NZ754837B2 - Orthogonal cas9 proteins for rna-guided gene regulation and editing - Google Patents
Orthogonal cas9 proteins for rna-guided gene regulation and editing Download PDFInfo
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Abstract
Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes. This allows the mediation of different RNA-guided activities at multiple targets concurrently, providing coordinated control over cellular behaviour. The orthogonal Cas9 proteins can be nuclease null, a nickase, or a nuclease. A-guided activities at multiple targets concurrently, providing coordinated control over cellular behaviour. The orthogonal Cas9 proteins can be nuclease null, a nickase, or a nuclease.
Description
(12) Granted patent specificaon (19) NZ (11) 754837 (13) B2
(47) Publicaon date: 2021.12.24
(54) ORTHOGONAL CAS9 PROTEINS FOR RNA-GUIDED GENE REGULATION AND EDITING
(51) Internaonal Patent Classificaon(s):
A61K 48/00 C07H 21/04 C12P 19/34
(22) Filing date: (73) Owner(s):
2014.07.08 PRESIDENT AND FELLOWS OF HARVARD CO
LLEGE
(23) Complete specificaon filing date:
2014.07.08 (74) Contact:
AJ PARK
(62) Divided out of 716605
(72) Inventor(s):
(30) Internaonal Priority Data: CHURCH, George M.
US 61/844,844 2013.07.10 MALI, Prashant
ESVELT, Kevin
(57) Abstract:
Methods of modulang expression of a target nucleic acid in a cell are provided including use of
mulple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding
genes or simultaneously and independently edit corresponding genes. This allows the mediaon of
different RNA-guided acvies at mulple targets concurrently, providing coordinated control over
cellular behaviour. The orthogonal Cas9 proteins can be nuclease null, a nickase, or a nuclease.
NZ 754837 B2
ORTHOGONAL CAS9 PROTEINS FOR RNA-GUIDED GENE REGULATION AND
EDITING
RELATED APPLICATION DATA
This application is a divisional of New Zealand patent application 716605, which is the
national phase entry in New Zealand of PCT international application (published
as ), and claims priority to U.S. Provisional Patent Application No. 61/844,844 filed
on July 10, 2013 and is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENT INTERESTS
This invention was made with government support under Grant No. P50 HG005550 from the
National Institutes of health and DE-FG02-02ER63445 from the Department of Energy. The
government has certain rights in the invention.
BACKGROUND
Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas
proteins to direct degradation of complementary sequences present within invading foreign nucleic
acid. See Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor
RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-
crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.
Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586
(2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus
CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282
(2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea:
versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297
(2011). A recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that
crRNA (“CRISPR RNA”) fused to a normally trans-encoded tracrRNA (“trans-activating CRISPR
RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences
matching the crRNA. Expressing a gRNA homologous to a target site results in Cas9 recruitment and
degradation of the target DNA. See H. Deveau et al., Phage response to CRISPR-encoded resistance
in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008).
SUMMARY
In a first aspect the present invention provides an in vitro or ex-vivo method of altering two or
more target nucleic acids in a cell comprising
introducing into the cell a first foreign nucleic acid encoding two or more RNAs
complementary to the two or more target nucleic acids,
introducing into the cell a second foreign nucleic acid encoding two or more orthogonal RNA
guided DNA binding protein nucleases that respectively bind to the two or more target nucleic acids
and are guided by the two or more RNAs,
wherein the RNAs and the orthogonal RNA guided DNA nucleases are expressed,
wherein two or more co-localization complexes form between an RNA, an orthogonal RNA
guided DNA binding protein nuclease and a target nucleic acid, and
wherein the two or more RNA guided DNA binding protein nucleases cut the two or more
target nucleic acids.
In a second aspect the present invention provides a cell comprising
a first foreign nucleic acid encoding two or more RNAs complementary to two or more
respective target nucleic acids,
a second foreign nucleic acid encoding two or more orthogonal RNA guided DNA binding
protein nucleases,
wherein the cell is configured to express the two or more RNAs and the two or more
orthogonal RNA guided DNA binding protein nucleases,
wherein the cell comprises two or more co-localization complexes with each including
an RNA, an orthogonal RNA guided DNA binding protein nuclease and a target nucleic acid, and
with the proviso that said cell is not present in a human being.
Aspects of the present disclosure are directed to a complex of a guide RNA, a DNA binding
protein and a double stranded DNA target sequence. According to certain aspects, DNA binding
proteins within the scope of the present disclosure include a protein that forms a complex with the
guide RNA and with the guide RNA guiding the complex to a double stranded DNA sequence wherein
the complex binds to the DNA sequence. This aspect of the present disclosure may be referred to as
co-localization of the RNA and DNA binding protein to or with the double stranded DNA. In this
manner, a DNA binding protein-guide RNA complex may be used to localize a transcriptional
regulator protein or domain at target DNA so as to regulate expression of target DNA. According to
one aspect, two or more or a plurality of orthogonal RNA guided DNA binding proteins or a set of
orthogonal RNA guided DNA binding proteins, may be used to simultaneously and independently
regulate genes in DNA in a cell. According to one aspect, two or more or a plurality of orthogonal
RNA guided DNA binding proteins or a set of orthogonal RNA guided DNA binding proteins, may be
used to simultaneously and independently edit genes in DNA in a cell. It is to be understood that
where reference is made to a DNA binding protein or an RNA guided DNA binding proteins, such
reference includes an orthogonal DNA binding protein or an orthogonal RNA guided DNA binding
protein. Such orthogonal DNA binding proteins or orthogonal RNA guided DNA binding proteins
may have nuclease activity, they may have nickase activity or they may be nuclease null.
According to certain aspects, a method of modulating expression of a target nucleic acid in a
cell is described including introducing into the cell a first foreign nucleic acid encoding one or more
RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes
the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a RNA guided
nuclease-null DNA binding protein, that binds to the DNA and is guided by the one or more RNAs,
introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or
domain, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein, and the
transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the RNA
guided nuclease-null DNA binding protein and the transcriptional regulator protein or domain co-
localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression
of the target nucleic acid.
According to one aspect, the foreign nucleic acid encoding an RNA guided nuclease-null DNA
binding protein further encodes the transcriptional regulator protein or domain fused to the RNA
guided nuclease-null DNA binding protein. According to one aspect, the foreign nucleic acid
encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign
nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding
domain fused to the transcriptional regulator protein or domain.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA is between about 10 to about 500 nucleotides. According to
one aspect, the RNA is between about 20 to about 100 nucleotides.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional
activator. According to one aspect, the transcriptional regulator protein or domain upregulates
expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or
domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition.
According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional repressor.
According to one aspect, the transcriptional regulator protein or domain downregulates expression of
the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain
downregulates expression of the target nucleic acid to treat a disease or detrimental condition.
According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
According to one aspect, the one or more RNAs is a guide RNA. According to one aspect, the
one or more RNAs is a tracrRNA-crRNA fusion.
According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or
exogenous DNA.
According to certain aspects, a method of modulating expression of a target nucleic acid in a
cell is described including introducing into the cell a first foreign nucleic acid encoding one or more
RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes
the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a RNA guided
nuclease-null DNA binding proteins of a Type II CRISPR System, that binds to the DNA and is guided
by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a
transcriptional regulator protein or domain, wherein the one or more RNAs, the RNA guided nuclease-
null DNA binding protein of a Type II CRISPR System, and the transcriptional regulator protein or
domain are expressed, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding
protein of a Type II CRISPR System and the transcriptional regulator protein or domain co-localize to
the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target
nucleic acid.
According to one aspect, the foreign nucleic acid encoding an RNA guided nuclease-null DNA
binding protein of a Type II CRISPR System further encodes the transcriptional regulator protein or
domain fused to the RNA guided nuclease-null DNA binding protein of a Type II CRISPR System.
According to one aspect, the foreign nucleic acid encoding one or more RNAs further encodes a target
of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein
or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or
domain.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA is between about 10 to about 500 nucleotides. According to
one aspect, the RNA is between about 20 to about 100 nucleotides.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional
activator. According to one aspect, the transcriptional regulator protein or domain upregulates
expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or
domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition.
According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
According to one aspect, the one or more RNAs is a guide RNA. According to one aspect, the
one or more RNAs is a tracrRNA-crRNA fusion.
According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or
exogenous DNA.
According to certain aspects, a method of modulating expression of a target nucleic acid in a
cell is described including introducing into the cell a first foreign nucleic acid encoding one or more
RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes
the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null
Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a
third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or
more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are
expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional
regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or
domain regulates expression of the target nucleic acid.
According to one aspect, the foreign nucleic acid encoding a nuclease-null Cas9 protein
further encodes the transcriptional regulator protein or domain fused to the nuclease-null Cas9 protein.
According to one aspect, the foreign nucleic acid encoding one or more RNAs further encodes a target
of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein
or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or
domain.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA is between about 10 to about 500 nucleotides. According to
one aspect, the RNA is between about 20 to about 100 nucleotides.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional
activator. According to one aspect, the transcriptional regulator protein or domain upregulates
expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or
domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition.
According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
According to one aspect, the one or more RNAs is a guide RNA. According to one aspect, the
one or more RNAs is a tracrRNA-crRNA fusion.
According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or
exogenous DNA.
According to one aspect a cell is described that includes a first foreign nucleic acid encoding
one or more RNAs complementary to DNA, wherein the DNA includes a target nucleic acid, a second
foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein, and a third foreign
nucleic acid encoding a transcriptional regulator protein or domain wherein the one or more RNAs, the
RNA guided nuclease-null DNA binding protein and the transcriptional regulator protein or domain
are members of a co-localization complex for the target nucleic acid.
According to one aspect, the foreign nucleic acid encoding an RNA guided nuclease-null DNA
binding protein further encodes the transcriptional regulator protein or domain fused to an RNA guided
nuclease-null DNA binding protein. According to one aspect, the foreign nucleic acid encoding one or
more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding
the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the
transcriptional regulator protein or domain.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA is between about 10 to about 500 nucleotides. According to
one aspect, the RNA is between about 20 to about 100 nucleotides.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional
activator. According to one aspect, the transcriptional regulator protein or domain upregulates
expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or
domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition.
According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
According to one aspect, the one or more RNAs is a guide RNA. According to one aspect, the
one or more RNAs is a tracrRNA-crRNA fusion.
According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or
exogenous DNA.
According to certain aspects, the RNA guided nuclease-null DNA binding protein is an RNA
guided nuclease-null DNA binding protein of a Type II CRISPR System. According to certain aspects,
the RNA guided nuclease-null DNA binding protein is a nuclease-null Cas9 protein.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
that includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with
each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into
the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase,
which may be an orthogonal RNA guided DNA binding protein nickase, and being guided by the two
or more RNAs, wherein the two or more RNAs and the at least one RNA guided DNA binding protein
nickase are expressed and wherein the at least one RNA guided DNA binding protein nickase co-
localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic
acid resulting in two or more adjacent nicks.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
that includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with
each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into
the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase
of a Type II CRISPR System and being guided by the two or more RNAs, wherein the two or more
RNAs and the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System are
expressed and wherein the at least one RNA guided DNA binding protein nickase of a Type II
CRISPR System co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the
DNA target nucleic acid resulting in two or more adjacent nicks.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
that includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with
each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into
the cell a second foreign nucleic acid encoding at least one Cas9 protein nickase having one inactive
nuclease domain and being guided by the two or more RNAs, wherein the two or more RNAs and the
at least one Cas9 protein nickase are expressed and wherein the at least one Cas9 protein nickase co-
localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic
acid resulting in two or more adjacent nicks.
