first and second recombinase
recognition sites, respectively. The first guide RNA target
sequence can be upstream or downstream of the first recombinase
recognition site or can overlap with the first recombinase
recognition site. Likewise, the second guide RNA target sequence
can be upstream or downstream of the second recombinase recognition
site or can overlap with the second recombinase recognition site.
In addition, the first guide RNA target sequence can be upstream of
the flanked transcription terminator or polyadenylation signal or
can overlap with the transcription terminator or polyadenylation
signal. Likewise, the second guide RNA target sequence can be
downstream of the flanked transcription terminator or
polyadenylation signal or can overlap with the transcription
terminator or polyadenylation signal.
[0108] Any suitable reporter proteins can be used. In some
reporters, the one or more reporter proteins comprise a
beta-galactosidase protein. In other reporters, there are two or
more different reporter proteins. Optionally, the two or more
reporter proteins can be different types of reporter proteins. For
example, the two or more different reporter proteins can include at
least one fluorescent reporter protein as defined elsewhere herein
and at least one non-fluorescent reporter protein. Examples of
fluorescent reporter proteins are provided elsewhere herein.
Non-fluorescent reporter proteins include, for example, reporter
proteins that can be used in histochemical or bioluminescent
assays, such as beta-galactosidase, luciferase (e.g., Renilla
luciferase, firefly luciferase, and NanoLuc luciferase), and
beta-glucuronidase. Some reporters include both a reporter protein
that can be detected in a flow cytometry assay (e.g., a fluorescent
reporter protein such as a blue fluorescent protein (BFP), an
enhanced BFP (eBFP), a green fluorescent protein (GFP), or an
enhanced GFP (eGFP)) and a reporter protein that can be detected in
a histochemical assay (e.g., beta-galactosidase protein) to provide
additional functionality and enable different types of assays to be
performed to detect and measure CRISPR activity in vivo. One
example of such a histochemical assay is visualization of in situ
beta-galactosidase expression histochemically through hydrolysis of
X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside), which
yields a blue precipitate, or using fluorogenic substrates such as
beta-methyl umbelliferyl galactoside (MUG) and fluorescein
digalactoside (FDG).
[0109] When two or more reporter proteins are included in the
CRISPR reporter, the coding sequence for the two or more reporter
proteins can comprise a multicistronic nucleic acid. Multicistronic
expression constructs simultaneously express two or more separate
proteins from the same mRNA (i.e., a transcript produced from the
same promoter). Suitable strategies for multicistronic expression
of proteins include, for example, the use of a 2A peptide and the
use of an internal ribosome entry site (IRES). For example, such
nucleic acids can comprise coding sequences for two or more
reporter proteins separated by an intervening internal ribosome
entry site (IRES) or an intervening 2A peptide coding sequence. As
one example, such multicistronic vectors can use one or more
internal ribosome entry sites (IRES) to allow for initiation of
translation from an internal region of an mRNA. As another example,
such multicistronic vectors can use one or more 2A peptides. These
peptides are small "self-cleaving" peptides, generally having a
length of 18-22 amino acids and produce equimolar levels of
multiple genes from the same mRNA. Ribosomes skip the synthesis of
a glycyl-prolyl peptide bond at the C-terminus of a 2A peptide,
leading to the "cleavage" between a 2A peptide and its immediate
downstream peptide. See, e.g., Kim et al. (2011) PLoS One 6(4):
e18556, herein incorporated by reference in its entirety for all
purposes. The "cleavage" occurs between the glycine and proline
residues found on the C-terminus, meaning the upstream cistron will
have a few additional residues added to the end, while the
downstream cistron will start with the proline. As a result, the
"cleaved-off" downstream peptide has proline at its N-terminus.
2A-mediated cleavage is a universal phenomenon in all eukaryotic
cells. 2A peptides have been identified from picornaviruses, insect
viruses and type C rotaviruses. See, e.g., Szymczak et al. (2005)
Expert Opin Biol Ther 5:627-638, herein incorporated by reference
in its entirety for all purposes. Examples of 2A peptides that can
be used include Thosea asigna virus 2A (T2A); porcine teschovirus-1
2A (P2A); equine rhinitis A virus (ERAV) 2A (E2A); and FMDV 2A
(F2A). Exemplary T2A, P2A, E2A, and F2A sequences include the
following: T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 3); P2A
(ATNFSLLKQAGDVEENPGP; SEQ ID NO: 4); E2A (QCTNYALLKLAGDVESNPGP; SEQ
ID NO: 5); and F2A (VKQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 6). GSG
residues can be added to the 5' end of any of these peptides to
improve cleavage efficiency.
[0110] The CRISPR reporter can be operably linked to any suitable
promoter for expression in vivo within a non-human animal. The
non-human animal can be any suitable non-human animal as described
elsewhere herein. As one example, the CRISPR reporter can be
operably linked to an endogenous promoter at a target genomic
locus, such as a Rosa26 promoter at an endogenous Rosa26 locus.
Alternatively, the CRISPR reporter can be operably linked to an
exogenous promoter. The promoter can be, for example, a
constitutively active promoter, a conditional promoter, an
inducible promoter, a temporally restricted promoter (e.g., a
developmentally regulated promoter), or a spatially restricted
promoter (e.g., a cell-specific or tissue-specific promoter). Such
promoters are well-known and are discussed elsewhere herein.
[0111] The CRISPR reporters disclosed herein can comprise other
components as well. Some CRISPR reporters further comprise a 3'
splicing sequence at the 5' end of the CRISPR reporter and/or a
second polyadenylation signal following the coding sequences for
the reporter proteins at the 3' end of the CRISPR reporter. Some
CRISPR reporters can further comprise a selection cassette
comprising, for example, the coding sequence for a drug resistance
protein. Alternatively, some CRISPR reporters disclosed herein do
not comprise a selection cassette. Examples of suitable selection
markers include neomycin phosphotransferase (neon), hygromycin B
phosphotransferase (hyg.sub.r), puromycin-N-acetyltransferase
(puro.sub.r), blasticidin S deaminase (bsr.sub.r), xanthine/guanine
phosphoribosyl transferase (gpt), and herpes simplex virus
thymidine kinase (HSV-k). Optionally, the selection cassette can be
flanked by recombinase recognition sites for a site-specific
recombinase. If the CRISPR reporter also comprises recombinase
recognition sites flanking the polyadenylation signal for use as a
positive control as described above, optionally a different set of
recombinase recognition sites recognized by a different recombinase
are used to flank the selection cassette.
[0112] CRISPR reporters can also comprise a transcription
terminator or polyadenylation signal in the reporter cassette
(i.e., following or downstream of the coding sequence for the one
or more reporter proteins). In some CRISPR reporters, the
transcription terminator or polyadenylation signal flanked by the
first and second guide RNA target sequences upstream of the
reporter cassette comprising the coding sequence for the one or
more reporter proteins is different from the transcription
terminator or polyadenylation signal following the coding sequence
for the one or more reporter proteins. In other CRISPR reporters,
the transcription terminator or polyadenylation signal flanked by
the first and second guide RNA target sequences upstream of the
reporter cassette comprising the coding sequence for the one or
more reporter proteins is the same as the transcription terminator
or polyadenylation signal following the coding sequence for the one
or more reporter proteins.
[0113] Alternatively or in addition to the CRISPR reporter elements
described above, the CRISPR reporters provided herein can comprise
a guide RNA target sequence and a coding sequence for a reporter
protein that can be converted to the coding sequence for a
different reporter protein. Upon cleavage of the guide RNA target
sequence by a CRISPR/Cas nuclease and repair of the reporter
protein coding sequence with an exogenous donor nucleic acid, the
coding sequence for the reporter protein is converted to the coding
sequence for a different reporter protein. Optionally, the coding
sequence for original reporter protein can be changed into a coding
sequence for the different reporter protein by changing a single
codon. For example, some such CRISPR reporters do not comprise a
polyadenylation signal or transcription terminator upstream of the
coding sequence for the reporter protein. Other such CRISPR
reporters do comprise a polyadenylation signal or transcription
terminator upstream of the coding sequence for the reporter protein
as described above. Some such CRISPR reporters comprise a single
reporter protein coding sequence. See, e.g., FIG. 3 and SEQ ID NOS:
18, 57, and 60. Other such CRISPR reporters comprise coding
sequences for two or more reporter proteins as described above.
See, e.g., FIG. 1A and SEQ ID NOS: 17, 58, and 59. Some such CRISPR
reporters can further comprise a selection cassette comprising, for
example, the coding sequence for a drug resistance protein.
Alternatively, some such CRISPR reporters do not comprise a
selection cassette.
[0114] Any suitable guide RNA target sequence can be used as
described elsewhere herein. As one example, the guide RNA target
sequence can comprise the sequence set forth in SEQ ID NO: 42 or
56. The guide RNA sequence can be within the coding sequence for
the reporter protein, optionally within a defined distance from the
region of the coding sequence to be altered upon recombination with
the exogenous donor sequence to convert the coding sequence to a
coding sequence for a different reporter protein. Alternatively,
the guide RNA sequence can be outside of and adjacent to the coding
sequence for the reporter protein. For example, the guide RNA
target sequence can be within 1, 5, 10, 50, 100, 200, 300, 400,
500, or 1000 base pairs (bp) or between about 1-100, 1-200, 1-300,
1-400, 1-500, 1-1000, 5-1000, 10-5000, 50-1000, 100-1000, 200-1000,
300-1000, 400-1000, or 500-1000 bp from the 5' or 3' end of the
coding sequence for the reporter protein or from the region of the
coding sequence to be altered. In a specific example, the guide RNA
target sequence can be within about 1000 or within about 500 base
pairs of the region to be altered or can overlap with the region to
be altered. Alternatively, the guide RNA target sequence can be
within about 1, 2, 3, 4, 5, or 10 kb, or between about 1-2, 1-3,
1-4, 1-5, or 1-10 kb from the 5' or 3' end of the coding sequence
for the reporter protein or from the region of the coding sequence
to be altered. For example, the guide RNA target sequence can be
between about 1 bp to 1 kb, 1 bp to 2 kb, 1 bp to 3 kb, 1 bp to 4
kb, 1 bp to 5 kb, or 1 bp to 10 kb from the 5' or 3' end of the
coding sequence for the reporter protein or the region of the
coding sequence to be altered.
[0115] Any suitable reporter protein as described elsewhere herein,
such as a fluorescent reporter protein, can be used. In a specific
example, the reporter protein can be converted to a different
reporter protein through changing a single codon. For example,
enhanced GFP (eGFP) emits green fluorescence, but a single amino
acid substitution of histidine to tyrosine at position 66 (Y66H)
results in a spectral shift to blue fluorescence (i.e., conversion
from eGFP to eBFP). Likewise, the reverse substitution in eBFP
(H66Y) can convert eBFP to eGFP. Other examples of mutations in
eGFP that can result in spectral shifts include T203Y (yellow
derivatives) and Y66W (cyan derivatives). Yet other examples of
mutations in eGFP can convert eGFP to BFP (e.g., mutating the LTYG
at positions 64-67 of eGFP to FX). In one specific example, the
original reporter protein is GFP or eGFP and is converted into BFP
or eBFP, or vice versa. In another specific example, the original
reporter protein is GFP or eGFP and is converted into CFP or eCFP,
or vice versa. In another specific example, the original reporter
protein is GFP or eGFP and is converted into YFP or eYFP, or vice
versa.
[0116] One exemplary CRISPR reporter comprises from 5' to 3': (a) a
3' splicing sequence; (b) a first polyadenylation signal flanked
by: (i) first and second recombinase recognition sites for a first
recombinase (e.g., loxP sites for a Cre recombinase); and (ii)
first and second guide RNA target sequences (e.g., guide RNA target
sequences each comprising SEQ ID NO: 41); (c) a reporter cassette,
comprising from 5' to 3': (i) a beta-galactosidase coding sequence;
(ii) a P2A coding sequence; (iii) a fluorescent protein (e.g.,
green fluorescent protein (GFP), blue fluorescent protein (BFP),
enhanced GFP (eGFP), or enhanced BFP (eBFP)) coding sequence
(optionally comprising a third guide RNA target sequence); and (iv)
a second polyadenylation signal. Optionally, the CRISPR reporter
comprises guide RNA target sequences comprising SEQ ID NOS: 41, 43,
44, 45, 46, and 47. The first and second guide RNA target sequences
can comprise, for example, SEQ ID NOS: 43 and 46, SEQ ID NOS: 43
and 47, SEQ ID NOS: 44 and 46, SEQ ID NOS: 44 and 47, SEQ ID NOS:
45 and 46, SEQ ID NOS: 45 and 47, SEQ ID NOS: 43 and 41, SEQ ID
NOS: 44 and 41, SEQ ID NOS: 45 and 41, SEQ ID NOS: 41 and 46, or
SEQ ID NOS: 41 and 47. Alternatively, each can comprise SEQ ID NO:
41. Optionally, the CRISPR reporter further comprises one or more
guide RNA target sequences within the first polyadenylation signal.
Examples of such guide RNA target sequences include SEQ ID NOS:
48-52. Optionally, the first polyadenylation signal and the second
polyadenylation signal are different. Optionally, the CRISPR
reporter can further comprise a selection cassette (e.g., 3' of the
reporter cassette), wherein the selection cassette is flanked by
recombinase recognition sites for a second recombinase (e.g., FRT
sites for a Flp recombinase). The selection cassette can comprise,
for example, from 5' to 3': (i) a third polyadenylation signal
(e.g., in antisense orientation); and (ii) the coding sequence for
a drug resistance gene (e.g., in antisense orientation) operably
linked to a promoter (e.g., a neomycin resistance gene (neomycin
phosphotransferase) coding sequence operably linked to a
constitutive promoter such as a human ubiquitin promoter).
Alternatively, the selection cassette can be flanked by recombinase
recognition sites for a second recombinase (e.g., FRT sites for a
Flp recombinase) and comprises from 5' to 3': (i) the coding
sequence for a drug resistance gene (e.g., in sense orientation)
operably linked to a promoter (e.g., a neomycin resistance gene
(neomycin phosphotransferase) coding sequence operably linked to a
constitutive promoter such as a human ubiquitin promoter and an EM7
promoter); and (ii) a third polyadenylation signal (e.g., Pgk
polyadenylation signal in sense orientation). See, e.g., FIG. 1A
and SEQ ID NO: 17. Optionally, the CRISPR reporter can be the
CRISPR reporter comprising the selection cassette following
treatment with the recombinase and excision of the selection
cassette. See, e.g., FIG. 1A and SEQ ID NO: 17 following treatment
with Flp recombinase and excision of the neomycin selection
cassette. Optionally, the CRISPR reporter can be the CRISPR
reporter (with or without the selection cassette) following
treatment with a recombinase and excision of the first
polyadenylation signal. See, e.g., FIG. 1A and SEQ ID NO: 17
following treatment with Cre recombinase and excision of the Pgk
polyadenylation signal, resulting in SEQ ID NO: 58 (or SEQ ID NO:
59 upon conversion from eBFP to eGFP).
[0117] Another exemplary CRISPR reporter comprises from 5' to 3':
(a) a 3' splicing sequence; (b) a first polyadenylation signal
comprising a guide RNA target sequence (e.g., SEQ ID NO: 48, 49,
50, 51, or 52) flanked by first and second recombinase recognition
sites for a first recombinase (e.g., loxP sites for a Cre
recombinase); (c) a reporter cassette, comprising from 5' to 3':
(i) a beta-galactosidase coding sequence; (ii) a P2A coding
sequence; (iii) a fluorescent protein (e.g., green fluorescent
protein (GFP), blue fluorescent protein (BFP), enhanced GFP (eGFP),
or enhanced BFP (eBFP)) coding sequence (optionally comprising a
second guide RNA target sequence); and (iv) a second
polyadenylation signal. Optionally, the first polyadenylation
signal and the second polyadenylation signal are different.
Optionally, the CRISPR reporter can further comprise a selection
cassette (e.g., 3' of the reporter cassette), wherein the selection
cassette is flanked by recombinase recognition sites for a second
recombinase (e.g., FRT sites for a Flp recombinase). The selection
cassette can comprise, for example, from 5' to 3': (i) a third
polyadenylation signal (e.g., in antisense orientation); and (ii)
the coding sequence for a drug resistance gene (e.g., in antisense
orientation) operably linked to a promoter (e.g., a neomycin
resistance gene (neomycin phosphotransferase) coding sequence
operably linked to a constitutive promoter such as a human
ubiquitin promoter). Alternatively, the selection cassette can be
flanked by recombinase recognition sites for a second recombinase
(e.g., FRT sites for a Flp recombinase) and comprises from 5' to
3': (i) the coding sequence for a drug resistance gene (e.g., in
sense orientation) operably linked to a promoter (e.g., a neomycin
resistance gene (neomycin phosphotransferase) coding sequence
operably linked to a constitutive promoter such as a human
ubiquitin promoter and an EM7 promoter); and (ii) a third
polyadenylation signal (e.g., Pgk polyadenylation signal in sense
orientation). See, e.g., FIG. 1A and SEQ ID NO: 17. Optionally, the
CRISPR reporter can be the CRISPR reporter comprising the selection
cassette following treatment with the recombinase and excision of
the selection cassette. See, e.g., FIG. 1A and SEQ ID NO: 17
following treatment with Flp recombinase and excision of the
neomycin selection cassette. Optionally, the CRISPR reporter can be
the CRISPR reporter (with or without the selection cassette)
following treatment with a recombinase and excision of the first
polyadenylation signal. See, e.g., FIG. 1A and SEQ ID NO: 17
following treatment with Cre recombinase and excision of the Pgk
polyadenylation signal, resulting in SEQ ID NO: 58 (or SEQ ID NO:
59 upon conversion from eBFP to eGFP).
[0118] An exemplary CRISPR reporter for testing only
CRISPR/Cas-induced recombination comprises from 5' to 3': (a) a 3'
splicing sequence; (b) a first recombinase recognition site (e.g.,
loxP site for Cre recombinase): (c) a first polyadenylation signal
(e.g., a triple polyadenylation signal); (d) a drug resistance gene
operably linked to a promoter (e.g., a neomycin resistance gene
operably linked to an EM7 promoter); (e) a second recombinase
recognition site; (f) a fluorescent protein coding sequence (e.g.,
green fluorescent protein (GFP), blue fluorescent protein (BFP),
enhanced GFP (eGFP), or enhanced BFP (eBFP)) comprising a guide RNA
target sequence; and (g) a second polyadenylation signal (e.g., a
SV40 polyadenylation signal). See, e.g., FIG. 3 and SEQ ID NO: 18.
Optionally, the CRISPR reporter can be the CRISPR reporter
following treatment with the recombinase and excision of the
selection cassette and first polyadenylation signal. See, e.g.,
FIG. 3 and SEQ ID NO: 18 following treatment with Cre recombinase
and excision of the neomycin selection cassette and triple
polyadenylation signal, resulting in SEQ ID NO: 57 (or SEQ ID NO:
60 upon conversion from eBFP to eGFP).
[0119] The CRISPR reporters described herein can be in any form.
For example, a CRISPR reporter can be in a plasmid or vector, such
as a viral vector. Likewise, the CRISPR reporter can be operably
linked to a promoter in an expression construct capable of
directing expression of the reporter proteins upon removal of the
upstream polyadenylation signal. Likewise, a CRISPR reporter can be
in a targeting vector as defined elsewhere herein. For example, the
targeting vector can comprise homology arms flanking the CRISPR
reporter, wherein the homology arms are suitable for directing
recombination with a desired target genomic locus to facilitate
genomic integration.
[0120] Likewise, the CRISPR reporters described herein can be in
vitro, they can be within a cell (e.g., an embryonic stem cell) ex
vivo (e.g., genomically integrated or extrachromosomal), or they
can be in an organism (e.g., a non-human animal) in vivo (e.g.,
genomically integrated or extrachromosomal). If ex vivo, the CRISPR
reporter can be in any type of cell from any organism, such as a
totipotent cell such as an embryonic stem cell (e.g., a mouse or a
rat embryonic stem cell) or an induced pluripotent stem cell (e.g.,
a human induced pluripotent stem cell). If in vivo, the CRISPR
reporter can be in any type of organism (e.g., a non-human animal
as described further below).
[0121] B. Cells and Non-Human Animals Comprising CRISPR
Reporters
[0122] Cells and non-human animals comprising the CRISPR reporters
described herein are also provided. The CRISPR reporter can be
stably integrated into the genome (i.e., into a chromosome) of the
cell or non-human animal or it can be located outside of a
chromosome (e.g., extrachromosomally replicating DNA). Optionally,
the CRISPR reporter is stably integrated into the genome. The
stably integrated CRISPR reporter can be randomly integrated into
the genome of the non-human animal (i.e., transgenic), or it can be
integrated into a predetermined region of the genome of the
non-human animal (i.e., knock in). Optionally, the CRISPR reporter
is stably integrated into a predetermined region of the genome,
such as a safe harbor locus. The target genomic locus at which the
CRISPR reporter is stably integrated can be heterozygous for the
CRISPR reporter or homozygous for the CRISPR reporter. A diploid
organism has two alleles at each genetic locus. Each pair of
alleles represents the genotype of a specific genetic locus.
Genotypes are described as homozygous if there are two identical
alleles at a particular locus and as heterozygous if the two
alleles differ.
[0123] The cells provided herein can be, for example, eukaryotic
cells, which include, for example, fungal cells (e.g., yeast),
plant cells, animal cells, mammalian cells, non-human mammalian
cells, and human cells. The term "animal" includes mammals, fishes,
and birds. A mammalian cell can be, for example, a non-human
mammalian cell, a human cell, a rodent cell, a rat cell, a mouse
cell, or a hamster cell. Other non-human mammals include, for
example, non-human primates, monkeys, apes, cats, dogs, rabbits,
horses, bulls, deer, bison, livestock (e.g., bovine species such as
cows, steer, and so forth; ovine species such as sheep, goats, and
so forth; and porcine species such as pigs and boars). Birds
include, for example, chickens, turkeys, ostrich, geese, ducks, and
so forth. Domesticated animals and agricultural animals are also
included. The term "non-human" excludes humans.
[0124] The cells can also be any type of undifferentiated or
differentiated state. For example, a cell can be a totipotent cell,
a pluripotent cell (e.g., a human pluripotent cell or a non-human
pluripotent cell such as a mouse embryonic stem (ES) cell or a rat
ES cell), or a non-pluripotent cell. Totipotent cells include
undifferentiated cells that can give rise to any cell type, and
pluripotent cells include undifferentiated cells that possess the
ability to develop into more than one differentiated cell types.
Such pluripotent and/or totipotent cells can be, for example, ES
cells or ES-like cells, such as an induced pluripotent stem (iPS)
cells. ES cells include embryo-derived totipotent or pluripotent
cells that are capable of contributing to any tissue of the
developing embryo upon introduction into an embryo. ES cells can be
derived from the inner cell mass of a blastocyst and are capable of
differentiating into cells of any of the three vertebrate germ
layers (endoderm, ectoderm, and mesoderm).
[0125] Examples of human pluripotent cells include human ES cells,
human adult stem cells, developmentally restricted human progenitor
cells, and human induced pluripotent stem (iPS) cells, such as
primed human iPS cells and naive human iPS cells. Induced
pluripotent stem cells include pluripotent stem cells 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 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, e.g., Takahashi and Yamanaka (2006) Cell
126:663-676, herein incorporated by reference in its entirety for
all purposes. Primed human ES cells and primed human iPS cells
include cells that express characteristics similar to those of
post-implantation epiblast cells and are committed for lineage
specification and differentiation. Naive human ES cells and naive
human iPS cells include cells that express characteristics similar
to those of ES cells of the inner cell mass of a pre-implantation
embryo and are not committed for lineage specification. See, e.g.,
Nichols and Smith (2009) Cell Stem Cell 4:487-492, herein
incorporated by reference in its entirety for all purposes.
[0126] The cells provided herein can also be germ cells (e.g.,
sperm or oocytes). The cells can be mitotically competent cells or
mitotically-inactive cells, meiotically competent cells or
meiotically-inactive cells. Similarly, the cells can also be
primary somatic cells or cells that are not a primary somatic cell.
Somatic cells include any cell that is not a gamete, germ cell,
gametocyte, or undifferentiated stem cell. For example, the cells
can be liver cells, kidney cells, hematopoietic cells, endothelial
cells, epithelial cells, fibroblasts, mesenchymal cells,
keratinocytes, blood cells, melanocytes, monocytes, mononuclear
cells, monocytic precursors, B cells, erythroid-megakaryocytic
cells, eosinophils, macrophages, T cells, islet beta cells,
exocrine cells, pancreatic progenitors, endocrine progenitors,
adipocytes, preadipocytes, neurons, glial cells, neural stem cells,
neurons, hepatoblasts, hepatocytes, cardiomyocytes, skeletal
myoblasts, smooth muscle cells, ductal cells, acinar cells, alpha
cells, beta cells, delta cells, PP cells, cholangiocytes, white or
brown adipocytes, or ocular cells (e.g., trabecular meshwork cells,
retinal pigment epithelial cells, retinal microvascular endothelial
cells, retinal pericyte cells, conjunctival epithelial cells,
conjunctival fibroblasts, iris pigment epithelial cells,
keratocytes, lens epithelial cells, non-pigment ciliary epithelial
cells, ocular choroid fibroblasts, photoreceptor cells, ganglion
cells, bipolar cells, horizontal cells, or amacrine cells).
[0127] Suitable cells provided herein also include primary cells.
Primary cells include cells or cultures of cells that have been
isolated directly from an organism, organ, or tissue. Primary cells
include cells that are neither transformed nor immortal. They
include any cell obtained from an organism, organ, or tissue which
was not previously passed in tissue culture or has been previously
passed in tissue culture but is incapable of being indefinitely
passed in tissue culture. Such cells can be isolated by
conventional techniques and include, for example, somatic cells,
hematopoietic cells, endothelial cells, epithelial cells,
fibroblasts, mesenchymal cells, keratinocytes, melanocytes,
monocytes, mononuclear cells, adipocytes, preadipocytes, neurons,
glial cells, hepatocytes, skeletal myoblasts, and smooth muscle
cells. For example, primary cells can be derived from connective
tissues, muscle tissues, nervous system tissues, or epithelial
tissues.
[0128] Other suitable cells provided herein include immortalized
cells. Immortalized cells include cells from a multicellular
organism that would normally not proliferate indefinitely but, due
to mutation or alteration, have evaded normal cellular senescence
and instead can keep undergoing division. Such mutations or
alterations can occur naturally or be intentionally induced.
Examples of immortalized cells include Chinese hamster ovary (CHO)
cells, human embryonic kidney cells (e.g., HEK 293 cells or 293T
cells), and mouse embryonic fibroblast cells (e.g., 3T3 cells).
Numerous types of immortalized cells are well known. Immortalized
or primary cells include cells that are typically used for
culturing or for expressing recombinant genes or proteins.
[0129] The cells provided herein also include one-cell stage
embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage
embryos can be from any genetic background (e.g., BALB/c, C57BL/6,
129, or a combination thereof for mice), can be fresh or frozen,
and can be derived from natural breeding or in vitro
fertilization.
[0130] The cells provided herein can be normal, healthy cells, or
can be diseased or mutant-bearing cells.
[0131] Non-human animals comprising a CRISPR reporter as described
herein can be made by the methods described elsewhere herein. The
term "animal" includes mammals, fishes, and birds. Mammals include,
for example, humans, non-human primates, monkeys, apes, cats, dogs,
horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice,
rats, hamsters, and guinea pigs), and livestock (e.g., bovine
species such as cows and steer; ovine species such as sheep and
goats; and porcine species such as pigs and boars). Birds include,
for example, chickens, turkeys, ostrich, geese, and ducks.
Domesticated animals and agricultural animals are also included.
The term "non-human animal" excludes humans. Preferred non-human
animals include, for example, rodents, such as mice and rats.
[0132] The non-human animals can be from any genetic background.
For example, suitable mice can be from a 129 strain, a C57BL/6
strain, a mix of 129 and C57BL/6, a BALB/c strain, or a Swiss
Webster strain. Examples of 129 strains include 129P1, 129P2,
129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4,
129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, and
129T2. See, e.g., Festing et al. (1999) Mammalian Genome 10:836,
herein incorporated by reference in its entirety for all purposes.
Examples of C57BL strains include C57BL/A, C57BL/An, C57BL/GrFa,
C57BL/Kal_wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,
C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. Suitable mice can also be
from a mix of an aforementioned 129 strain and an aforementioned
C57BL/6 strain (e.g., 50% 129 and 50% C57BL/6). Likewise, suitable
mice can be from a mix of aforementioned 129 strains or a mix of
aforementioned BL/6 strains (e.g., the 129S6 (129/SvEvTac)
strain).
[0133] Similarly, rats can be from any rat strain, including, for
example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar
rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or
a Fischer rat strain such as Fisher F344 or Fisher F6. Rats can
also be obtained from a strain derived from a mix of two or more
strains recited above. For example, a suitable rat can be from a DA
strain or an ACI strain. The ACI rat strain is characterized as
having black agouti, with white belly and feet and an RT1.sup.av1
haplotype. Such strains are available from a variety of sources
including Harlan Laboratories. The Dark Agouti (DA) rat strain is
characterized as having an agouti coat and an RT1.sup.av1
haplotype. Such rats are available from a variety of sources
including Charles River and Harlan Laboratories. In some cases,
suitable rats can be from an inbred rat strain. See, e.g., US
2014/0235933, herein incorporated by reference in its entirety for
all purposes.
[0134] C. Target Genomic Loci
[0135] The CRISPR reporters described herein can be genomically
integrated at a target genomic locus in a cell or a non-human
animal. Any target genomic locus capable of expressing a gene can
be used.
[0136] An example of a target genomic locus into which the CRISPR
reporters described herein can be stably integrated is a safe
harbor locus in the genome of the non-human animal. 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 (i.e., without any
deleterious effects on the host cell). See, e.g., Sadelain et al.
(2012) Nat. Rev. Cancer 12:51-58, herein incorporated by reference
in its entirety for all purposes. Optionally, the safe harbor locus
is one in which expression of the inserted gene sequence is not
perturbed by any read-through expression from neighboring genes.
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.
[0137] 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, e.g., Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. USA
94:3789-3794, herein incorporated by reference in its entirety for
all purposes. In addition, the Rosa26 locus can be targeted with
high efficiency, and disruption of the Rosa26 gene produces no
overt phenotype. Other examples of safe harbor loci include CCR5,
HPRT, AAVS1, and albumin. See, e.g., U.S. Pat. Nos. 7,888,121;
7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526;
and US Patent Publication Nos. 2003/0232410; 2005/0208489;
2005/0026157; 2006/0063231; 2008/0159996; 2010/00218264;
2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591;
2013/0177983; 2013/0177960; and 2013/0122591, each of which is
herein incorporated by reference in its entirety for all purposes.
Biallelic targeting of safe harbor loci such as the Rosa26 locus
has no negative consequences, so different genes or reporters can
be targeted to the two Rosa26 alleles. In one example, a CRISPR
reporter is integrated into an intron of the Rosa26 locus, such as
the first intron of the Rosa26 locus.
[0138] D. CRISPR/Cas Systems
[0139] 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, for example, a type I, a type
II, or a type III system. Alternatively, a CRISPR/Cas system can be
a type V system (e.g., subtype V-A or subtype V-B). CRISPR/Cas
systems used in the compositions and methods disclosed herein can
be 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, non-naturally occurring CRISPR/Cas systems
can employ CRISPR complexes comprising a gRNA and a Cas protein
that do not naturally occur together, a Cas protein that does not
occur naturally, or a gRNA that does not occur naturally.
[0140] (1) Cas Proteins and Polynucleotides Encoding Cas
Proteins
[0141] Cas proteins generally comprise at least one RNA recognition
or binding domain that 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. Some such domains (e.g.,
DNase domains) can be from a native Cas protein. Other such domains
can be added to make a modified Cas protein. A nuclease domain
possesses catalytic activity for nucleic acid cleavage, which
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. For example, a wild type
Cas9 protein will typically create a blunt cleavage product.
Alternatively, a wild type Cpf1 protein (e.g., FnCpf1) can result
in a cleavage product with a 5-nucleotide 5' overhang, with the
cleavage occurring after the 18th base pair from the PAM sequence
on the non-targeted strand and after the 23rd base on the targeted
strand. A Cas protein can have full cleavage activity to create a
double-strand break at a target genomic locus (e.g., a
double-strand break with blunt ends), or it can be a nickase that
creates a single-strand break at a target genomic locus.
[0142] 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 Cu1966,
and homologs or modified versions thereof.
[0143] An exemplary Cas protein is a Cas9 protein or a protein
derived from Cas9. Cas9 proteins are from a type II CRISPR/Cas
system and 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. Exemplary Cas9 proteins are from Streptococcus
pyogenes, Streptococcus thermophilus, Streptococcus sp.,
Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces
pristinaespiralis, Streptomyces viridochromogenes, Streptomyces
viridochromogenes, Streptosporangium roseum, Streptosporangium
roseum, Alicyclobacillus 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, Acaryochloris marina, Neisseria meningitidis, or
Campylobacter jejuni. Additional examples of the Cas9 family
members are described in WO 2014/131833, herein incorporated by
reference in its entirety for all purposes. Cas9 from S. pyogenes
(SpCas9) (assigned SwissProt accession number Q99ZW2) is an
exemplary Cas9 protein. Cas9 from S. aureus (SaCas9) (assigned
UniProt accession number J7RUA5) is another exemplary Cas9 protein.
Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProt accession
number QOP897) is another exemplary Cas9 protein. See, e.g., Kim et
al. (2017) Nat. Comm. 8:14500, herein incorporated by reference in
its entirety for all purposes. SaCas9 is smaller than SpCas9, and
CjCas9 is smaller than both SaCas9 and SpCas9. An exemplary Cas9
protein is set forth in SEQ ID NO: 53 (encoded by SEQ ID NO:
54).
[0144] Another example of a Cas protein is a Cpf1 (CRISPR from
Prevotella and Francisella 1) protein. Cpf1 is a large protein
(about 1300 amino acids) that contains a RuvC-like nuclease domain
homologous to the corresponding domain of Cas9 along with a
counterpart to the characteristic arginine-rich cluster of Cas9.
However, Cpf1 lacks the HNH nuclease domain that is present in Cas9
proteins, and the RuvC-like domain is contiguous in the Cpf1
sequence, in contrast to Cas9 where it contains long inserts
including the HNH domain. See, e.g., Zetsche et al. (2015) Cell
163(3):759-771, herein incorporated by reference in its entirety
for all purposes. Exemplary Cpf1 proteins are from Francisella
tularensis 1, Francisella tularensis subsp. novicida, Prevotella
albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio
proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2_33_10,
Parcubacteria bacterium GW2011 GWC2_44_17, Smithella sp. SCADC,
Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,
Candidatus methanoplasma termitum, Eubacterium eligens, Moraxella
bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006,
Porphyromonas crevioricanis 3, Prevotella disiens, and
Porphyromonas macacae. Cpf1 from Francisella novicida U112 (FnCpf1;
assigned UniProt accession number A0Q7Q2) is an exemplary Cpf1
protein.
[0145] 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 with respect to
catalytic activity of wild type or modified Cas proteins. Active
variants or fragments with respect to catalytic activity 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.
[0146] Cas proteins can be modified to increase or decrease one or
more of nucleic acid binding affinity, nucleic acid binding
specificity, and 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 or a property of the Cas protein.
[0147] One example of a modified Cas protein is the modified
SpCas9-HF1 protein, which is a high-fidelity variant of
Streptococcus pyogenes Cas9 harboring alterations
(N497A/R661A/Q695A/Q926A) designed to reduce non-specific DNA
contacts. See, e.g., Kleinstiver et al. (2016) Nature
529(7587):490-495, herein incorporated by reference in its entirety
for all purposes. Another example of a modified Cas protein is the
modified eSpCas9 variant (K848A/K1003A/R1060A) designed to reduce
off-target effects. See, e.g., Slaymaker et al. (2016) Science
351(6268):84-88, herein incorporated by reference in its entirety
for all purposes. Other SpCas9 variants include K855A and
K810A/K1003A/R1060A.
[0148] Cas proteins can comprise at least one nuclease domain, such
as a DNase domain. For example, a wild type Cpf1 protein generally
comprises a RuvC-like domain that cleaves both strands of target
DNA, perhaps in a dimeric configuration. Cas proteins can also
comprise at least two nuclease domains, such as DNase domains. For
example, a wild type Cas9 protein generally comprises 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, herein incorporated by reference in its
entirety for all purposes.
[0149] One or more of the nuclease domains can be deleted or
mutated so that they are no longer functional or have reduced
nuclease activity. For example, if one of the nuclease domains is
deleted or mutated in a Cas9 protein, the resulting Cas9 protein
can be referred to as a nickase and can generate a single-strand
break at a guide RNA 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). 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), H840A (histidine
to alanine at amino acid position 840), or N863A (asparagine to
alanine at amino acid position N863) 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 for all purposes. Such mutations can be generated
using methods such as site-directed mutagenesis, PCR-mediated
mutagenesis, or total gene synthesis. Examples of other mutations
creating nickases can be found, for example, in WO 2013/176772 and
WO 2013/142578, each of which is herein incorporated by reference
in its entirety for all purposes.
[0150] Examples of inactivating mutations in the catalytic domains
of Staphylococcus aureus Cas9 proteins are also known. For example,
the Staphylococcus aureus Cas9 enzyme (SaCas9) may comprise a
substitution at position N580 (e.g., N580A substitution) and a
substitution at position D10 (e.g., D10A substitution) to generate
a nuclease-inactive Cas protein. See, e.g., WO 2016/106236, herein
incorporated by reference in its entirety for all purposes.
[0151] Examples of inactivating mutations in the catalytic domains
of Cpf1 proteins are also known. With reference to Cpf1 proteins
from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6
(AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella
bovoculi 237 (MbCpf1 Cpf1), such mutations can include mutations at
positions 908, 993, or 1263 of AsCpf1 or corresponding positions in
Cpf1 orthologs, or positions 832, 925, 947, or 1180 of LbCpf1 or
corresponding positions in Cpf1 orthologs. Such mutations can
include, for example one or more of mutations D908A, E993A, and
D1263A of AsCpf1 or corresponding mutations in Cpf1 orthologs, or
D832A, E925A, D947A, and D1180A of LbCpf1 or corresponding
mutations in Cpf1 orthologs. See, e.g., US 2016/0208243, herein
incorporated by reference in its entirety for all purposes.
[0152] Cas proteins can also be operably linked to heterologous
polypeptides as fusion proteins. For example, a Cas protein can be
fused to a cleavage domain or an epigenetic modification domain.
See WO 2014/089290, herein incorporated by reference in its
entirety for all purposes. 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.
[0153] As one example, a Cas protein can be fused to one or more
heterologous polypeptides that provide for subcellular
localization. Such heterologous polypeptides can include, for
example, one or more nuclear localization signals (NLS) such as the
monopartite SV40 NLS and/or a bipartite alpha-importin 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,
herein incorporated by reference in its entirety for all purposes.
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. Optionally, the Cas
protein can comprise two or more NLSs, including an NLS (e.g., an
alpha-importin NLS or a monopartite NLS) at the N-terminus and an
NLS (e.g., an SV40 NLS or a bipartite NLS) at the C-terminus. A Cas
protein can also comprise two or more NLSs at the N-terminus and/or
two or more NLSs at the C-terminus.
[0154] Cas proteins can also be operably linked to a
cell-penetrating domain or protein transduction 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, e.g., WO
2014/089290 and WO 2013/176772, each of which is herein
incorporated by reference in its entirety for all purposes. The
cell-penetrating domain can be located at the N-terminus, the
C-terminus, or anywhere within the Cas protein.
[0155] Cas proteins can also be operably linked to 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, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent
proteins (e.g., eCFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan),
red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed
monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer,
HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry,
Jred), orange fluorescent proteins (e.g., 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.
[0156] Cas proteins can also be tethered to exogenous donor nucleic
acids or labeled nucleic acids. Such tethering (i.e., physical
linking) can be achieved through covalent interactions or
noncovalent interactions, and the tethering can be direct (e.g.,
through direct fusion or chemical conjugation, which can be
achieved by modification of cysteine or lysine residues on the
protein or intein modification), or can be achieved through one or
more intervening linkers or adapter molecules such as streptavidin
or aptamers. See, e.g., Pierce et al. (2005) Mini Rev. Med. Chem.
5(1):41-55; Duckworth et al. (2007) Angew. Chem. Int. Ed. Engl.
46(46):8819-8822; Schaeffer and Dixon (2009) Australian J. Chem.
62(10):1328-1332; Goodman et al. (2009) Chembiochem.
10(9):1551-1557; and Khatwani et al. (2012) Bioorg. Med. Chem.
20(14):4532-4539, each of which is herein incorporated by reference
in its entirety for all purposes. Noncovalent strategies for
synthesizing protein-nucleic acid conjugates include
biotin-streptavidin and nickel-histidine methods. Covalent
protein-nucleic acid conjugates can be synthesized by connecting
appropriately functionalized nucleic acids and proteins using a
wide variety of chemistries. Some of these chemistries involve
direct attachment of the oligonucleotide to an amino acid residue
on the protein surface (e.g., a lysine amine or a cysteine thiol),
while other more complex schemes require post-translational
modification of the protein or the involvement of a catalytic or
reactive protein domain. Methods for covalent attachment of
proteins to nucleic acids can include, for example, chemical
cross-linking of oligonucleotides to protein lysine or cysteine
residues, expressed protein-ligation, chemoenzymatic methods, and
the use of photoaptamers. The exogenous donor nucleic acid or
labeled nucleic acid can be tethered to the C-terminus, the
N-terminus, or to an internal region within the Cas protein.
Optionally, the exogenous donor nucleic acid or labeled nucleic
acid is tethered to the C-terminus or the N-terminus of the Cas9
protein. Likewise, the Cas protein can be tethered to the 5' end,
the 3' end, or to an internal region within the exogenous donor
nucleic acid or labeled nucleic acid. That is, the exogenous donor
nucleic acid or labeled nucleic acid can be tethered in any
orientation and polarity. Optionally, the Cas protein is tethered
to the 5' end or the 3' end of the exogenous donor nucleic acid or
labeled nucleic acid.
[0157] 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 can be codon optimized
for efficient translation into protein in a particular cell or
organism. For example, the nucleic acid encoding the Cas protein
can be modified to substitute codons having a higher frequency of
usage in a human cell, a non-human cell, a mammalian cell, a rodent
cell, a mouse cell, a rat cell, or any other host cell of interest,
as compared to the naturally occurring polynucleotide sequence.
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.
[0158] Cas proteins provided as mRNAs can be modified for improved
stability and/or immunogenicity properties. The modifications may
be made to one or more nucleosides within the mRNA. Examples of
chemical modifications to mRNA nucleobases include pseudouridine,
1-methyl-pseudouridine, and 5-methyl-cytidine. For example, capped
and polyadenylated Cas mRNA containing N1-methyl pseudouridine can
be used. Likewise, Cas mRNAs can be modified by depletion of
uridine using synonymous codons.
[0159] 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 vector comprising a DNA encoding a
gRNA. Alternatively, it can be in a vector or plasmid that is
separate from the vector comprising the DNA encoding the gRNA.
Promoters that can be used in an expression construct include
promoters active, for example, in one or more of a eukaryotic cell,
a human cell, a non-human cell, a mammalian cell, a non-human
mammalian cell, a rodent cell, a mouse cell, a rat cell, a
pluripotent cell, an embryonic stem (ES) cell, an adult stem cell,
a developmentally restricted progenitor cell, an induced
pluripotent stem (iPS) cell, or a one-cell stage embryo. Such
promoters can be, for example, conditional promoters, inducible
promoters, constitutive promoters, or tissue-specific promoters.
Optionally, the promoter can be a bidirectional promoter driving
expression of both a Cas protein in one direction and a guide RNA
in the other direction. Such bidirectional promoters can consist of
(1) a complete, conventional, unidirectional Pol III promoter that
contains 3 external control elements: a distal sequence element
(DSE), a proximal sequence element (PSE), and a TATA box; and (2) a
second basic Pol III promoter that includes a PSE and a TATA box
fused to the 5' terminus of the DSE in reverse orientation. For
example, in the H1 promoter, the DSE is adjacent to the PSE and the
TATA box, and the promoter can be rendered bidirectional by
creating a hybrid promoter in which transcription in the reverse
direction is controlled by appending a PSE and TATA box derived
from the U6 promoter. See, e.g., US 2016/0074535, herein
incorporated by references in its entirety for all purposes. Use of
a bidirectional promoter to express genes encoding a Cas protein
and a guide RNA simultaneously allow for the generation of compact
expression cassettes to facilitate delivery.
[0160] (2) Guide RNAs
[0161] A "guide RNA" or "gRNA" is an RNA molecule that binds to a
Cas protein (e.g., Cas9 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 section or region of a molecule,
such as a contiguous stretch of nucleotides in an RNA. Some gRNAs,
such as those for Cas9, can comprise two separate RNA molecules: an
"activator-RNA" (e.g., tracrRNA) and a "targeter-RNA" (e.g., CRISPR
RNA or crRNA). 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 2013/176772, WO
2014/065596, WO 2014/089290, WO 2014/093622, WO 2014/099750, WO
2013/142578, and WO 2014/131833, each of which is herein
incorporated by reference in its entirety for all purposes. For
Cas9, for example, a single-guide RNA can comprise a crRNA fused to
a tracrRNA (e.g., via a linker). For Cpf1, for example, only a
crRNA is needed to achieve binding to and/or cleavage of a target
sequence. The terms "guide RNA" and "gRNA" include both
double-molecule (i.e., modular) gRNAs and single-molecule
gRNAs.
[0162] 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") molecule. A crRNA comprises
both the DNA-targeting segment (single-stranded) of the gRNA and a
stretch of nucleotides (i.e., the crRNA tail) that forms one half
of the dsRNA duplex of the protein-binding segment of the gRNA. An
example of a crRNA tail, located downstream (3') of the
DNA-targeting segment, comprises, consists essentially of, or
consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 37). Any of the
DNA-targeting segments (i.e., guide sequences or guides) disclosed
herein (e.g., SEQ ID NO: 2, 14, 20, 22, 24, 26, 28, 29, 30, 31, 35,
36, or 55) can be joined to the 5' end of SEQ ID NO: 37 to form a
crRNA.
[0163] 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. An example of a tracrRNA sequence
comprises, consists essentially of, or consists of
TABLE-US-00002 (SEQ ID NO: 38)
AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG
CACCGAGUCGGUGCUUU.
[0164] In systems in which both a crRNA and a tracrRNA are needed,
the crRNA and the corresponding tracrRNA hybridize to form a gRNA.
In systems in which only a crRNA is needed, the crRNA can be the
gRNA. The crRNA additionally provides the single-stranded
DNA-targeting segment that targets a guide RNA target sequence by
hybridizing to the opposite strand (i.e., the complementary
strand). 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, e.g., 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 in its entirety for all purposes.
[0165] The DNA-targeting segment (crRNA) of a given gRNA comprises
a nucleotide sequence that is complementary to a sequence (i.e.,
the complementary strand of the guide RNA recognition sequence on
the strand opposite of the guide RNA target 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 CRISPR/Cas 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., WO 2014/131833, herein
incorporated by reference in its entirety for all purposes). 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 Cas protein.
[0166] The DNA-targeting segment can have a length of at least
about 12 nucleotides, at least about 15 nucleotides, at least about
17 nucleotides, at least about 18 nucleotides, at least about 19
nucleotides, at least about 20 nucleotides, at least about 25
nucleotides, at least about 30 nucleotides, at least about 35
nucleotides, or at least about 40 nucleotides. Such DNA-targeting
segments can have a length from about 12 nucleotides to about 100
nucleotides, from about 12 nucleotides to about 80 nucleotides,
from about 12 nucleotides to about 50 nucleotides, from about 12
nucleotides to about 40 nucleotides, from about 12 nucleotides to
about 30 nucleotides, from about 12 nucleotides to about 25
nucleotides, or from about 12 nucleotides to about 20 nucleotides.
For example, the DNA targeting segment can be from about 15
nucleotides to about 25 nucleotides (e.g., from about 17
nucleotides to about 20 nucleotides, or about 17 nucleotides, about
18 nucleotides, about 19 nucleotides, or about 20 nucleotides).
See, e.g., US 2016/0024523, herein incorporated by reference in its
entirety for all purposes. For Cas9 from S. pyogenes, a typical
DNA-targeting segment is between 16 and 20 nucleotides in length or
between 17 and 20 nucleotides in length. For Cas9 from S. aureus, a
typical DNA-targeting segment is between 21 and 23 nucleotides in
length. For Cpf1, a typical DNA-targeting segment is at least 16
nucleotides in length or at least 18 nucleotides in length.
[0167] 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, consist essentially of, 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,
e.g., Deltcheva et al. (2011) Nature 471:602-607; WO 2014/093661,
each of which is herein incorporated by reference in its entirety
for all purposes. 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, herein incorporated by reference in its
entirety for all purposes.
[0168] The percent complementarity between the DNA-targeting
segment and the complementary strand of the guide RNA recognition
sequence within the target DNA can be at least 60% (e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 97%, at least 98%, at least 99%,
or 100%). The percent complementarity between the DNA-targeting
segment and the complementary strand of the guide RNA recognition
sequence within the target DNA can be at least 60% over about 20
contiguous nucleotides. As an example, the percent complementarity
between the DNA-targeting segment and the complementary strand of
the guide RNA recognition sequence within the target DNA is 100%
over the 14 contiguous nucleotides at the 5' end of the
complementary strand of the guide RNA recognition sequence within
the complementary strand of the target DNA and as low as 0% over
the remainder. In such a case, the DNA-targeting segment can be
considered to be 14 nucleotides in length. As another example, the
percent complementarity between the DNA-targeting segment and the
complementary strand of the guide RNA recognition sequence within
the target DNA is 100% over the seven contiguous nucleotides at the
5' end of the complementary strand of the guide RNA recognition
sequence within the complementary strand of the target DNA and as
low as 0% over the remainder. In such a case, the DNA-targeting
segment can be considered to be 7 nucleotides in length. In some
guide RNAs, at least 17 nucleotides within the DNA-targeting
segment are complementary to the target DNA. For example, the
DNA-targeting segment can be 20 nucleotides in length and can
comprise 1, 2, or 3 mismatches with the complementary strand of the
guide RNA recognition sequence. Optionally, the mismatches are not
adjacent to a protospacer adjacent motif (PAM) sequence (e.g., the
mismatches are in the 5' end of the DNA-targeting segment, or the
mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, or 19 base pairs away from the PAM sequence).
[0169] 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.
[0170] Single-guide RNAs have the DNA-targeting segment and a
scaffold sequence (i.e., the protein-binding or Cas-binding
sequence of the guide RNA). For example, such guide RNAs have a 5'
DNA-targeting segment and a 3' scaffold sequence. Exemplary
scaffold sequences comprise, consist essentially of, or consist
of:
TABLE-US-00003 (version 1; SEQ ID NO: 39)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGCU; (version 2; SEQ ID NO: 7)
GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUU
AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (version 3; SEQ ID NO: 8)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGC; and (version 4; SEQ ID NO: 9)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC
CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC,
Guide RNAs targeting any guide RNA target sequence can include, for
example, a DNA-targeting segment on the 5' end of the guide RNA
fused to any of the exemplary guide RNA scaffold sequences on the
3' end of the guide RNA. That is, any of the DNA-targeting segments
(i.e., guide sequences or guides) disclosed herein (e.g., SEQ ID
NO: 2, 14, 20, 22, 24, 26, 28, 29, 30, 31, 35, 36, or 55) can be
joined to the 5' end of any one of SEQ ID NOS: 39, 7, 8, or 9 to
form a single guide RNA (chimeric guide RNA). Guide RNA versions 1,
2, 3, and 4 as disclosed elsewhere herein refer to DNA-targeting
segments (i.e., guide sequences or guides) joined with scaffold
versions 1, 2, 3, and 4, respectively.
[0171] 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, and so forth); a modification or sequence
that provides a binding site for proteins (e.g., proteins that act
on DNA, including DNA methyltransferases, DNA demethylases, histone
acetyltransferases, histone deacetylases, and the like); and
combinations thereof. Other examples of modifications include
engineered stem loop duplex structures, engineered bulge regions,
engineered hairpins 3' of the stem loop duplex structure, or any
combination thereof. See, e.g., US 2015/0376586, herein
incorporated by reference in its entirety for all purposes. A bulge
can be an unpaired region of nucleotides within the duplex made up
of the crRNA-like region and the minimum tracrRNA-like region. A
bulge can comprise, on one side of the duplex, an unpaired
5'-XXXY-3' where X is any purine and Y can be a nucleotide that can
form a wobble pair with a nucleotide on the opposite strand, and an
unpaired nucleotide region on the other side of the duplex.
[0172] Unmodified nucleic acids can be prone to degradation.
Exogenous nucleic acids can also induce an innate immune response.
Modifications can help introduce stability and reduce
immunogenicity. Guide RNAs can comprise modified nucleosides and
modified nucleotides including, for example, one or more of the
following: (1) alteration or replacement of one or both of the
non-linking phosphate oxygens and/or of one or more of the linking
phosphate oxygens in the phosphodiester backbone linkage; (2)
alteration or replacement of a constituent of the ribose sugar such
as alteration or replacement of the 2' hydroxyl on the ribose
sugar; (3) replacement of the phosphate moiety with dephospho
linkers; (4) modification or replacement of a naturally occurring
nucleobase; (5) replacement or modification of the ribose-phosphate
backbone; (6) modification of the 3' end or 5' end of the
oligonucleotide (e.g., removal, modification or replacement of a
terminal phosphate group or conjugation of a moiety); and (7)
modification of the sugar. Other possible guide RNA modifications
include modifications of or replacement of uracils or poly-uracil
tracts. See, e.g., WO 2015/048577 and US 2016/0237455, each of
which is herein incorporated by reference in its entirety for all
purposes. Similar modifications can be made to Cas-encoding nucleic
acids, such as Cas mRNAs.
[0173] As one example, nucleotides at the 5' or 3' end of a guide
RNA can include phosphorothioate linkages (e.g., the bases can have
a modified phosphate group that is a phosphorothioate group). For
example, a guide RNA can include phosphorothioate linkages between
the 2, 3, or 4 terminal nucleotides at the 5' or 3' end of the
guide RNA. As another example, nucleotides at the 5' and/or 3' end
of a guide RNA can have 2'-O-methyl modifications. For example, a
guide RNA can include 2'-O-methyl modifications at the 2, 3, or 4
terminal nucleotides at the 5' and/or 3' end of the guide RNA
(e.g., the 5' end). See, e.g., WO 2017/173054 A1 and Finn et al.
(2018) Cell Reports 22:1-9, each of which is herein incorporated by
reference in its entirety for all purposes. In one specific
example, the guide RNA comprises 2'-O-methyl analogs and 3'
phosphorothioate internucleotide linkages at the first three 5' and
3' terminal RNA residues. In another specific example, the guide
RNA is modified such that all 2'OH groups that do not interact with
the Cas9 protein are replaced with 2'-O-methyl analogs, and the
tail region of the guide RNA, which has minimal interaction with
Cas9, is modified with 5' and 3' phosphorothioate internucleotide
linkages. See, e.g., Yin et al. (2017) Nat. Biotech.
35(12):1179-1187, herein incorporated by reference in its entirety
for all purposes.
[0174] 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 gRNA. 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 one DNA
molecule or as separate DNA molecules encoding the crRNA and
tracrRNA, respectively.
[0175] When a gRNA is provided in the form of DNA, the gRNA can be
transiently, conditionally, or constitutively expressed in the
cell. 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 vector comprising a heterologous nucleic acid, such as a
nucleic acid encoding a Cas protein. Alternatively, it can be in a
vector or a plasmid that is separate from the vector comprising the
nucleic acid encoding the Cas protein. Promoters that can be used
in such expression constructs include promoters active, for
example, in one or more of a eukaryotic cell, a human cell, a
non-human cell, a mammalian cell, a non-human mammalian cell, a
rodent cell, a mouse cell, a rat cell, a hamster cell, a rabbit
cell, a pluripotent cell, an embryonic stem (ES) cell, an adult
stem cell, a developmentally restricted progenitor cell, an induced
pluripotent stem (iPS) cell, or a one-cell stage embryo. Such
promoters can be, for example, conditional promoters, inducible
promoters, constitutive promoters, or tissue-specific promoters.
Such promoters can also be, for example, bidirectional promoters.
Specific examples of suitable promoters include an RNA polymerase
III promoter, such as a human U6 promoter, a rat U6 polymerase III
promoter, or a mouse U6 polymerase III promoter.
[0176] 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, e.g., WO
2014/089290 and WO 2014/065596, each of which is herein
incorporated by reference in its entirety for all purposes). Guide
RNAs can also be a synthetically produced molecule prepared by
chemical synthesis.
[0177] (3) Guide RNA Recognition Sequences and Guide RNA Target
Sequences
[0178] The term "guide RNA recognition sequence" includes nucleic
acid sequences present in a target DNA to which a DNA-targeting
segment of a gRNA will bind, provided sufficient conditions for
binding exist. The term guide RNA recognition sequence as used
herein encompasses both strands of the target double-stranded DNA
(i.e., the sequence on the complementary strand to which the guide
RNA hybridizes and the corresponding sequence on the
non-complementary strand adjacent to the protospacer adjacent motif
(PAM)). The term "guide RNA target sequence" as used herein refers
specifically to the sequence on the non-complementary strand
adjacent to the PAM (i.e., upstream or 5' of the PAM). That is, the
guide RNA target sequence refers to the sequence on the
non-complementary strand corresponding to the sequence to which the
guide RNA hybridizes on the complementary strand. A guide RNA
target sequence is equivalent to the DNA-targeting segment of a
guide RNA, but with thymines instead of uracils. As one example, a
guide RNA target sequence for a Cas9 enzyme would refer to the
sequence on the non-complementary strand adjacent to the 5'-NGG-3'
PAM. Guide RNA recognition sequences include sequences to which a
guide RNA is designed to have complementarity, where hybridization
between the complementary strand of a guide RNA recognition
sequence and a DNA-targeting segment of a guide RNA promotes the
formation of a CRISPR complex. Full complementarity is not
necessarily required, provided that there is sufficient
complementarity to cause hybridization and promote formation of a
CRISPR complex. Guide RNA recognition sequences or guide RNA target
sequences also include cleavage sites for Cas proteins, described
in more detail below. A guide RNA recognition sequence or a guide
RNA 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.
[0179] The guide RNA recognition 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 (see, e.g., Molecular Cloning: A
Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory
Press 2001), herein incorporated by reference in its entirety for
all purposes). 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."
[0180] The Cas protein can cleave the nucleic acid at a site within
or outside of the nucleic acid sequence present in the target DNA
to which the DNA-targeting 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 the complementary strand of a guide RNA
recognition 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 to which the DNA-targeting
segment of the gRNA will bind, the cleavage site is still
considered to be within the "guide RNA recognition sequence" or
guide RNA 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 (i.e., overhangs)). Staggered ends can be produced,
for example, by using two Cas proteins, each of which produces a
single-strand break at a different cleavage site on a different
strand, thereby producing a double-strand break. For example, a
first nickase can create a single-strand break on the first strand
of double-stranded DNA (dsDNA), and a second nickase can create a
single-strand break on the second strand of dsDNA such that
overhanging sequences are created. In some cases, the guide RNA
recognition sequence or guide RNA target sequence of the nickase on
the first strand is separated from the guide RNA recognition
sequence or guide RNA 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.
[0181] Site-specific binding and/or cleavage of target DNA by Cas
proteins 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 guide RNA target sequence on the
non-complementary strand opposite of the strand to which the guide
RNA hybridizes. Optionally, the guide RNA target sequence can be
flanked on the 3' end by the PAM. Alternatively, the guide RNA
target sequence can be flanked on the 5' end by the PAM. For
example, the cleavage site of Cas proteins 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'-N.sub.1GG-3',
where N.sub.1 is any DNA nucleotide and is immediately 3' of the
guide RNA recognition sequence of the non-complementary strand of
the target DNA (i.e., immediately 3' of the guide RNA target
sequence). As such, the PAM sequence of the complementary strand
would be 5'-CCN.sub.2-3', where N.sub.2 is any DNA nucleotide and
is immediately 5' of the guide RNA recognition sequence of the
complementary strand of the target DNA. In some such cases, N.sub.1
and N.sub.2 can be complementary and the N.sub.1-N.sub.2 base pair
can be any base pair (e.g., N.sub.1=C and N.sub.2=G; N.sub.1=G and
N.sub.2=C; N.sub.1=A and N.sub.2=T; or N.sub.1=T, and N.sub.2=A).