According to the methods of altering a DNA target nucleic acid, the two or more adjacent
nicks are on the same strand of the double stranded DNA. According to one aspect, the two or more
adjacent nicks are on the same strand of the double stranded DNA and result in homologous
recombination. According to one aspect, the two or more adjacent nicks are on different strands of the
double stranded DNA. According to one aspect, the two or more adjacent nicks are on different
strands of the double stranded DNA and create double stranded breaks. According to one aspect, the
two or more adjacent nicks are on different strands of the double stranded DNA and create double
stranded breaks resulting in nonhomologous end joining. According to one aspect, the two or more
adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one
another. According to one aspect, the two or more adjacent nicks are on different strands of the double
stranded DNA and are offset with respect to one another and create double stranded breaks. According
to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and
are offset with respect to one another and create double stranded breaks resulting in nonhomologous
end joining. According to one aspect, the method further includes introducing into the cell a third
foreign nucleic acid encoding a donor nucleic acid sequence wherein the two or more nicks results in
homologous recombination of the target nucleic acid with the donor nucleic acid sequence.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each
RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell
a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase and
being guided by the two or more RNAs, and wherein the two or more RNAs and the at least one RNA
guided DNA binding protein nickase are expressed and wherein the at least one RNA guided DNA
binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and
nicks the DNA target nucleic acid resulting in two or more adjacent nicks, and wherein the two or
more adjacent nicks are on different strands of the double stranded DNA and create double stranded
breaks resulting in fragmentation of the target nucleic acid thereby preventing expression of the target
nucleic acid.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each
RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell
a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase of a
Type II CRISPR system and being guided by the two or more RNAs, and wherein the two or more
RNAs and the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System are
expressed and wherein the at least one RNA guided DNA binding protein nickase of a Type II
CRISPR System co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the
DNA target nucleic acid resulting in two or more adjacent nicks, and wherein the two or more adjacent
nicks are on different strands of the double stranded DNA and create double stranded breaks resulting
in fragmentation of the target nucleic acid thereby preventing expression of the target nucleic acid.
According to one aspect, a method of altering a DNA target nucleic acid in a cell is described
including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each
RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell
a second foreign nucleic acid encoding at least one Cas9 protein nickase having one inactive nuclease
domain and being guided by the two or more RNAs, and wherein the two or more RNAs and the at
least one Cas9 protein nickase are expressed and wherein the at least one Cas9 protein nickase co-
localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic
acid resulting in two or more adjacent nicks, and wherein the two or more adjacent nicks are on
different strands of the double stranded DNA and create double stranded breaks resulting in
fragmentation of the target nucleic acid thereby preventing expression of the target nucleic acid.
According to one aspect, a cell is described including a first foreign nucleic acid encoding two
or more RNAs with each RNA being complementary to an adjacent site in a DNA target nucleic acid,
and a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase, and
wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase are
members of a co-localization complex for the DNA target nucleic acid.
According to one aspect, the RNA guided DNA binding protein nickase is an RNA guided
DNA binding protein nickase of a Type II CRISPR System. According to one aspect, the RNA guided
DNA binding protein nickase is a Cas9 protein nickase having one inactive nuclease domain.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA includes between about 10 to about 500 nucleotides.
According to one aspect, the RNA includes between about 20 to about 100 nucleotides.
According to one aspect, the target nucleic acid is associated with a disease or detrimental
condition.
According to one aspect, the two or more RNAs are guide RNAs. According to one aspect,
the two or more RNAs are tracrRNA-crRNA fusions.
According to one aspect, the DNA target nucleic acid is genomic DNA, mitochondrial DNA,
viral DNA, or exogenous DNA.
According to one aspect, methods may include the simultaneous use of orthogonal RNA
guided DNA binding protein nickases, orthogonal RNA guided DNA binding protein nucleases,
orthogonal RNA guided nuclease null DNA binding proteins. Accordingly, in the same cell,
alterations created by nicking or cutting the DNA and translational mediation can be carried out.
Further, one or more or a plurality of exogenous donor nucleic acids may also be added to a cell using
methods known to those of skill in the art of introducing nucleic acids into cells, such as
electroporation, and the one or more or a plurality of exogenous donor nucleic acids may be introduced
into the DNA of the cell by recombination, such as homologous recombination, or other mechanisms
known to those of skill in the art. Accordingly, the use of a plurality of orthogonal RNA guided DNA
binding proteins described herein allows a single cell to be altered by nicking or cutting, allows donor
nucleic acids to be introduced into the DNA in the cell and allows genes to be transcriptionally
regulated.
Further features and advantages of certain embodiments of the present invention will become
more fully apparent in the following description of embodiments and drawings thereof, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other features and advantages of the present embodiments will be more
fully understood from the following detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
Figure 1A and Figure 1B are schematics of RNA-guided transcriptional activation. Figure 1C
is a design of a reporter construct. Figure 1D shows data demonstrating that Cas9N-VP64 fusions
display RNA-guided transcriptional activation as assayed by both fluorescence-activated cell sorting
(FACS) and immunofluorescence assays (IF). Figure 1E shows assay data by FACS and IF
demonstrating gRNA sequence-specific transcriptional activation from reporter constructs in the
presence of Cas9N, MS2-VP64 and gRNA bearing the appropriate MS2 aptamer binding sites. Figure
1F depicts data demonstrating transcriptional induction by individual gRNAs and multiple gRNAs.
Figure 2A depicts a methodology for evaluating the landscape of targeting by Cas9-gRNA
complexes and TALEs. Figure 2B depicts data demonstrating that a Cas9-gRNA complex is on
average tolerant to 1-3 mutations in its target sequences. Figure 2C depicts data demonstrating that the
Cas9-gRNA complex is largely insensitive to point mutations, except those localized to the PAM
sequence. Figure 2D depicts heat plot data demonstrating that introduction of 2 base mismatches
significantly impairs the Cas9-gRNA complex activity. Figure 2E depicts data demonstrating that an
18-mer TALE reveals is on average tolerant to 1-2 mutations in its target sequence. Figure 2F depicts
data demonstrating the 18-mer TALE is, similar to the Cas9-gRNA complexes, largely insensitive to
single base mismatched in its target. Figure 2G depicts heat plot data demonstrating that introduction
of 2 base mismatches significantly impairs the 18-mer TALE activity.
Figure 3A depicts a schematic of a guide RNA design. Figure 3B depicts data showing
percentage rate of non-homologous end joining for off-set nicks leading to 5’ overhangs and off-set
nicks leading to 5’ overhangs. Figure 3C depicts data showing percentage rate of targeting for off-set
nicks leading to 5’ overhangs and off-set nicks leading to 5’ overhangs.
Figure 4A is a schematic of a metal coordinating residue in RuvC PDB ID: 4EP4 (blue)
position D7 (left), a schematic of HNH endonuclease domains from PDB IDs: 3M7K (orange) and
4H9D (cyan) including a coordinated Mg-ion (gray sphere) and DNA from 3M7K (purple) (middle)
and a list of mutants analyzed (right). Figure 4B depicts data showing undetectable nuclease activity
for Cas9 mutants m3 and m4, and also their respective fusions with VP64. Figure 4C is a higher-
resolution examination of the data in Figure 4B.
Figure 5A is a schematic of a homologous recombination assay to determine Cas9-gRNA
activity. Figure 5B depicts guide RNAs with random sequence insertions and percentage rate of
homologous recombination
Figure 6A is a schematic of guide RNAs for the OCT4 gene. Figure 6B depicts transcriptional
activation for a promoter-luciferase reporter construct. Figure 6C depicts transcriptional activation via
qPCR of endogenous genes.
Figure 7A is a schematic of guide RNAs for the REX1 gene. Figure 7B depicts transcriptional
activation for a promoter-luciferase reporter construct. Figure 7C depicts transcriptional activation via
qPCR of endogenous genes.
Figure 8A depicts in schematic a high level specificity analysis processing flow for calculation
of normalized expression levels. Figure 8B depicts data of distributions of percentages of binding sites
by numbers of mismatches generated within a biased construct library. Left: Theoretical distribution.
Right: Distribution observed from an actual TALE construct library. Figure 8C depicts data of
distributions of percentages of tag counts aggregated to binding sites by numbers of mismatches. Left:
Distribution observed from the positive control sample. Right: Distribution observed from a sample in
which a non-control TALE was induced.
Figure 9A depicts data for analysis of the targeting landscape of a Cas9-gRNA complex
showing tolerance to 1-3 mutations in its target sequence. Figure 9B depicts data for analysis of the
targeting landscape of a Cas9-gRNA complex showing insensitivity to point mutations, except those
localized to the PAM sequence. Figure 9C depicts heat plot data for analysis of the targeting
landscape of a Cas9-gRNA complex showing that introduction of 2 base mismatches significantly
impairs activity. Figure 9D depicts data from a nuclease mediated HR assay confirming that the
predicted PAM for the S. pyogenes Cas9 is NGG and also NAG.
Figure 10A depicts data from a nuclease mediated HR assay confirming that 18-mer TALEs
tolerate multiple mutations in their target sequences. Figure 10B depicts data from analysis of the
targeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-mer). Figure 10C depicts
data for 10-mer TALEs show near single-base mismatch resolution. Figure 10D depicts heat plot data
for 10-mer TALEs show near single-base mismatch resolution.
Figure 11A depicts designed guide RNAs. Figure 11B depicts percentage rate of non-
homologous end joining for various guide RNAs
Figures 12A-12F depict comparison and characterization of putatively orthogonal Cas9
proteins. Figure 12A: Repeat sequences of SP, ST1, NM, and TD. Bases are colored to indicate the
degree of conservation. Figure 12B: Plasmids used for characterization of Cas9 proteins in E. coli.
Figure 12C: Functional PAMs are depleted from the library when spacer and protospacer match due to
Cas9 cutting. Figure 12D: Cas9 does not cut when the targeting plasmid spacer and the library
protospacer do not match. Figure 12D: Nonfunctional PAMs are never cut or depleted. Figure 12F:
Selection scheme to identify PAMs. Cells expressing a Cas9 protein and one of two spacer-containing
targeting plasmids were transformed with one of two libraries with corresponding protospacers and
subjected to antibiotic selection. Surviving uncleaved plasmids were subjected to deep sequencing.
Cas9-mediated PAM depletion was quantified by comparing the relative abundance of each sequence
within the matched versus the mismatched protospacer libraries.
Figures 13A-13F depict depletion of functional protospacer-adjacent motifs (PAMs) from
libraries by Cas9 proteins. The log frequency of each base at every position for matched spacer-
protospacer pairs is plotted relative to control conditions in which spacer and protospacer do not
match. Results reflect the mean depletion of libraries by NM (Figure 13A), ST1 (Figure 13B), and TD
(Figure 13C) based on two distinct protospacer sequences (Figure 13D). Depletion of specific
sequences for each protospacer are plotted separately for each Cas9 protein (Figur 13E-13F).
Figures 14A-14B depict transcriptional repression mediated by NM. Figure 14A: Reporter
plasmid used to quantify repression. Figure 14B: Normalized cellular fluorescence for matched and
mismatched spacer-protospacer pairs. Error bars represent the standard deviation across five
replicates.
Figure 15 depicts orthogonal recognition of crRNAs in E. coli. Cells with all combinations of
Cas9 and crRNA were challenged with a plasmid bearing a matched or mismatched protospacer and
appropriate PAM. Sufficient cells were plated to reliably obtain colonies from matching spacer and
protospacer pairings and total colony counts used to calculate the fold depletion.
Figures 16A-16B depict Cas9-mediated gene editing in human cells. Figure 16A: A
homologous recombination assay was used to quantify gene editing efficiency. Cas9-mediated double-
strand breaks within the protospacer stimulated repair of the interrupted GFP cassette using the donor
template, yielding cells with intact GFP. Three different templates were used in order to provide the
correct PAM for each Cas9. Fluorescent cells were quantified by flow cytometry. Figure 16B: Cell
sorting results for NM, ST1, and TD in combination with each of their respective sgRNAs. The
protospacer and PAM sequence for each Cas9 are displayed above each set. Repair efficiencies are
indicated in the upper-right corner of each plot.
Figures 17A-17B depict transcriptional activation in human cells. Figure 17A: Reporter
constructs for transcriptional activation featured a minimal promoter driving tdTomato. Protospacer
and PAM sequences were placed upstream of the minimal promoter. Nuclease-null Cas9-VP64 fusion
proteins binding to the protospacers resulted in transcriptional activation and enhanced fluorescence.
Figure 17B: Cells transfected with all combinations of Cas9 activator and sgRNA and tdTomato
fluorescent visualized. Transcriptional activation occurred only when each Cas9 was paired with its
own sgRNA.
DETAILED DESCRIPTION
Supporting references listed herein may be referred to by superscript. It is to be understood
that the superscript refers to the reference as if fully set forth to support a particular statement.
The CRISPR-Cas systems of bacteria and archaea confer acquired immunity by incorporating
fragments of viral or plasmid DNA into CRISPR loci and utilizing the transcribed crRNAs to guide
1, 2
nucleases to degrade homologous sequences . In Type II CRISPR systems, a ternary complex of
Cas9 nuclease with crRNA and tracrRNA (trans-activating crRNA) binds to and cleaves dsDNA
protospacer sequences that match the crRNA spacer and also contain a short protospacer-adjacent
3, 4
motif (PAM) . Fusing the crRNA and tracrRNA produces a single guide RNA (sgRNA) that is
sufficient to target Cas9 .
As an RNA-guided nuclease and nickase, Cas9 has been adapted for targeted gene editing
and selection in a variety of organisms. While these successes are arguably transformative, nuclease-
null Cas9 variants are useful for regulatory purposes, as the ability to localize proteins and RNA to
nearly any set of dsDNA sequences affords tremendous versatility for controlling biological systems
17 18
. Beginning with targeted gene repression through promoter and 5'-UTR obstruction in bacteria ,
Cas9-mediated regulation is extended to transcriptional activation by means of VP64 recruitment in
human cells. According to certain aspects the DNA binding proteins described herein, including the
orthogonal RNA guided DNA binding proteins such as orthogonal Cas9 proteins, may be used with
transcriptional activators, repressors, fluorescent protein labels, chromosome tethers, and numerous
other tools known to those of skill in the art. According to this aspect, use of orthogonal Cas9 allows
genetic modification using any and all of transcriptional activators, repressors, fluorescent protein
labels, chromosome tethers, and numerous other tools known to those of skill in the art. Accordingly,
aspect of the present disclosure are directed to the use of orthogonal Cas9 proteins for multiplexed
RNA-guided transcriptional activation, repression, and gene editing.
Embodiments of the present disclosure are directed to characterizing and demonstrating
orthogonality between multiple Cas9 proteins in bacteria and human cells. Such orthogonal RNA
guided DNA binding proteins may be used in a plurality or set to simultaneously and independently
regulate transcription, label or edit a plurality of genes in DNA of individual cells.
According to one aspect, a plurality of orthogonal Cas9 proteins are identified from within a
single family of CRISPR systems. Though clearly related, exemplary Cas9 proteins from S. pyogenes,
N. meningitidis, S. thermophilus, and T. denticola range from 3.25 to 4.6 kb in length and recognize
completely different PAM sequences.
Embodiments of the present disclosure are based on the use of DNA binding proteins to co-
localize transcriptional regulator proteins or domains to DNA in a manner to regulate a target nucleic
acid. Such DNA binding proteins are readily known to those of skill in the art to bind to DNA for
various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins
included within the scope of the present disclosure include those which may be guided by RNA,
referred to herein as guide RNA. According to this aspect, the guide RNA and the RNA guided DNA
binding protein form a co-localization complex at the DNA. According to certain aspects, the DNA
binding protein may be a nuclease-null DNA binding protein. According to this aspect, the nuclease-
null DNA binding protein may result from the alteration or modification of a DNA binding protein
having nuclease activity. Such DNA binding proteins having nuclease activity are known to those of
skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as
Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II
CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology,
Vol. 9, June 2011, pp. 467-477 including all supplementary information hereby incorporated by
reference in its entirety.
According to a certain aspect, methods are described to identify two or more or a plurality or a
set of orthogonal DNA binding proteins, such as orthogonal RNA guided DNA binding proteins, such
as orthogonal RNA guided DNA binding proteins of a Type II CRISPR system, such as orthogonal
cas9 proteins, each of which may be nuclease active or nuclease null. According to certain aspects,
two or more or a plurality or a set of orthogonal DNA binding proteins may be used with
corresponding guide RNAs to simultaneously and independently regulate genes or edit nucleic acids
within a cell. According to certain aspects, nucleic acids may be introduced into the cell which encode
for the two or more or a plurality or a set of orthogonal DNA binding proteins, the corresponding guide
RNAs and two or more or a plurality or a set of corresponding transcriptional regulators or domains.
In this manner, many genes may be target in parallel within the same cell for regulation or editing.
Methods of editing genomic DNA are well known to those of skill in the art.
Exemplary DNA binding proteins having nuclease activity function to nick or cut double
stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more
polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have
two separate nuclease domains with each domain responsible for cutting or nicking a particular strand
of the double stranded DNA. Exemplary polypeptide sequences having nuclease activity known to
those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease
domain. Accordingly, exemplary DNA binding proteins are those that in nature contain one or more
of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain. According to certain
aspects, the DNA binding protein is altered or otherwise modified to inactivate the nuclease activity.
Such alteration or modification includes altering one or more amino acids to inactivate the nuclease
activity or the nuclease domain. Such modification includes removing the polypeptide sequence or
polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide
sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from
the DNA binding protein. Other modifications to inactivate nuclease activity will be readily apparent
to one of skill in the art based on the present disclosure. Accordingly, a nuclease-null DNA binding
protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a
polypeptide sequence or sequences to inactivate nuclease activity. The nuclease-null DNA binding
protein retains the ability to bind to DNA even though the nuclease activity has been inactivated.
Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for
DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease
activity. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences
required for DNA binding but may have one or more or all of the nuclease sequences exhibiting
nuclease activity inactivated.
According to one aspect, a DNA binding protein having two or more nuclease domains may be
modified or altered to inactivate all but one of the nuclease domains. Such a modified or altered DNA
binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding
protein cuts or nicks only one strand of double stranded DNA. When guided by RNA to DNA, the
DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase.
An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II
CRISPR System which lacks nuclease activity. An exemplary DNA binding protein is a nuclease-null
Cas9 protein. An exemplary DNA binding protein is a Cas9 protein nickase.
In S. pyogenes, Cas9 generates a blunt-ended double-stranded break 3bp upstream of the
protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an
HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves
the non-complementary strand. See Jinke et al., Science 337, 816-821 (2012) hereby incorporated by
reference in its entirety. Cas9 proteins are known to exist in many Type II CRISPR systems including
the following as identified in the supplementary information to Makarova et al., Nature Reviews,
Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis C7; Corynebacterium
diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato;
Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R;
Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia
farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus
B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavida
DSM 17836 uid43465; Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bd1;
Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX
H1; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium
psychrophilum JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM
13941; Roseiflexus RS1; Synechocystis PCC6803; Elusimicrobium minutum Pei191; uncultured
Termite group 1 bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC
10987; Listeria innocua;Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius
UCC118; Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae
2603; Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus
MGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst
CH1; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes M1
GAS; Streptococcus pyogenes MGAS5005; Streptococcus pyogenes MGAS2096; Streptococcus
pyogenes MGAS9429; Streptococcus pyogenes MGAS10270; Streptococcus pyogenes MGAS6180;
Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1; Streptococcus pyogenes
MGAS10750; Streptococcus pyogenes NZ131; Streptococcus thermophiles CNRZ1066; Streptococcus
thermophiles LMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum A3 Loch
Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinum Ba4 657; Clostridium botulinum
F Langeland; Clostridium cellulolyticum H10; Finegoldia magna ATCC 29328; Eubacterium rectale
ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile 163K; Mycoplasma penetrans;
Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112; Bradyrhizobium BTAi1;
Nitrobacter hamburgensis X14; Rhodopseudomonas palustris BisB18; Rhodopseudomonas palustris
BisB5; Parvibaculum lavamentivorans DS-1; Dinoroseobacter shibae DFL 12; Gluconacetobacter
diazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5 JGI; Azospirillum B510
uid46085; Rhodospirillum rubrum ATCC 11170; Diaphorobacter TPSY uid29975; Verminephrobacter
eiseniae EF01-2; Neisseria meningitides 053442; Neisseria meningitides alpha14; Neisseria
meningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacter jejuni doylei 269 97;
Campylobacter jejuni 81116; Campylobacter jejuni; Campylobacter lari RM2100; Helicobacter
hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187; Pseudoalteromonas atlantica T6c;
Shewanella pealeana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes
130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica;
Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418;
and Treponema denticola ATCC 35405. Accordingly, aspects of the present disclosure are directed to
a Cas9 protein present in a Type II CRISPR system, which has been rendered nuclease null or which
has been rendered a nickase as described herein.
The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. The S.
pyogenes Cas9 protein sequence that is the subject of experiments described herein is shown below.
See Deltcheva et al., Nature 471, 602-607 (2011) hereby incorporated by reference in its entirety.
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE
ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG
NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD
VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN
LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA
GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE
VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL
SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG
RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL
HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER
MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH
IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS
KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF
ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA
YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE
QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD-
According to certain aspects of methods of RNA-guided genome regulation described herein,
Cas9 is altered to reduce, substantially reduce or eliminate nuclease activity. Such a Cas9 may be an
orthogonal Cas9, such as when more than one Cas9 proteins are envisioned. In this context, two or
more or a plurality or set of orthogonal Cas9 proteins may be used in the methods described herein.
According to one aspect, Cas9 nuclease activity is reduced, substantially reduced or eliminated by
altering the RuvC nuclease domain or the HNH nuclease domain. According to one aspect, the RuvC
nuclease domain is inactivated. According to one aspect, the HNH nuclease domain is inactivated.
According to one aspect, the RuvC nuclease domain and the HNH nuclease domain are inactivated.
According to an additional aspect, Cas9 proteins are described where the RuvC nuclease domain and
the HNH nuclease domain are inactivated. According to an additional aspect, nuclease-null Cas9
proteins are described insofar as the RuvC nuclease domain and the HNH nuclease domain are
inactivated. According to an additional aspect, a Cas9 nickase is described where either the RuvC
nuclease domain or the HNH nuclease domain is inactivated, thereby leaving the remaining nuclease
domain active for nuclease activity. In this manner, only one strand of the double stranded DNA is cut
or nicked.
According to an additional aspect, nuclease-null Cas9 proteins are described where one or
more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins.
According to one aspect, the amino acids include D10 and H840. See Jinke et al., Science 337, 816-
821 (2012). According to an additional aspect, the amino acids include D839 and N863. According to
one aspect, one or more or all of D10, H840, D839 and H863 are substituted with an amino acid which
reduces, substantially eliminates or eliminates nuclease activity. According to one aspect, one or more
or all of D10, H840, D839 and H863 are substituted with alanine. According to one aspect, a Cas9
protein having one or more or all of D10, H840, D839 and H863 substituted with an amino acid which
reduces, substantially eliminates or eliminates nuclease activity, such as alanine, is referred to as a
nuclease-null Cas9 or Cas9N and exhibits reduced or eliminated nuclease activity, or nuclease activity
is absent or substantially absent within levels of detection. According to this aspect, nuclease activity
for a Cas9N may be undetectable using known assays, i.e. below the level of detection of known
assays.
According to one aspect, the nuclease null Cas9 protein includes homologs and orthologs
thereof which retain the ability of the protein to bind to the DNA and be guided by the RNA.
According to one aspect, the nuclease null Cas9 protein includes the sequence as set forth for naturally
occurring Cas9 from S. pyogenes and having one or more or all of D10, H840, D839 and H863
substituted with alanine and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA
binding protein.
According to one aspect, the nuclease null Cas9 protein includes the sequence as set forth for
naturally occurring Cas9 from S. pyogenes excepting the protein sequence of the RuvC nuclease
domain and the HNH nuclease domain and also protein sequences having at least 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as
an RNA guided DNA binding protein. In this manner, aspects of the present disclosure include the
protein sequence responsible for DNA binding, for example, for co-localizing with guide RNA and
binding to DNA and protein sequences homologous thereto, and need not include the protein
sequences for the RuvC nuclease domain and the HNH nuclease domain (to the extent not needed for
DNA binding), as these domains may be either inactivated or removed from the protein sequence of
the naturally occurring Cas9 protein to produce a nuclease null Cas9 protein.