In the case of Cas9 from S. aureus, the PAM can be NNGRRT or NNGRR,
where N can A, G, C, or T, and R can be G or A. In the case of Cas9
from C. jejuni, the PAM can be, for example, NNNNACAC or NNNNRYAC,
where N can be A, G, C, or T, and R can be G or A. In some cases
(e.g., for FnCpf1), the PAM sequence can be upstream of the 5' end
and have the sequence 5'-TTN-3'.
[0182] Examples of guide RNA target sequences or guide RNA target
sequences in addition to a PAM sequence are provided below. For
example, the guide RNA target sequence can be a 20-nucleotide DNA
sequence immediately preceding an NGG motif recognized by a Cas9
protein. Examples of such guide RNA target sequences plus a PAM
sequence are GN.sub.19NGG (SEQ ID NO: 10) or N.sub.20NGG (SEQ ID
NO: 11). See, e.g., WO 2014/165825, herein incorporated by
reference in its entirety for all purposes. The guanine at the 5'
end can facilitate transcription by RNA polymerase in cells. Other
examples of guide RNA target sequences plus a PAM sequence can
include two guanine nucleotides at the 5' end (e.g., GGN.sub.20NGG;
SEQ ID NO: 12) to facilitate efficient transcription by T7
polymerase in vitro. See, e.g., WO 2014/065596, herein incorporated
by reference in its entirety for all purposes. Other guide RNA
target sequences plus a PAM sequence can have between 4-22
nucleotides in length of SEQ ID NOS: 10-12, including the 5' G or
GG and the 3' GG or NGG. Yet other guide RNA target sequences can
have between 14 and 20 nucleotides in length of SEQ ID NOS:
10-12.
[0183] The guide RNA recognition sequence or guide RNA target
sequence can be any nucleic acid sequence endogenous or exogenous
to a cell. The guide RNA recognition sequence or guide RNA 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 can
include both.
III. Methods of Assessing CRISPR/Cas Activity In Vivo
[0184] Various methods are provided for assessing CRISPR/Cas
delivery to and for assessing CRISPR/Cas activity in tissues and
organs of a live animal. Such methods make use of non-human animals
comprising a CRISPR reporter as described elsewhere herein.
[0185] A. Methods of Testing Ability of CRISPR/Cas to Excise or
Disrupt a Target Genomic Nucleic Acid In Vivo or Ex Vivo
[0186] Various methods are provided for assessing
CRISPR/Cas-induced NHEJ activity in vivo using non-human animals
comprising a CRISPR reporter as described elsewhere herein. Such
methods can comprise introducing into the non-human animal: (i) a
first guide RNA designed to target the first guide RNA target
sequence in the CRISPR reporter; (ii) a second guide RNA designed
to target the second guide RNA target sequence in the CRISPR
reporter; and (iii) a Cas protein (e.g., a Cas9 protein); and (b)
measuring the activity or expression of at least one of the one or
more different reporter proteins. Optionally, the first guide RNA
and the second guide RNA can be identical, and the first guide RNA
target sequence and the second guide RNA target sequence can be
identical. Alternatively, the first guide RNA and the second guide
RNA can be different, and the first guide RNA target sequence and
the second guide RNA target sequence can be different. Activity or
expression of the reporter proteins will be induced when the first
guide RNA forms a complex with the Cas protein and directs the Cas
protein to the CRISPR reporter, the second guide RNA forms a
complex with the Cas protein and directs the Cas protein to the
CRISPR reporter, the first Cas/guide RNA complex cleaves the first
guide RNA target sequence, and the second Cas/guide RNA complex
cleaves the second guide RNA target sequence, resulting in excision
of the polyadenylation signal or transcription terminator upstream
of the one or more reporter proteins. Alternatively, if the first
or second guide RNA target sequence or a third guide RNA target
sequence is at or near the poly A recognition motif (canonical
polyadenylation hexamer AATAAA), a single guide RNA can be
introduced instead of the pair of first and second guide RNAs, and
the single guide RNA forms a complex with the Cas protein and
directs the Cas protein to the CRISPR reporter, the Cas/guide RNA
complex cleaves the guide RNA target sequence at or near the
canonical polyadenylation hexamer, resulting in disruption of the
canonical polyadenylation hexamer and consequently disruption of
the polyadenylation signal or transcription terminator upstream of
the one or more reporter proteins.
[0187] Likewise, the various methods provided above for assessing
CRISPR/Cas activity in vivo can also be used to assess CRISPR/Cas
activity ex vivo using cells comprising a CRISPR reporter as
described elsewhere herein.
[0188] Guide RNAs and Cas proteins can be introduced into the cell
or non-human animal via any delivery method (e.g., AAV, LNP, or
HDD) and any route of administration as disclosed elsewhere herein.
In particular methods, the guide RNA (or guide RNAs) is delivered
via AAV-mediated delivery. For example, AAV8 can be used if the
liver is being targeted. In one specific example, Cas9, gRNAs, and
optionally exogenous donor nucleic acid (e.g., ssODN) are delivered
via AAV8 as disclosed elsewhere herein. In another specific
example, Cas9 mRNA and gRNAs (in the form of RNA) and optionally
exogenous donor nucleic acid are delivered via LNP as disclosed
elsewhere herein.
[0189] Methods for assessing modification of the target genomic
locus are provided elsewhere herein and are well known. Assessment
of modification of the target genomic locus can be in any cell
type, any tissue type, or any organ type as disclosed elsewhere
herein. In some methods, modification of the target genomic locus
is assessed in liver cells.
[0190] B. Methods of Optimizing Ability of CRISPR/Cas to Excise a
Target Genomic Nucleic Acid In Vivo or Ex Vivo
[0191] Various methods are provided for optimizing delivery of
CRISPR/Cas to a cell or non-human animal or optimizing
CRISPR/Cas-induced NHEJ activity in vivo or ex vivo. Such methods
can comprise, for example: (a) performing the method of testing the
ability of CRISPR/Cas to excise the polyadenylation signal or
transcription terminator in the CRISPR reporter as described
elsewhere herein a first time in a first non-human animal; (b)
changing a variable and performing the method a second time with
the changed variable in a second non-human animal (i.e., of the
same species); and (c) comparing the activity or expression of at
least one of the one or more reporter proteins in step (a) with the
activity or expression of the at least one of the one or more
different proteins in step (b), and selecting the method resulting
in the higher activity or expression of the at least one of the one
or more different reporter proteins (i.e., the method resulting in
higher efficacy).
[0192] Alternatively, the method selected in step (c) can be the
method resulting in targeted modification of the CRISPR reporter or
increased activity or expression of the at least one of the one or
more different reporter proteins with higher efficacy, higher
precision, higher consistency, or higher specificity. Higher
efficacy refers to higher levels of modification of the target
locus in the CRISPR reporter (e.g., a higher percentage of cells is
targeted within a particular target cell type, within a particular
target tissue, or within a particular target organ). Higher
precision refers to more precise modification of the target locus
in the CRISPR reporter (e.g., a higher percentage of targeted cells
having the same modification or having the desired modification
without extra unintended insertions and deletions (e.g., NHEJ
indels)). Higher consistency refers to more consistent modification
of the target locus in the CRISPR reporter among different types of
targeted cells, tissues, or organs if more than one type of cell,
tissue, or organ is being targeted (e.g., modification of a greater
number of cell types within a target organ). If a particular organ
is being targeted, higher consistency can also refer to more
consistent modification throughout all locations within the organ.
Higher specificity can refer to higher specificity with respect to
the locus targeted within the CRISPR reporter, higher specificity
with respect to the cell type targeted, higher specificity with
respect to the tissue type targeted, or higher specificity with
respect to the organ targeted. For example, increased locus
specificity refers to less modification of off-target genomic loci
(e.g., a lower percentage of targeted cells having modifications at
unintended, off-target genomic loci instead of or in addition to
modification of the target locus in the CRISPR reporter). Likewise,
increased cell type, tissue, or organ type specificity refers to
less modification of off-target cell types, tissue types, or organ
types if a particular cell type, tissue type, or organ type is
being targeted (e.g., when a particular organ is targeted (e.g.,
the liver), there is less modification of cells in organs or
tissues that are not intended targets).
[0193] The variable that is changed can be any parameter. As one
example, the changed variable can be the packaging or the delivery
method by which one or more or all of the guide RNAs and the Cas
protein are introduced into the cell or non-human animal. Examples
of delivery methods, such as LNP, HDD, and AAV, are disclosed
elsewhere herein. As another example, the changed variable can be
the route of administration for introduction of one or more or all
of the guide RNAs and the Cas protein into the non-human animal.
Examples of routes of administration, such as intravenous,
intravitreal, intraparenchymal, and nasal instillation, are
disclosed elsewhere herein.
[0194] As another example, the changed variable can be the
concentration or amount of one or more or all of the guide RNAs
introduced and the Cas protein introduced. As another example, the
changed variable can be the concentration or the amount of guide
RNAs introduced relative to the concentration or the amount of Cas
protein introduced.
[0195] As another example, the changed variable can be the timing
of introducing one or more or all of the guide RNAs and the Cas
protein relative to the timing of measuring expression or activity
of the one or more reporter proteins. As another example, the
changed variable can be the number of times or frequency with which
one or more or all of the guide RNA and the Cas protein are
introduced. As another example, the changed variable can be the
timing of introduction of guide RNAs relative to the timing of
introduction of Cas protein.
[0196] As another example, the changed variable can be the form in
which one or more or all of the guide RNAs and the Cas protein are
introduced. For example, the guide RNAs can be introduced in the
form of DNA or in the form of RNA. The Cas protein can be
introduced in the form of DNA, RNA, or protein. Similarly, each of
the components can comprise various combinations of modifications
for stability, to reduce off-target effects, to facilitate
delivery, and so forth. As another example, the changed variable
can be one or more or all of the guide RNAs that are introduced
(e.g., introducing a different guide RNA with a different sequence)
and the Cas protein that is introduced (e.g., introducing a
different Cas protein with a different sequence, or a nucleic acid
with a different sequence but encoding the same Cas protein amino
acid sequence).
[0197] C. Methods of Testing Ability of CRISPR/Cas to Induce
Recombination of a Target Genomic Nucleic Acid with an Exogenous
Donor Nucleic Acid In Vivo or Ex Vivo
[0198] Various methods are provided for assessing
CRISPR/Cas-induced recombination activity in vivo using non-human
animals comprising a CRISPR reporter as described elsewhere herein.
If the CRISPR reporter is a CRISPR reporter comprising a
polyadenylation signal upstream of the coding sequences for the
reporter protein(s), optionally the polyadenylation signal has been
removed through CRISPR/Cas-mediated excision or
recombinase-mediated excision. Such methods can comprise
introducing into the non-human animal: (i) a guide RNA designed to
target a guide RNA target sequence in the CRISPR reporter; (ii) a
Cas protein (e.g., a Cas9 protein); and (iii) an exogenous donor
nucleic acid capable of recombining with the CRISPR reporter and
changing the coding sequence for a reporter protein within the
CRISPR reporter into a coding sequence for a different reporter
protein; and (b) measuring the activity or expression of the
different reporter protein. The guide RNA target sequence can be,
for example, within the coding sequence for the reporter protein
being modified. Optionally, the Cas protein can be tethered to the
exogenous donor nucleic acid as described elsewhere herein.
Activity or expression of the reporter proteins will be induced
when the guide RNA forms a complex with the Cas protein and directs
the Cas protein to the CRISPR reporter, the Cas/guide RNA complex
cleaves the guide RNA target sequence, and the CRISPR reporter
recombines with the exogenous donor nucleic acid to convert the
coding sequence for the reporter protein within the CRISPR reporter
into a coding sequence for a different reporter protein. The
exogenous donor nucleic acid can recombine with the CRISPR
reporter, for example, via homology-directed repair (HDR) or via
NHEJ-mediated insertion. Any type of exogenous donor nucleic acid
can be used, examples of which are provided elsewhere herein.
[0199] Likewise, the various methods provided above for assessing
CRISPR/Cas activity in vivo can also be used to assess CRISPR/Cas
activity ex vivo using cells comprising a CRISPR reporter as
described elsewhere herein.
[0200] In one example, the reporter protein is BFP or eBFP, and the
exogenous donor nucleic acid comprises mutations to convert the BFP
or eBFP coding sequence into a GFP or eGFP coding sequence by
altering a single codon. An exemplary guide RNA targeting this
region of the BFP or eBFP coding sequence comprises the targeting
sequence set forth in SEQ ID NO: 42. The sequence for an exemplary
exogenous donor nucleic acid is set forth in SEQ ID NO: 15 or SEQ
ID NO: 16. In another example, the reporter protein is GFP or eGFP,
and the exogenous donor nucleic acid comprises mutations to convert
the GFP or eGFP coding sequence into a BFP or eBFP coding sequence
by altering a single codon.
[0201] Guide RNAs, Cas proteins, and exogenous donor nucleic acids
can be introduced into the cell or non-human animal via any
delivery method (e.g., AAV, LNP, or HDD) and any route of
administration as disclosed elsewhere herein. In particular
methods, the guide RNA (or guide RNAs) is delivered via
AAV-mediated delivery. For example, AAV8 can be used if the liver
is being targeted.
[0202] Methods for assessing modification of the target genomic
locus are provided elsewhere herein and are well known. Assessment
of modification of the target genomic locus can be in any cell
type, any tissue type, or any organ type as disclosed elsewhere
herein. In some methods, modification of the target genomic locus
is assessed in liver cells.
[0203] (1) Exogenous Donor Nucleic Acids
[0204] The methods and compositions disclosed herein utilize
exogenous donor nucleic acids to modify the CRISPR reporter (i.e.,
the target genomic locus) following cleavage of the CRISPR reporter
with a Cas protein. In such methods, the Cas protein cleaves the
CRISPR reporter to create a single-strand break (nick) or
double-strand break, and the exogenous donor nucleic acid
recombines the target nucleic acid via non-homologous end joining
(NHEJ)-mediated ligation or through a homology-directed repair
event. Optionally, repair with the exogenous donor nucleic acid
removes or disrupts the guide RNA target sequence or the Cas
cleavage site so that alleles that have been targeted cannot be
re-targeted by the Cas protein.
[0205] Exogenous donor nucleic acids can comprise deoxyribonucleic
acid (DNA) or ribonucleic acid (RNA), they can be single-stranded
or double-stranded, and they can be in linear or circular form. For
example, an exogenous donor nucleic acid can be a single-stranded
oligodeoxynucleotide (ssODN). See, e.g., Yoshimi et al. (2016) Nat.
Commun. 7:10431, herein incorporated by reference in its entirety
for all purposes. An exemplary exogenous donor nucleic acid is
between about 50 nucleotides to about 5 kb in length, is between
about 50 nucleotides to about 3 kb in length, or is between about
50 to about 1,000 nucleotides in length. Other exemplary exogenous
donor nucleic acids are between about 40 to about 200 nucleotides
in length. For example, an exogenous donor nucleic acid can be
between about 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,
120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or
190-200 nucleotides in length. Alternatively, an exogenous donor
nucleic acid can be between about 50-100, 100-200, 200-300,
300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000
nucleotides in length. Alternatively, an exogenous donor nucleic
acid can be between about 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4,
4-4.5, or 4.5-5 kb in length. Alternatively, an exogenous donor
nucleic acid can be, for example, no more than 5 kb, 4.5 kb, 4 kb,
3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 nucleotides, 800
nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400
nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, or
50 nucleotides in length. Exogenous donor nucleic acids (e.g.,
targeting vectors) can also be longer.
[0206] In one example, an exogenous donor nucleic acid is an ssODN
that is between about 80 nucleotides and about 200 nucleotides in
length. In another example, an exogenous donor nucleic acids is an
ssODN that is between about 80 nucleotides and about 3 kb in
length. Such an ssODN can have homology arms, for example, that are
each between about 40 nucleotides and about 60 nucleotides in
length. Such an ssODN can also have homology arms, for example,
that are each between about 30 nucleotides and 100 nucleotides in
length. The homology arms can be symmetrical (e.g., each 40
nucleotides or each 60 nucleotides in length), or they can be
asymmetrical (e.g., one homology arm that is 36 nucleotides in
length, and one homology arm that is 91 nucleotides in length).
[0207] Exogenous donor nucleic acids can include modifications or
sequences that provide for additional desirable features (e.g.,
modified or regulated stability; tracking or detecting with a
fluorescent label; a binding site for a protein or protein complex;
and so forth). Exogenous donor nucleic acids can comprise one or
more fluorescent labels, purification tags, epitope tags, or a
combination thereof. For example, an exogenous donor nucleic acid
can comprise one or more fluorescent labels (e.g., fluorescent
proteins or other fluorophores or dyes), such as at least 1, at
least 2, at least 3, at least 4, or at least 5 fluorescent labels.
Exemplary fluorescent labels include fluorophores such as
fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX,
Cy3, Cy5, Cy5.5, Pacific Blue,
5-(and-6)-carboxytetramethylrhodamine (TAMRA), and Cy7. A wide
range of fluorescent dyes are available commercially for labeling
oligonucleotides (e.g., from Integrated DNA Technologies). Such
fluorescent labels (e.g., internal fluorescent labels) can be used,
for example, to detect an exogenous donor nucleic acid that has
been directly integrated into a cleaved target nucleic acid having
protruding ends compatible with the ends of the exogenous donor
nucleic acid. The label or tag can be at the 5' end, the 3' end, or
internally within the exogenous donor nucleic acid. For example, an
exogenous donor nucleic acid can be conjugated at 5' end with the
IR700 fluorophore from Integrated DNA Technologies (5'IRDYE.RTM.
700).
[0208] Exogenous donor nucleic acids can also comprise nucleic acid
inserts including segments of DNA to be integrated at target
genomic loci. Integration of a nucleic acid insert at a target
genomic locus can result in addition of a nucleic acid sequence of
interest to the target genomic locus, deletion of a nucleic acid
sequence of interest at the target genomic locus, or replacement of
a nucleic acid sequence of interest at the target genomic locus
(i.e., deletion and insertion). Some exogenous donor nucleic acids
are designed for insertion of a nucleic acid insert at a target
genomic locus without any corresponding deletion at the target
genomic locus. Other exogenous donor nucleic acids are designed to
delete a nucleic acid sequence of interest at a target genomic
locus without any corresponding insertion of a nucleic acid insert.
Yet other exogenous donor nucleic acids are designed to delete a
nucleic acid sequence of interest at a target genomic locus and
replace it with a nucleic acid insert.
[0209] The nucleic acid insert or the corresponding nucleic acid at
the target genomic locus being deleted and/or replaced can be
various lengths. An exemplary nucleic acid insert or corresponding
nucleic acid at the target genomic locus being deleted and/or
replaced is between about 1 nucleotide to about 5 kb in length or
is between about 1 nucleotide to about 1,000 nucleotides in length.
For example, a nucleic acid insert or a corresponding nucleic acid
at the target genomic locus being deleted and/or replaced can be
between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,
70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,
150-160, 160-170, 170-180, 180-190, or 190-120 nucleotides in
length. Likewise, a nucleic acid insert or a corresponding nucleic
acid at the target genomic locus being deleted and/or replaced can
be between 1-100, 100-200, 200-300, 300-400, 400-500, 500-600,
600-700, 700-800, 800-900, or 900-1000 nucleotides in length.
Likewise, a nucleic acid insert or a corresponding nucleic acid at
the target genomic locus being deleted and/or replaced can be
between about 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, or
4.5-5 kb in length or longer.
[0210] The nucleic acid insert can comprise a sequence that is
homologous or orthologous to all or part of sequence targeted for
replacement. For example, the nucleic acid insert can comprise a
sequence that comprises one or more point mutations (e.g., 1, 2, 3,
4, 5, or more) compared with a sequence targeted for replacement at
the target genomic locus. Optionally, such point mutations can
result in a conservative amino acid substitution (e.g.,
substitution of aspartic acid [Asp, D] with glutamic acid [Glu, E])
in the encoded polypeptide.
[0211] (2) Donor Nucleic Acids for
Non-Homologous-End-Joining-Mediated Insertion
[0212] Some exogenous donor nucleic acids have short
single-stranded regions at the 5' end and/or the 3' end that are
complementary to one or more overhangs created by
Cas-protein-mediated cleavage at the target genomic locus. These
overhangs can also be referred to as 5' and 3' homology arms. For
example, some exogenous donor nucleic acids have short
single-stranded regions at the 5' end and/or the 3' end that are
complementary to one or more overhangs created by
Cas-protein-mediated cleavage at 5' and/or 3' target sequences at
the target genomic locus. Some such exogenous donor nucleic acids
have a complementary region only at the 5' end or only at the 3'
end. For example, some such exogenous donor nucleic acids have a
complementary region only at the 5' end complementary to an
overhang created at a 5' target sequence at the target genomic
locus or only at the 3' end complementary to an overhang created at
a 3' target sequence at the target genomic locus. Other such
exogenous donor nucleic acids have complementary regions at both
the 5' and 3' ends. For example, other such exogenous donor nucleic
acids have complementary regions at both the 5' and 3' ends e.g.,
complementary to first and second overhangs, respectively,
generated by Cas-mediated cleavage at the target genomic locus. For
example, if the exogenous donor nucleic acid is double-stranded,
the single-stranded complementary regions can extend from the 5'
end of the top strand of the donor nucleic acid and the 5' end of
the bottom strand of the donor nucleic acid, creating 5' overhangs
on each end. Alternatively, the single-stranded complementary
region can extend from the 3' end of the top strand of the donor
nucleic acid and from the 3' end of the bottom strand of the
template, creating 3' overhangs.
[0213] The complementary regions can be of any length sufficient to
promote ligation between the exogenous donor nucleic acid and the
target nucleic acid. Exemplary complementary regions are between
about 1 to about 5 nucleotides in length, between about 1 to about
25 nucleotides in length, or between about 5 to about 150
nucleotides in length. For example, a complementary region can be
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
Alternatively, the complementary region can be about 5-10, 10-20,
20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,
110-120, 120-130, 130-140, or 140-150 nucleotides in length, or
longer.
[0214] Such complementary regions can be complementary to overhangs
created by two pairs of nickases. Two double-strand breaks with
staggered ends can be created by using first and second nickases
that cleave opposite strands of DNA to create a first double-strand
break, and third and fourth nickases that cleave opposite strands
of DNA to create a second double-strand break. For example, a Cas
protein can be used to nick first, second, third, and fourth guide
RNA target sequences corresponding with first, second, third, and
fourth guide RNAs. The first and second guide RNA target sequences
can be positioned to create a first cleavage site such that the
nicks created by the first and second nickases on the first and
second strands of DNA create a double-strand break (i.e., the first
cleavage site comprises the nicks within the first and second guide
RNA target sequences). Likewise, the third and fourth guide RNA
target sequences can be positioned to create a second cleavage site
such that the nicks created by the third and fourth nickases on the
first and second strands of DNA create a double-strand break (i.e.,
the second cleavage site comprises the nicks within the third and
fourth guide RNA target sequences). Optionally, the nicks within
the first and second guide RNA target sequences and/or the third
and fourth guide RNA target sequences can be off-set nicks that
create overhangs. The offset window can be, for example, at least
about 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp,
90 bp, 100 bp or more. See Ran et al. (2013) Cell 154:1380-1389;
Mali et al. (2013) Nat. Biotech. 31:833-838; and Shen et al. (2014)
Nat. Methods 11:399-404, each of which is herein incorporated by
reference in its entirety for all purposes. In such cases, a
double-stranded exogenous donor nucleic acid can be designed with
single-stranded complementary regions that are complementary to the
overhangs created by the nicks within the first and second guide
RNA target sequences and by the nicks within the third and fourth
guide RNA target sequences. Such an exogenous donor nucleic acid
can then be inserted by non-homologous-end-joining-mediated
ligation.
[0215] (3) Donor Nucleic Acids for Insertion by Homology-Directed
Repair
[0216] Some exogenous donor nucleic acids comprise homology arms.
If the exogenous donor nucleic acid also comprises a nucleic acid
insert, the homology arms can flank the nucleic acid insert. 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 exogenous donor nucleic acid.
The 5' and 3' homology arms correspond to regions within the target
genomic locus, which are referred to herein as "5' target sequence"
and "3' target sequence," respectively.
[0217] 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 in the exogenous donor nucleic acid 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 exogenous donor nucleic
acid (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. Exemplary homology
arms are between about 25 nucleotides to about 2.5 kb in length,
are between about 25 nucleotides to about 1.5 kb in length, or are
between about 25 to about 500 nucleotides in length. For example, a
given homology arm (or each of the homology arms) and/or
corresponding target sequence can comprise corresponding regions of
homology that are between about 25-30, 30-40, 40-50, 50-60, 60-70,
70-80, 80-90, 90-100, 100-150, 150-200, 200-250, 250-300, 300-350,
350-400, 400-450, or 450-500 nucleotides in length, such that the
homology arms have sufficient homology to undergo homologous
recombination with the corresponding target sequences within the
target nucleic acid. Alternatively, a given homology arm (or each
homology arm) and/or corresponding target sequence can comprise
corresponding regions of homology that are between about 0.5 kb to
about 1 kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb,
or about 2 kb to about 2.5 kb in length. For example, the homology
arms can each be about 750 nucleotides in length. The homology arms
can be symmetrical (each about the same size in length), or they
can be asymmetrical (one longer than the other).
[0218] When a CRISPR/Cas system is used in combination with an
exogenous donor nucleic acid, the 5' and 3' target sequences are
optionally located in sufficient proximity to the Cas cleavage site
(e.g., within sufficient proximity to a the guide RNA target
sequence) so as to promote the occurrence of a homologous
recombination event between the target sequences and the homology
arms upon a single-strand break (nick) or double-strand break at
the Cas cleavage site. The term "Cas cleavage site" includes a DNA
sequence at which a nick or double-strand break is created by a Cas
enzyme (e.g., a Cas9 protein complexed with a guide RNA). The
target sequences within the targeted locus that correspond to the
5' and 3' homology arms of the exogenous donor nucleic acid are
"located in sufficient proximity" to a Cas 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 single-strand break or double-strand break at
the Cas cleavage site. Thus, the target sequences corresponding to
the 5' and/or 3' homology arms of the exogenous donor nucleic acid
can be, for example, within at least 1 nucleotide of a given Cas
cleavage site or within at least 10 nucleotides to about 1,000
nucleotides of a given Cas cleavage site. As an example, the Cas
cleavage site can be immediately adjacent to at least one or both
of the target sequences.
[0219] The spatial relationship of the target sequences that
correspond to the homology arms of the exogenous donor nucleic acid
and the Cas cleavage site can vary. For example, target sequences
can be located 5' to the Cas cleavage site, target sequences can be
located 3' to the Cas cleavage site, or the target sequences can
flank the Cas cleavage site.
[0220] D. Methods of Optimizing Ability of CRISPR/Cas to Induce
Recombination of a Target Genomic Nucleic Acid with an Exogenous
Donor Nucleic Acid In Vivo or Ex Vivo
[0221] Various methods are provided for optimizing delivery of
CRISPR/Cas to a cell or non-human animal or optimizing
CRISPR/Cas-induced recombination activity in vivo or ex vivo. Such
methods can comprise, for example: (a) performing the method of
testing CRISPR/Cas-induced recombination of a target genomic locus
with an exogenous donor nucleic acid as described elsewhere herein
a first time in a first non-human animal; (b) changing a variable
and performing the method a second time with the changed variable
in a second non-human animal (i.e., of the same species); and (c)
comparing the activity or expression of the reporter protein in
step (a) with the activity or expression of the reporter protein in
step (b), and selecting the method resulting in the higher activity
or expression the reporter protein (i.e., selecting the method
resulting in higher efficacy).
[0222] Alternatively, the method selected in step (c) can be the
method resulting in targeted modification of the CRISPR reporter or
increased activity or expression of the reporter protein with
higher efficacy, higher precision, higher consistency, or higher
specificity. Higher efficacy refers to higher levels of
modification of the target locus in the CRISPR reporter (e.g., a
higher percentage of cells is targeted within a particular target
cell type, within a particular target tissue, or within a
particular target organ). Higher precision refers to more precise
modification of the target locus in the CRISPR reporter (e.g., a
higher percentage of targeted cells having the same modification or
having the desired modification without extra unintended insertions
and deletions (e.g., NHEJ indels)). Higher consistency refers to
more consistent modification of the target locus in the CRISPR
reporter among different types of targeted cells, tissues, or
organs if more than one type of cell, tissue, or organ is being
targeted (e.g., modification of a greater number of cell types
within a target organ). If a particular organ is being targeted,
higher consistency can also refer to more consistent modification
throughout all locations within the organ. Higher specificity can
refer to higher specificity with respect to the locus targeted
within the CRISPR reporter, higher specificity with respect to the
cell type targeted, higher specificity with respect to the tissue
type targeted, or higher specificity with respect to the organ
targeted. For example, increased locus specificity refers to less
modification of off-target genomic loci (e.g., a lower percentage
of targeted cells having modifications at unintended, off-target
genomic loci instead of or in addition to modification of the
target locus in the CRISPR reporter). Likewise, increased cell
type, tissue, or organ type specificity refers to less modification
of off-target cell types, tissue types, or organ types if a
particular cell type, tissue type, or organ type is being targeted
(e.g., when a particular organ is targeted (e.g., the liver), there
is less modification of cells in organs or tissues that are not
intended targets).
[0223] The variable that is changed can be any parameter. As one
example, the changed variable can be the packaging or the delivery
method by which one or more or all of the guide RNA, the exogenous
donor nucleic acid, and the Cas protein are introduced into the
cell or non-human animal. Examples of delivery methods, such as
LNP, HDD, and AAV, are disclosed elsewhere herein. As another
example, the changed variable can be the route of administration
for introduction of one or more or all of the guide RNA, the
exogenous donor nucleic acid, and the Cas protein into the
non-human animal. Examples of routes of administration, such as
intravenous, intravitreal, intraparenchymal, and nasal
instillation, are disclosed elsewhere herein.
[0224] As another example, the changed variable can be the
concentration or amount of one or more or all of the guide RNA
introduced, the Cas protein introduced, and the exogenous donor
nucleic acid introduced. As another example, the changed variable
can be the concentration or the amount of guide RNA introduced
relative to the concentration or the amount of Cas protein
introduced, the concentration or the amount of guide RNA introduced
relative to the concentration or the amount of exogenous donor
nucleic acid introduced, or the concentration or the amount of
exogenous donor nucleic acid introduced relative to the
concentration or the amount of Cas protein introduced.
[0225] As another example, the changed variable can be the timing
of introducing one or more or all of the guide RNA, exogenous donor
nucleic acid, and the Cas protein relative to the timing of
measuring expression or activity of the one or more reporter
proteins. As another example, the changed variable can be the
number of times or frequency with which one or more or all of the
guide RNA, exogenous donor nucleic acid, and the Cas protein are
introduced. As another example, the changed variable can be the
timing of introduction of guide RNA relative to the timing of
introduction of Cas protein, the timing of introduction of guide
RNA relative to the timing of introduction of exogenous donor
nucleic acid, or the timing of introduction of exogenous donor
nucleic acid relative to the timing of introduction of Cas
protein.
[0226] As another example, the changed variable can be the form in
which one or more or all of the guide RNA, the exogenous donor
nucleic acid, and the Cas protein are introduced. For example, the
guide RNA can be introduced in the form of DNA or in the form of
RNA. The Cas protein can be introduced in the form of DNA, RNA, or
protein. The exogenous donor nucleic acid can be DNA, RNA,
single-stranded, double-stranded, linear, circular, and so forth.
Similarly, each of the components can comprise various combinations
of modifications for stability, to reduce off-target effects, to
facilitate delivery, and so forth. As another example, the changed
variable can be one or more or all of the guide RNA that is
introduced (e.g., introducing a different guide RNA with a
different sequence), the exogenous donor nucleic acid that is
introduced (e.g., introducing a different exogenous donor nucleic
acid with a different sequence), and the Cas protein that is
introduced (e.g., introducing a different Cas protein with a
different sequence, or a nucleic acid with a different sequence but
encoding the same Cas protein amino acid sequence).
[0227] E. Introducing Guide RNAs and Cas Proteins into Cells and
Non-Human Animals
[0228] The methods disclosed herein comprise introducing into a
cell or non-human animal one or more or all of guide RNAs,
exogenous donor nucleic acids, and Cas proteins. "Introducing"
includes presenting to the cell or non-human animal the nucleic
acid or protein in such a manner that the nucleic acid or protein
gains access to the interior of the cell or to the interior of
cells within the non-human animal. The introducing can be
accomplished by any means, and two or more of the components (e.g.,
two of the components, or all of the components) can be introduced
into the cell or non-human animal simultaneously or sequentially in
any combination. For example, a Cas protein can be introduced into
a cell or non-human animal before introduction of a guide RNA, or
it can be introduced following introduction of the guide RNA. As
another example, an exogenous donor nucleic acid can be introduced
prior to the introduction of a Cas protein and a guide RNA, or it
can be introduced following introduction of the Cas protein and the
guide RNA (e.g., the exogenous donor nucleic acid can be
administered about 1, 2, 3, 4, 8, 12, 24, 36, 48, or 72 hours
before or after introduction of the Cas protein and the guide RNA).
See, e.g., US 2015/0240263 and US 2015/0110762, each of which is
herein incorporated by reference in its entirety for all purposes.
In addition, two or more of the components can be introduced into
the cell or non-human animal by the same delivery method or
different delivery methods. Similarly, two or more of the
components can be introduced into a non-human animal by the same
route of administration or different routes of administration.
[0229] A guide RNA can be introduced into the cell in the form of
an RNA (e.g., in vitro transcribed RNA) or in the form of a DNA
encoding the guide RNA. When introduced in the form of a DNA, the
DNA encoding a guide RNA can be operably linked to a promoter
active in the cell. For example, a guide RNA may be delivered via
AAV and expressed in vivo under a U6 promoter. Such DNAs can be in
one or more expression constructs. For example, such expression
constructs can be components of a single nucleic acid molecule.
Alternatively, they can be separated in any combination among two
or more nucleic acid molecules (i.e., DNAs encoding one or more
CRISPR RNAs and DNAs encoding one or more tracrRNAs can be
components of a separate nucleic acid molecules).
[0230] Likewise, 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. For example, the nucleic acid encoding the Cas
protein can be modified to substitute codons having a higher
frequency of usage in a bacterial cell, a yeast cell, a human cell,
a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a
rat cell, or any other host cell of interest, as compared to the
naturally occurring polynucleotide sequence. 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.
[0231] Nucleic acids encoding Cas proteins or guide RNAs 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 vector comprising
a DNA encoding one or more gRNAs. Alternatively, it can be in a
vector or plasmid that is separate from the vector comprising the
DNA encoding one or more gRNAs. Suitable promoters that can be used
in an expression construct include promoters active, for example,
in one or more of a eukaryotic cell, a human cell, a non-human
cell, a mammalian cell, a non-human mammalian cell, a rodent cell,
a mouse cell, a rat cell, a hamster cell, a rabbit cell, a
pluripotent cell, an embryonic stem (ES) cell, an adult stem cell,
a developmentally restricted progenitor cell, an induced
pluripotent stem (iPS) cell, or a one-cell stage embryo. Such
promoters can be, for example, conditional promoters, inducible
promoters, constitutive promoters, or tissue-specific promoters.
Optionally, the promoter can be a bidirectional promoter driving
expression of both a Cas protein in one direction and a guide RNA
in the other direction. Such bidirectional promoters can consist of
(1) a complete, conventional, unidirectional Pol III promoter that
contains 3 external control elements: a distal sequence element
(DSE), a proximal sequence element (PSE), and a TATA box; and (2) a
second basic Pol III promoter that includes a PSE and a TATA box
fused to the 5' terminus of the DSE in reverse orientation. For
example, in the H1 promoter, the DSE is adjacent to the PSE and the
TATA box, and the promoter can be rendered bidirectional by
creating a hybrid promoter in which transcription in the reverse
direction is controlled by appending a PSE and TATA box derived
from the U6 promoter. See, e.g., US 2016/0074535, herein
incorporated by references in its entirety for all purposes. Use of
a bidirectional promoter to express genes encoding a Cas protein
and a guide RNA simultaneously allows for the generation of compact
expression cassettes to facilitate delivery.
[0232] Exogenous donor nucleic acids, guide RNAs, and Cas proteins
(or nucleic acids encoding guide RNAs or Cas proteins) can be
provided in compositions comprising a carrier increasing the
stability of the exogenous donor nucleic acid, guide RNA, or Cas
protein (e.g., prolonging the period under given conditions of
storage (e.g., -20.degree. C., 4.degree. C., or ambient
temperature) for which degradation products remain below a
threshold, such below 0.5% by weight of the starting nucleic acid
or protein; or increasing the stability in vivo). Non-limiting
examples of such carriers include poly(lactic acid) (PLA)
microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres,
liposomes, micelles, inverse micelles, lipid cochleates, and lipid
microtubules.
[0233] Various methods and compositions are provided herein to
allow for introduction of a nucleic acid or protein into a cell or
non-human animal. Methods for introducing nucleic acids into
various cell types are known in the art and include, for example,
stable transfection methods, transient transfection methods, and
virus-mediated methods.
[0234] Transfection protocols as well as protocols for introducing
nucleic acid sequences into cells may vary. Non-limiting
transfection methods include chemical-based transfection methods
using liposomes; nanoparticles; calcium phosphate (Graham et al.
(1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc. Natl.
Acad. Sci. USA 74 (4): 1590-4, and Kriegler, M (1991). Transfer and
Expression: A Laboratory Manual. New York: W. H. Freeman and
Company. pp. 96-97); dendrimers; or cationic polymers such as
DEAE-dextran or polyethylenimine. Non-chemical methods include
electroporation, Sono-poration, and optical transfection.
Particle-based transfection includes the use of a gene gun, or
magnet-assisted transfection (Bertram (2006) Current Pharmaceutical
Biotechnology 7, 277-28). Viral methods can also be used for
transfection.
[0235] Introduction of nucleic acids or proteins into a cell can
also be mediated by electroporation, by intracytoplasmic injection,
by viral infection, by adenovirus, by adeno-associated virus, by
lentivirus, by retrovirus, by transfection, by lipid-mediated
transfection, or by nucleofection. Nucleofection is an improved
electroporation technology that enables nucleic acid substrates to
be delivered not only to the cytoplasm but also through the nuclear
membrane and into the nucleus. In addition, use of nucleofection in
the methods disclosed herein typically requires much fewer cells
than regular electroporation (e.g., only about 2 million compared
with 7 million by regular electroporation). In one example,
nucleofection is performed using the LONZA.RTM. NUCLEOFECTOR.TM.
system.
[0236] Introduction of nucleic acids or proteins into a cell (e.g.,
a zygote) can also be accomplished by microinjection. In zygotes
(i.e., one-cell stage embryos), microinjection can be into the
maternal and/or paternal pronucleus or into the cytoplasm. If the
microinjection is into only one pronucleus, the paternal pronucleus
is preferable due to its larger size. Microinjection of an mRNA is
preferably into the cytoplasm (e.g., to deliver mRNA directly to
the translation machinery), while microinjection of a Cas protein
or a polynucleotide encoding a Cas protein or encoding an RNA is
preferable into the nucleus/pronucleus. Alternatively,
microinjection can be carried out by injection into both the
nucleus/pronucleus and the cytoplasm: a needle can first be
introduced into the nucleus/pronucleus and a first amount can be
injected, and while removing the needle from the one-cell stage
embryo a second amount can be injected into the cytoplasm. If a Cas
protein is injected into the cytoplasm, the Cas protein optionally
comprises a nuclear localization signal to ensure delivery to the
nucleus/pronucleus. Methods for carrying out microinjection are
well known. See, e.g., Nagy et al. (Nagy A, Gertsenstein M,
Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo.
Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press);
see also Meyer et al. (2010) Proc. Natl. Acad. Sci. USA
107:15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA
109:9354-9359.
[0237] Other methods for introducing nucleic acid or proteins into
a cell or non-human animal can include, for example, vector
delivery, particle-mediated delivery, exosome-mediated delivery,
lipid-nanoparticle-mediated delivery,
cell-penetrating-peptide-mediated delivery, or
implantable-device-mediated delivery. As specific examples, a
nucleic acid or protein can be introduced into a cell or non-human
animal in a carrier such as a poly(lactic acid) (PLA) microsphere,
a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome,
a micelle, an inverse micelle, a lipid cochleate, or a lipid
microtubule. Some specific examples of delivery to a non-human
animal include hydrodynamic delivery, virus-mediated delivery
(e.g., adeno-associated virus (AAV)-mediated delivery), and
lipid-nanoparticle-mediated delivery.
[0238] Introduction of nucleic acids and proteins into cells or
non-human animals can be accomplished by hydrodynamic delivery
(HDD). Hydrodynamic delivery has emerged as a method for
intracellular DNA delivery in vivo. For gene delivery to
parenchymal cells, only essential DNA sequences need to be injected
via a selected blood vessel, eliminating safety concerns associated
with current viral and synthetic vectors. When injected into the
bloodstream, DNA is capable of reaching cells in the different
tissues accessible to the blood. Hydrodynamic delivery employs the
force generated by the rapid injection of a large volume of
solution into the incompressible blood in the circulation to
overcome the physical barriers of endothelium and cell membranes
that prevent large and membrane-impermeable compounds from entering
parenchymal cells. In addition to the delivery of DNA, this method
is useful for the efficient intracellular delivery of RNA,
proteins, and other small compounds in vivo. See, e.g., Bonamassa
et al. (2011) Pharm. Res. 28(4):694-701, herein incorporated by
reference in its entirety for all purposes.
[0239] Introduction of nucleic acids can also be accomplished by
virus-mediated delivery, such as AAV-mediated delivery or
lentivirus-mediated delivery. Other exemplary viruses/viral vectors
include retroviruses, adenoviruses, vaccinia viruses, poxviruses,
and herpes simplex viruses. The viruses can infect dividing cells,
non-dividing cells, or both dividing and non-dividing cells. Such
viruses can also be engineered to have reduced immunity. The
viruses can be replication-competent or can be
replication-defective (e.g., defective in one or more genes
necessary for additional rounds of virion replication and/or
packaging). Viruses can cause transient expression, long-lasting
expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3
months), or permanent expression (e.g., of Cas9 and/or gRNA).
Exemplary viral titers (e.g., AAV titers) include 10.sup.12,
10.sup.13, 10.sup.14, 10.sup.15, and 10.sup.16 vector
genomes/mL.
[0240] The ssDNA AAV genome consists of two open reading frames,
Rep and Cap, flanked by two inverted terminal repeats that allow
for synthesis of the complementary DNA strand. When constructing an
AAV transfer plasmid, the transgene is placed between the two ITRs,
and Rep and Cap can be supplied in trans. In addition to Rep and
Cap, AAV can require a helper plasmid containing genes from
adenovirus. These genes (E4, E2a, and VA) mediated AAV replication.
For example, the transfer plasmid, Rep/Cap, and the helper plasmid
can be transfected into HEK293 cells containing the adenovirus gene
E1+ to produce infectious AAV particles. Alternatively, the Rep,
Cap, and adenovirus helper genes may be combined into a single
plasmid. Similar packaging cells and methods can be used for other
viruses, such as retroviruses.
[0241] Multiple serotypes of AAV have been identified. These
serotypes differ in the types of cells they infect (i.e., their
tropism), allowing preferential transduction of specific cell
types. Serotypes for CNS tissue include AAV1, AAV2, AAV4, AAV5,
AAV8, and AAV9. Serotypes for heart tissue include AAV1, AAV8, and
AAV9. Serotypes for kidney tissue include AAV2. Serotypes for lung
tissue include AAV4, AAV5, AAV6, and AAV9. Serotypes for pancreas
tissue include AAV8. Serotypes for photoreceptor cells include
AAV2, AAV5, and AAV8. Serotypes for retinal pigment epithelium
tissue include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes for
skeletal muscle tissue include AAV1, AAV6, AAV7, AAV8, and AAV9.
Serotypes for liver tissue include AAV7, AAV8, and AAV9, and
particularly AAV8.
[0242] Tropism can be further refined through pseudotyping, which
is the mixing of a capsid and a genome from different viral
serotypes. For example AAV2/5 indicates a virus containing the
genome of serotype 2 packaged in the capsid from serotype 5. Use of
pseudotyped viruses can improve transduction efficiency, as well as
alter tropism. Hybrid capsids derived from different serotypes can
also be used to alter viral tropism. For example, AAV-DJ contains a
hybrid capsid from eight serotypes and displays high infectivity
across a broad range of cell types in vivo. AAV-DJ8 is another
example that displays the properties of AAV-DJ but with enhanced
brain uptake. AAV serotypes can also be modified through mutations.
Examples of mutational modifications of AAV2 include Y444F, Y500F,
Y730F, and S662V. Examples of mutational modifications of AAV3
include Y705F, Y731F, and T492V. Examples of mutational
modifications of AAV6 include S663V and T492V. Other
pseudotyped/modified AAV variants include AAV2/1, AAV2/6, AAV2/7,
AAV2/8, AAV2/9, AAV2.5, AAV8.2, and AAV/SASTG.
[0243] To accelerate transgene expression, self-complementary AAV
(scAAV) variants can be used. Because AAV depends on the cell's DNA
replication machinery to synthesize the complementary strand of the
AAV's single-stranded DNA genome, transgene expression may be
delayed. To address this delay, scAAV containing complementary
sequences that are capable of spontaneously annealing upon
infection can be used, eliminating the requirement for host cell
DNA synthesis. However, single-stranded AAV (ssAAV) vectors can
also be used.
[0244] To increase packaging capacity, longer transgenes may be
split between two AAV transfer plasmids, the first with a 3' splice
donor and the second with a 5' splice acceptor. Upon co-infection
of a cell, these viruses form concatemers, are spliced together,
and the full-length transgene can be expressed. Although this
allows for longer transgene expression, expression is less
efficient. Similar methods for increasing capacity utilize
homologous recombination. For example, a transgene can be divided
between two transfer plasmids but with substantial sequence overlap
such that co-expression induces homologous recombination and
expression of the full-length transgene.
[0245] Introduction of nucleic acids and proteins can also be
accomplished by lipid nanoparticle (LNP)-mediated delivery. For
example, LNP-mediated delivery can be used to deliver a combination
of Cas mRNA and guide RNA or a combination of Cas protein and guide
RNA. Delivery through such methods results in transient Cas
expression, and the biodegradable lipids improve clearance, improve
tolerability, and decrease immunogenicity. Lipid formulations can
protect biological molecules from degradation while improving their
cellular uptake. Lipid nanoparticles are particles comprising a
plurality of lipid molecules physically associated with each other
by intermolecular forces. These include microspheres (including
unilamellar and multilamellar vesicles, e.g., liposomes), a
dispersed phase in an emulsion, micelles, or an internal phase in a
suspension. Such lipid nanoparticles can be used to encapsulate one
or more nucleic acids or proteins for delivery. Formulations which
contain cationic lipids are useful for delivering polyanions such
as nucleic acids. Other lipids that can be included are neutral
lipids (i.e., uncharged or zwitterionic lipids), anionic lipids,
helper lipids that enhance transfection, and stealth lipids that
increase the length of time for which nanoparticles can exist in
vivo. Examples of suitable cationic lipids, neutral lipids, anionic
lipids, helper lipids, and stealth lipids can be found in WO
2016/010840 A1, herein incorporated by reference in its entirety
for all purposes. An exemplary lipid nanoparticle can comprise a
cationic lipid and one or more other components. In one example,
the other component can comprise a helper lipid such as
cholesterol. In another example, the other components can comprise
a helper lipid such as cholesterol and a neutral lipid such as
DSPC. In another example, the other components can comprise a
helper lipid such as cholesterol, an optional neutral lipid such as
DSPC, and a stealth lipid such as 5010, 5024, 5027, 5031, or
5033.
[0246] The LNP may contain one or more or all of the following: (i)
a lipid for encapsulation and for endosomal escape; (ii) a neutral
lipid for stabilization; (iii) a helper lipid for stabilization;
and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell
Reports 22:1-9 and WO 2017/173054 A1, each of which is herein
incorporated by reference in its entirety for all purposes. In
certain LNPs, the cargo can include a guide RNA or a nucleic acid
encoding a guide RNA. In certain LNPs, the cargo can include an
exogenous donor nucleic acid. In certain LNPs, the cargo can
include a guide RNA or a nucleic acid encoding a guide RNA and a
Cas protein or a nucleic acid encoding a Cas protein. In certain
LNPs, the cargo can include a guide RNA or a nucleic acid encoding
a guide RNA, a Cas protein or a nucleic acid encoding a Cas
protein, and an exogenous donor nucleic acid.
[0247] The lipid for encapsulation and endosomal escape can be a
cationic lipid. The lipid can also be a biodegradable lipid, such
as a biodegradable ionizable lipid. One example of a suitable lipid
is Lipid A or LP01, which is
(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-
)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called
3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl-
)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn
et al. (2018) Cell Reports 22:1-9 and WO 2017/173054 A1, each of
which is herein incorporated by reference in its entirety for all
purposes. Another example of a suitable lipid is Lipid B, which is
((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bi-
s(decanoate), also called
((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bi-
s(decanoate). Another example of a suitable lipid is Lipid C, which
is
2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1-
,3-diyl(9Z,9Z',12Z,12Z')-bis(octadeca-9,12-dienoate). Another
example of a suitable lipid is Lipid D, which is
3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl
3-octylundecanoate. Other suitable lipids include
heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate
(also known as Dlin-MC3-DMA (MC3))).
[0248] Some such lipids suitable for use in the LNPs described
herein are biodegradable in vivo. For example, LNPs comprising such
a lipid include those where at least 75% of the lipid is cleared
from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6,
7, or 10 days. As another example, at least 50% of the LNP is
cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4,
5, 6, 7, or 10 days.
[0249] Such lipids may be ionizable depending upon the pH of the
medium they are in. For example, in a slightly acidic medium, the
lipids may be protonated and thus bear a positive charge.
Conversely, in a slightly basic medium, such as, for example, blood
where pH is approximately 7.35, the lipids may not be protonated
and thus bear no charge. In some embodiments, the lipids may be
protonated at a pH of at least about 9, 9.5, or 10. The ability of
such a lipid to bear a charge is related to its intrinsic pKa. For
example, the lipid may, independently, have a pKa in the range of
from about 5.8 to about 6.2.
[0250] Neutral lipids function to stabilize and improve processing
of the LNPs. Examples of suitable neutral lipids include a variety
of neutral, uncharged or zwitterionic lipids. Examples of neutral
phospholipids suitable for use in the present disclosure include,
but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol),
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC),
dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC),
phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),
dilauryloylphosphatidylcholine (DLPC),
dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl
phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl
phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl
phosphatidylcholine (PSPC),
1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),
1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC),
palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl
choline, dioleoyl phosphatidylethanolamine (DOPE),
dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine
(DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl
phosphatidylethanolamine (DPPE), palmitoyloleoyl
phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and
combinations thereof. For example, the neutral phospholipid may be
selected from the group consisting of distearoylphosphatidylcholine
(DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE).
[0251] Helper lipids include lipids that enhance transfection. The
mechanism by which the helper lipid enhances transfection can
include enhancing particle stability. In certain cases, the helper
lipid can enhance membrane fusogenicity. Helper lipids include
steroids, sterols, and alkyl resorcinols. Examples of suitable
helper lipids suitable include cholesterol, 5-heptadecylresorcinol,
and cholesterol hemisuccinate. In one example, the helper lipid may
be cholesterol or cholesterol hemisuccinate.
[0252] Stealth lipids include lipids that alter the length of time
the nanoparticles can exist in vivo. Stealth lipids may assist in
the formulation process by, for example, reducing particle
aggregation and controlling particle size. Stealth lipids may
modulate pharmacokinetic properties of the LNP. Suitable stealth
lipids include lipids having a hydrophilic head group linked to a
lipid moiety.
[0253] The hydrophilic head group of stealth lipid can comprise,
for example, a polymer moiety selected from polymers based on PEG
(sometimes referred to as poly(ethylene oxide)), poly(oxazoline),
poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),
polyaminoacids, and poly N-(2-hydroxypropyl)methacrylamide. The
term PEG means any polyethylene glycol or other polyalkylene ether
polymer. In certain LNP formulations, the PEG, is a PEG-2K, also
termed PEG 2000, which has an average molecular weight of about
2,000 daltons. See, e.g., WO 2017/173054 A1, herein incorporated by
reference in its entirety for all purposes.
[0254] The lipid moiety of the stealth lipid may be derived, for
example, from diacylglycerol or diacylglycamide, including those
comprising a dialkylglycerol or dialkylglycamide group having alkyl
chain length independently comprising from about C4 to about C40
saturated or unsaturated carbon atoms, wherein the chain may
comprise one or more functional groups such as, for example, an
amide or ester. The dialkylglycerol or dialkylglycamide group can
further comprise one or more substituted alkyl groups.
[0255] As one example, the stealth lipid may be selected from
PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG),
PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE),
PEG-dilaurylglycamide, PEG-dimyristylglycamide,
PEG-dipalmitoylglycamide, and PEG-distearoylglycamide,
PEG-cholesterol
(1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-
-[omega]-methyl-poly(ethylene glycol), PEG-DMB
(3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene
glycol)ether),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (PEG2k-DMG),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypoly
ethylene glycol (PEG2k-DSG), poly(ethylene
glycol)-2000-dimethacrylate (PEG2k-DMA), and
1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene
glycol)-2000] (PEG2k-DSA). In one particular example, the stealth
lipid may be PEG2k-DMG.
[0256] The LNPs can comprise different respective molar ratios of
the component lipids in the formulation. The mol-% of the CCD lipid
may be, for example, from about 30 mol-% to about 60 mol-%, from
about 35 mol-% to about 55 mol-%, from about 40 mol-% to about 50
mol-%, from about 42 mol-% to about 47 mol-%, or about 45%. The
mol-% of the helper lipid may be, for example, from about 30 mol-%
to about 60 mol-%, from about 35 mol-% to about 55 mol-%, from
about 40 mol-% to about 50 mol-%, from about 41 mol-% to about 46
mol-%, or about 44 mol-%. The mol-% of the neutral lipid may be,
for example, from about 1 mol-% to about 20 mol-%, from about 5
mol-% to about 15 mol-%, from about 7 mol-% to about 12 mol-%, or
about 9 mol-%. The mol-% of the stealth lipid may be, for example,
from about 1 mol-% to about 10 mol-%, from about 1 mol-% to about 5
mol-%, from about 1 mol-% to about 3 mol-%, about 2 mol-%, or about
1 mol-%.
[0257] The LNPs can have different ratios between the positively
charged amine groups of the biodegradable lipid (N) and the
negatively charged phosphate groups (P) of the nucleic acid to be
encapsulated. This may be mathematically represented by the
equation N/P. For example, the N/P ratio may be from about 0.5 to
about 100, from about 1 to about 50, from about 1 to about 25, from
about 1 to about 10, from about 1 to about 7, from about 3 to about
5, from about 4 to about 5, about 4, about 4.5, or about 5.
[0258] In some LNPs, the cargo can comprise Cas mRNA and gRNA. The
Cas mRNA and gRNAs can be in different ratios. For example, the LNP
formulation can include a ratio of Cas mRNA to gRNA nucleic acid
ranging from about 25:1 to about 1:25, ranging from about 10:1 to
about 1:10, ranging from about 5:1 to about 1:5, or about 1:1.
Alternatively, the LNP formulation can include a ratio of Cas mRNA
to gRNA nucleic acid from about 1:1 to about 1:5, or about 10:1.
Alternatively, the LNP formulation can include a ratio of Cas mRNA
to gRNA nucleic acid of about 1:10, 25:1, 10:1, 5:1, 3:1, 1:1, 1:3,
1:5, 1:10, or 1:25.
[0259] In some LNPs, the cargo can comprise exogenous donor nucleic
acid and gRNA. The exogenous donor nucleic acid and gRNAs can be in
different ratios. For example, the LNP formulation can include a
ratio of exogenous donor nucleic acid to gRNA nucleic acid ranging
from about 25:1 to about 1:25, ranging from about 10:1 to about
1:10, ranging from about 5:1 to about 1:5, or about 1:1.
Alternatively, the LNP formulation can include a ratio of exogenous
donor nucleic acid to gRNA nucleic acid from about 1:1 to about
1:5, about 5:1 to about 1:1, about 10:1, or about 1:10.
Alternatively, the LNP formulation can include a ratio of exogenous
donor nucleic acid to gRNA nucleic acid of about 1:10, 25:1, 10:1,
5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:25.
[0260] A specific example of a suitable LNP has a
nitrogen-to-phosphate (N/P) ratio of 4.5 and contains biodegradable
cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2
molar ratio. The biodegradable cationic lipid can be
(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-
)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called
3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl-
)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn
et al. (2018) Cell Reports 22:1-9, herein incorporated by reference
in its entirety for all purposes. Another specific example of a
suitable LNP contains Dlin-MC3-DMA (MC3), cholesterol, DSPC, and
PEG-DMG in a 50:38.5:10:1.5 molar ratio.
[0261] The mode of delivery can be selected to decrease
immunogenicity. For example, a Cas protein and a gRNA may be
delivered by different modes (e.g., bi-modal delivery). These
different modes may confer different pharmacodynamics or
pharmacokinetic properties on the subject delivered molecule (e.g.,
Cas or nucleic acid encoding, gRNA or nucleic acid encoding, or
exogenous donor nucleic acid/repair template). For example, the
different modes can result in different tissue distribution,
different half-life, or different temporal distribution. Some modes
of delivery result in more persistent expression and presence of
the molecule, whereas other modes of delivery are transient and
less persistent (e.g., delivery of an RNA or a protein). Delivery
of Cas proteins in a more transient manner, for example as mRNA or
protein, can ensure that the Cas/gRNA complex is only present and
active for a short period of time and can reduce immunogenicity
caused by peptides from the bacterially-derived Cas enzyme being
displayed on the surface of the cell by MEW molecules. Such
transient delivery can also reduce the possibility of off-target
modifications.
[0262] Administration in vivo can be by any suitable route
including, for example, parenteral, intravenous, oral,
subcutaneous, intra-arterial, intracranial, intrathecal,
intraperitoneal, topical, intranasal, or intramuscular. Systemic
modes of administration include, for example, oral and parenteral
routes. Examples of parenteral routes include intravenous,
intraarterial, intraosseous, intramuscular, intradermal,
subcutaneous, intranasal, and intraperitoneal routes. A specific
example is intravenous infusion. Nasal instillation and
intravitreal injection are other specific examples. Local modes of
administration include, for example, intrathecal,
intracerebroventricular, intraparenchymal (e.g., localized
intraparenchymal delivery to the striatum (e.g., into the caudate
or into the putamen), cerebral cortex, precentral gyms, hippocampus
(e.g., into the dentate gyrus or CA3 region), temporal cortex,
amygdala, frontal cortex, thalamus, cerebellum, medulla,
hypothalamus, tectum, tegmentum, or substantia nigra), intraocular,
intraorbital, subconjuctival, intravitreal, subretinal, and
transscleral routes. Significantly smaller amounts of the
components (compared with systemic approaches) may exert an effect
when administered locally (for example, intraparenchymal or
intravitreal) compared to when administered systemically (for
example, intravenously). Local modes of administration may also
reduce or eliminate the incidence of potentially toxic side effects
that may occur when therapeutically effective amounts of a
component are administered systemically.
[0263] Administration in vivo can be by any suitable route
including, for example, parenteral, intravenous, oral,
subcutaneous, intra-arterial, intracranial, intrathecal,
intraperitoneal, topical, intranasal, or intramuscular. A specific
example is intravenous infusion. Compositions comprising the guide
RNAs and/or Cas proteins (or nucleic acids encoding the guide RNAs
and/or Cas proteins) can be formulated using one or more
physiologically and pharmaceutically acceptable carriers, diluents,
excipients or auxiliaries. The formulation can depend on the route
of administration chosen. The term "pharmaceutically acceptable"
means that the carrier, diluent, excipient, or auxiliary is
compatible with the other ingredients of the formulation and not
substantially deleterious to the recipient thereof.