For purposes of the present disclosure, Figure 4A depicts metal coordinating residues in
known protein structures with homology to Cas9. Residues are labeled based on position in Cas9
sequence. Left: RuvC structure, PDB ID: 4EP4 (blue) position D7, which corresponds to D10 in the
Cas9 sequence, is highlighted in a Mg-ion coordinating position. Middle: Structures of HNH
endonuclease domains from PDB IDs: 3M7K (orange) and 4H9D (cyan) including a coordinated Mg-
ion (gray sphere) and DNA from 3M7K (purple). Residues D92 and N113 in 3M7K and 4H9D
positions D53 and N77, which have sequence homology to Cas9 amino acids D839 and N863, are
shown as sticks. Right: List of mutants made and analyzed for nuclease activity: Cas9 wildtype;
Cas9 which substitutes alanine for D10; Cas9 which substitutes alanine for D10 and alanine for
m1 m2
H840; Cas9 which substitutes alanine for D10, alanine for H840, and alanine for D839; and Cas9
m3 m4
which substitutes alanine for D10, alanine for H840, alanine for D839, and alanine for N863.
As shown in Figure 4B, the Cas9 mutants: m3 and m4, and also their respective fusions with
VP64 showed undetectable nuclease activity upon deep sequencing at targeted loci. The plots show the
mutation frequency versus genomic position, with the red lines demarcating the gRNA target. Figure
4C is a higher-resolution examination of the data in Figure 4B and confirms that the mutation
landscape shows comparable profile as unmodified loci.
According to one aspect, an engineered Cas9-gRNA system is described which enables RNA-
guided genome regulation in human cells by tethering transcriptional activation domains to either a
nuclease-null Cas9 or to guide RNAs. According to one aspect of the present disclosure, one or more
transcriptional regulatory proteins or domains (such terms are used interchangeably) are joined or
otherwise connected to a nuclease-deficient Cas9 or one or more guide RNA (gRNA). The
transcriptional regulatory domains correspond to targeted loci. Accordingly, aspects of the present
disclosure include methods and materials for localizing transcriptional regulatory domains to targeted
loci by fusing, connecting or joining such domains to either Cas9N or to the gRNA.
According to one aspect, a Cas9N-fusion protein capable of transcriptional activation is
described. According to one aspect, a VP64 activation domain (see Zhang et al., Nature Biotechnology
29, 149-153 (2011) hereby incorporated by reference in its entirety) is joined, fused, connected or
otherwise tethered to the C terminus of Cas9N. According to one method, the transcriptional
regulatory domain is provided to the site of target genomic DNA by the Cas9N protein. According to
one method, a Cas9N fused to a transcriptional regulatory domain is provided within a cell along with
one or more guide RNAs. The Cas9N with the transcriptional regulatory domain fused thereto bind at
or near target genomic DNA. The one or more guide RNAs bind at or near target genomic DNA. The
transcriptional regulatory domain regulates expression of the target gene. According to a specific
aspect, a Cas9N-VP64 fusion activated transcription of reporter constructs when combined with
gRNAs targeting sequences near the promoter, thereby displaying RNA-guided transcriptional
activation.
According to one aspect, a gRNA-fusion protein capable of transcriptional activation is
described. According to one aspect, a VP64 activation domain is joined, fused, connected or otherwise
tethered to the gRNA. According to one method, the transcriptional regulatory domain is provided to
the site of target genomic DNA by the gRNA. According to one method, a gRNA fused to a
transcriptional regulatory domain is provided within a cell along with a Cas9N protein. The Cas9N
binds at or near target genomic DNA. The one or more guide RNAs with the transcriptional regulatory
protein or domain fused thereto bind at or near target genomic DNA. The transcriptional regulatory
domain regulates expression of the target gene. According to a specific aspect, a Cas9N protein and a
gRNA fused with a transcriptional regulatory domain activated transcription of reporter constructs,
thereby displaying RNA-guided transcriptional activation.
The gRNA tethers capable of transcriptional regulation were constructed by identifying which
regions of the gRNA will tolerate modifications by inserting random sequences into the gRNA and
assaying for Cas9 function. gRNAs bearing random sequence insertions at either the 5’ end of the
crRNA portion or the 3’ end of the tracrRNA portion of a chimeric gRNA retain functionality, while
insertions into the tracrRNA scaffold portion of the chimeric gRNA result in loss of function. See Fig.
5A-5B summarizing gRNA flexibility to random base insertions. Figure 5A is a schematic of a
homologous recombination (HR) assay to determine Cas9-gRNA activity. As shown in Figure 5B,
gRNAs bearing random sequence insertions at either the 5’ end of the crRNA portion or the 3’ end of
the tracrRNA portion of a chimeric gRNA retain functionality, while insertions into the tracrRNA
scaffold portion of the chimeric gRNA result in loss of function. The points of insertion in the gRNA
sequence are indicated by red nucleotides. Without wishing to be bound by scientific theory, the
increased activity upon random base insertions at the 5’ end may be due to increased half-life of the
longer gRNA.
To attach VP64 to the gRNA, two copies of the MS2 bacteriophage coat-protein binding RNA
stem-loop were appended to the 3’ end of the gRNA. See Fusco et al., Current Biology: CB13, 161-
167 (2003) hereby incorporated by reference in its entirety. These chimeric gRNAs were expressed
together with Cas9N and MS2-VP64 fusion protein. Sequence-specific transcriptional activation from
reporter constructs was observed in the presence of all 3 components.
Figure 1A is a schematic of RNA-guided transcriptional activation. As shown in Figure 1A, to
generate a Cas9N-fusion protein capable of transcriptional activation, the VP64 activation domain was
directly tethered to the C terminus of Cas9N. As shown in Figure 1B, to generate gRNA tethers
capable of transcriptional activation, two copies of the MS2 bacteriophage coat-protein binding RNA
stem-loop were appended to the 3’ end of the gRNA. These chimeric gRNAs were expressed together
with Cas9N and MS2-VP64 fusion protein. Figure 1C shows design of reporter constructs used to
assay transcriptional activation. The two reporters bear distinct gRNA target sites, and share a control
TALE-TF target site. As shown in Figure 1D, Cas9N-VP64 fusions display RNA-guided
transcriptional activation as assayed by both fluorescence-activated cell sorting (FACS) and
immunofluorescence assays (IF). Specifically, while the control TALE-TF activated both reporters, the
Cas9N-VP64 fusion activates reporters in a gRNA sequence specific manner. As shown in Figure 1E,
gRNA sequence-specific transcriptional activation from reporter constructs only in the presence of all
3 components: Cas9N, MS2-VP64 and gRNA bearing the appropriate MS2 aptamer binding sites was
observed by both FACS and IF.
According to certain aspects, methods are described for regulating endogenous genes using
Cas9N, one or more gRNAs and a transcriptional regulatory protein or domain. According to one
aspect, an endogenous gene can be any desired gene, refered to herein as a target gene. According to
one exemplary aspect, genes target for regulation included ZFP42 (REX1) and POU5F1 (OCT4),
which are both tightly regulated genes involved in maintenance of pluripotency. As shown in Figure
1F, 10 gRNAs targeting a ~5kb stretch of DNA upstream of the transcription start site (DNase
hypersensitive sites are highlighted in green) were designed for the REX1 gene. Transcriptional
activation was assayed using either a promoter-luciferase reporter construct (see Takahashi et al., Cell
131 861-872 (2007) hereby incorporated by reference in its entirety) or directly via qPCR of the
endogenous genes.
Figures 6A-6D are directed to RNA-guided OCT4 regulation using Cas9N-VP64. As shown in
Figure 6A, 21 gRNAs targeting a ~5kb stretch of DNA upstream of the transcription start site were
designed for the OCT4 gene. The DNase hypersensitive sites are highlighted in green. Figure 6B
shows transcriptional activation using a promoter-luciferase reporter construct. Figure 6C shows
transcriptional activation directly via qPCR of the endogenous genes. While introduction of individual
gRNAs modestly stimulated transcription, multiple gRNAs acted synergistically to stimulate robust
multi-fold transcriptional activation.
Figures 7A-7C are directed to RNA-guided REX1 regulation using Cas9N, MS2-VP64 and
gRNA+2X-MS2 aptamers. As shown in Figure 7A, 10 gRNAs targeting a ~5kb stretch of DNA
upstream of the transcription start site were designed for the REX1 gene. The DNase hypersensitive
sites are highlighted in green. Figure 7B shows transcriptional activation using a promoter-luciferase
reporter construct. Figure 7C shows transcriptional activation directly via qPCR of the endogenous
genes. While introduction of individual gRNAs modestly stimulated transcription, multiple gRNAs
acted synergistically to stimulate robust multi-fold transcriptional activation. In one aspect, the absence
of the 2X-MS2 aptamers on the gRNA does not result in transcriptional activation. See Maeder et al.,
Nature Methods 10, 243-245 (2013) and Perez-Pinera et al., Nature Methods 10, 239-242 (2013) each
of which are hereby incorporated by reference in its entirety.
Accordingly, methods are directed to the use of multiple guide RNAs with a Cas9N protein
and a transcriptional regulatory protein or domain to regulate expression of a target gene.
Both the Cas9 and gRNA tethering approaches were effective, with the former displaying
~1.5–2 fold higher potency. This difference is likely due to the requirement for 2-component as
opposed to 3-component complex assembly. However, the gRNA tethering approach in principle
enables different effector domains to be recruited by distinct gRNAs so long as each gRNA uses a
different RNA-protein interaction pair. See Karyer-Bibens et al., Biology of the Cell / Under the
Auspices of the European Cell Biology Organization 100, 125-138 (2008) hereby incorporated by
reference in its entirety. According to one aspect of the present disclosure, different target genes may
be regulated using specific guide RNA and a generic Cas9N protein, i.e. the same or a similar Cas9N
protein for different target genes. According to one aspect, methods of multiplex gene regulation are
described using the same or similar Cas9N.
Methods of the present disclosure are also directed to editing target genes using the Cas9N
proteins and guide RNAs described herein to provide multiplex genetic and epigenetic engineering of
human cells. With Cas9-gRNA targeting being an issue (see Jiang et al., Nature Biotechnology 31,
233-239 (2013) hereby incorporated by reference in its entirety), methods are described for in-depth
interrogation of Cas9 affinity for a very large space of target sequence variations. Accordingly, aspects
of the present disclosure provide direct high-throughput readout of Cas9 targeting in human cells,
while avoiding complications introduced by dsDNA cut toxicity and mutagenic repair incurred by
specificity testing with native nuclease-active Cas9.
Further aspects of the present disclosure are directed to the use of DNA binding proteins or
systems in general for the transcriptional regulation of a target gene. One of skill in the art will readily
identify exemplary DNA binding systems based on the present disclosure. Such DNA binding systems
need not have any nuclease activity, as with the naturally occurring Cas9 protein. Accordingly, such
DNA binding systems need not have nuclease activity inactivated. One exemplary DNA binding
system is TALE. According to one aspect, TALE specificity was evaluated using the methodology
shown in Figure 2A. A construct library in which each element of the library comprises a minimal
promoter driving a dTomato fluorescent protein is designed. Downstream of the transcription start site
m, a 24bp (A/C/G) random transcript tag is inserted, while two TF binding sites are placed upstream of
the promoter: one is a constant DNA sequence shared by all library elements, and the second is a
variable feature that bears a ‘biased’ library of binding sites which are engineered to span a large
collection of sequences that present many combinations of mutations away from the target sequence
the programmable DNA targeting complex was designed to bind. This is achieved using degenerate
oligonucleotides engineered to bear nucleotide frequencies at each position such that the target
sequence nucleotide appears at a 79% frequency and each other nucleotide occurs at 7% frequency.