[0264] The frequency of administration and the number of dosages
can be depend on the half-life of the exogenous donor nucleic
acids, guide RNAs, or Cas proteins (or nucleic acids encoding the
guide RNAs or Cas proteins) and the route of administration among
other factors. The introduction of nucleic acids or proteins into
the cell or non-human animal can be performed one time or multiple
times over a period of time. For example, the introduction can be
performed at least two times over a period of time, at least three
times over a period of time, at least four times over a period of
time, at least five times over a period of time, at least six times
over a period of time, at least seven times over a period of time,
at least eight times over a period of time, at least nine times
over a period of times, at least ten times over a period of time,
at least eleven times, at least twelve times over a period of time,
at least thirteen times over a period of time, at least fourteen
times over a period of time, at least fifteen times over a period
of time, at least sixteen times over a period of time, at least
seventeen times over a period of time, at least eighteen times over
a period of time, at least nineteen times over a period of time, or
at least twenty times over a period of time.
[0265] F. Measuring CRISPR/Cas Activity In Vivo
[0266] The methods disclosed herein can further comprise detecting
or measuring expression or activity of one or more reporter
proteins encoded by the CRISPR reporter. The methods for detecting
or measuring expression or activity will depend on the reporter
protein.
[0267] For example, for fluorescent reporter proteins, the
detecting or measuring can comprise spectrophotometry or flow
cytometry assays or fluorescence microscopy of cells isolated from
the non-human animal or macro-photography assays or in vivo imaging
of the non-human animal itself.
[0268] For luciferase reporter proteins, the assay can comprise a
luciferase reporter assay comprising breaking open cells isolated
from the non-human animal to release all the proteins (including
the luciferase), adding luciferin (for firefly luciferase) or
coelenterazine (for Renilla luciferase) and all the necessary
cofactors, and measuring the enzymatic activity using a
luminometer. Luciferin is converted to oxyluciferin by the
luciferase enzyme. Some of the energy released by this reaction is
in the form of light. Alternatively, bioluminescence imaging of the
non-human animal can be performed following injection of the
luciferase substrate (e.g., luciferin or coelenterazine) into the
non-human animal. Such assays enable noninvasive optical imaging of
living animals with high sensitivity.
[0269] For beta-galactosidase reporter proteins, the assay can
comprise histochemical staining of cells or tissues isolated from
the non-human animal. Beta-galactosidase catalyzes the hydrolysis
of X-Gal producing a blue precipitate that can be easily visualized
under a microscope, thereby providing a simple and convenient
method for the visual detection of LacZ expression within cells or
tissues.
[0270] Other reporter proteins and assays for detecting or
measuring expression or activity of such reporter proteins are well
known.
[0271] Alternatively, the methods disclosed herein can further
comprise identifying a cell having a modified CRISPR reporter in
which the polyadenylation signal or transcription terminator
sequence has been excised or identifying a cell having a modified
CRISPR reporter in which the coding sequence for one reporter
protein has been altered and converted into the coding sequence for
a different reporter protein. Various methods can be used to
identify cells having a targeted genetic modification. The
screening can comprise a quantitative assay for assessing
modification of allele (MOA) of a parental chromosome. For example,
the quantitative assay can be carried out via a quantitative PCR,
such as a real-time PCR (qPCR). The real-time PCR can utilize a
first primer set that recognizes the target locus and a second
primer set that recognizes a non-targeted reference locus. The
primer set can comprise a fluorescent probe that recognizes the
amplified sequence. Other examples of suitable quantitative assays
include fluorescence-mediated in situ hybridization (FISH),
comparative genomic hybridization, isothermic DNA amplification,
quantitative hybridization to an immobilized probe(s), INVADER.RTM.
Probes, TAQMAN.RTM. Molecular Beacon probes, or ECLIPSE.TM. probe
technology (see, e.g., US 2005/0144655, herein incorporated by
reference in its entirety for all purposes).
[0272] Next-generation sequencing (NGS) can also be used for
screening. Next-generation sequencing can also be referred to as
"NGS" or "massively parallel sequencing" or "high throughput
sequencing." NGS can be used as a screening tool in addition to the
MOA assays to define the exact nature of the targeted genetic
modification and whether it is consistent across cell types or
tissue types or organ types.
[0273] Assessing modification of the target genomic locus in a
non-human animal can be in any cell type from any tissue or organ.
For example, detecting or measuring expression or activity of one
or more reporter proteins encoded by the CRISPR reporter can be
assessed in multiple cell types from the same tissue or organ or in
cells from multiple locations within the tissue or organ. This can
provide information about which cell types within a target tissue
or organ are being modified or which sections of a tissue or organ
are being reached by the CRISPR/Cas and modified. As another
example, detecting or measuring expression or activity of one or
more reporter proteins encoded by the CRISPR reporter can be
assessed in multiple types of tissue or in multiple organs. In
methods in which a particular tissue or organ is being targeted,
this can provide information about how effectively that tissue or
organ is being targeted and whether there are off-target effects in
other tissues or organs.
[0274] As one example, the CRISPR reporters disclosed herein can be
a CRISPR reporter comprising both a lacZ gene and a gene encoding a
fluorescent reporter protein and can be used to detect both NHEJ
and homology-directed repair (HDR). For example, primary
hepatocytes can be harvested from non-human animals comprising the
CRISPR reporters to evaluate strategies to induce NHEJ and HDR in
this cell type. Cas9 can be introduced, for example, as either an
AAV, mRNA, or protein, and the gRNA can be introduced as either
single guide RNA (modified and unmodified) or modular (duplex) RNA.
DNA repair templates can be introduced as symmetric or asymmetric
single-strand, symmetric or asymmetric double strand, or AAV
vector. As a specific example, lacZ staining can be completed to
assess the success of NHEJ, and fluorescent microscopy and FACs
analysis can then be used to evaluate HDR efficiencies. Information
gathered ex vivo can then applied to the adult non-human animal
(e.g., mouse). Cas9, guide RNA, and repair template may be
introduced in any of the states listed above.
IV. Methods of Making Non-Human Animals Comprising a CRISPR
Reporter
[0275] Various methods are provided for making a non-human animal
comprising a CRISPR reporter as disclosed elsewhere herein. Any
convenient method or protocol for producing a genetically modified
organism is suitable for producing such a genetically modified
non-human animal. See, e.g., Cho et al. (2009) Current Protocols in
Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010)
Brain Struct. Funct. 214(2-3):91-109, each of which is herein
incorporated by reference in its entirety for all purposes. Such
genetically modified non-human animals can be generated, for
example, through gene knock-in at a targeted locus (e.g., a safe
harbor locus such as Rosa26) or through use of a randomly
integrating transgene. See, e.g., WO 2014/093622 and WO
2013/176772, each of which is herein incorporated by reference in
its entirety for all purposes. Methods of targeting a construct to
the Rosa26 locus are described, for example, in US 2012/0017290, US
2011/0265198, and US 2013/0236946, each of which is herein
incorporated by reference in its entirety for all purposes.
[0276] For example, the method of producing a non-human animal
comprising a CRISPR reporter as disclosed elsewhere herein can
comprise: (1) modifying the genome of a pluripotent cell to
comprise a CRISPR reporter; (2) identifying or selecting the
genetically modified pluripotent cell comprising the CRISPR
reporter; (3) introducing the genetically modified pluripotent cell
into a non-human animal host embryo; and (4) implanting and
gestating the host embryo in a surrogate mother. Optionally, the
host embryo comprising modified pluripotent cell (e.g., a non-human
ES cell) can be incubated until the blastocyst stage before being
implanted into and gestated in the surrogate mother to produce an
FO non-human animal. The surrogate mother can then produce an FO
generation non-human animal comprising a CRISPR reporter.
[0277] The methods can further comprise identifying a cell or
animal having a modified target genomic locus. Various methods can
be used to identify cells and animals having a targeted genetic
modification.
[0278] The screening step can comprise, for example, a quantitative
assay for assessing modification of allele (MOA) of a parental
chromosome. For example, the quantitative assay can be carried out
via a quantitative PCR, such as a real-time PCR (qPCR). The
real-time PCR can utilize a first primer set that recognizes the
target locus and a second primer set that recognizes a non-targeted
reference locus. The primer set can comprise a fluorescent probe
that recognizes the amplified sequence.
[0279] Other examples of suitable quantitative assays include
fluorescence-mediated in situ hybridization (FISH), comparative
genomic hybridization, isothermic DNA amplification, quantitative
hybridization to an immobilized probe(s), INVADER.RTM. Probes,
TAQMAN.RTM. Molecular Beacon probes, or ECLIPSE.TM. probe
technology (see, e.g., US 2005/0144655, incorporated herein by
reference in its entirety for all purposes).
[0280] An example of a suitable pluripotent cell is an embryonic
stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The
modified pluripotent cell can be generated, for example, by (a)
introducing into the cell one or more targeting vectors comprising
an insert nucleic acid flanked by 5' and 3' homology arms
corresponding to 5' and 3' target sites, wherein the insert nucleic
acid comprises a CRISPR reporter; and (b) identifying at least one
cell comprising in its genome the insert nucleic acid integrated at
the target genomic locus. Alternatively, the modified pluripotent
cell can be generated by (a) introducing into the cell: (i) a
nuclease agent, wherein the nuclease agent induces a nick or
double-strand break at a target sequence within the target genomic
locus; and (ii) one or more targeting vectors 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 target
sequence, wherein the insert nucleic acid comprises a CRISPR
reporter; and (c) identifying at least one cell comprising a
modification (e.g., integration of the insert nucleic acid) at the
target genomic locus. Any nuclease agent that induces a nick or
double-strand break into a desired target sequence can be used.
Examples of suitable nucleases include a Transcription
Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease
(ZFN), a meganuclease, and Clustered Regularly Interspersed Short
Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems or
components of such systems (e.g., CRISPR/Cas9). See, e.g., US
2013/0309670 and US 2015/0159175, each of which is herein
incorporated by reference in its entirety for all purposes.
[0281] The donor cell can be introduced into a host embryo at any
stage, such as the blastocyst stage or the pre-morula stage (i.e.,
the 4 cell stage or the 8 cell stage). Progeny that are capable of
transmitting the genetic modification though the germline are
generated. See, e.g., U.S. Pat. No. 7,294,754, herein incorporated
by reference in its entirety for all purposes.
[0282] Alternatively, the method of producing the non-human animals
described elsewhere herein can comprise: (1) modifying the genome
of a one-cell stage embryo to comprise the CRISPR reporter using
the methods described above for modifying pluripotent cells; (2)
selecting the genetically modified embryo; and (3) implanting and
gestating the genetically modified embryo into a surrogate mother.
Progeny that are capable of transmitting the genetic modification
though the germline are generated.
[0283] Nuclear transfer techniques can also be used to generate the
non-human mammalian animals. Briefly, methods for nuclear transfer
can include the steps of: (1) enucleating an oocyte or providing an
enucleated oocyte; (2) isolating or providing a donor cell or
nucleus to be combined with the enucleated oocyte; (3) inserting
the cell or nucleus into the enucleated oocyte to form a
reconstituted cell; (4) implanting the reconstituted cell into the
womb of an animal to form an embryo; and (5) allowing the embryo to
develop. In such methods, oocytes are generally retrieved from
deceased animals, although they may be isolated also from either
oviducts and/or ovaries of live animals. Oocytes can be matured in
a variety of well-known media prior to enucleation. Enucleation of
the oocyte can be performed in a number of well-known manners.
Insertion of the donor cell or nucleus into the enucleated oocyte
to form a reconstituted cell can be by microinjection of a donor
cell under the zona pellucida prior to fusion. Fusion may be
induced by application of a DC electrical pulse across the
contact/fusion plane (electrofusion), by exposure of the cells to
fusion-promoting chemicals, such as polyethylene glycol, or by way
of an inactivated virus, such as the Sendai virus. A reconstituted
cell can be activated by electrical and/or non-electrical means
before, during, and/or after fusion of the nuclear donor and
recipient oocyte. Activation methods include electric pulses,
chemically induced shock, penetration by sperm, increasing levels
of divalent cations in the oocyte, and reducing phosphorylation of
cellular proteins (as by way of kinase inhibitors) in the oocyte.
The activated reconstituted cells, or embryos, can be cultured in
well-known media and then transferred to the womb of an animal.
See, e.g., US 2008/0092249, WO 1999/005266, US 2004/0177390, WO
2008/017234, and U.S. Pat. No. 7,612,250, each of which is herein
incorporated by reference in its entirety for all purposes.
[0284] The various methods provided herein allow for the generation
of a genetically modified non-human FO animal wherein the cells of
the genetically modified FO animal comprise the CRISPR reporter. It
is recognized that depending on the method used to generate the FO
animal, the number of cells within the FO animal that have the
CRISPR reporter will vary. The introduction of the donor ES cells
into a pre-morula stage embryo from a corresponding organism (e.g.,
an 8-cell stage mouse embryo) via for example, the VELOCIMOUSE.RTM.
method allows for a greater percentage of the cell population of
the FO animal to comprise cells having the nucleotide sequence of
interest comprising the targeted genetic modification. For example,
at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the
cellular contribution of the non-human FO animal can comprise a
cell population having the targeted modification.
[0285] The cells of the genetically modified FO animal can be
heterozygous for the CRISPR reporter or can be homozygous for the
CRISPR reporter.
[0286] All patent filings, websites, other publications, accession
numbers and the like cited above or below are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual item were specifically and individually
indicated to be so incorporated by reference. If different versions
of a sequence are associated with an accession number at different
times, the version associated with the accession number at the
effective filing date of this application is meant. The effective
filing date means the earlier of the actual filing date or filing
date of a priority application referring to the accession number if
applicable. Likewise, if different versions of a publication,
website or the like are published at different times, the version
most recently published at the effective filing date of the
application is meant unless otherwise indicated. Any feature, step,
element, embodiment, or aspect of the invention can be used in
combination with any other unless specifically indicated otherwise.
Although the present invention has been described in some detail by
way of illustration and example for purposes of clarity and
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
BRIEF DESCRIPTION OF THE SEQUENCES
[0287] The nucleotide and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three-letter code for amino
acids. The nucleotide sequences follow the standard convention of
beginning at the 5' end of the sequence and proceeding forward
(i.e., from left to right in each line) to the 3' end. Only one
strand of each nucleotide sequence is shown, but the complementary
strand is understood to be included by any reference to the
displayed strand. When a nucleotide sequence encoding an amino acid
sequence is provided, it is understood that codon degenerate
variants thereof that encode the same amino acid sequence are also
provided. The amino acid sequences follow the standard convention
of beginning at the amino terminus of the sequence and proceeding
forward (i.e., from left to right in each line) to the carboxy
terminus.
TABLE-US-00004 TABLE 2 Description of Sequences. SEQ ID NO Type
Description 1 DNA Complementary Strand of Pgk Poly(A) Excision
Guide RNA Recognition Sequence v1 2 RNA Pgk Poly(A) Excision Guide
Sequence v1 (gRNA#16; pA removal guide) 3 Protein T2A 4 Protein P2A
5 Protein E2A 6 Protein F2A 7 RNA Generic Guide RNA Scaffold v.2 8
RNA Generic Guide RNA Scaffold v.3 9 RNA Generic Guide RNA Scaffold
v.4 10 DNA Generic Guide RNA Target Sequence Plus PAM v.1 11 DNA
Generic Guide RNA Target Sequence Plus PAM v.2 12 DNA Generic Guide
RNA Target Sequence Plus PAM v.3 13 DNA Complementary Strand of
eBFP Guide RNA Recognition Sequence 14 RNA eBFP Guide Sequence 15
DNA eBFP to eGFP Repair Donor - ssODN F 16 DNA eBFP to eGFP Repair
Donor - ssODN R 17 DNA LSL-LacZ-P2A-eBFP/eGFP Reporter Allele
(MAID2634) 18 DNA LSL-eBFP/eGFP Reporter Allele (MAID2652) 19 DNA
Complementary Strand of Pgk Poly(A) Excision Guide RNA Recognition
Sequence v2 20 RNA Pgk Poly(A) Excision Guide Sequence v2
(LSL_LacZ_gU2) 21 DNA Complementary Strand of Pgk Poly(A) Excision
Guide RNA Recognition Sequence v3 22 RNA Pgk Poly(A) Excision Guide
Sequence v3 (LSL_LacZ_gU3) 23 DNA Complementary Strand of Pgk
Poly(A) Excision Guide RNA Recognition Sequence v4 24 RNA Pgk
Poly(A) Excision Guide Sequence v4 (LSL_LacZ_gU1) 25 DNA
Complementary Strand of Pgk Poly(A) Excision Guide RNA Recognition
Sequence v5 26 RNA Pgk Poly(A) Excision Guide Sequence v5
(LSL_LacZ_gD2) 27 DNA Complementary Strand of Pgk Poly(A) Excision
Guide RNA Recognition Sequence v6 28 RNA Pgk Poly(A) Excision Guide
Sequence v6 (LSL_LacZ_gD1) 29 RNA Pgk Poly(A) Disruption Guide
Sequence v1 (cGM2) 30 RNA Pgk Poly(A) Disruption Guide Sequence v2
(cGM) 31 RNA Pgk Poly(A) Disruption Guide Sequence v3 (cGM3) 32 DNA
Complementary Strand of Pgk Poly(A) Disruption Guide RNA
Recognition Sequence v1 (cGM2) 33 DNA Complementary Strand of Pgk
Poly(A) Disruption Guide RNA Recognition Sequence v2 (cGM) 34 DNA
Complementary Strand of Pgk Poly(A) Disruption Guide RNA
Recognition Sequence v3 (cGM3) 35 RNA Pgk Poly(A) Disruption Guide
Sequence v4 cGM4 36 RNA Pgk Poly(A) Disruption Guide Sequence v5
cGM5 37 RNA crRNA Tail 38 RNA TracrRNA 39 RNA Generic Guide RNA
Scaffold v.1 40 RNA Cre mRNA 41 DNA Pgk Poly(A) Excision Guide RNA
Target Sequence v1 (gRNA#16; pA removal guide) 42 DNA eBFP Guide
RNA Target Sequence 43 DNA Pgk Poly(A) Excision Guide RNA Target
Sequence v2 (LSL_LacZ_ gU2) 44 DNA Pgk Poly(A) Excision Guide RNA
Target Sequence v3 (LSL_LacZ_gU3) 45 DNA Pgk Poly(A) Excision Guide
RNA Target Sequence v4 (LSL_LacZ_gU1) 46 DNA Pgk Poly(A) Excision
Guide RNA Target Sequence v5 (LSL_LacZ_gD2) 47 DNA Pgk Poly(A)
Excision Guide RNA Target Sequence v6 (LSL_LacZ_gD1) 48 DNA Pgk
Poly(A) Disruption Guide RNA Target Sequence v1 (cGM2) 49 DNA Pgk
Poly(A) Disruption Guide RNA Target Sequence v2 (cGM) 50 DNA Pgk
Poly(A) Disruption Guide RNA Target Sequence v3 (cGM3) 51 DNA Pgk
Poly(A) Disruption Guide RNA Target Sequence v4 cGM4 52 DNA Pgk
Poly(A) Disruption Guide RNA Target Sequence v5 cGM5 53 Protein
Cas9 Protein 54 DNA Cas9 Coding Sequence 55 RNA eBFP Guide Sequence
56 DNA eBFP Guide Target Sequence 57 DNA L-eBFP CRISPR Reporter
Allele (MAID20090) 58 DNA L-LacZ: eBFP CRISPR Reporter Allele
(MAID2809) 59 DNA L-LacZ: eGFP CRISPR Reporter Allele 60 DNA L-eGFP
CRISPR Reporter Allele
EXAMPLES
Example 1. Validation of CRISPR Reporters
[0288] The CRISPR/Cas9 technology is a promising new therapeutic
modality. Assessing the efficiency of mutation generation or
targeted gene modification by an introduced CRISPR/Cas9 agent in
vivo currently relies on difficult molecular assays, such as
single-strand DNase sensitivity assays, digital PCR, or next
generation sequencing.
[0289] CRISPR/Cas9, an RNA-guided DNA endonuclease, catalyzes the
double strand break (DSB) of DNA at the binding site of its RNA
guide. The RNA guide can consist of a 42-nucleotide CRISPR RNA
(crRNA) that joins with an 87-nucleotide trans-activating RNA
(tracrRNA). The tracrRNA is complementary to and base pairs with
the crRNA to form a functional crRNA/tracrRNA guide. This duplex
RNA becomes bound to the Cas9 protein to form an active
ribonucleoprotein (RNP) that can interrogate the genome for the
specific seed sequence. A secondary requirement for strand breakage
is that the Cas9 protein must recognize a protospacer adjacent
motif (PAM) directly adjacent to the 3' CRISPR target sequence.
Alternatively, an active RNP complex can also be formed by
replacing the crRNA/tracrRNA duplex with a single chimeric RNA
(sgRNA). This sgRNA can be formed by fusing the 20 nucleotide seed
sequence directly to the processed tracrRNA sequence. The sgRNA can
interact with both the Cas9 protein and the DNA in the same way and
with similar efficiency as the crRNA/tracrRNA duplex would. This
bacterial natural defense mechanism has been shown to function
effectively in mammalian cells and activate break induced
endogenous repair pathways. When a double strand break occurs in
the genome, repair pathways will attempt to fix the DNA by either
the error prone non-homologous end joining (NHEJ) pathway or
homology-directed repair (HDR) if an appropriate template is
available. We can leverage these pathways to facilitate site
specific deletion of genomic regions or HDR in mammalian cells.
[0290] To provide better assays of CRISPR/Cas9 delivery to and
activity in tissues and organs of a live animal, mice were
developed carrying genetic alleles that have the ability to report
CRISPR/Cas9-induced non-homologous end joining (NHEJ) (e.g.,
Cas9-mediated excision caused by a pair of Cas9-mediated cleavage
events) and/or CRISPR/Cas9-induced homology-directed repair (HDR)
using a donor sequence to convert eBFP to eGFP (or eGFP to eBFP)
following a Cas9-mediated single-strand or double-strand cleavage
event. The CRISPR reporter alleles described in this example are
based on modification of the mouse Gt(ROSA)26Sor (Rosa26) locus.
The Rosa26 locus exhibits strong and ubiquitous expression of a
long non-coding RNA of unknown function. Mice with a homozygous
deletion of Rosa26 are viable, healthy, and fertile. The first
general property of the first CRISPR reporter allele described
herein is that CRISPR/Cas9-induced excision of a polyadenylation
signal will activate expression of reporter proteins being
expressed from the Rosa26 promoter, and the reporter proteins can
then be detected by enzymatic activity or by fluorescence (or an
immune assay or other means). The second general property of the
first CRISPR reporter allele described herein (and the property of
the second CRISPR reporter allele described herein) is that
CRISPR/Cas9-induced recombination of a gene encoding a first
fluorescent reporter protein with a donor sequence will convert the
first fluorescent reporter protein being expressed from the Rosa26
promoter into a different second fluorescent reporter protein which
can then be detected by fluorescence. The CRISPR reporter alleles
described in this example were targeted to the first intron of the
Rosa26 locus (see FIG. 2) and take advantage of the strong
universal expression of the Rosa26 locus and the ease of targeting
the Rosa26 locus.
[0291] The first CRISPR reporter allele (i.e., the LSL-LacZ:eBFP
CRISPR reporter allele) is depicted in FIG. 1A and in SEQ ID NO:
17. It uses lacZ as a high definition histological reporter and
eBFP (or alternatively eGFP) as a fluorescent reporter of the
extent and location of CRISPR/Cas9-mediated NHEJ action (e.g.,
excision of a target sequence) in the liver or other target organs
and uses eGFP (or alternatively eBFP) as a fluorescent reporter of
the extent and location of delivered CRISPR/Cas9-induced HDR
following recombination with a donor nucleic acid in the liver or
other target organs. In addition, if only one organ is being
targeted, the CRISPR reporter allele can also be used to assess
off-target effects in other organs and tissues. Accordingly, the
CRISPR reporter allele can be used to test and optimize CRISPR/Cas9
delivery methods in vivo. The components of the first CRISPR
reporter allele from 5' to 3' are shown in Table 3 and in SEQ ID
NO: 17. The sequence of the first CRISPR reporter allele after
treatment with Cre recombinase to remove the first Pgk
polyadenylation signal is set forth in SEQ ID NO: 58. The sequence
of the Cre-treated reporter allele after conversion of eBFP to eGFP
(e.g., after treatment with ssODN as described in more detail
below) is set forth in SEQ ID NO: 59.
TABLE-US-00005 TABLE 3 LSL-LacZ: eBFP CRISPR Reporter Allele.
Nucleotide Region(s) Within SEQ Component ID NO: 17 First loxP site
248-281 Pgk poly(A) excision guide RNA target sequence 305-327 v1A
for excising polyadenylation signal First Pgk polyadenylation
signal 336-796 Poly(A) recognition motif AATAAA 371-376 Multiple
Pgk poly(A) disruption guide RNA target 337-356 sequences within
the Pgk polyadenylation signal 385-404 405-424 452-471 571-590 Pgk
poly(A) excision guide RNA target sequence 797-819 v1B for excising
polyadenylation signal Second loxP site 820-853 LacZ gene 862-3930
P2A coding sequence 3931-3996 eBFP coding sequence 3997-4713 eBFP
guide RNA target sequence within eBFP coding 4193-4212 sequence for
converting eBFP to eGFP SV40 polyadenylation signal 4748-4969 Frt
site 4976-5023 Human ubiquitin C promoter 5030-6242 EM7 promoter
6243-6309 Sequence encoding neomycin phosphotransferase for
6310-7113 resistance to neomycin family antibiotics (e.g. G418)
Second Pgk polyadenylation signal 7122-7598 Second Frt site
7609-7656
[0292] Alternate guide RNA target sequences flanking the first Pgk
polyadenylation signal can also be used. A table summarizing
different guide RNA target sequences in the first CRISPR reporter,
their nucleotide positions in the first CRISPR reporter, and the
guide sequences within the corresponding guide RNAs targeting those
guide RNA target sequences are provided in Table 4. Table 4 also
provide the positions of the first and second loxP sites, the first
Pgk polyadenylation signal, and the eBFP coding sequence for
reference.
TABLE-US-00006 TABLE 4 Locations of Guide RNA Target Sequences in
LSL-LacZ: eBFP CRISPR Reporter Allele. Nucleotide Region Within
Guide RNA Target Sequence or Other Region SEQ ID NO: Guide Sequence
in Guide RNA (SEQ ID NO) 17 (SEQ ID NO) Pgk poly(A) excision guide
RNA target sequence 215-234 Pgk poly(A) excision guide sequence v2
v2 (SEQ ID NO: 43) (SEQ ID NO: 20) Pgk poly(A) excision guide RNA
target sequence 243-262 Pgk poly(A) excision guide sequence v4 v4
(SEQ ID NO: 45) (SEQ ID NO: 24) First loxP site 248-281 N/A Pgk
poly(A) excision guide RNA target sequence 263-282 Pgk poly(A)
excision guide sequence v3 v3 (SEQ ID NO: 44) (SEQ ID NO: 22) Pgk
poly(A) excision guide RNA target sequence 305-327 Pgk poly(A)
excision guide sequence v1A (SEQ ID NO: 41) v1A (SEQ ID NO: 2)
First Pgk polyadenylation signal 336-796 N/A Pgk Poly(A) disruption
guide RNA target 357-356 Pgk Poly(A) disruption guide sequence
sequence v4 (SEQ ID NO: 51) v4 (SEQ ID NO: 35) Pgk Poly(A)
disruption guide RNA target 385-404 Pgk Poly(A) disruption guide
sequence sequence v5 (SEQ ID NO: 52) v5 (SEQ ID NO: 36) Pgk Poly(A)
disruption guide RNA target 405-424 Pgk Poly(A) disruption guide
sequence sequence v1 (SEQ ID NO: 48) v1 (SEQ ID NO: 29) Pgk Poly(A)
disruption guide RNA target 452-471 Pgk Poly(A) disruption guide
sequence sequence v2 (SEQ ID NO: 49) v2 (SEQ ID NO: 30) Pgk Poly(A)
disruption guide RNA target 571-590 Pgk Poly(A) disruption guide
sequence sequence v3 (SEQ ID NO: 50) v3 (SEQ ID NO: 31) Pgk poly(A)
excision guide RNA target sequence 794-813 Pgk poly(A) excision
guide sequence v5 v5 (SEQ ID NO: 46) (SEQ ID NO: 26) Pgk poly(A)
excision guide RNA target sequence 797-819 Pgk poly(A) excision
guide sequence v1B v1B (SEQ ID NO: 41) (SEQ ID NO: 2) Pgk poly(A)
excision guide RNA target sequence 803-822 Pgk poly(A) excision
guide sequence v6 v6 (SEQ ID NO: 47) (SEQ ID NO: 28) Second loxP
site 820-853 N/A eBFP coding sequence 3997-4713 N/A eBFP Guide RNA
target sequence 4193-4212 eBFP guide sequence (SEQ ID NO: 14) (SEQ
ID NO: 42)
[0293] The first Pgk polyadenylation signal normally blocks
expression of the beta-galactosidase protein from the lacZ gene and
the eBFP protein. Upon excision of the first Pgk polyadenylation
signal following cleavage of the guide RNA target sequences
flanking the first Pgk polyadenylation signal (SEQ ID NO: 41 for
each, or SEQ ID NO: 41, 43, 44, or 45 for the first guide RNA
target sequence and SEQ ID NO: 41, 46, or 47 for the second guide
RNA target sequence) by a Cas9 nuclease, the lacZ gene and the eBFP
coding sequence will normally be expressed. A beta-galactosidase
protein and an eBFP protein are then expressed and can be used to
quantify the cells modified by the CRISPR/Cas9-induced excision via
NHEJ.
[0294] Upon recognition and cleavage of the guide RNA target
sequence within the eBFP coding sequence (SEQ ID NO: 42) by a Cas9
nuclease and induction of repair with a donor sequence, the eBFP
coding sequence in the CRISPR reporter allele can be repaired by
homology-directed repair to convert it into an eGFP coding
sequence. An eGFP protein is then expressed and can be used to
quantify the cells modified by the combination of the CRISPR/Cas9
and the donor sequence via HDR. The guide sequence of a guide RNA
used to target the eBFP coding sequence comprises the sequence set
forth in SEQ ID NO: 14, and a donor nucleic acid that can be used
to repair the eBFP coding sequence and convert it to an eGFP coding
sequence is set forth in SEQ ID NO: 15 or SEQ ID NO: 16.