See Patwardhan et al., Nature Biotechnology 30, 265-270 (2012) hereby incorporated by reference in
its entirety. The reporter library is then sequenced to reveal the associations between the 24bp
dTomato transcript tags and their corresponding ‘biased’ target site in the library element. The large
diversity of the transcript tags assures that sharing of tags between different targets will be extremely
rare, while the biased construction of the target sequences means that sites with few mutations will be
associated with more tags than sites with more mutations. Next, transcription of the dTomato reporter
genes is stimulated with either a control-TF engineered to bind the shared DNA site, or the target-TF
that was engineered to bind the target site. The abundance of each expressed transcript tag is measured
in each sample by conducting RNAseq on the stimulated cells, which is then mapped back to their
corresponding binding sites using the association table established earlier. The control-TF is expected
to excite all library members equally since its binding site is shared across all library elements, while
the target-TF is expected to skew the distribution of the expressed members to those that are
preferentially targeted by it. This assumption is used in step 5 to compute a normalized expression
level for each binding site by dividing the tag counts obtained for the target-TF by those obtained for
the control-TF.
Specifically, the methodology shown in Figure 2A involves:
Step 1: Map barcode to corresponding target site in the library;
Step 2: stimulate library by either a:
1) control-TF that binds the shared site; or
2) TALE-TF/gRNA+Cas9-TF (target-TF) that binds the target site.
Step 3: Perform RNAseq and determine expressed barcodes for each.
Step 4: Map back expressed barcodes to corresponding binding sites.
Step 5: Compute relative enrichment of target-TF vs. control-TF barcodes.
As shown in Figure 2B, the targeting landscape of a Cas9-gRNA complex reveals that it is on
average tolerant to 1-3 mutations in its target sequences. As shown in Figure 2C, the Cas9-gRNA
complex is also largely insensitive to point mutations, except those localized to the PAM sequence.
Notably this data reveals that the predicted PAM for the S. pyogenes Cas9 is not just NGG but also
NAG. As shown in Figure 2D, introduction of 2 base mismatches significantly impairs the Cas9-
gRNA complex activity, however only when these are localized to the 8-10 bases nearer the 3’ end of
the gRNA target sequence (in the heat plot the target sequence positions are labeled from 1-23 starting
from the 5’ end).
The mutational tolerance of another widely used genome editing tool, TALE domains, was
determined using the transcriptional specificity assay described herein. As shown in Figure 2E, the
TALE off-targeting data for an 18-mer TALE reveals that it can tolerate on average 1-2 mutations in
its target sequence, and fails to activate a large majority of 3 base mismatch variants in its targets. As
shown in Figure 2F, the 18-mer TALE is, similar to the Cas9-gRNA complexes, largely insensitive to
single base mismatched in its target. As shown in Figure 2G, introduction of 2 base mismatches
significantly impairs the 18-mer TALE activity. TALE activity is more sensitive to mismatches nearer
the 5’ end of its target sequence (in the heat plot the target sequence positions are labeled from 1-18
starting from the 5’ end).
Results were confirmed using targeted experiments in a nuclease assay which is the subject of
Figures 10A-10D directed to evaluating the landscape of targeting by TALEs of different sizes. As
shown in Figure 10A, using a nuclease mediated HR assay, it was confirmed that 18-mer TALEs
tolerate multiple mutations in their target sequences. As shown in Figure 10B, using the approach
described in Fig. 2A, the targeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-
mer) was analyzed. Shorter TALEs (14-mer and 10-mer) are progressively more specific in their
targeting but also reduced in activity by nearly an order of magnitude. As shown in Figures 10C and
10D, 10-mer TALEs show near single-base mismatch resolution, losing almost all activity against
targets bearing 2 mismatches (in the heat plot the target sequence positions are labeled from 1-10
starting from the 5’ end). Taken together, these data imply that engineering shorter TALEs can yield
higher specificity in genome engineering applications, while the requirement for FokI dimerization in
TALE nuclease applications is essential to avoid off-target effect. See Kim et al., Proceedings of the
National Academy of Sciences of the United States of America 93, 1156-1160 (1996) and Pattanayak et
al., Nature Methods 8, 765-770 (2011) each of which are hereby incorporated by reference in its
entirety.
Figures 8A-8C are directed to high level specificity analysis processing flow for calculation of
normalized expression levels illustrated with examples from experimental data. As shown in Figure
8A, construct libraries are generated with a biased distribution of binding site sequences and random
sequence 24bp tags that will be incorporated into reporter gene transcripts (top). The transcribed tags
are highly degenerate so that they should map many-to-one to Cas9 or TALE binding sequences. The
construct libraries are sequenced (3 level, left) to establish which tags co-occur with binding sites,
resulting in an association table of binding sites vs. transcribed tags (4 level, left). Multiple construct
libraries built for different binding sites may be sequenced at once using library barcodes (indicated
here by the light blue and light yellow colors; levels 1-4, left). A construct library is then transfected
into a cell population and a set of different Cas9/gRNA or TALE transcription factors are induced in
samples of the populations (2 level, right). One sample is always induced with a fixed TALE
activator targeted to a fixed binding site sequence within the construct (top level, green box); this
sample serves as a positive control (green sample, also indicated by a + sign). cDNAs generated from
the reporter mRNA molecules in the induced samples are then sequenced and analyzed to obtain tag
rd th
counts for each tag in a sample (3 and 4 level, right). As with the construct library sequencing,
multiple samples, including the positive control, are sequenced and analyzed together by appending
sample barcodes. Here the light red color indicates one non-control sample that has been sequenced
and analyzed with the positive control (green). Because only the transcribed tags and not the construct
binding sites appear in each read, the binding site vs. tag association table obtained from construct
library sequencing is then used to tally up total counts of tags expressed from each binding site in each
level). The tallies for each non-positive control sample are then converted to normalized
sample (5
expression levels for each binding site by dividing them by the tallies obtained in the positive control
sample. Examples of plots of normalized expression levels by numbers of mismatches are provided in
Figures 2B and 2E, and in Figure 9A and Figure 10B. Not covered in this overall process flow are
several levels of filtering for erroneous tags, for tags not associable with a construct library, and for
tags apparently shared with multiple binding sites. Figure 8B depicts example distributions of
percentages of binding sites by numbers of mismatches generated within a biased construct library.
Left: Theoretical distribution. Right: Distribution observed from an actual TALE construct library.
Figure 8C depicts example distributions of percentages of tag counts aggregated to binding sites by
numbers of mismatches. Left: Distribution observed from the positive control sample. Right:
Distribution observed from a sample in which a non-control TALE was induced. As the positive
control TALE binds to a fixed site in the construct, the distribution of aggregated tag counts closely
reflects the distribution of binding sites in Figure 8B, while the distribution is skewed to the left for the
non-control TALE sample because sites with fewer mismatches induce higher expression levels.
Below: Computing the relative enrichment between these by dividing the tag counts obtained for the
target-TF by those obtained for the control-TF reveals the average expression level versus the number
of mutations in the target site.
These results are further reaffirmed by specificity data generated using a different Cas9-gRNA
complex. As shown in Figure 9A, a different Cas9-gRNA complex is tolerant to 1-3 mutations in its
target sequence. As shown in Figure 9B, the Cas9-gRNA complex is also largely insensitive to point
mutations, except those localized to the PAM sequence. As shown in Figure 9C, introduction of 2
base mismatches however significantly impairs activity (in the heat plot the target sequence positions
are labeled from 1-23 starting from the 5’ end). As shown in Figure 9D, it was confirmed using a
nuclease mediated HR assay that the predicted PAM for the S. pyogenes Cas9 is NGG and also NAG.
According to certain aspects, binding specificity is increased according to methods described
herein. Because synergy between multiple complexes is a factor in target gene activation by Cas9N-
VP64, transcriptional regulation applications of Cas9N is naturally quite specific as individual off-
target binding events should have minimal effect. According to one aspect, off-set nicks are used in
methods of genome-editing. A large majority of nicks seldom result in NHEJ events, (see Certo et al.,
Nature Methods 8, 671-676 (2011) hereby incorporated by reference in its entirety) thus minimizing
the effects of off-target nicking. In contrast, inducing off-set nicks to generate double stranded breaks
(DSBs) is highly effective at inducing gene disruption. According to certain aspects, 5’ overhangs
generate more significant NHEJ events as opposed to 3’ overhangs. Similarly, 3’ overhangs favor HR
over NHEJ events, although the total number of HR events is significantly lower than when a 5’
overhang is generated. Accordingly, methods are described for using nicks for homologous
recombination and off-set nicks for generating double stranded breaks to minimize the effects of off-
target Cas9-gRNA activity.
Figures 3A-3C are directed to multiplex off-set nicking and methods for reducing the off-
target binding with the guide RNAs. As shown in Figure 3A, the traffic light reporter was used to
simultaneously assay for HR and NHEJ events upon introduction of targeted nicks or breaks. DNA
cleavage events resolved through the HDR pathway restore the GFP sequence, whereas mutagenic
NHEJ causes frameshifts rendering the GFP out of frame and the downstream mCherry sequence in
frame. For the assay, 14 gRNAs covering a 200bp stretch of DNA: 7 targeting the sense strand (U1-7)
and 7 the antisense strand (D1-7) were designed. Using the Cas9D10A mutant, which nicks the
complementary strand, different two-way combinations of the gRNAs were used to induce a range of
programmed 5’ or 3’ overhangs (the nicking sites for the 14 gRNAs are indicated). As shown in
Figure 3B, inducing off-set nicks to generate double stranded breaks (DSBs) is highly effective at
inducing gene disruption. Notably off-set nicks leading to 5’ overhangs result in more NHEJ events as
opposed to 3’ overhangs. As shown in Figure 3C, generating 3’ overhangs also favors the ratio of HR
over NHEJ events, but the total number of HR events is significantly lower than when a 5’ overhang is
generated.
Figurea 11A-11B are directed to Cas9D10A nickase mediated NHEJ. As shown in Figure
11A, the traffic light reporter was used to assay NHEJ events upon introduction of targeted nicks or
double-stranded breaks. Briefly, upon introduction of DNA cleavage events, if the break goes through
mutagenic NHEJ, the GFP is translated out of frame and the downstream mCherry sequences are
rendered in frame resulting in red fluorescence. 14 gRNAs covering a 200bp stretch of DNA: 7
targeting the sense strand (U1-7) and 7 the antisense strand (D1-7) were designed. As shown in Figure
11B, it was observed that unlike the wild-type Cas9 which results in DSBs and robust NHEJ across all
targets, most nicks (using the Cas9D10A mutant) seldom result in NHEJ events. All 14 sites are
located within a contiguous 200bp stretch of DNA and over 10-fold differences in targeting
efficiencies were observed.
According to certain aspects, methods are described herein of modulating expression of a
target nucleic acid in a cell that include introducing one or more, two or more or a plurality of foreign
nucleic acids into the cell. The foreign nucleic acids introduced into the cell encode for a guide RNA
or guide RNAs, a nuclease-null Cas9 protein or proteins and a transcriptional regulator protein or
domain. Together, a guide RNA, a nuclease-null Cas9 protein and a transcriptional regulator protein
or domain are referred to as a co-localization complex as that term is understood by one of skill in the
art to the extent that the guide RNA, the nuclease-null Cas9 protein and the transcriptional regulator
protein or domain bind to DNA and regulate expression of a target nucleic acid. According to certain
additional aspects, the foreign nucleic acids introduced into the cell encode for a guide RNA or guide
RNAs and a Cas9 protein nickase. Together, a guide RNA and a Cas9 protein nickase are referred to
as a co-localization complex as that term is understood by one of skill in the art to the extent that the
guide RNA and the Cas9 protein nickase bind to DNA and nick a target nucleic acid.
Cells according to the present disclosure include any cell into which foreign nucleic acids can
be introduced and expressed as described herein. It is to be understood that the basic concepts of the
present disclosure described herein are not limited by cell type. Cells according to the present
disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archael
cells, eubacterial cells and the like. Cells include eukaryotic cells such as yeast cells, plant cells, and
animal cells. Particular cells include mammalian cells. Further, cells include any in which it would be
beneficial or desirable to regulate a target nucleic acid. Such cells may include those which are
deficient in expression of a particular protein leading to a disease or detrimental condition. Such
diseases or detrimental conditions are readily known to those of skill in the art. According to the
present disclosure, the nucleic acid responsible for expressing the particular protein may be targeted by
the methods described herein and a transcriptional activator resulting in upregulation of the target
nucleic acid and corresponding expression of the particular protein. In this manner, the methods
described herein provide therapeutic treatment.
Target nucleic acids include any nucleic acid sequence to which a co-localization complex as
described herein can be useful to either regulate or nick. Target nucleic acids include genes. For
purposes of the present disclosure, DNA, such as double stranded DNA, can include the target nucleic
acid and a co-localization complex can bind to or otherwise co-localize with the DNA at or adjacent or
near the target nucleic acid and in a manner in which the co-localization complex may have a desired
effect on the target nucleic acid. Such target nucleic acids can include endogenous (or naturally
occurring) nucleic acids and exogenous (or foreign) nucleic acids. One of skill based on the present
disclosure will readily be able to identify or design guide RNAs and Cas9 proteins which co-localize to
a DNA including a target nucleic acid. One of skill will further be able to identify transcriptional
regulator proteins or domains which likewise co-localize to a DNA including a target nucleic acid.
DNA includes genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.
Foreign nucleic acids (i.e. those which are not part of a cell’s natural nucleic acid composition)
may be introduced into a cell using any method known to those skilled in the art for such introduction.
Such methods include transfection, transduction, viral transduction, microinjection, lipofection,
nucleofection, nanoparticle bombardment, transformation, conjugation and the like. One of skill in the
art will readily understand and adapt such methods using readily identifiable literature sources.
Transcriptional regulator proteins or domains which are transcriptional activators include
VP16 and VP64 and others readily identifiable by those skilled in the art based on the present
disclosure.
Diseases and detrimental conditions are those characterized by abnormal loss of expression of
a particular protein. Such diseases or detrimental conditions can be treated by upregulation of the
particular protein. Accordingly, methods of treating a disease or detrimental condition are described
where the co-localization complex as described herein associates or otherwise binds to DNA including
a target nucleic acid, and the transcriptional activator of the co-localization complex upregulates
expression of the target nucleic acid. For example upregulating PRDM16 and other genes promoting
brown fat differentiation and increased metabolic uptake can be used to treat metabolic syndrome or
obesity. Activating anti-inflammatory genes are useful in autoimmunity and cardiovascular disease.
Activating tumor suppressor genes is useful in treating cancer. One of skill in the art will readily
identify such diseases and detrimental conditions based on the present disclosure.
The following examples are set forth as being representative of the present disclosure. These
examples are not to be construed as limiting the scope of the present disclosure as these and other
equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying
claims.
EXAMPLE I
Cas9 Mutants
Sequences homologous to Cas9 with known structure were searched to identify candidate
mutations in Cas9 that could ablate the natural activity of its RuvC and HNH domains. Using HHpred
(world wide website toolkit.tuebingen.mpg.de/hhpred), the full sequence of Cas9 was queried against
the full Protein Data Bank (January 2013). This search returned two different HNH endonucleases that
had significant sequence homology to the HNH domain of Cas9; PacI and a putative endonuclease
(PDB IDs: 3M7K and 4H9D respectively). These proteins were examined to find residues involved in
magnesium ion coordination. The corresponding residues were then identified in the sequence
alignment to Cas9. Two Mg-coordinating side-chains in each structure were identified that aligned to
the same amino acid type in Cas9. They are 3M7K D92 and N113, and 4H9D D53 and N77. These
residues corresponded to Cas9 D839 and N863. It was also reported that mutations of PacI residues
D92 and N113 to alanine rendered the nuclease catalytically deficient. The Cas9 mutations D839A and
N863A were made based on this analysis. Additionally, HHpred also predicts homology between Cas9
and the N-terminus of a Thermus thermophilus RuvC (PDB ID: 4EP4). This sequence alignment
covers the previously reported mutation D10A which eliminates function of the RuvC domain in Cas9.
To confirm this as an appropriate mutation, the metal binding residues were determined as before. In
4EP4, D7 helps to coordinate a magnesium ion. This position has sequence homology corresponding
to Cas9 D10, confirming that this mutation helps remove metal binding, and thus catalytic activity
from the Cas9 RuvC domain.
EXAMPLE II
Plasmid Construction
The Cas9 mutants were generated using the Quikchange kit (Agilent technologies). The target
gRNA expression constructs were either (1) directly ordered as individual gBlocks from IDT and
cloned into the pCR-BluntII-TOPO vector (Invitrogen); or (2) custom synthesized by Genewiz; or (3)
assembled using Gibson assembly of oligonucleotides into the gRNA cloning vector (plasmid #41824).
The vectors for the HR reporter assay involving a broken GFP were constructed by fusion PCR
assembly of the GFP sequence bearing the stop codon and appropriate fragment assembled into the
EGIP lentivector from Addgene (plasmid #26777). These lentivectors were then used to establish the
GFP reporter stable lines. TALENs used in this study were constructed using standard protocols. See
Sanjana et al., Nature Protocols 7, 171-192 (2012) hereby incorporated by reference in its entirety.
Cas9N and MS2 VP64 fusions were performed using standard PCR fusion protocol procedures. The
promoter luciferase constructs for OCT4 and REX1 were obtained from Addgene (plasmid #17221 and
plasmid #17222).
EXAMPLE III
Cell culture and Transfections
HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin
(pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). Cells were maintained at
37°C and 5% CO in a humidified incubator.
Transfections involving nuclease assays were as follows: 0.4×10 cells were transfected with
2μg Cas9 plasmid, 2μg gRNA and/or 2μg DNA donor plasmid using Lipofectamine 2000 as per the
manufacturer’s protocols. Cells were harvested 3 days after transfection and either analyzed by FACS,
or for direct assay of genomic cuts the genomic DNA of ~1 X 10 cells was extracted using DNAeasy
kit (Qiagen). For these PCR was conducted to amplify the targeting region with genomic DNA derived
from the cells and amplicons were deep sequenced by MiSeq Personal Sequencer (Illumina) with
coverage >200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiencies.
For transfections involving transcriptional activation assays: 0.4×10 cells were transfected
with (1) 2μg Cas9N-VP64 plasmid, 2μg gRNA and/or 0.25μg of reporter construct; or (2) 2μg Cas9N
plasmid, 2μg MS2-VP64, 2μg gRNA-2XMS2aptamer and/or 0.25μg of reporter construct. Cells were
harvested 24-48hrs post transfection and assayed using FACS or immunofluorescence methods, or
their total RNA was extracted and these were subsequently analyzed by RT-PCR. Here standard
taqman probes from Invitrogen for OCT4 and REX1 were used, with normalization for each sample
performed against GAPDH.
For transfections involving transcriptional activation assays for specificity profile of Cas9-
gRNA complexes and TALEs: 0.4×10 cells were transfected with (1) 2μg Cas9N-VP64 plasmid, 2μg
gRNA and 0.25μg of reporter library; or (2) 2μg TALE-TF plasmid and 0.25μg of reporter library; or
(3) 2μg control-TF plasmid and 0.25μg of reporter library. Cells were harvested 24hrs post transfection
(to avoid the stimulation of reporters being in saturation mode). Total RNA extraction was performed
using RNAeasy-plus kit (Qiagen), and standard RT-pcr performed using Superscript-III (Invitrogen).
Libraries for next-generation sequencing were generated by targeted pcr amplification of the
transcript-tags.
EXAMPLE IV
Computational and Sequence Analysis for Calculation of Cas9-TF and TALE-TF Reporter Expression
Levels
The high-level logic flow for this process is depicted in Figure 8A, and additional details are
given here. For details on construct library composition, see Figures 8A (level 1) and 8B.
Sequencing: For Cas9 experiments, construct library (Figure 8A, level 3, left) and reporter gene
cDNA sequences (Figure 8A, level 3, right) were obtained as 150bp overlapping paired end reads on
an Illumina MiSeq, while for TALE experiments, corresponding sequences were obtained as 51bp
non-overlapping paired end reads on an Illumina HiSeq.
Construct library sequence processing: Alignment: For Cas9 experiments, novoalign V2.07.17 (world
wide website novocraft.com/main/index/php) was used to align paired reads to a set of 250bp reference
sequences that corresponded to 234bp of the constructs flanked by the pairs of 8bp library barcodes
(see Figure 8A, 3 level, left). In the reference sequences supplied to novoalign, the 23bp degenerate
Cas9 binding site regions and the 24bp degenerate transcript tag regions (see Figure 8A, first level)
were specified as Ns, while the construct library barcodes were explicitly provided. For TALE
experiments, the same procedures were used except that the reference sequences were 203bp in length
and the degenerate binding site regions were 18bp vs. 23bp in length. Validity checking: Novoalign
output for comprised files in which left and right reads for each read pair were individually aligned to
the reference sequences. Only read pairs that were both uniquely aligned to the reference sequence
were subjected to additional validity conditions, and only read pairs that passed all of these conditions
were retained. The validity conditions included: (i) Each of the two construct library barcodes must
align in at least 4 positions to a reference sequence barcode, and the two barcodes must to the barcode
pair for the same construct library. (ii) All bases aligning to the N regions of the reference sequence
must be called by novoalign as As, Cs, Gs or Ts. Note that for neither Cas9 nor TALE experiments
did left and right reads overlap in a reference N region, so that the possibility of ambiguous novoalign
calls of these N bases did not arise. (iii) Likewise, no novoalign-called inserts or deletions must appear
in these regions. (iv) No Ts must appear in the transcript tag region (as these random sequences were
generated from As, Cs, and Gs only). Read pairs for which any one of these conditions were violated
were collected in a rejected read pair file. These validity checks were implemented using custom perl
scripts.
Induced sample reporter gene cDNA sequence processing: Alignment: SeqPrep (downloaded from
world wide website github.com/jstjohn/SeqPrep) was first used to merge the overlapping read pairs to
the 79bp common segment, after which novoalign (version above) was used to align these 79bp
common segments as unpaired single reads to a set of reference sequences (see Figure 8A, 3 level,
right) in which (as for the construct library sequencing) the 24bp degenerate transcript tag was
specified as Ns while the sample barcodes were explicitly provided. Both TALE and Cas9 cDNA
sequence regions corresponded to the same 63bp regions of cDNA flanked by pairs of 8bp sample
barcode sequences. Validity checking: The same conditions were applied as for construct library
sequencing (see above) except that: (a) Here, due prior SeqPrep merging of read pairs, validity
processing did not have to filter for unique alignments of both reads in a read pair but only for unique
alignments of the merged reads. (b) Only transcript tags appeared in the cDNA sequence reads, so that
validity processing only applied these tag regions of the reference sequences and not also to a separate
binding site region.
Assembly of table of binding sites vs. transcript tag associations: Custom perl was used to generate
these tables from the validated construct library sequences (Figure 8A, 4 level, left). Although the
24bp tag sequences composed of A, C, and G bases should be essentially unique across a construct
library (probability of sharing = ~2.8e-11), early analysis of binding site vs. tag associations revealed
that a non-negligible fraction of tag sequences were in fact shared by multiple binding sequences,
likely mainly caused by a combination of sequence errors in the binding sequences, or oligo synthesis
errors in the oligos used to generate the construct libraries. In addition to tag sharing, tags found
associated with binding sites in validated read pairs might also be found in the construct library read
pair reject file if it was not clear, due to barcode mismatches, which construct library they might be
from. Finally, the tag sequences themselves might contain sequence errors. To deal with these sources
of error, tags were categorized with three attributes: (i) safe vs. unsafe, where unsafe meant the tag
could be found in the construct library rejected read pair file; shared vs. nonshared, where shared
meant the tag was found associated with multiple binding site sequences, and 2+ vs. 1-only, where 2+
meant that the tag appeared at least twice among the validated construct library sequences and so
presumed to be less likely to contain sequence errors. Combining these three criteria yielded 8 classes
of tags associated with each binding site, the most secure (but least abundant) class comprising only
safe, nonshared, 2+ tags; and the least secure (but most abundant) class comprising all tags regardless
of safety, sharing, or number of occurrences.