[0295] The second CRISPR reporter allele (i.e., the LSL-eBFP CRISPR
reporter allele) is depicted in FIG. 3 and SEQ ID NO: 18 and can be
used to directly assess CRISPR/Cas9-induced HDR. It uses eGFP (or
alternatively eBFP) as a fluorescent reporter of the extent and
location of CRISPR/Cas9-induced HDR following recombination with a
donor nucleic acid in the liver or other target organs. In
addition, if only one organ is being targeted, the CRISPR reporter
allele can also be used to assess off-target effects in other
organs and tissues. Accordingly, the CRISPR reporter allele can be
used to test and optimize CRISPR/Cas9 delivery methods in vivo. The
components of the second CRISPR reporter allele from 5' to 3' are
shown in the Table 5 and SEQ ID NO: 18. The sequence of the second
CRISPR reporter allele after treatment with Cre recombinase to
remove the selection cassette is set forth in SEQ ID NO: 57. The
sequence of the Cre-treated reporter allele after conversion of
eBFP to eGFP (e.g., after treatment with ssODN as described in more
detail below) is set forth in SEQ ID NO: 60).
TABLE-US-00007 TABLE 5 LSL-eBFP CRISPR Reporter Allele. Nucleotide
Region Within Component SEQ ID NO: 18 First loxP site 226-259 EM7
promoter 357-423 Sequence encoding neomycin phosphotransferase
424-1227 for resistance to neomycin family antibiotics (e.g. G418)
Triple polyadenylation signal 1236-2489 Second loxP site 2517-2550
eBFP coding sequence 2596-3315 Guide RNA target sequence within
eBFP coding 2795-2814 sequence for converting eBFP to eGFP SV40
polyadenylation signal 3350-3571
[0296] The triple polyadenylation signal blocks expression of the
eBFP protein. Upon excision of the polyadenylation signal by a Cre
recombinase, the eBFP coding sequence will be expressed. Upon
recognition and cleavage of the guide RNA target sequence within
the eBFP coding sequence (SEQ ID NO: 42) by a Cas9 nuclease and
induction of repair with a donor sequence, the eBFP coding sequence
in CRISPR reporter allele can be repaired by homology-directed
repair to convert it into an eGFP coding sequence. An eGFP protein
is then expressed and can be used to quantify the cells modified by
the combination of the CRISPR/Cas9 and the donor sequence via HDR.
The guide sequence of the guide RNA used to target the eBFP coding
sequence comprises the sequence set forth in SEQ ID NO: 14, and a
donor nucleic acid that can be used to repair the eBFP coding
sequence and convert it to an eGFP coding sequence is set forth in
SEQ ID NO: 15 or SEQ ID NO: 16.
[0297] LacZ is a naturally occurring gene in E. coli that encodes
the protein beta-galactosidase. This protein is responsible for the
breakdown of lactose by cleaving the bond between the two carbon
rings in lactose to produce glucose and galactose. Originally used
in E. coli, lacZ is an important gene in research as it can be used
as a histochemical reporter. When in the presence of lactose analog
X-Gal, beta-galactosidase will hydrolyze the substrate to produce a
blue color that is easily visualized under a microscope.
[0298] Enhanced Green Fluorescent Protein (eGFP) is a protein that
emits a green fluorescence when exposed to light in the blue to
ultraviolet range. eGFP was derived from the GFP originally
isolated from the jellyfish Aequorea victoria. Mutations were
engineered that increased fluorescence and photostability as well
as allowed proper folding at 37.degree. C. for use in mammalian
cells. The major excitation peak is 488 nanometers (nm) with peak
emission at 509 nm. A single mutation, Y66H, converts eGFP into
enhanced Blue Fluorescent Protein (eBFP) with major excitation peak
is shifted to 380 nm with peak emission at 448 nm. We have
engineered the expression of lacZ and a P2A linked eBFP into the
first intron of mouse Rosa26 locus with a preceding floxed poly(A)
sequence and appropriate splicing signals. Prior to floxing out the
poly(A), the lacZ and eBFP coding sequences will not be expressed
as the poly(A) region will block transcription. Upon floxing out
the poly(A), beta-galactosidase and eBFP proteins will be
constitutively expressed by the Rosa26 promoter. Alternatively, the
poly(A) can be removed using the Cas9 system to delete the region
by incorporating two sgRNA sites flanking the sequence. Pre- and
post-floxed targeted cells are first verified by TAQMAN.RTM.
quantitative Polymerase Chain Reaction (qPCR) to detect the single,
site specific, integration of the targeting vector at Rosa26.
[0299] The Lox-Stop-Lox (LSL) LacZ-2A-eBFP allele as depicted in
FIG. 1A (or alternatively, a LSL LacZ-2A-eGFP allele) can be an
effective NHEJ and HDR reporter in mouse embryonic stem cells
(mESCs) as well as in adult mouse tissue. By incorporating this
allele into Rosa26, lacZ or eBFP will not normally not be expressed
in cells and tissues. Introduction of the sgRNA to drop out the
poly(A) along with Cas9 protein can turn on lacZ and eBFP
expression. If no repair template is provided, this deletion can
take place using the NHEJ pathway. In this way, this allele can be
used to indicate when and where NHEJ has taken place within an
adult mouse. Further, a guide RNA can then be introduced to induce
double strand break in eBFP, and a repair donor containing an H66Y
mutation to convert eBFP to eGFP (or alternatively, for a LSL
LacZ-2A-eGFP allele, a repair donor containing a Y66H mutation to
convert eGFP to eBFP).
[0300] LacZ can be turned on in mESCs targeted with the CRISPR
allele as shown in FIG. 1A by deleting the poly(A) region by either
the Cre-Lox system or Cas9-induced NHEJ. To test the ability of
CRISPR/Cas9 to delete the poly(A) region in the LSL-LacZ:eBFP
CRISPR reporter allele, embryonic stem cells comprising the
reporter allele were electroporated with ribonucleoprotein
complexes of Cas9 and various guide RNAs designed to target
upstream and downstream of the poly(A) region. The cells were
plated onto 6-well plates (3.times.10.sup.5 cells) and stained for
LacZ for two hours 3 days post-electroporation. The results are
shown in FIGS. 4A-4E. The figures show untreated cells (FIG. 4A),
cells electroporated with a plasmid encoding Cre recombinase (FIG.
4B), and cells electroporated with ribonucleoprotein complexes
comprising Cas9 protein complexed together with synthetic sgRNAs
targeting guide RNA target sequences flanking the Pgk
polyadenylation signal upstream of the lacZ gene (FIGS. 4C-4E). As
shown in FIG. 4B compared to FIG. 4A, treatment with the Cre
recombinase plasmid activated expression of lacZ. Likewise, as
shown in FIGS. 4C-4E compared to FIG. 4A, treatment with the
CRISPR/Cas9 activated expression of lacZ, confirming that the
upstream polyadenylation signal had been excised. The cells in FIG.
4C were targeted with the gU3 and gD1 sgRNAs targeting Pgk poly(A)
excision guide RNA target sequences v3 and v6 (SEQ ID NOS: 44 and
47, respectively). These guide RNAs included the sequences set
forth in SEQ ID NOS: 22 and 28, respectively. The cells in FIG. 4D
were targeted with the gU3 and gD2 sgRNAs targeting Pgk poly(A)
excision guide RNA target sequences v3 and v5 (SEQ ID NOS: 44 and
46, respectively). These guide RNAs included the sequences set
forth in SEQ ID NOS: 22 and 26, respectively. The cells in FIG. 4E
were targeted with the gU2 and gD1 sgRNAs targeting Pgk poly(A)
excision guide RNA target sequences v2 and v6 (SEQ ID NOS: 43 and
47, respectively). These guide RNAs included the sequences set
forth in SEQ ID NOS: 20 and 28, respectively. Similar results were
obtained for the combination of the gU1 and gD1 sgRNAs, the
combination of the gU1 and gD2 sgRNAs, and the gU2 and the gD2
sgRNAs (data not shown).
[0301] Similar experiments were done in mouse ES cells comprising
the LSL-LacZ:eBFP CRISPR reporter allele by treating with three
other combinations of guide RNAs that target within the Pgk
polyadenylation signal (see FIG. 1B): cGM4+cGM5, cGM4+cGM, and
cGM4+cGM3. The guide RNA target sequences for cGM, cGM3, cGM4, and
cGM5 are set forth in SEQ ID NOS: 49, 50, 51, and 52, respectively,
and the guide RNA guide sequences are set forth in SEQ ID NOS: 30,
31, 35, and 36, respectively. The results are shown in FIG. 5. Cre
was used as a positive control, and Cas9-only or Cas9+a non-cutting
guide RNA (9172) were used as negative controls. Treatment with
Cas9 and each of the three combinations of guide RNAs activated
expression of lacZ, confirming that the upstream polyadenylation
signal had been excised. Likewise, a similar experiment was done
with guide RNA #16, which has target sequences both upstream and
downstream of the polyadenylation signal. The guide RNA target
sequence for gRNA #16 is set forth in SEQ ID NO: 41, and the guide
sequence of the guide RNA is set forth in SEQ ID NO: 2. As shown in
FIG. 6, treatment with Cas9 and guide RNA #16 activated expression
of lacZ, confirming that the upstream polyadenylation signal had
been excised.
[0302] To determine the effectiveness of lacZ as an NHEJ readout in
adult mice, mESCs targeted with the CRISPR reporter allele shown in
FIG. 1A were microinjected into 8-cell mouse embryos using the
VELOCIMOUSE.RTM. method. See, e.g., U.S. Pat. Nos. 7,576,259;
7,659,442; 7,294,754; US 2008/007800; and Poueymirou et al. (2007)
Nature Biotech. 25(1):91-99, each of which is herein incorporated
by reference in its entirety for all purposes. Specifically, a
small hole was created in the zona pellucida to facilitate the
injection of targeted mESCs. These injected 8-cell embryos were
transferred to surrogate mothers to produce live pups carrying the
transgene. Upon gestation in a surrogate mother, the injected
embryos produced FO mice that carry no detectable host embryo
contribution. The fully ES cell-derived mice were normal, healthy,
and fertile (with germline transmission). This allele can be used
to evaluate the off-target editing potential of the Cas9 system.
When Cas9 and sgRNA are introduced to a mouse via tail vein
injection of lipid nanoparticles (LNP) or adeno-associated virus
(AAV), they can edit the liver. Assessment of editing in other
tissues can also be undertaken using these delivery methods.
Post-injection, the various mouse tissues are harvested, and lacZ
staining is completed. All tissues that are edited by the
introduced Cas9 and sgRNA express lacZ, thus allowing a
determination if any unexpected tissues are affected by Cas9
editing. This system can be used to evaluate additional delivery
methods as well as the editing potential of various AAV
serotypes.
[0303] Mice comprising the LSL-LacZ:eBFP CRISPR reporter allele
integrated at the Rosa26 locus were dosed in the following groups:
(1) polyA removal guide RNA (gRNA #16)+Cas9 lipid nanoparticle
(LNP) (4 mice); (2) gU2 guide RNA+gD1 guide RNA+Cas9 LNP (4 mice);
(3) non-cutting guide RNA+Cas9 LNP (2 mice); and (4) Cre
recombinase LNP (1 mouse). The animals were dosed with 2 mg/kg LNP
in 200 .mu.L tris-saline sucrose buffer by tail vein injection.
Cas9 was in the form of mRNA, gRNA was in the form of RNA, and Cre
recombinase was in the form of mRNA. One week post-injection,
animals were harvested, and tissues were collected for whole mount
imaging/lacZ staining, for fixing in 10% formalin for
histology/immunohistochemistry, and for next-generation sequencing
(NGS). The tissues that were collected included liver, spleen,
pancreas, kidney, skeletal muscle, gonad, heart, lung, and brain.
Results of whole mount lacZ staining of livers after 72 hours in
beta-galactosidase are shown in FIG. 7. Briefly, the livers were
fixed in PFA for 30 minutes on ice and were then rinsed three times
for 20 minutes on ice. Staining with X-Gal was for 72 hours on ice,
with agitation and washing in PBS. Livers were fixed in formalin
overnight at 4.degree. C. and then washed with PBS. The Cre-treated
mice showed abundant lacZ staining, whereas the mice treated with
the non-cutting guide RNA showed no staining. The two test mouse
groups--polyA removal guide RNA or the combination of the gU2 and
gD1 guide RNAs--both showed lacZ staining, indicating that the
upstream polyadenylation signal had been excised in the livers of
the mice in vivo. These results are consistent with the lacZ
immunohistochemistry results shown in FIG. 8. Briefly,
formalin-fixed paraffin embedded liver sections were deparrafinized
followed by an antigen retrieval and then blocked. The slides were
then incubated with an antibody against LACZ and bound with a
secondary antibody conjugated to HRP (horseradish peroxidase).
These tissues were then incubated in DAB until the positive cells
exhibited a brown color. The slides were then scanned and positive
cells identified. Not surprisingly, the staining was lower than
that for Cre recombinase because the Cre recombinase allows for
easier excision of the Pgk pA, while the guide RNAs are relying on
the efficiency of NHEJ and the coinciding of the gRNA+Cas9 complex
forming at two different location for collapse to occur. Similarly,
these results are also consistent with the next-generation
sequencing results from livers harvested from the various mice as
shown in FIG. 11. As shown in FIG. 11, the editing efficiency
(i.e., number of reads with a deletion between the gRNA target
sequences) was higher for the gU2+gD1+Cas9 mice and the polyA
removal guide RNA+Cas9 mice than the negative control mice
(non-cutting guide RNA plus Cas9, or gU2+gD1 with no Cas9. Similar
to the whole mount staining and the immunohistochemistry, Cre
recombinase treatment resulted in even higher editing levels. These
whole mount staining, immunohistochemistry, and NGS results
indicate that the LSL-LacZ:eBFP CRISPR reporter can effectively be
used as a reporter for CRISPR/Cas-mediated NHEJ activity in vivo.
Different guide RNA target sequences can be engineered into the
reporter to test different guide RNAs.
[0304] To test the ability of CRISPR/Cas9 to convert eBFP to eGFP
via homology-directed repair in the LSL-LacZ:eBFP CRISPR reporter
allele, embryonic stem cells comprising a Cre-recombinase-treated
reporter allele genomically integrated at the Rosa26 locus (i.e.,
L-LacZ:eBFP CRISPR reporter allele with the upstream
polyadenylation signal has been removed) are electroporated with
Cas9 (20 .mu.g) and a guide RNA that targets a target sequence in
eBFP (2.5 .mu.g) in the form of a ribonucleoprotein complex and a
ssODN (35 .mu.g) designed to convert eBFP to eGFP. The guide RNA
target sequence is set forth in SEQ ID NO: 42, and the guide
sequence of the guide RNA is set forth in SEQ ID NO: 14. Either of
two ssODNs (F and R, representing "forward" and "reverse"
complementary single strands; SEQ ID NOS: 15 and 16, respectively)
are used.
[0305] A Lox-Stop-Lox (LSL) eBFP/eGFP allele as depicted in FIG. 3
can also be an effective HDR reporter in mouse embryonic stem cells
(mESCs) as well as in adult mouse tissue. By incorporating the
LSL-eBFP allele into Rosa26, eBFP will not normally be expressed in
cells and tissues. If the triple polyadenylation signal-neomycin
cassette is removed with Cre, eBFP can express. A guide RNA can be
introduced to induce a double strand break in eBFP, and a repair
donor containing the H66Y mutation can be introduced to convert
eBFP to eGFP (or alternatively, for the LSL-eGFP reporter, induce a
double strand break in eGFP and introduce a repair donor containing
the Y66H mutation to convert eGFP to eBFP).
[0306] To test the ability of CRISPR/Cas9 to convert eBFP to eGFP
via homology-directed repair in the LSL-eBFP CRISPR reporter allele
in which the upstream polyadenylation signal has been removed by
Cre recombinase (L-eBFP CRISPR reporter allele), embryonic stem
cells comprising the reporter allele were electroporated with Cas9
(20 .mu.g) and a guide RNA that targets a target sequence in eBFP
(2.5 .mu.g) in the form of a ribonucleoprotein complex and a ssODN
(35 .mu.g) designed to convert eBFP to eGFP. The guide RNA target
sequence is set forth in SEQ ID NO: 42, and the guide sequence of
the guide RNA is set forth in SEQ ID NO: 14. Two ssODNs (F and R,
representing "forward" and "reverse" complementary single strands;
SEQ ID NOS: 15 and 16, respectively) were used in separate
experiments. As shown in FIG. 9, the CRISPR/Cas9 and ssODN F
successfully converted eBFP to eGFP, and the eGFP is expressed. The
top row in FIG. 9 shows brightfield images, and the bottom row
shows fluorescence microscopy images (eGFP).
[0307] Similar experiments were performed in hematopoietic stem
cells and progenitors cells ex vivo. To test the ability of
CRISPR/Cas9 to convert eBFP to eGFP via homology-directed repair in
the LSL-eBFP CRISPR reporter allele in which the upstream
polyadenylation signal has been removed (L-eBFP CRISPR reporter
allele), bone marrow cells were extracted from mice containing the
reporter integrated at the Rosa26 locus. Briefly, the femurs and
tibias were harvested from the mice, and the bone marrow was
extracted. Hematopoietic and progenitor cells (HSPC) were further
isolated using the EasySep kit from STEMCELL. The cells were plated
in 24-well plates at a density between 250,000 to 1,000,000 cells
per well in StemSpan SFEM (STEM cell #09650) media comprising SCF
at 100 ng/mL, TPO at 100 ng/mL, Flt3L at 100 ng/mL, IL-6 at 50
ng/mL, and IL-3 at 30 ng/mL. The cells were electroporated with
Cas9, a guide RNA that targets a target sequence in eBFP, and an
ssODN designed to convert eBFP to eGFP. The guide RNA target
sequence is set forth in SEQ ID NO: 42, and the guide sequence of
the guide RNA is set forth in SEQ ID NO: 14. The sequences of the
ssODNs used are set forth in SEQ ID NOS: 15 and 16.
[0308] Specifically, Cas9-sgRNA RNPs were used and were generated
by incubating 200 ng to 1 .mu.g of sgRNA with 1 .mu.g Cas9 protein
(PNA Bio) for 10-15 minutes at room temperature and then
electroporating into 10,000 murine hematopoietic stem and
progenitor cells (HSPCs) after 1-3 hours in culture. The optimized
electroporation condition for murine HSPCs is 1700V, 20 ms, and 1
pulse using Neon transfection system (Thermo Fisher Scientific).
The following EP conditions were tested using 10 nM ssODN+RNP of
Cas9+sgRNA: (1) 1720V 10 pulse width, 2 pulse, and cell density:
5e6; (2) 1680V 20 pulse width, 1 pulse, and cell density: 6e7; and
(3) 1700V 20 pulse width, 1 pulse, and cell density: 1e8.
Brightfield and fluorescence microscopy images 48 hours after
electroporation (1720V 10 pulse width, 2 pulse, cell density: 5e6)
with Cas9/sgRNA RNP and ssODN FW or Cas9/sgRNA RNP and ssODN REV
are shown in FIG. 10A. Untreated cells were used as a negative
control. Brightfield images are shown in the top row, and
fluorescence microscopy images (eGFP) are shown in the bottom row.
Images of the cells 7 days after electroporation are shown in FIG.
10B. The first column shows brightfield images, and the second and
third columns show fluorescence microscopy images (eGFP). The
fluorescence is especially clear in some larger, differentiated
cells. As shown in FIGS. 10A and 10B, the CRISPR/Cas9 and ssODN
successfully converted eBFP to eGFP ex vivo in primary cells
isolated from the L-eBFP reporter mice, and the eGFP is
expressed.
[0309] To determine the effectiveness of eGFP as an HDR readout in
adult mice in vivo, mESCs targeted with the CRISPR reporter allele
shown in FIG. 1A or 3 or Cre-recombinase-treated versions of these
alleles were microinjected into 8-cell mouse embryos using the
VELOCIMOUSE.RTM. method. See, e.g., U.S. Pat. Nos. 7,576,259;
7,659,442; 7,294,754; US 2008/007800; and Poueymirou et al. (2007)
Nature Biotech. 25(1):91-99, each of which is herein incorporated
by reference in its entirety for all purposes. Specifically, a
small hole was created in the zona pellucida to facilitate the
injection of targeted mESC. These injected 8-cell embryos were
transferred to surrogate mothers to produce live pups carrying the
transgene. Upon gestation in a surrogate mother, the injected
embryos produced FO mice that carried no detectable host embryo
contribution. The fully ES cell-derived mice were normal, healthy,
and fertile (with germline transmission). The mice can be used to
evaluate the feasibility of correcting a mutation in an adult mouse
livers using the Cas9 system. To this end, primary hepatocytes are
harvested from LSL-eBFP/eGFP targeted mice and are used to assess
methods of correcting a point mutation in these non-dividing cells.
The most efficient approach can be determined. All materials are
then introduced into the mouse via tail vein injection of lipid
nanoparticles or adeno-associated virus or by another suitable
delivery method. Livers or other tissues of these mice are
harvested, and assessment of eGFP expression is performed to look
for correctly modified cells. Next-generation sequencing is also
performed to look for correctly modified cells. Next-generation
sequencing and RNAseq can provide information on the types of cells
in the liver or other tissues in which the CRISPR/Cas9 is active.