Computation of normalized expression levels: Custom perl code was used to implement the steps
indicated in Figure 8A, levels 5-6. First, tag counts obtained for each induced sample were aggregated
for each binding site, using the binding site vs. transcript tag table previously computed for the
construct library (see Figure 8C). For each sample, the aggregated tag counts for each binding site
were then divided by the aggregated tag counts for the positive control sample to generate normalized
expression levels. Additional considerations relevant to these calculations included:
1. For each sample, a subset of “novel” tags were found among the validity-checked cDNA gene
sequences that could not be found in the binding site vs. transcript tag association table. These tags
were ignored in the subsequent calculations.
2. The aggregations of tag counts described above were performed for each of the eight classes of
tags described above in binding site vs. transcript tag association table. Because the binding sites in
the construct libraries were biased to generate sequences similar to a central sequence frequently, but
sequences with increasing numbers of mismatches increasingly rarely, binding sites with few
mismatches generally aggregated to large numbers of tags, while binding sites with more mismatches
aggregated to smaller numbers. Thus, although use of the most secure tag class was generally
desirable, evaluation of binding sites with two or more mismatches might be based on small numbers
of tags per binding site, making the secure counts and ratios less statistically reliable even if the tags
themselves were more reliable. In such cases, all tags were used. Some compensation for this
consideration obtains from the fact that the number of separate aggregated tag counts for n
mismatching positions grew with the number of combinations of mismatching positions (equal to
), and so dramatically increases with n; thus the averages of aggregated tag counts for different
numbers n of mismatches (shown in Figs. 2B, 2E, and in Figs. 9A, 10B) are based on a statistically
very large set of aggregated tag counts for n ≥ 2.
3. Finally, the binding site built into the TALE construct libraries was 18bp and tag associations
were assigned based on these 18bp sequences, but some experiments were conducted with TALEs
programmed to bind central 14bp or 10bp regions within the 18bp construct binding site regions. In
computing expression levels for these TALEs, tags were aggregated to binding sites based on the
corresponding regions of the 18bp binding sites in the association table, so that binding site
mismatches outside of this region were ignored.
EXAMPLE V
Vector and Strain Construction
Cas9 sequences from S. thermophilus, N. meningitidis, and T. denticola were obtained from
NCBI and human codon optimized using JCAT (world wide website jcat.de) and modified to
facilitate DNA synthesis and expression in E. coli. 500 bp gBlocks (Integrated DNA Technologies,
Coralville IA) were joined by hierarchical overlap PCR and isothermal assembly . The resulting full-
length products were subcloned into bacterial and human expression vectors. Nuclease-null Cas9
cassettes (NM: D16A D587A H588A N611A, SP: D10A D839A H840A N863A, ST1: D9A D598A
H599A N622A, TD: D13A D878A H879A N902A) were constructed from these templates by standard
methods.
EXAMPLE VI
Bacterial plasmids
Cas9 was expressed in bacteria from a cloDF13/aadA plasmid backbone using the medium-
strength proC constitutive promoter. tracrRNA cassettes, including promoters and terminators from the
native bacterial loci, were synthesized as gBlocks and inserted downstream of the Cas9 coding
sequence for each vector for robust tracrRNA production. When the tracrRNA cassette was expected
to additionally contain a promoter in the opposite orientation, the lambda t1 terminator was inserted to
prevent interference with cas9 transcription. Bacterial targeting plasmids were based on a p15A/cat
backbone with the strong J23100 promoter followed by one of two 20 base pair spacer sequences (Fig.
13D) previously determined to function using SP. Spacer sequences were immediately followed by
one of the three 36 base pair repeat sequences depicted in Fig. 12A. The YFP reporter vector was
based on a pSC101/kan backbone with the pR promoter driving GFP and the T7 g10 RBS preceding
the EYFP coding sequence, with protospacer 1 and AAAAGATT PAM inserted into the non-template
strand in the 5' UTR. Substrate plasmids for orthogonality testing in bacteria were identical to library
plasmids (see below) but with the following PAMs: GAAGGGTT (NM), GGGAGGTT (SP),
GAAGAATT (ST1), AAAAAGGG (TD).
EXAMPLE VII
Mammalian vectors
Mammalian Cas9 expression vectors were based on pcDNA3.3-TOPO with C-terminal SV40
NLSs. sgRNAs for each Cas9 were designed by aligning crRNA repeats with tracrRNAs and fusing
the 5' crRNA repeat to the 3' tracrRNA so as to leave a stable stem for Cas9 interaction . sgRNA
expression constructs were generated by cloning 455 bp gBlocks into the pCR-BluntII-TOPO vector
backbone. Spacers were identical to those used in previous work . Lentivectors for the broken-GFP HR
reporter assay were modified from those previously described to include appropriate PAM sequences
for each Cas9 and used to establish the stable GFP reporter lines.
RNA-guided transcriptional activators consisted of nuclease-null Cas9 proteins fused to the
VP64 activator and corresponding reporter constucts bearing a tdTomato driven by a minimal promoter
were constructed.
EXAMPLE VIII
Library construction and transformation
Protospacer libraries were constructed by amplifying the pZE21 vector (ExpressSys,
Ruelzheim, Germany) using primers (IDT, Coralville, IA) encoding one of the two protospacer
sequences followed by 8 random bases and assembled by standard isothermal methods . Library
assemblies were initially transformed into NEBTurbo cells (New England Biolabs, Ipswich MA),
yielding >1E8 clones per library according to dilution plating, and purified by Midiprep (Qiagen,
Carlsbad CA). Electrocompetent NEBTurbo cells containing a Cas9 expression plasmid (DS-NMcas,
DS-ST1cas, or DS-TDcas) and a targeting plasmid (PM-NM!sp1, PM-NM!sp2, PM-ST1!sp1, PM-
ST1!sp2, PM-TD!sp1, or PM-TD!sp2) were transformed with 200 ng of each library and recovered for
2 hours at 37°C prior to dilution with media containing spectinomycin (50 µg/mL), chloramphenicol
(30 µg/mL), and kanamycin (50 µg/mL). Serial dilutions were plated to estimate post-transformation
library size. All libraries exceeded ~1E7 clones, indicative of complete coverage of the 65,536 random
PAM sequences.
EXAMPLE IX
High-throughput sequencing
Library DNA was harvested by spin columns (Qiagen, Carlsbad CA) after 12 hours of
antibiotic selection. Intact PAMs were amplified with barcoded primers and sequences obtained from
overlapping 25bp paired-end reads on an Illumina MiSeq. MiSeq yielded 18,411,704 total reads or
9,205,852 paired-end reads with an average quality score >34 for each library. Paired end reads were
merged and filtered for perfect alignment to each other, their protospacer, and the plasmid backbone.
The remaining 7,652,454 merged filtered reads were trimmed to remove plasmid backbone and
protospacer sequences, then used to generate position weight matrices for each PAM library. Each
library combination received at least 450,000 high-quality reads.
EXAMPLE X
Sequence processing
To calculate the fold depletion for each candidate PAM, we employed two scripts to filter the
data. patternProp (usage: python patternProp.py [PAM] file.fastq) returns the number and fraction of
reads matching each 1-base derivative of the indicated PAM. patternProp3 returns the fraction of reads
matching each 1-base derivative relative to the total number of reads for the library. Spreadsheets
detailing depletion ratios for each calculated PAM were used to identify the minimal fold depletion
among all 1-base derivatives and thereby classify PAMs.
EXAMPLE XI
Repression and orthogonality assays in bacteria
Cas9-mediated repression was assayed by transforming the NM expression plasmid and the
YFP reporter plasmid with each of the two corresponding targeting plasmids. Colonies with matching
or mismatched spacer and protospacer were picked and grown in 96-well plates. Fluorescence at
495/528 nm and absorbance at 600nm were measured using a Synergy Neo microplate reader (BioTek,
Winooski VT).
Orthogonality tests were performed by preparing electrocompetent NEBTurbo cells bearing all
combinations of Cas9 and targeting plasmids and transforming them with matched or mismatched
substrate plasmids bearing appropriate PAMs for each Cas9. Sufficient cells and dilutions were plated
to ensure that at least some colonies appeared even for correct Cas9 + targeting + matching
protospacer combinations, which typically arise due to mutational inactivation of the Cas9 or the
crRNA. Colonies were counted and fold depletion calculated for each.
EXAMPLE XII
Cell culture and transfections
HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin
(pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). Cells were maintained at
37°C and 5% CO in a humidified incubator.
Transfections involving nuclease assays were as follows: 0.4×10 cells were transfected with
2μg Cas9 plasmid, 2μg gRNA and/or 2μg DNA donor plasmid using Lipofectamine 2000 as per the
manufacturer’s protocols. Cells were harvested 3 days after transfection and either analyzed by FACS,
or for direct assay of genomic cuts the genomic DNA of ~1 X 10 cells was extracted using DNAeasy
kit (Qiagen).
For transfections involving transcriptional activation assays: 0.4×10 cells were transfected
with 2μg Cas9 VP64 plasmid, 2μg gRNA and/or 0.25μg of reporter construct. Cells were harvested
24-48hrs post transfection and assayed using FACS or immunofluorescence methods, or their total
RNA was extracted and these were subsequently analyzed by RT-PCR.
EXAMPLE XIII
Selecting putatively orthogonal Cas9 proteins
Cas9 RNA binding and sgRNA specificity is primarily determined by the 36 base pair repeat
sequence in crRNA. Known Cas9 genes were examined for highly divergent repeats in their adjacent
CRISPR loci. Streptococcus pyogenes and Streptococcus thermophilus CRISPR1 Cas9 proteins (SP
6, 22
and ST1) and two additional Cas9 proteins from Neisseria meningitidis (NM) and Treponema
denticola (TD) were selected whose loci harbor repeats that differ by at least 13 nucleotides from one
another and from those of SP and ST1 (Fig. 12A).
EXAMPLE XIV
PAM characterization
Cas9 proteins will only target dsDNA sequences flanked by a 3' PAM sequence specific to the
Cas9 of interest. Of the four Cas9 variants, only SP has an experimentally characterized PAM, while
the ST1 PAM and, very recently, the NM PAM were deduced bioinformatically. SP is readily
targetable due to its short PAM of NGG , while ST1 and NM targeting are less radily targetable
22, 23
because of PAMs of NNAGAAW and NNNNGATT, respectively . Bioinformatic approaches
inferred more stringent PAM requirements for Cas9 activity than are empirically necessary for effector
cleavage due to the spacer acquisition step. Because the PAM sequence is the most frequent target of
mutation in escape phages, redundancy in the acquired PAM would preclude resistance. A library-
based approach was adopted to comprehensively characterize these sequences in bacteria using high-
throughput sequencing.
Genes encoding ST1, NM, and TD were assembled from synthetic fragments and cloned into
bacterial expression plasmids along with their associated tracrRNAs (Fig. 12B). Two SP-functional
spacers were selected for incorporation into the six targeting plasmids. Each targeting plasmid
encodes a constitutively expressed crRNA in which one of the two spacers is followed by the 36 base-
pair repeat sequence specific to a Cas9 protein (Fig. 12B). Plasmid libraries containing one of the two
protospacers followed by all possible 8 base pair PAM sequences were generated by PCR and
assembly . Each library was electroporated into E. coli cells harboring Cas9 expression and targeting
plasmids, for a total of 12 combinations of Cas9 protein, spacer, and protospacer. Surviving library
plasmids were selectively amplified by barcoded PCR and sequenced by MiSeq to distinguish between
functional PAM sequences, which are depleted only when the spacer and protospacer match (Fig. 12C-
12D), from nonfunctional PAMs, which are never depleted (Fig. 12D-12E). To graphically depict the
importance of each nucleotide at every position, the log relative frequency of each base for matched
spacer-protospacer pairs relative to the corresponding mismatched case was plotted (Figs. 13A-13F).