Sequence CWU 1
1
60120DNAArtificial SequenceSynthetic 1gctggactac gtcgtgtgcc
20220RNAArtificial SequenceSynthetic 2cgaccugaug cagcucucgg
20318PRTArtificial SequenceSynthetic 3Glu Gly Arg Gly Ser Leu Leu
Thr Cys Gly Asp Val Glu Glu Asn Pro1 5 10 15Gly Pro419PRTArtificial
SequenceSynthetic 4Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp
Val Glu Glu Asn1 5 10 15Pro Gly Pro520PRTArtificial
SequenceSynthetic 5Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly
Asp Val Glu Ser1 5 10 15Asn Pro Gly Pro 20622PRTArtificial
SequenceSynthetic 6Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu
Ala Gly Asp Val1 5 10 15Glu Ser Asn Pro Gly Pro 20782RNAArtificial
SequenceSynthetic 7guuggaacca uucaaaacag cauagcaagu uaaaauaagg
cuaguccguu aucaacuuga 60aaaaguggca ccgagucggu gc 82876RNAArtificial
SequenceSynthetic 8guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc
cguuaucaac uugaaaaagu 60ggcaccgagu cggugc 76986RNAArtificial
SequenceSynthetic 9guuuaagagc uaugcuggaa acagcauagc aaguuuaaau
aaggcuaguc cguuaucaac 60uugaaaaagu ggcaccgagu cggugc
861023DNAArtificial SequenceSyntheticmisc_feature(2)..(21)n = A, T,
C, or G 10gnnnnnnnnn nnnnnnnnnn ngg 231123DNAArtificial
SequenceSyntheticmisc_feature(1)..(21)n = A, T, C, or G
11nnnnnnnnnn nnnnnnnnnn ngg 231225DNAArtificial
SequenceSyntheticmisc_feature(3)..(23)n = A, T, C, or G
12ggnnnnnnnn nnnnnnnnnn nnngg 251320DNAArtificial SequenceSynthetic
13cgacttcgtg acgtgcggta 201420RNAArtificial SequenceSynthetic
14gcugaagcac ugcacgccau 2015123DNAArtificial SequenceSynthetic
15ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
60tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
120tcc 12316123DNAArtificial SequenceSynthetic 16ggacttgaag
aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc 60gtaggtcagg
gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat 120gaa
123177807DNAArtificial SequenceSyntheticmisc_feature(1)..(120)Mouse
Upstream Sequencemisc_feature(215)..(234)Pgk Excision sgRNA v2
Target Sitemisc_feature(243)..(262)Pgk Excision sgRNA v4 Target
Sitemisc_feature(248)..(281)LoxPmisc_feature(263)..(282)Pgk
Excision sgRNA v3 Target Sitemisc_feature(305)..(327)Pgk Excision
sgRNA v1A Target Sitemisc_feature(336)..(796)Pgk
Poly(A)misc_feature(337)..(356)Pgk Disruption sgRNA v4 (cGM4)
Target Sitemisc_feature(371)..(376)Pgk Poly(A) Recognition
Motifmisc_feature(385)..(404)Pgk Disruption sgRNA v5 (cGM5) Target
Sitemisc_feature(405)..(424)Pgk Disruption sgRNA v1 (cGM2) Target
Sitemisc_feature(452)..(471)Pgk Disruption sgRNA v2 (cGM) Target
Sitemisc_feature(571)..(590)Pgk Disruption sgRNA v3 (cGM3) Target
Sitemisc_feature(794)..(813)Pgk Excision sgRNA v5 Target
Sitemisc_feature(797)..(819)Pgk Excision sgRNA v1B Target
Sitemisc_feature(803)..(822)Pgk Excision sgRNA v6 Target
Sitemisc_feature(820)..(853)LoxPmisc_feature(862)..(3930)LacZmisc_feature-
(3931)..(3996)P2Amisc_feature(3997)..(4713)eBFPmisc_feature(4193)..(4212)e-
BFP sgRNA Target Sitemisc_feature(4748)..(4969)SV40
Poly(A)misc_feature(4976)..(5023)Frtmisc_feature(5030)..(6242)Human
Ubiquitin C Promotermisc_feature(6243)..(6309)EM7
Promotermisc_feature(6310)..(7113)Neomycin
Phosphotransferasemisc_feature(7122)..(7598)Pgk
Poly(A)misc_feature(7609)..(7656)Frtmisc_feature(7663)..(7807)Mouse
Downstream Sequence 17ggagtgttgc aatacctttc tgggagttct ctgctgcctc
ctggcttctg aggaccgccc 60tgggcctggg agaatccctt ccccctcttc cctcgtgatc
tgcaactcca gtctttctag 120ttgaccagct cggcggtgac ctgcacgtct
agggcgcagt agtccagggt ttccttgatg 180atgtcatact tatcctgtcc
cttttttttc cacagggcgc gccactagtg gatccggaac 240ccttaatata
acttcgtata atgtatgcta tacgaagtta ttaggtccct cgacctgcag
300gaatcgacct gatgcagctc tcggaggggg gatccgctgt aagtctgcag
aaattgatga 360tctattaaac aataaagatg tccactaaaa tggaagtttt
tcctgtcata ctttgttaag 420aagggtgaga acagagtacc tacattttga
atggaaggat tggagctacg ggggtggggg 480tggggtggga ttagataaat
gcctgctctt tactgaaggc tctttactat tgctttatga 540taatgtttca
tagttggata tcataattta aacaagcaaa accaaattaa gggccagctc
600attcctccca ctcatgatct atagatctat agatctctcg tgggatcatt
gtttttctct 660tgattcccac tttgtggttc taagtactgt ggtttccaaa
tgtgtcagtt tcatagcctg 720aagaacgaga tcagcagcct ctgttccaca
tacacttcat tctcagtatt gttttgccaa 780gttctaattc catcagcgac
ctgatgcagc tctcggagga taacttcgta taatgtatgc 840tatacgaagt
tatccgccac catgggtacc gatttaaatg atccagtggt cctgcagagg
900agagattggg agaatcccgg tgtgacacag ctgaacagac tagccgccca
ccctcccttt 960gcttcttgga gaaacagtga ggaagctagg acagacagac
caagccagca actcagatct 1020ttgaacgggg agtggagatt tgcctggttt
ccggcaccag aagcggtgcc ggaaagctgg 1080ctggagtgcg atcttcctga
ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac 1140ggttacgatg
cgcccatcta caccaacgtg acctatccca ttacggtcaa tccgccgttt
1200gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga
tgaaagctgg 1260ctacaggaag gccagacgcg aattattttt gatggcgtta
actcggcgtt tcatctgtgg 1320tgcaacgggc gctgggtcgg ttacggccag
gacagtcgtt tgccgtctga atttgacctg 1380agcgcatttt tacgcgccgg
agaaaaccgc ctcgcggtga tggtgctgcg ctggagtgac 1440ggcagttatc
tggaagatca ggatatgtgg cggatgagcg gcattttccg tgacgtctcg
1500ttgctgcata aaccgactac acaaatcagc gatttccatg ttgccactcg
ctttaatgat 1560gatttcagcc gcgctgtact ggaggctgaa gttcagatgt
gcggcgagtt gcgtgactac 1620ctacgggtaa cagtttcttt atggcagggt
gaaacgcagg tcgccagcgg caccgcgcct 1680ttcggcggtg aaattatcga
tgagcgtggt ggttatgccg atcgcgtcac actacgtctg 1740aacgtcgaaa
acccgaaact gtggagcgcc gaaatcccga atctctatcg tgcggtggtt
1800gaactgcaca ccgccgacgg cacgctgatt gaagcagaag cctgcgatgt
cggtttccgc 1860gaggtgcgga ttgaaaatgg tctgctgctg ctgaacggca
agccgttgct gattcgaggc 1920gttaaccgtc acgagcatca tcctctgcat
ggtcaggtca tggatgagca gacgatggtg 1980caggatatcc tgctgatgaa
gcagaacaac tttaacgccg tgcgctgttc gcattatccg 2040aaccatccgc
tgtggtacac gctgtgcgac cgctacggcc tgtatgtggt ggatgaagcc
2100aatattgaaa cccacggcat ggtgccaatg aatcgtctga ccgatgatcc
gcgctggcta 2160ccggcgatga gcgaacgcgt aacgcgaatg gtgcagcgcg
atcgtaatca cccgagtgtg 2220atcatctggt cgctggggaa tgaatcaggc
cacggcgcta atcacgacgc gctgtatcgc 2280tggatcaaat ctgtcgatcc
ttcccgcccg gtgcagtatg aaggcggcgg agccgacacc 2340acggccaccg
atattatttg cccgatgtac gcgcgcgtgg atgaagacca gcccttcccg
2400gctgtgccga aatggtccat caaaaaatgg ctttcgctac ctggagagac
gcgcccgctg 2460atcctttgcg aatacgccca cgcgatgggt aacagtcttg
gcggtttcgc taaatactgg 2520caggcgtttc gtcagtatcc ccgtttacag
ggcggcttcg tctgggactg ggtggatcag 2580tcgctgatta aatatgatga
aaacggcaac ccgtggtcgg cttacggcgg tgattttggc 2640gatacgccga
acgatcgcca gttctgtatg aacggtctgg tctttgccga ccgcacgccg
2700catccagcgc tgacggaagc aaaacaccag cagcagtttt tccagttccg
tttatccggg 2760caaaccatcg aagtgaccag cgaatacctg ttccgtcata
gcgataacga gctcctgcac 2820tggatggtgg cgctggatgg taagccgctg
gcaagcggtg aagtgcctct ggatgtcgct 2880ccacaaggta aacagttgat
tgaactgcct gaactaccgc agccggagag cgccgggcaa 2940ctctggctca
cagtacgcgt agtgcaaccg aacgcgaccg catggtcaga agccgggcac
3000atcagcgcct ggcagcagtg gcgtctggcg gaaaacctca gtgtgacgct
ccccgccgcg 3060tcccacgcca tcccgcatct gaccaccagc gaaatggatt
tttgcatcga gctgggtaat 3120aagcgttggc aatttaaccg ccagtcaggc
tttctttcac agatgtggat tggcgataaa 3180aaacaactgc tgacgccgct
gcgcgatcag ttcacccgtg caccgctgga taacgacatt 3240ggcgtaagtg
aagcgacccg cattgaccct aacgcctggg tcgaacgctg gaaggcggcg
3300ggccattacc aggccgaagc agcgttgttg cagtgcacgg cagatacact
tgctgatgcg 3360gtgctgatta cgaccgctca cgcgtggcag catcagggga
aaaccttatt tatcagccgg 3420aaaacctacc ggattgatgg tagtggtcaa
atggcgatta ccgttgatgt tgaagtggcg 3480agcgatacac cgcatccggc
gcggattggc ctgaactgcc agctggcgca ggtagcagag 3540cgggtaaact
ggctcggatt agggccgcaa gaaaactatc ccgaccgcct tactgccgcc
3600tgttttgacc gctgggatct gccattgtca gacatgtata ccccgtacgt
cttcccgagc 3660gaaaacggtc tgcgctgcgg gacgcgcgaa ttgaattatg
gcccacacca gtggcgcggc 3720gacttccagt tcaacatcag ccgctacagt
caacagcaac tgatggaaac cagccatcgc 3780catctgctgc acgcggaaga
aggcacatgg ctgaatatcg acggtttcca tatggggatt 3840ggtggcgacg
actcctggag cccgtcagta tcggcggaat tccagctgag cgccggtcgc
3900taccattacc agttggtctg gtgtcaaaaa ggaagcggag ctactaactt
cagcctgctg 3960aagcaggctg gagacgtgga ggagaaccct ggacctgtga
gcaagggcga ggagctgttc 4020accggggtgg tgcccatcct ggtcgagctg
gacggcgacg taaacggcca caagttcagc 4080gtgtccggcg agggcgaggg
cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 4140accaccggca
agctgcccgt gccctggccc accctcgtga ccaccctgac ccatggcgtg
4200cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
gtccgccatg 4260cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
acgacggcaa ctacaagacc 4320cgcgccgagg tgaagttcga gggcgacacc
ctggtgaacc gcatcgagct gaagggcatc 4380gacttcaagg aggacggcaa
catcctgggg cacaagctgg agtacaacta caacagccac 4440aacgtctata
tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
4500cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
cacccccatc 4560ggcgacggcc ccgtgctgct gcccgacaac cactacctga
gcacccagtc cgccctgagc 4620aaagacccca acgagaagcg cgatcacatg
gtcctgctgg agttcgtgac cgccgccggg 4680atcactctcg gcatggacga
gctgtacaag taataattct agagtcgggg cggccggccg 4740cttcgagcag
acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
4800tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
aaccattata 4860agctgcaata aacaagttaa caacaacaat tgcattcatt
ttatgtttca ggttcagggg 4920gaggtgtggg aggtttttta aagcaagtaa
aacctctaca aatgtggtac tcgaggaagt 4980tcctattccg aagttcctat
tctctagaaa gtataggaac ttcatgcatg gcctccgcgc 5040cgggttttgg
cgcctcccgc gggcgccccc ctcctcacgg cgagcgctgc cacgtcagac
5100gaagggcgca gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc
ggcccgctgc 5160tcataagact cggccttaga accccagtat cagcagaagg
acattttagg acgggacttg 5220ggtgactcta gggcactggt tttctttcca
gagagcggaa caggcgagga aaagtagtcc 5280cttctcggcg attctgcgga
gggatctccg tggggcggtg aacgccgatg attatataag 5340gacgcgccgg
gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg
5400tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg
gccggggctt 5460tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg
agagaccgcc aagggctgta 5520gtctgggtcc gcgagcaagg ttgccctgaa
ctgggggttg gggggagcgc agcaaaatgg 5580cggctgttcc cgagtcttga
atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa 5640caaggtgggg
ggcatggtgg gcggcaagaa cccaaggtct tgaggccttc gctaatgcgg
5700gaaagctctt attcgggtga gatgggctgg ggcaccatct ggggaccctg
acgtgaagtt 5760tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg
ggcggcagtt atggcggtgc 5820cgttgggcag tgcacccgta cctttgggag
cgcgcgccct cgtcgtgtcg tgacgtcacc 5880cgttctgttg gcttataatg
cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt 5940tctccgtcgc
aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc
6000ggacctctgg tgaggggagg gataagtgag gcgtcagttt ctttggtcgg
ttttatgtac 6060ctatcttctt aagtagctga agctccggtt ttgaactatg
cgctcggggt tggcgagtgt 6120gttttgtgaa gttttttagg caccttttga
aatgtaatca tttgggtcaa tatgtaattt 6180tcagtgttag actagtaaat
tgtccgctaa attctggccg tttttggctt ttttgttaga 6240cgtgttgaca
attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
6300aactaaacca tgggatcggc cattgaacaa gatggattgc acgcaggttc
tccggccgct 6360tgggtggaga ggctattcgg ctatgactgg gcacaacaga
caatcggctg ctctgatgcc 6420gccgtgttcc ggctgtcagc gcaggggcgc
ccggttcttt ttgtcaagac cgacctgtcc 6480ggtgccctga atgaactgca
ggacgaggca gcgcggctat cgtggctggc cacgacgggc 6540gttccttgcg
cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
6600ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
gaaagtatcc 6660atcatggctg atgcaatgcg gcggctgcat acgcttgatc
cggctacctg cccattcgac 6720caccaagcga aacatcgcat cgagcgagca
cgtactcgga tggaagccgg tcttgtcgat 6780caggatgatc tggacgaaga
gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 6840aaggcgcgca
tgcccgacgg cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg
6900aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
gctgggtgtg 6960gcggaccgct atcaggacat agcgttggct acccgtgata
ttgctgaaga gcttggcggc 7020gaatgggctg accgcttcct cgtgctttac
ggtatcgccg ctcccgattc gcagcgcatc 7080gccttctatc gccttcttga
cgagttcttc tgaggggatc cgctgtaagt ctgcagaaat 7140tgatgatcta
ttaaacaata aagatgtcca ctaaaatgga agtttttcct gtcatacttt
7200gttaagaagg gtgagaacag agtacctaca ttttgaatgg aaggattgga
gctacggggg 7260tgggggtggg gtgggattag ataaatgcct gctctttact
gaaggctctt tactattgct 7320ttatgataat gtttcatagt tggatatcat
aatttaaaca agcaaaacca aattaagggc 7380cagctcattc ctcccactca
tgatctatag atctatagat ctctcgtggg atcattgttt 7440ttctcttgat
tcccactttg tggttctaag tactgtggtt tccaaatgtg tcagtttcat
7500agcctgaaga acgagatcag cagcctctgt tccacataca cttcattctc
agtattgttt 7560tgccaagttc taattccatc agacctcgac ctgcagcccc
tagtcgacga agttcctatt 7620ccgaagttcc tattctctag aaagtatagg
aacttcgcta gctaaaattg gagggacaag 7680acttcccaca gattttcggt
tttgtcggga agttttttaa taggggcaaa taaggaaaat 7740gggaggatag
gtagtcatct ggggttttat gcagcaaaac tacaggttat tattgcttgt 7800gatccgc
7807183716DNAArtificial
SequenceSyntheticmisc_feature(1)..(172)Mouse Upstream
Sequencemisc_feature(226)..(259)LoxPmisc_feature(357)..(423)EM7
Promotermisc_feature(424)..(1227)Neomycin
Phosphotransferasemisc_feature(1236)..(2489)Triple
Poly(A)misc_feature(2517)..(2550)LoxPmisc_feature(2596)..(3315)eBFPmisc_f-
eature(2795)..(2814)eBFP sgRNA Target
Sitemisc_feature(3350)..(3571)SV40
Poly(A)misc_feature(3572)..(3716)Mouse Downstream Sequence
18ctgcagtgga gtaggcgggg agaaggccgc acccttctcc ggagggggga ggggagtgtt
60gcaatacctt tctgggagtt ctctgctgcc tcctggcttc tgaggaccgc cctgggcctg
120ggagaatccc ttccccctct tccctcgtga tctgcaactc cagtctttct
agttgaccag 180ctcggcggtg acctgcacgt ctagggcgca gtagtccagg
gtttccttga tgatgtcata 240cttatcctgt cccttttttt tccacagggc
gcgccactag tggatccgga acccttaata 300taacttcgta taatgtatgc
tatacgaagt tattaggtcc ctcgacctgc aggaattgtt 360gacaattaat
catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
420accatgggat cggccattga acaagatgga ttgcacgcag gttctccggc
cgcttgggtg 480gagaggctat tcggctatga ctgggcacaa cagacaatcg
gctgctctga tgccgccgtg 540ttccggctgt cagcgcaggg gcgcccggtt
ctttttgtca agaccgacct gtccggtgcc 600ctgaatgaac tgcaggacga
ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 660tgcgcagctg
tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
720gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
atccatcatg 780gctgatgcaa tgcggcggct gcatacgctt gatccggcta
cctgcccatt cgaccaccaa 840gcgaaacatc gcatcgagcg agcacgtact
cggatggaag ccggtcttgt cgatcaggat 900gatctggacg aagagcatca
ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 960cgcatgcccg
acggcgatga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
1020atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
tgtggcggac 1080cgctatcagg acatagcgtt ggctacccgt gatattgctg
aagagcttgg cggcgaatgg 1140gctgaccgct tcctcgtgct ttacggtatc
gccgctcccg attcgcagcg catcgccttc 1200tatcgccttc ttgacgagtt
cttctgaggg gatccgctgt aagtctgcag aaattgatga 1260tctattaaac
aataaagatg tccactaaaa tggaagtttt tcctgtcata ctttgttaag
1320aagggtgaga acagagtacc tacattttga atggaaggat tggagctacg
ggggtggggg 1380tggggtggga ttagataaat gcctgctctt tactgaaggc
tctttactat tgctttatga 1440taatgtttca tagttggata tcataattta
aacaagcaaa accaaattaa gggccagctc 1500attcctccca ctcatgatct
atagatctat agatctctcg tgggatcatt gtttttctct 1560tgattcccac
tttgtggttc taagtactgt ggtttccaaa tgtgtcagtt tcatagcctg
1620aagaacgaga tcagcagcct ctgttccaca tacacttcat tctcagtatt
gttttgccaa 1680gttctaattc catcagaagc ttgcagatct gcgactctag
aggatctgcg actctagagg 1740atcataatca gccataccac atttgtagag
gttttacttg ctttaaaaaa cctcccacac 1800ctccccctga acctgaaaca
taaaatgaat gcaattgttg ttgttaactt gtttattgca 1860gcttataatg
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
1920tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
tgtctggatc 1980tgcgactcta gaggatcata atcagccata ccacatttgt
agaggtttta cttgctttaa 2040aaaacctccc acacctcccc ctgaacctga
aacataaaat gaatgcaatt gttgttgtta 2100acttgtttat tgcagcttat
aatggttaca aataaagcaa tagcatcaca aatttcacaa 2160ataaagcatt
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
2220atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat
ttgtagaggt 2280tttacttgct ttaaaaaacc tcccacacct ccccctgaac
ctgaaacata aaatgaatgc 2340aattgttgtt gttaacttgt ttattgcagc
ttataatggt tacaaataaa gcaatagcat 2400cacaaatttc acaaataaag
catttttttc actgcattct agttgtggtt tgtccaaact 2460catcaatgta
tcttatcatg tctggatccc catcaagctg atccggaacc cttaatataa
2520cttcgtataa tgtatgctat acgaagttat taggtccctc gacctgcagc
ccaagctagt 2580gcccgggccg ccaccatggt gagcaagggc gaggagctgt
tcaccggggt ggtgcccatc 2640ctggttgagc tggacggcga cgtaaacggc
cacaagttca gcgtgtccgg cgagggcgag 2700ggcgatgcca cctacggcaa
gctgaccctg aagttcatct gcaccaccgg caagctgccc 2760gtgccctggc
ccaccctcgt gaccaccctg acccatggcg tgcagtgctt cagccgctac
2820cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
ctacgtccag 2880gagcgcacca tcttcttcaa ggacgacggc aactacaaga
cccgcgccga ggtgaagttc 2940gagggcgaca ccctggtgaa ccgcatcgag
ctgaagggca tcgacttcaa ggaggacggc 3000aacatcctgg ggcacaagct
ggagtacaac tacaacagcc acaacgtcta tatcatggcc 3060gacaagcaga
agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
3120agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
ccccgtgctg 3180ctgcccgaca
accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
3240cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
cggcatggac 3300gagctgtaca agtaataatt ctagagtcgg ggcggccggc
cgcttcgagc agacatgata 3360agatacattg atgagtttgg acaaaccaca
actagaatgc agtgaaaaaa atgctttatt 3420tgtgaaattt gtgatgctat
tgctttattt gtaaccatta taagctgcaa taaacaagtt 3480aacaacaaca
attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
3540taaagcaagt aaaacctcta caaatgtggt actcgagtaa aattggaggg
acaagacttc 3600ccacagattt tcggttttgt cgggaagttt tttaataggg
gcaaataagg aaaatgggag 3660gataggtagt catctggggt tttatgcagc
aaaactacag gttattattg cttgtg 37161920DNAArtificial
SequenceSynthetic 19cccgcgcggt gatcacctag 202020RNAArtificial
SequenceSynthetic 20gggcgcgcca cuaguggauc 202120DNAArtificial
SequenceSynthetic 21catacgatat gcttcaataa 202220RNAArtificial
SequenceSynthetic 22guaugcuaua cgaaguuauu 202320DNAArtificial
SequenceSynthetic 23taatatgctt caatataatt 202420RNAArtificial
SequenceSynthetic 24auuauacgaa guuauauuaa 202520DNAArtificial
SequenceSynthetic 25gtcgctggac tacgtcgaga 202620RNAArtificial
SequenceSynthetic 26cagcgaccug augcagcucu 202720DNAArtificial
SequenceSynthetic 27ataggaggct ctcgacgtag 202820RNAArtificial
SequenceSynthetic 28uauccuccga gagcugcauc 202920RNAArtificial
SequenceSynthetic 29ccuucuuaac aaaguaugac 203020RNAArtificial
SequenceSynthetic 30uggaaggauu ggagcuacgg 203120RNAArtificial
SequenceSynthetic 31aacaagcaaa accaaauuaa 203220DNAArtificial
SequenceSynthetic 32ggaagaattg tttcatactg 203320DNAArtificial
SequenceSynthetic 33accttcctaa cctcgatgcc 203420DNAArtificial
SequenceSynthetic 34ttgttcgttt tggtttaatt 203520RNAArtificial
SequenceSynthetic 35caauuucugc agacuuacag 203620RNAArtificial
SequenceSynthetic 36aggaaaaacu uccauuuuag 203716RNAArtificial
SequenceSynthetic 37guuuuagagc uaugcu 163867RNAArtificial
SequenceSynthetic 38agcauagcaa guuaaaauaa ggcuaguccg uuaucaacuu
gaaaaagugg caccgagucg 60gugcuuu 673977RNAArtificial
SequenceSynthetic 39guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc
cguuaucaac uugaaaaagu 60ggcaccgagu cggugcu 77401053RNAArtificial
SequenceSynthetic 40augcccaaga agaagaggaa gguguccaau uuacugaccg
uacaccaaaa uuugccugca 60uuaccggucg augcaacgag ugaugagguu cgcaagaacc
ugauggacau guucagggau 120cgccaggcgu uuucugagca uaccuggaaa
augcuucugu ccguuugccg gucgugggcg 180gcauggugca aguugaauaa
ccggaaaugg uuucccgcag aaccugaaga uguucgcgau 240uaucuucuau
aucuucaggc gcgcggucug gcaguaaaaa cuauccagca acauuugggc
300cagcuaaaca ugcuucaucg ucgguccggg cugccacgac caagugacag
caaugcuguu 360ucacugguua ugcggcggau ccgaaaagaa aacguugaug
ccggugaacg ugcaaaacag 420gcucuagcgu ucgaacgcac ugauuucgac
cagguucguu cacucaugga aaauagcgau 480cgcugccagg auauacguaa
ucuggcauuu cuggggauug cuuauaacac ccuguuacgu 540auagccgaaa
uugccaggau caggguuaaa gauaucucac guacugacgg ugggagaaug
600uuaauccaua uuggcagaac gaaaacgcug guuagcaccg cagguguaga
gaaggcacuu 660agccuggggg uaacuaaacu ggucgagcga uggauuuccg
ucucuggugu agcugaugau 720ccgaauaacu accuguuuug ccgggucaga
aaaaauggug uugccgcgcc aucugccacc 780agccagcuau caacucgcgc
ccuggaaggg auuuuugaag caacucaucg auugauuuac 840ggcgcuaagg
augacucugg ucagagauac cuggccuggu cuggacacag ugcccguguc
900ggagccgcgc gagauauggc ccgcgcugga guuucaauac cggagaucau
gcaagcuggu 960ggcuggacca auguaaauau ugucaugaac uauauccgua
accuggauag ugaaacaggg 1020gcaauggugc gccugcugga agauggcgau uag
10534120DNAArtificial SequenceSynthetic 41cgacctgatg cagctctcgg
204220DNAArtificial SequenceSynthetic 42gctgaagcac tgcacgccat
204320DNAArtificial SequenceSynthetic 43gggcgcgcca ctagtggatc
204420DNAArtificial SequenceSynthetic 44gtatgctata cgaagttatt
204520DNAArtificial SequenceSynthetic 45attatacgaa gttatattaa
204620DNAArtificial SequenceSynthetic 46cagcgacctg atgcagctct
204720DNAArtificial SequenceSynthetic 47tatcctccga gagctgcatc
204820DNAArtificial SequenceSynthetic 48ccttcttaac aaagtatgac
204920DNAArtificial SequenceSynthetic 49tggaaggatt ggagctacgg
205020DNAArtificial SequenceSynthetic 50aacaagcaaa accaaattaa
205120DNAArtificial SequenceSynthetic 51caatttctgc agacttacag
205220DNAArtificial SequenceSynthetic 52aggaaaaact tccattttag
20531391PRTArtificial SequenceSynthetic 53Met Asp Lys Pro Lys Lys
Lys Arg Lys Val Lys Tyr Ser Ile Gly Leu1 5 10 15Asp Ile Gly Thr Asn
Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 20 25 30Lys Val Pro Ser
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 35 40 45Ser Ile Lys
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 50 55 60Thr Ala
Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr65 70 75
80Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
85 90 95Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
Phe 100 105 110Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile
Phe Gly Asn 115 120 125Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
Pro Thr Ile Tyr His 130 135 140Leu Arg Lys Lys Leu Val Asp Ser Thr
Asp Lys Ala Asp Leu Arg Leu145 150 155 160Ile Tyr Leu Ala Leu Ala
His Met Ile Lys Phe Arg Gly His Phe Leu 165 170 175Ile Glu Gly Asp
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 180 185 190Ile Gln
Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 195 200
205Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
210 215 220Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
Glu Lys225 230 235 240Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
Ser Leu Gly Leu Thr 245 250 255Pro Asn Phe Lys Ser Asn Phe Asp Leu
Ala Glu Asp Ala Lys Leu Gln 260 265 270Leu Ser Lys Asp Thr Tyr Asp
Asp Asp Leu Asp Asn Leu Leu Ala Gln 275 280 285Ile Gly Asp Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 290 295 300Asp Ala Ile
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr305 310 315
320Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
325 330 335Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
Pro Glu 340 345 350Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
Gly Tyr Ala Gly 355 360 365Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
Phe Tyr Lys Phe Ile Lys 370 375 380Pro Ile Leu Glu Lys Met Asp Gly
Thr Glu Glu Leu Leu Val Lys Leu385 390 395 400Asn Arg Glu Asp Leu
Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 405 410 415Ile Pro His
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 420 425 430Gln
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 435 440
445Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
450 455 460Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile465 470 475 480Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
Gly Ala Ser Ala Gln 485 490 495Ser Phe Ile Glu Arg Met Thr Asn Phe
Asp Lys Asn Leu Pro Asn Glu 500 505 510Lys Val Leu Pro Lys His Ser
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 515 520 525Asn Glu Leu Thr Lys
Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 530 535 540Ala Phe Leu
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe545 550 555
560Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
565 570 575Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val
Glu Asp 580 585 590Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
Leu Lys Ile Ile 595 600 605Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu
Asn Glu Asp Ile Leu Glu 610 615 620Asp Ile Val Leu Thr Leu Thr Leu
Phe Glu Asp Arg Glu Met Ile Glu625 630 635 640Glu Arg Leu Lys Thr
Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 645 650 655Gln Leu Lys
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 660 665 670Leu
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 675 680
685Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
690 695 700His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala
Gln Val705 710 715 720Ser Gly Gln Gly Asp Ser Leu His Glu His Ile
Ala Asn Leu Ala Gly 725 730 735Ser Pro Ala Ile Lys Lys Gly Ile Leu
Gln Thr Val Lys Val Val Asp 740 745 750Glu Leu Val Lys Val Met Gly
Arg His Lys Pro Glu Asn Ile Val Ile 755 760 765Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 770 775 780Arg Glu Arg
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser785 790 795
800Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
805 810 815Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr
Val Asp 820 825 830Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
Val Asp His Ile 835 840 845Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
Ile Asp Asn Lys Val Leu 850 855 860Thr Arg Ser Asp Lys Asn Arg Gly
Lys Ser Asp Asn Val Pro Ser Glu865 870 875 880Glu Val Val Lys Lys
Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 885 890 895Lys Leu Ile
Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 900 905 910Gly
Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 915 920
925Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
930 935 940Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg
Glu Val945 950 955 960Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser
Asp Phe Arg Lys Asp 965 970 975Phe Gln Phe Tyr Lys Val Arg Glu Ile
Asn Asn Tyr His His Ala His 980 985 990Asp Ala Tyr Leu Asn Ala Val
Val Gly Thr Ala Leu Ile Lys Lys Tyr 995 1000 1005Pro Lys Leu Glu
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1010 1015 1020Asp Val
Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys 1025 1030
1035Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe
1040 1045 1050Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys
Arg Pro 1055 1060 1065Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
Val Trp Asp Lys 1070 1075 1080Gly Arg Asp Phe Ala Thr Val Arg Lys
Val Leu Ser Met Pro Gln 1085 1090 1095Val Asn Ile Val Lys Lys Thr
Glu Val Gln Thr Gly Gly Phe Ser 1100 1105 1110Lys Glu Ser Ile Leu
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala 1115 1120 1125Arg Lys Lys
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1130 1135 1140Pro
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys 1145 1150
1155Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile
1160 1165 1170Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile
Asp Phe 1175 1180 1185Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
Asp Leu Ile Ile 1190 1195 1200Lys Leu Pro Lys Tyr Ser Leu Phe Glu
Leu Glu Asn Gly Arg Lys 1205 1210 1215Arg Met Leu Ala Ser Ala Gly
Glu Leu Gln Lys Gly Asn Glu Leu 1220 1225 1230Ala Leu Pro Ser Lys
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His 1235 1240 1245Tyr Glu Lys
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1250 1255 1260Leu
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 1265 1270
1275Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn
1280 1285 1290Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp
Lys Pro 1295 1300 1305Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
Phe Thr Leu Thr 1310 1315 1320Asn Leu Gly Ala Pro Ala Ala Phe Lys
Tyr Phe Asp Thr Thr Ile 1325 1330 1335Asp Arg Lys Arg Tyr Thr Ser
Thr Lys Glu Val Leu Asp Ala Thr 1340 1345 1350Leu Ile His Gln Ser
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp 1355 1360 1365Leu Ser Gln
Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys 1370 1375 1380Ala
Gly Gln Ala Lys Lys Lys Lys 1385 1390544173DNAArtificial
SequenceSynthetic 54atggacaagc ccaagaaaaa gcggaaagtg aagtacagca
tcggcctgga catcggcacc 60aactctgtgg gctgggccgt gatcaccgac gagtacaagg
tgcccagcaa gaaattcaag 120gtgctgggca acaccgacag gcacagcatc
aagaagaacc tgatcggcgc cctgctgttc 180gacagcggcg aaacagccga
ggccaccaga ctgaagagaa ccgccagaag aagatacacc 240aggcggaaga
acaggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
300gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
caagaagcac 360gagagacacc ccatcttcgg caacatcgtg gacgaggtgg
cctaccacga gaagtacccc 420accatctacc acctgagaaa gaaactggtg
gacagcaccg acaaggccga cctgagactg 480atctacctgg ccctggccca
catgatcaag ttcagaggcc acttcctgat cgagggcgac 540ctgaaccccg
acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
600cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
tatcctgtct 660gccagactga gcaagagcag aaggctggaa aatctgatcg
cccagctgcc cggcgagaag 720aagaacggcc tgttcggcaa cctgattgcc
ctgagcctgg gcctgacccc caacttcaag 780agcaacttcg acctggccga
ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 840gacctggaca
acctgctggc ccagatcggc gaccagtacg ccgacctgtt cctggccgcc
900aagaacctgt ctgacgccat cctgctgagc gacatcctga gagtgaacac
cgagatcacc 960aaggcccccc tgagcgcctc tatgatcaag agatacgacg
agcaccacca ggacctgacc 1020ctgctgaaag ctctcgtgcg gcagcagctg
cctgagaagt acaaagaaat cttcttcgac 1080cagagcaaga acggctacgc
cggctacatc gatggcggcg ctagccagga agagttctac 1140aagttcatca
agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
1200aacagagagg acctgctgag aaagcagaga accttcgaca acggcagcat
cccccaccag 1260atccacctgg gagagctgca cgctatcctg agaaggcagg
aagattttta cccattcctg 1320aaggacaacc gggaaaagat cgagaagatc
ctgaccttca ggatccccta ctacgtgggc 1380cccctggcca gaggcaacag
cagattcgcc tggatgacca gaaagagcga ggaaaccatc 1440accccctgga
acttcgagga agtggtggac aagggcgcca gcgcccagag cttcatcgag
1500agaatgacaa acttcgataa gaacctgccc aacgagaagg tgctgcccaa
gcacagcctg 1560ctgtacgagt acttcaccgt gtacaacgag ctgaccaaag
tgaaatacgt gaccgaggga 1620atgagaaagc ccgccttcct gagcggcgag
cagaaaaagg ccatcgtgga cctgctgttc 1680aagaccaaca
gaaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
1740tgcttcgact ccgtggaaat ctccggcgtg gaagatagat tcaacgcctc
cctgggcaca 1800taccacgatc tgctgaaaat tatcaaggac aaggacttcc
tggataacga agagaacgag 1860gacattctgg aagatatcgt gctgaccctg
acactgtttg aggaccgcga gatgatcgag 1920gaaaggctga aaacctacgc
tcacctgttc gacgacaaag tgatgaagca gctgaagaga 1980aggcggtaca
ccggctgggg caggctgagc agaaagctga tcaacggcat cagagacaag
2040cagagcggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
ccggaacttc 2100atgcagctga tccacgacga cagcctgaca ttcaaagagg
acatccagaa agcccaggtg 2160tccggccagg gcgactctct gcacgagcat
atcgctaacc tggccggcag ccccgctatc 2220aagaagggca tcctgcagac
agtgaaggtg gtggacgagc tcgtgaaagt gatgggcaga 2280cacaagcccg
agaacatcgt gatcgagatg gctagagaga accagaccac ccagaaggga
2340cagaagaact cccgcgagag gatgaagaga atcgaagagg gcatcaaaga
gctgggcagc 2400cagatcctga aagaacaccc cgtggaaaac acccagctgc
agaacgagaa gctgtacctg 2460tactacctgc agaatggccg ggatatgtac
gtggaccagg aactggacat caacagactg 2520tccgactacg atgtggacca
tatcgtgcct cagagctttc tgaaggacga ctccatcgat 2580aacaaagtgc
tgactcggag cgacaagaac agaggcaaga gcgacaacgt gccctccgaa
2640gaggtcgtga agaagatgaa gaactactgg cgacagctgc tgaacgccaa
gctgattacc 2700cagaggaagt tcgataacct gaccaaggcc gagagaggcg
gcctgagcga gctggataag 2760gccggcttca tcaagaggca gctggtggaa
accagacaga tcacaaagca cgtggcacag 2820atcctggact cccggatgaa
cactaagtac gacgaaaacg ataagctgat ccgggaagtg 2880aaagtgatca
ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
2940aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
cgccgtcgtg 3000ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
agttcgtgta cggcgactac 3060aaggtgtacg acgtgcggaa gatgatcgcc
aagagcgagc aggaaatcgg caaggctacc 3120gccaagtact tcttctacag
caacatcatg aactttttca agaccgaaat caccctggcc 3180aacggcgaga
tcagaaagcg ccctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
3240tgggataagg gcagagactt cgccacagtg cgaaaggtgc tgagcatgcc
ccaagtgaat 3300atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
aagagtctat cctgcccaag 3360aggaacagcg acaagctgat cgccagaaag
aaggactggg accccaagaa gtacggcggc 3420ttcgacagcc ctaccgtggc
ctactctgtg ctggtggtgg ctaaggtgga aaagggcaag 3480tccaagaaac
tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
3540tttgagaaga accctatcga ctttctggaa gccaagggct acaaagaagt
gaaaaaggac 3600ctgatcatca agctgcctaa gtactccctg ttcgagctgg
aaaacggcag aaagagaatg 3660ctggcctctg ccggcgaact gcagaaggga
aacgagctgg ccctgcctag caaatatgtg 3720aacttcctgt acctggcctc
ccactatgag aagctgaagg gcagccctga ggacaacgaa 3780cagaaacagc
tgtttgtgga acagcataag cactacctgg acgagatcat cgagcagatc
3840agcgagttct ccaagagagt gatcctggcc gacgccaatc tggacaaggt
gctgtctgcc 3900tacaacaagc acagggacaa gcctatcaga gagcaggccg
agaatatcat ccacctgttc 3960accctgacaa acctgggcgc tcctgccgcc
ttcaagtact ttgacaccac catcgaccgg 4020aagaggtaca ccagcaccaa
agaggtgctg gacgccaccc tgatccacca gagcatcacc 4080ggcctgtacg
agacaagaat cgacctgtct cagctgggag gcgacaagag acctgccgcc
4140actaagaagg ccggacaggc caaaaagaag aag 41735523RNAArtificial
SequenceSynthetic 55gcugaagcac ugcacgccau ggg 235623DNAArtificial
SequenceSynthetic 56gctgaagcac tgcacgccat ggg 23571499DNAArtificial
SequenceSyntheticmisc_feature(1)..(172)Mouse Upstream
Sequencemisc_feature(300)..(333)LoxPmisc_feature(379)..(1098)eBFPmisc_fea-
ture(1133)..(1354)SV40 Poly(A)misc_feature(1355)..(1499)Mouse
Downstream Sequence 57ctgcagtgga gtaggcgggg agaaggccgc acccttctcc
ggagggggga ggggagtgtt 60gcaatacctt tctgggagtt ctctgctgcc tcctggcttc
tgaggaccgc cctgggcctg 120ggagaatccc ttccccctct tccctcgtga
tctgcaactc cagtctttct agttgaccag 180ctcggcggtg acctgcacgt
ctagggcgca gtagtccagg gtttccttga tgatgtcata 240cttatcctgt
cccttttttt tccacagggc gcgccactag tggatccgga acccttaata
300taacttcgta taatgtatgc tatacgaagt tattaggtcc ctcgacctgc
agcccaagct 360agtgcccggg ccgccaccat ggtgagcaag ggcgaggagc
tgttcaccgg ggtggtgccc 420atcctggttg agctggacgg cgacgtaaac
ggccacaagt tcagcgtgtc cggcgagggc 480gagggcgatg ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg 540cccgtgccct
ggcccaccct cgtgaccacc ctgacccatg gcgtgcagtg cttcagccgc
600taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
aggctacgtc 660caggagcgca ccatcttctt caaggacgac ggcaactaca
agacccgcgc cgaggtgaag 720ttcgagggcg acaccctggt gaaccgcatc
gagctgaagg gcatcgactt caaggaggac 780ggcaacatcc tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg 840gccgacaagc
agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
900ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
cggccccgtg 960ctgctgcccg acaaccacta cctgagcacc cagtccgccc
tgagcaaaga ccccaacgag 1020aagcgcgatc acatggtcct gctggagttc
gtgaccgccg ccgggatcac tctcggcatg 1080gacgagctgt acaagtaata
attctagagt cggggcggcc ggccgcttcg agcagacatg 1140ataagataca
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
1200atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
caataaacaa 1260gttaacaaca acaattgcat tcattttatg tttcaggttc
agggggaggt gtgggaggtt 1320ttttaaagca agtaaaacct ctacaaatgt
ggtactcgag taaaattgga gggacaagac 1380ttcccacaga ttttcggttt
tgtcgggaag ttttttaata ggggcaaata aggaaaatgg 1440gaggataggt
agtcatctgg ggttttatgc agcaaaacta caggttatta ttgcttgtg
1499587235DNAArtificial
SequenceSyntheticmisc_feature(1)..(120)Mouse Upstream
Sequencemisc_feature(248)..(281)LoxPmisc_feature(290)..(3358)LacZmisc_fea-
ture(3359)..(3424)P2Amisc_feature(3425)..(4141)eBFPmisc_feature(4176)..(43-
97)SV40
Poly(A)misc_feature(4404)..(4451)Frtmisc_feature(4458)..(5670)Ubiq-
uitin Promotermisc_feature(5671)..(5737)EM7
Promotermisc_feature(5738)..(6541)Neomycin
Phosphotransferasemisc_feature(6550)..(7026)Pgk
Poly(A)misc_feature(7037)..(7084)Frtmisc_feature(7091)..(7235)Mouse
Downstream Sequence 58ggagtgttgc aatacctttc tgggagttct ctgctgcctc
ctggcttctg aggaccgccc 60tgggcctggg agaatccctt ccccctcttc cctcgtgatc
tgcaactcca gtctttctag 120ttgaccagct cggcggtgac ctgcacgtct
agggcgcagt agtccagggt ttccttgatg 180atgtcatact tatcctgtcc
cttttttttc cacagggcgc gccactagtg gatccggaac 240ccttaatata
acttcgtata atgtatgcta tacgaagtta tccgccacca tgggtaccga
300tttaaatgat ccagtggtcc tgcagaggag agattgggag aatcccggtg
tgacacagct 360gaacagacta gccgcccacc ctccctttgc ttcttggaga
aacagtgagg aagctaggac 420agacagacca agccagcaac tcagatcttt
gaacggggag tggagatttg cctggtttcc 480ggcaccagaa gcggtgccgg
aaagctggct ggagtgcgat cttcctgagg ccgatactgt 540cgtcgtcccc
tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
600ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
gttgttactc 660gctcacattt aatgttgatg aaagctggct acaggaaggc
cagacgcgaa ttatttttga 720tggcgttaac tcggcgtttc atctgtggtg
caacgggcgc tgggtcggtt acggccagga 780cagtcgtttg ccgtctgaat
ttgacctgag cgcattttta cgcgccggag aaaaccgcct 840cgcggtgatg
gtgctgcgct ggagtgacgg cagttatctg gaagatcagg atatgtggcg
900gatgagcggc attttccgtg acgtctcgtt gctgcataaa ccgactacac
aaatcagcga 960tttccatgtt gccactcgct ttaatgatga tttcagccgc
gctgtactgg aggctgaagt 1020tcagatgtgc ggcgagttgc gtgactacct
acgggtaaca gtttctttat ggcagggtga 1080aacgcaggtc gccagcggca
ccgcgccttt cggcggtgaa attatcgatg agcgtggtgg 1140ttatgccgat
cgcgtcacac tacgtctgaa cgtcgaaaac ccgaaactgt ggagcgccga
1200aatcccgaat ctctatcgtg cggtggttga actgcacacc gccgacggca
cgctgattga 1260agcagaagcc tgcgatgtcg gtttccgcga ggtgcggatt
gaaaatggtc tgctgctgct 1320gaacggcaag ccgttgctga ttcgaggcgt
taaccgtcac gagcatcatc ctctgcatgg 1380tcaggtcatg gatgagcaga
cgatggtgca ggatatcctg ctgatgaagc agaacaactt 1440taacgccgtg
cgctgttcgc attatccgaa ccatccgctg tggtacacgc tgtgcgaccg
1500ctacggcctg tatgtggtgg atgaagccaa tattgaaacc cacggcatgg
tgccaatgaa 1560tcgtctgacc gatgatccgc gctggctacc ggcgatgagc
gaacgcgtaa cgcgaatggt 1620gcagcgcgat cgtaatcacc cgagtgtgat
catctggtcg ctggggaatg aatcaggcca 1680cggcgctaat cacgacgcgc
tgtatcgctg gatcaaatct gtcgatcctt cccgcccggt 1740gcagtatgaa
ggcggcggag ccgacaccac ggccaccgat attatttgcc cgatgtacgc
1800gcgcgtggat gaagaccagc ccttcccggc tgtgccgaaa tggtccatca
aaaaatggct 1860ttcgctacct ggagagacgc gcccgctgat cctttgcgaa
tacgcccacg cgatgggtaa 1920cagtcttggc ggtttcgcta aatactggca
ggcgtttcgt cagtatcccc gtttacaggg 1980cggcttcgtc tgggactggg
tggatcagtc gctgattaaa tatgatgaaa acggcaaccc 2040gtggtcggct
tacggcggtg attttggcga tacgccgaac gatcgccagt tctgtatgaa
2100cggtctggtc tttgccgacc gcacgccgca tccagcgctg acggaagcaa
aacaccagca 2160gcagtttttc cagttccgtt tatccgggca aaccatcgaa
gtgaccagcg aatacctgtt 2220ccgtcatagc gataacgagc tcctgcactg
gatggtggcg ctggatggta agccgctggc 2280aagcggtgaa gtgcctctgg
atgtcgctcc acaaggtaaa cagttgattg aactgcctga 2340actaccgcag
ccggagagcg ccgggcaact ctggctcaca gtacgcgtag tgcaaccgaa
2400cgcgaccgca tggtcagaag ccgggcacat cagcgcctgg cagcagtggc
gtctggcgga 2460aaacctcagt gtgacgctcc ccgccgcgtc ccacgccatc
ccgcatctga ccaccagcga 2520aatggatttt tgcatcgagc tgggtaataa
gcgttggcaa tttaaccgcc agtcaggctt 2580tctttcacag atgtggattg
gcgataaaaa acaactgctg acgccgctgc gcgatcagtt 2640cacccgtgca
ccgctggata acgacattgg cgtaagtgaa gcgacccgca ttgaccctaa
2700cgcctgggtc gaacgctgga aggcggcggg ccattaccag gccgaagcag
cgttgttgca 2760gtgcacggca gatacacttg ctgatgcggt gctgattacg
accgctcacg cgtggcagca 2820tcaggggaaa accttattta tcagccggaa
aacctaccgg attgatggta gtggtcaaat 2880ggcgattacc gttgatgttg
aagtggcgag cgatacaccg catccggcgc ggattggcct 2940gaactgccag
ctggcgcagg tagcagagcg ggtaaactgg ctcggattag ggccgcaaga
3000aaactatccc gaccgcctta ctgccgcctg ttttgaccgc tgggatctgc
cattgtcaga 3060catgtatacc ccgtacgtct tcccgagcga aaacggtctg
cgctgcggga cgcgcgaatt 3120gaattatggc ccacaccagt ggcgcggcga
cttccagttc aacatcagcc gctacagtca 3180acagcaactg atggaaacca
gccatcgcca tctgctgcac gcggaagaag gcacatggct 3240gaatatcgac
ggtttccata tggggattgg tggcgacgac tcctggagcc cgtcagtatc
3300ggcggaattc cagctgagcg ccggtcgcta ccattaccag ttggtctggt
gtcaaaaagg 3360aagcggagct actaacttca gcctgctgaa gcaggctgga
gacgtggagg agaaccctgg 3420acctgtgagc aagggcgagg agctgttcac
cggggtggtg cccatcctgg tcgagctgga 3480cggcgacgta aacggccaca
agttcagcgt gtccggcgag ggcgagggcg atgccaccta 3540cggcaagctg
accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
3600cctcgtgacc accctgaccc atggcgtgca gtgcttcagc cgctaccccg
accacatgaa 3660gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
gtccaggagc gcaccatctt 3720cttcaaggac gacggcaact acaagacccg
cgccgaggtg aagttcgagg gcgacaccct 3780ggtgaaccgc atcgagctga
agggcatcga cttcaaggag gacggcaaca tcctggggca 3840caagctggag
tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
3900cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
tgcagctcgc 3960cgaccactac cagcagaaca cccccatcgg cgacggcccc
gtgctgctgc ccgacaacca 4020ctacctgagc acccagtccg ccctgagcaa
agaccccaac gagaagcgcg atcacatggt 4080cctgctggag ttcgtgaccg
ccgccgggat cactctcggc atggacgagc tgtacaagta 4140ataattctag
agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga
4200gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
aaatttgtga 4260tgctattgct ttatttgtaa ccattataag ctgcaataaa
caagttaaca acaacaattg 4320cattcatttt atgtttcagg ttcaggggga
ggtgtgggag gttttttaaa gcaagtaaaa 4380cctctacaaa tgtggtactc
gaggaagttc ctattccgaa gttcctattc tctagaaagt 4440ataggaactt
catgcatggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct
4500cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg
atccttccgc 4560ccggacgctc aggacagcgg cccgctgctc ataagactcg
gccttagaac cccagtatca 4620gcagaaggac attttaggac gggacttggg
tgactctagg gcactggttt tctttccaga 4680gagcggaaca ggcgaggaaa
agtagtccct tctcggcgat tctgcggagg gatctccgtg 4740gggcggtgaa
cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc
4800gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc
acttggtgag 4860tagcgggctg ctgggctggc cggggctttc gtggccgccg
ggccgctcgg tgggacggaa 4920gcgtgtggag agaccgccaa gggctgtagt
ctgggtccgc gagcaaggtt gccctgaact 4980gggggttggg gggagcgcag
caaaatggcg gctgttcccg agtcttgaat ggaagacgct 5040tgtgaggcgg
gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc
5100caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga
tgggctgggg 5160caccatctgg ggaccctgac gtgaagtttg tcactgactg
gagaactcgg tttgtcgtct 5220gttgcggggg cggcagttat ggcggtgccg
ttgggcagtg cacccgtacc tttgggagcg 5280cgcgccctcg tcgtgtcgtg
acgtcacccg ttctgttggc ttataatgca gggtggggcc 5340acctgccggt
aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
5400ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga
taagtgaggc 5460gtcagtttct ttggtcggtt ttatgtacct atcttcttaa
gtagctgaag ctccggtttt 5520gaactatgcg ctcggggttg gcgagtgtgt
tttgtgaagt tttttaggca ccttttgaaa 5580tgtaatcatt tgggtcaata
tgtaattttc agtgttagac tagtaaattg tccgctaaat 5640tctggccgtt
tttggctttt ttgttagacg tgttgacaat taatcatcgg catagtatat
5700cggcatagta taatacgaca aggtgaggaa ctaaaccatg ggatcggcca
ttgaacaaga 5760tggattgcac gcaggttctc cggccgcttg ggtggagagg
ctattcggct atgactgggc 5820acaacagaca atcggctgct ctgatgccgc
cgtgttccgg ctgtcagcgc aggggcgccc 5880ggttcttttt gtcaagaccg
acctgtccgg tgccctgaat gaactgcagg acgaggcagc 5940gcggctatcg
tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
6000tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
tcctgtcatc 6060tcaccttgct cctgccgaga aagtatccat catggctgat
gcaatgcggc ggctgcatac 6120gcttgatccg gctacctgcc cattcgacca
ccaagcgaaa catcgcatcg agcgagcacg 6180tactcggatg gaagccggtc
ttgtcgatca ggatgatctg gacgaagagc atcaggggct 6240cgcgccagcc
gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt
6300cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
gcttttctgg 6360attcatcgac tgtggccggc tgggtgtggc ggaccgctat
caggacatag cgttggctac 6420ccgtgatatt gctgaagagc ttggcggcga
atgggctgac cgcttcctcg tgctttacgg 6480tatcgccgct cccgattcgc
agcgcatcgc cttctatcgc cttcttgacg agttcttctg 6540aggggatccg
ctgtaagtct gcagaaattg atgatctatt aaacaataaa gatgtccact
6600aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag
tacctacatt 6660ttgaatggaa ggattggagc tacgggggtg ggggtggggt
gggattagat aaatgcctgc 6720tctttactga aggctcttta ctattgcttt
atgataatgt ttcatagttg gatatcataa 6780tttaaacaag caaaaccaaa
ttaagggcca gctcattcct cccactcatg atctatagat 6840ctatagatct
ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta
6900ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca
gcctctgttc 6960cacatacact tcattctcag tattgttttg ccaagttcta
attccatcag acctcgacct 7020gcagccccta gtcgacgaag ttcctattcc
gaagttccta ttctctagaa agtataggaa 7080cttcgctagc taaaattgga
gggacaagac ttcccacaga ttttcggttt tgtcgggaag 7140ttttttaata
ggggcaaata aggaaaatgg gaggataggt agtcatctgg ggttttatgc
7200agcaaaacta caggttatta ttgcttgtga tccgc 7235597235DNAArtificial
SequenceSyntheticmisc_feature(1)..(120)Mouse Upstream
Sequencemisc_feature(248)..(281)LoxPmisc_feature(290)..(3358)LacZmisc_fea-
ture(3359)..(3424)P2Amisc_feature(3425)..(4141)eGFPmisc_feature(4176)..(43-
97)SV40
Poly(A)misc_feature(4404)..(4451)Frtmisc_feature(4458)..(5670)Ubiq-
uitin Promotermisc_feature(5671)..(5737)EM7
Promotermisc_feature(5738)..(6541)Neomycin
Phosphotransferasemisc_feature(6550)..(7026)Pgk
Poly(A)misc_feature(7037)..(7084)Frtmisc_feature(7091)..(7235)Mouse
Downstream Sequence 59ggagtgttgc aatacctttc tgggagttct ctgctgcctc
ctggcttctg aggaccgccc 60tgggcctggg agaatccctt ccccctcttc cctcgtgatc
tgcaactcca gtctttctag 120ttgaccagct cggcggtgac ctgcacgtct
agggcgcagt agtccagggt ttccttgatg 180atgtcatact tatcctgtcc
cttttttttc cacagggcgc gccactagtg gatccggaac 240ccttaatata
acttcgtata atgtatgcta tacgaagtta tccgccacca tgggtaccga
300tttaaatgat ccagtggtcc tgcagaggag agattgggag aatcccggtg
tgacacagct 360gaacagacta gccgcccacc ctccctttgc ttcttggaga
aacagtgagg aagctaggac 420agacagacca agccagcaac tcagatcttt
gaacggggag tggagatttg cctggtttcc 480ggcaccagaa gcggtgccgg
aaagctggct ggagtgcgat cttcctgagg ccgatactgt 540cgtcgtcccc
tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
600ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
gttgttactc 660gctcacattt aatgttgatg aaagctggct acaggaaggc
cagacgcgaa ttatttttga 720tggcgttaac tcggcgtttc atctgtggtg
caacgggcgc tgggtcggtt acggccagga 780cagtcgtttg ccgtctgaat
ttgacctgag cgcattttta cgcgccggag aaaaccgcct 840cgcggtgatg
gtgctgcgct ggagtgacgg cagttatctg gaagatcagg atatgtggcg
900gatgagcggc attttccgtg acgtctcgtt gctgcataaa ccgactacac
aaatcagcga 960tttccatgtt gccactcgct ttaatgatga tttcagccgc
gctgtactgg aggctgaagt 1020tcagatgtgc ggcgagttgc gtgactacct
acgggtaaca gtttctttat ggcagggtga 1080aacgcaggtc gccagcggca
ccgcgccttt cggcggtgaa attatcgatg agcgtggtgg 1140ttatgccgat
cgcgtcacac tacgtctgaa cgtcgaaaac ccgaaactgt ggagcgccga
1200aatcccgaat ctctatcgtg cggtggttga actgcacacc gccgacggca
cgctgattga 1260agcagaagcc tgcgatgtcg gtttccgcga ggtgcggatt
gaaaatggtc tgctgctgct 1320gaacggcaag ccgttgctga ttcgaggcgt
taaccgtcac gagcatcatc ctctgcatgg 1380tcaggtcatg gatgagcaga
cgatggtgca ggatatcctg ctgatgaagc agaacaactt 1440taacgccgtg
cgctgttcgc attatccgaa ccatccgctg tggtacacgc tgtgcgaccg
1500ctacggcctg tatgtggtgg atgaagccaa tattgaaacc cacggcatgg
tgccaatgaa 1560tcgtctgacc gatgatccgc gctggctacc ggcgatgagc
gaacgcgtaa cgcgaatggt 1620gcagcgcgat cgtaatcacc cgagtgtgat
catctggtcg ctggggaatg aatcaggcca 1680cggcgctaat cacgacgcgc
tgtatcgctg gatcaaatct gtcgatcctt cccgcccggt 1740gcagtatgaa
ggcggcggag ccgacaccac ggccaccgat attatttgcc cgatgtacgc
1800gcgcgtggat gaagaccagc ccttcccggc tgtgccgaaa tggtccatca
aaaaatggct 1860ttcgctacct ggagagacgc gcccgctgat cctttgcgaa
tacgcccacg cgatgggtaa 1920cagtcttggc ggtttcgcta aatactggca
ggcgtttcgt cagtatcccc gtttacaggg 1980cggcttcgtc tgggactggg
tggatcagtc gctgattaaa tatgatgaaa acggcaaccc 2040gtggtcggct
tacggcggtg attttggcga tacgccgaac gatcgccagt tctgtatgaa
2100cggtctggtc tttgccgacc gcacgccgca tccagcgctg acggaagcaa
aacaccagca 2160gcagtttttc cagttccgtt tatccgggca aaccatcgaa
gtgaccagcg aatacctgtt 2220ccgtcatagc gataacgagc tcctgcactg
gatggtggcg
ctggatggta agccgctggc 2280aagcggtgaa gtgcctctgg atgtcgctcc
acaaggtaaa cagttgattg aactgcctga 2340actaccgcag ccggagagcg
ccgggcaact ctggctcaca gtacgcgtag tgcaaccgaa 2400cgcgaccgca
tggtcagaag ccgggcacat cagcgcctgg cagcagtggc gtctggcgga
2460aaacctcagt gtgacgctcc ccgccgcgtc ccacgccatc ccgcatctga
ccaccagcga 2520aatggatttt tgcatcgagc tgggtaataa gcgttggcaa
tttaaccgcc agtcaggctt 2580tctttcacag atgtggattg gcgataaaaa
acaactgctg acgccgctgc gcgatcagtt 2640cacccgtgca ccgctggata
acgacattgg cgtaagtgaa gcgacccgca ttgaccctaa 2700cgcctgggtc
gaacgctgga aggcggcggg ccattaccag gccgaagcag cgttgttgca
2760gtgcacggca gatacacttg ctgatgcggt gctgattacg accgctcacg
cgtggcagca 2820tcaggggaaa accttattta tcagccggaa aacctaccgg
attgatggta gtggtcaaat 2880ggcgattacc gttgatgttg aagtggcgag
cgatacaccg catccggcgc ggattggcct 2940gaactgccag ctggcgcagg
tagcagagcg ggtaaactgg ctcggattag ggccgcaaga 3000aaactatccc
gaccgcctta ctgccgcctg ttttgaccgc tgggatctgc cattgtcaga
3060catgtatacc ccgtacgtct tcccgagcga aaacggtctg cgctgcggga
cgcgcgaatt 3120gaattatggc ccacaccagt ggcgcggcga cttccagttc
aacatcagcc gctacagtca 3180acagcaactg atggaaacca gccatcgcca
tctgctgcac gcggaagaag gcacatggct 3240gaatatcgac ggtttccata
tggggattgg tggcgacgac tcctggagcc cgtcagtatc 3300ggcggaattc
cagctgagcg ccggtcgcta ccattaccag ttggtctggt gtcaaaaagg
3360aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
agaaccctgg 3420acctgtgagc aagggcgagg agctgttcac cggggtggtg
cccatcctgg tcgagctgga 3480cggcgacgta aacggccaca agttcagcgt
gtccggcgag ggcgagggcg atgccaccta 3540cggcaagctg accctgaagt
tcatctgcac caccggcaag ctgcccgtgc cctggcccac 3600cctcgtgacc
accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
3660gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
gcaccatctt 3720cttcaaggac gacggcaact acaagacccg cgccgaggtg
aagttcgagg gcgacaccct 3780ggtgaaccgc atcgagctga agggcatcga
cttcaaggag gacggcaaca tcctggggca 3840caagctggag tacaactaca
acagccacaa cgtctatatc atggccgaca agcagaagaa 3900cggcatcaag
gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
3960cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
ccgacaacca 4020ctacctgagc acccagtccg ccctgagcaa agaccccaac
gagaagcgcg atcacatggt 4080cctgctggag ttcgtgaccg ccgccgggat
cactctcggc atggacgagc tgtacaagta 4140ataattctag agtcggggcg
gccggccgct tcgagcagac atgataagat acattgatga 4200gtttggacaa
accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
4260tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
acaacaattg 4320cattcatttt atgtttcagg ttcaggggga ggtgtgggag
gttttttaaa gcaagtaaaa 4380cctctacaaa tgtggtactc gaggaagttc
ctattccgaa gttcctattc tctagaaagt 4440ataggaactt catgcatggc
ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct 4500cctcacggcg
agcgctgcca cgtcagacga agggcgcagc gagcgtcctg atccttccgc
4560ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac
cccagtatca 4620gcagaaggac attttaggac gggacttggg tgactctagg
gcactggttt tctttccaga 4680gagcggaaca ggcgaggaaa agtagtccct
tctcggcgat tctgcggagg gatctccgtg 4740gggcggtgaa cgccgatgat
tatataagga cgcgccgggt gtggcacagc tagttccgtc 4800gcagccggga
tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag
4860tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg
tgggacggaa 4920gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
gagcaaggtt gccctgaact 4980gggggttggg gggagcgcag caaaatggcg
gctgttcccg agtcttgaat ggaagacgct 5040tgtgaggcgg gctgtgaggt
cgttgaaaca aggtgggggg catggtgggc ggcaagaacc 5100caaggtcttg
aggccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggg
5160caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg
tttgtcgtct 5220gttgcggggg cggcagttat ggcggtgccg ttgggcagtg
cacccgtacc tttgggagcg 5280cgcgccctcg tcgtgtcgtg acgtcacccg
ttctgttggc ttataatgca gggtggggcc 5340acctgccggt aggtgtgcgg
taggcttttc tccgtcgcag gacgcagggt tcgggcctag 5400ggtaggctct
cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
5460gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag
ctccggtttt 5520gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt
tttttaggca ccttttgaaa 5580tgtaatcatt tgggtcaata tgtaattttc
agtgttagac tagtaaattg tccgctaaat 5640tctggccgtt tttggctttt
ttgttagacg tgttgacaat taatcatcgg catagtatat 5700cggcatagta
taatacgaca aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga
5760tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
atgactgggc 5820acaacagaca atcggctgct ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc 5880ggttcttttt gtcaagaccg acctgtccgg
tgccctgaat gaactgcagg acgaggcagc 5940gcggctatcg tggctggcca
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 6000tgaagcggga
agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
6060tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
ggctgcatac 6120gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
catcgcatcg agcgagcacg 6180tactcggatg gaagccggtc ttgtcgatca
ggatgatctg gacgaagagc atcaggggct 6240cgcgccagcc gaactgttcg
ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt 6300cgtgacccat
ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
6360attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
cgttggctac 6420ccgtgatatt gctgaagagc ttggcggcga atgggctgac
cgcttcctcg tgctttacgg 6480tatcgccgct cccgattcgc agcgcatcgc
cttctatcgc cttcttgacg agttcttctg 6540aggggatccg ctgtaagtct
gcagaaattg atgatctatt aaacaataaa gatgtccact 6600aaaatggaag
tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt
6660ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat
aaatgcctgc 6720tctttactga aggctcttta ctattgcttt atgataatgt
ttcatagttg gatatcataa 6780tttaaacaag caaaaccaaa ttaagggcca
gctcattcct cccactcatg atctatagat 6840ctatagatct ctcgtgggat
cattgttttt ctcttgattc ccactttgtg gttctaagta 6900ctgtggtttc
caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc
6960cacatacact tcattctcag tattgttttg ccaagttcta attccatcag
acctcgacct 7020gcagccccta gtcgacgaag ttcctattcc gaagttccta
ttctctagaa agtataggaa 7080cttcgctagc taaaattgga gggacaagac
ttcccacaga ttttcggttt tgtcgggaag 7140ttttttaata ggggcaaata
aggaaaatgg gaggataggt agtcatctgg ggttttatgc 7200agcaaaacta
caggttatta ttgcttgtga tccgc 7235601499DNAArtificial
SequenceSyntheticmisc_feature(1)..(172)Mouse Upstream
Sequencemisc_feature(300)..(333)LoxPmisc_feature(379)..(1098)eGFPmisc_fea-
ture(1133)..(1354)SV40 Poly(A)misc_feature(1355)..(1499)Mouse
Downstream Sequence 60ctgcagtgga gtaggcgggg agaaggccgc acccttctcc
ggagggggga ggggagtgtt 60gcaatacctt tctgggagtt ctctgctgcc tcctggcttc
tgaggaccgc cctgggcctg 120ggagaatccc ttccccctct tccctcgtga
tctgcaactc cagtctttct agttgaccag 180ctcggcggtg acctgcacgt
ctagggcgca gtagtccagg gtttccttga tgatgtcata 240cttatcctgt
cccttttttt tccacagggc gcgccactag tggatccgga acccttaata
300taacttcgta taatgtatgc tatacgaagt tattaggtcc ctcgacctgc
agcccaagct 360agtgcccggg ccgccaccat ggtgagcaag ggcgaggagc
tgttcaccgg ggtggtgccc 420atcctggttg agctggacgg cgacgtaaac
ggccacaagt tcagcgtgtc cggcgagggc 480gagggcgatg ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg 540cccgtgccct
ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
600taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
aggctacgtc 660caggagcgca ccatcttctt caaggacgac ggcaactaca
agacccgcgc cgaggtgaag 720ttcgagggcg acaccctggt gaaccgcatc
gagctgaagg gcatcgactt caaggaggac 780ggcaacatcc tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg 840gccgacaagc
agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
900ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
cggccccgtg 960ctgctgcccg acaaccacta cctgagcacc cagtccgccc
tgagcaaaga ccccaacgag 1020aagcgcgatc acatggtcct gctggagttc
gtgaccgccg ccgggatcac tctcggcatg 1080gacgagctgt acaagtaata
attctagagt cggggcggcc ggccgcttcg agcagacatg 1140ataagataca
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
1200atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
caataaacaa 1260gttaacaaca acaattgcat tcattttatg tttcaggttc
agggggaggt gtgggaggtt 1320ttttaaagca agtaaaacct ctacaaatgt
ggtactcgag taaaattgga gggacaagac 1380ttcccacaga ttttcggttt
tgtcgggaag ttttttaata ggggcaaata aggaaaatgg 1440gaggataggt
agtcatctgg ggttttatgc agcaaaacta caggttatta ttgcttgtg 1499
* * * * *