NM and ST1 recognize PAMs that are less stringent and more complex than earlier
bioinformatic predictions, suggesting that requirements for spacer acquisition are more stringent than
those for effector cleavage. NM primarily requires a single G nucleotide positioned five bases from
the 3' end of the protospacer (Fig. 13A), while ST1 and TD each require at least three specific bases
(Fig. 5b-c). Sorting results by position allowed quantification of depletion of any PAM sequence from
each protospacer library (Figs. 13D-13F). All three enzymes cleaved protospacer 2 more effectively
than protospacer 1 for nearly all PAMs, with ST1 exhibiting an approximately 10-fold disparity.
However, there was also considerable PAM-dependent variation in this interaction. For example, NM
cleaved protospacers 1 and 2 approximately equally when they were followed by sequences matching
TNNNGNNN, but was 10-fold more active in cleaving protospacer 2 when the PAMs matched
ANNNGNNN.
Results highlight the difficulty of defining a single acceptable PAM for a given Cas9. Not
only do activity levels depend on the protospacer sequence, but specific combinations of unfavorable
PAM bases can significantly reduce activity even when the primary base requirements are met. We
initially identified PAMs as patterns that underwent >100-fold average depletion with the lower-
activity protospacer 1 and >50-fold depletion of all derivatives with one additional base fixed (Table 1,
plain text). While these levels are presumably sufficient to defend against targets in bacteria, particular
combinations of deleterious mutations dramatically reduced activity. For example, NM depleted
sequences matching NCCAGGTN by only 4-fold. A more stringent threshold requiring >500-fold
depletion of matching sequences and >200-fold depletion of one-base derivatives was defined for
applications requiring high affinity (Table 1, bolded).
Table 1
EXAMPLE XV
Transcriptional regulation in bacteria
A nuclease-null variant of SP has been demonstrated to repress targeted genes in bacteria with
an efficacy dependent upon the position of the targeted protospacer and PAM . Because the PAM of
NM occurs more frequently than that of SP, nuclease-null version may be similarly capable of targeted
repression. The catalytic residues of the RuvC and HNH nuclease domains were identified by
sequence homology and inactivated to generate a putative nuclease-null NM. To create a suitable
reporter, protospacer 1 was inserted with an appropriate PAM into the non-template strand within the
'UTR of a YFP reporter plasmid (Fig. 14A). These constructs were cotransformed into E. coli
together with each of the two NM targeting plasmids used previously and measured their comparative
fluorescence. Cells with matching spacer and protospacer exhibited ~22-fold weaker fluorescence
than the corresponding mismatched case (Fig. 14B). These results suggest that NM can function as an
easily targeted repressor to control transcription in bacteria, substantially increasing the number of
endogenous genes that can be subjected to Cas9-mediated repression.
EXAMPLE XVI
Orthogonality in bacteria
A set of Cas9 proteins were selected for their disparate crRNA repeat sequences. To verify
that they are in fact orthogonal, each Cas9 expression plasmid was co-transformed with all four
targeting plasmids containing spacer 2. These cells were challenged by transformation of substrate
plasmids containing either protospacer 1 or protospacer 2 and a suitable PAM. Plasmid depletion was
observed exclusively when each Cas9 was paired with its own crRNA, demonstrating that all four
constructs are indeed orthogonal in bacteria (Fig. 15).
EXAMPLE XVII
Genome editing in human cells
These Cas9 variants were then used to engineer human cells. Single guide RNAs (sgRNAs)
were constructed from the corresponding crRNAs and tracrRNAs for NM and ST1, the two smaller
Cas9 orthologs, by examining complementary regions between crRNA and tracrRNA and fusing the
two sequences via a stem-loop at various fusion junctions analogous to those of the sgRNAs created
for SP. In certain instances where multiple successive uracils caused Pol III termination in the
expression system, multiple single-base mutants were generated. The complete 3' tracrRNA sequence
was always included, as truncations are known to be detrimental . All sgRNAs were assayed for
activity along with their corresponding Cas9 protein using a previously described homologous
recombination assay in 293 cells . Briefly, a genomically integrated non-fluorescent GFP reporter line
was constructed for each Cas9 protein in which the GFP coding sequence was interrupted by an insert
encoding a stop codon and protospacer sequence with functional PAM. Reporter lines were
transfected with expression vectors encoding a Cas9 protein and corresponding sgRNA along with a
repair donor capable of restoring fluorescence upon nuclease-induced homologous recombination (Fig.
16A). ST1- and NM-mediated editing was observed at levels comparable to those induced by SP. The
ST1 sgRNA with 5 successive uracils functioned efficiently, suggesting that Pol III termination did not
occur at levels sufficient to impair activity. Full-length crRNA-tracrRNA fusions were active in all
instances and are useful for chimeric sgRNA design. Both NM and ST1 are capable of efficient gene
editing in human cells using chimeric guide RNAs.
EXAMPLE XVIII
Cas9 orthogonality in mammalian cells
Having discovered highly effective sgRNAs for NM and ST1 activity in human cells, it was
verified that none of the three proteins can be guided by the sgRNAs of the others. The same
homologous recombination assay was used to measure the comparative efficiency of NM, SP, and ST1
in combination with each of the three sgRNAs. All three Cas9 proteins were determined to be fully
orthogonal to one another, demonstrating that they are capable of targeting distinct and non-
overlapping sets of sequences within the same cell (Fig. 16B). To contrast the roles of sgRNA and
PAM in orthogonal targeting, a variety of downstream PAM sequences with SP and ST1 and their
respective sgRNAs were tested. Both a matching sgRNA and a valid PAM are required for activity,
with the orthogonality determined almost entirely by the specific sgRNA affinity for the corresponding
Cas9.
EXAMPLE XIX
Transcriptional activation in human cells
NM and ST1 mediate transcriptional activation in human cells. Nuclease-null NM and ST1
genes were fused to the VP64 activator domain at their C-termini to yield putative RNA-guided
activators modeled after the SP activator. Reporter constructs for activation consisted of a protospacer
with an appropriate PAM inserted upstream of the tdTomato coding region. Vectors expressing an
RNA-guided transcriptional activator, an sgRNA, and an appropriate reporter were cotransfected and
the extent of transcriptional activation measured by FACS (Fig. 17A). In each case, robust
transcriptional activation by all three Cas9 variants was observed (Fig. 17B). Each Cas9 activator
stimulated transcription only when paired with its corresponding sgRNA.
EXAMPLE XX
Discussion
Using two distinct protospacers for comprehensive PAM characterization allowed
investigation of the complexities governing protospacer and PAM recognition. Differential
protospacer cleavage efficiencies exhibited a consistent trend across diverse Cas9 proteins, although
the magnitude of the disparity varied considerably between orthologs. This pattern suggests that
sequence-dependent differences in D-loop formation or stabilization determine the basal targeting
efficiency for each protospacer, but that additional Cas9 or repeat-dependent factors also play a role.
Similarly, numerous factors preclude efforts to describe PAM recognition with a single sequence
motif. Individual bases adjacent to the primary PAM recognition determinants can combine to
dramatically decrease overall affinity. Indeed, certain PAMs appear to interact nonlinearly with the
spacer or protospacer to determine the overall activity. Moreover, different affinity levels may be
required for distinct activities across disparate cell types. Finally, experimentally identified PAMs
required fewer bases than those inferred from bioinformatic analyses, suggesting that spacer
acquisition requirements differ from those for effector cleavage.
This difference is most significant for the Cas9 protein from Neisseria meningitidis, which has
fewer PAM requirements relative to both its bioinformatic prediction and to the currently popular Cas9
from S. pyogenes. Its discovery considerably expands the number of sequences that can be readily
targeted with a Cas9 protein. At 3.25 kbp in length, it is also 850 bp smaller than SP, a significant
advantage when gene delivery capabilities are limiting. Most notably, both NM and ST1 are small
enough to fit into an AAV vector for therapeutic applications, while NM may represent a more suitable
starting point for directed evolution efforts designed to alter PAM recognition or specificity.
The following references are hereby incorporated by reference in their entireties for all
purposes.
References
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The term “comprising” as used in this specification and claims means “consisting at least in part
of”. When interpreting statements in this specification, and claims which include the term
“comprising”, it is to be understood that other features that are additional to the features prefaced by
this term in each statement or claim may also be present. Related terms such as “comprise” and
“comprised” are to be interpreted in similar manner.
In this specification where reference has been made to patent specifications, other external documents,
or other sources of information, this is generally for the purpose of providing a context for discussing
the features of the invention. Unless specifically stated otherwise, reference to such external
documents is not to be construed as an admission that such documents, or such sources of information,
in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
In the description in this specification reference may be made to subject matter that is not within the
scope of the claims of the current application. That subject matter should be readily identifiable by a
person skilled in the art and may assist in putting into practice the invention as defined in the claims of
this application.
Claims (24)
1. An in vitro or ex-vivo method of altering two or more target nucleic acids in a cell comprising 5 introducing into the cell a first foreign nucleic acid encoding two or more RNAs complementary to the two or more target nucleic acids, introducing into the cell a second foreign nucleic acid encoding two or more orthogonal RNA guided DNA binding protein nucleases that respectively bind to the two or more target nucleic acids and are guided by the two or more RNAs, 10 wherein the RNAs and the orthogonal RNA guided DNA nucleases are expressed, wherein two or more co-localization complexes form between an RNA, an orthogonal RNA guided DNA binding protein nuclease and a target nucleic acid, and wherein the two or more RNA guided DNA binding protein nucleases cut the two or more target nucleic acids. 15
2. The method of claim 1 wherein the cell is a eukaryotic cell.
3. The method of claim 1 wherein the cell is a human cell.
4. The method of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.
5. The method of claim 1 wherein the RNA is between about 10 to about 500 nucleotides. 20
6. The method of claim 1 wherein the RNA is between about 20 to about 100 nucleotides.
7. The method of claim 1 wherein each of the two or more RNAs is a guide RNA.
8. The method of claim 1 wherein each of the two or more RNAs is a tracrRNA-crRNA fusion. 25
9. The method of claim 1 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
10. The method of claim 1 wherein the orthogonal RNA guided DNA binding protein nuclease is a DNA binding protein of a Type II Crispr system.
11. The method of claim 1 wherein the orthogonal RNA guided DNA binding protein 30 nuclease is an orthogonal Cas9 nuclease.
12. A cell comprising a first foreign nucleic acid encoding two or more RNAs complementary to two or more respective target nucleic acids, a second foreign nucleic acid encoding two or more orthogonal RNA guided DNA binding protein nucleases, 5 wherein the cell is configured to express the two or more RNAs and the two or more orthogonal RNA guided DNA binding protein nucleases, wherein the cell comprises two or more co-localization complexes with each including an RNA, an orthogonal RNA guided DNA binding protein nuclease and a target nucleic acid, and with the proviso that said cell is not present in a human being. 10
13. The cell of claim 12 wherein the cell is a eukaryotic cell.
14. The cell of claim 12 wherein the cell is a human cell.
15. The cell of claim 12 wherein the cell is a yeast cell, a plant cell or an animal cell.
16. The cell of claim 12 wherein the RNA includes between about 10 to about 500 nucleotides. 15
17. The cell of claim 12 wherein the RNA includes between about 20 to about 100 nucleotides.
18. The cell of claim 12 wherein each of the two or more RNAs is a guide RNA.
19. The cell of claim 12 wherein each of the two or more RNAs is a tracrRNA-crRNA fusion.
20. 20. The cell of claim 12 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
21. The cell of claim 12 wherein the RNA guided DNA binding protein nuclease is a DNA binding protein nuclease of a Type II CRISPR system.
22. The cell of claim 12 wherein the RNA guided DNA binding protein is an orthogonal 25 Cas9 protein nuclease.
23. A method as claimed in any one of claims 1-11 substantially as herein described and with reference to any example thereof.
24. A cell as claimed in any one of claims 12-22 substantially as herein described and with reference to any example thereof.
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