Patent Publication Number: US-2021171944-A1

Title: Compositions and methods for modulating gene expression

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 62/595,410 filed Dec. 6, 2017; U.S. Provisional Application No. 62/617,328 filed Jan. 15, 2018; and U.S. Provisional Application No. 62/753,531 filed Oct. 31, 2018, all of which are incorporated herein in their entirety by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with government support under Contract Nos. 5R44 AG053084 and R56 AG051638 awarded by the National Institute of Health. The government has certain rights in the invention. 
    
    
     FIELD OF THE DISCLOSURE 
     The present disclosure relates to compositions and methods for modulating gene expression and in particular to compositions and methods for increasing expression of Klotho. 
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING 
     This application contains a Sequence Listing in computer readable form (filename: 7411618.00005 Sequence Listing_ST25: 317,964 bytes—ASCII text file: created Nov. 30, 2018), which is incorporated by reference in its entirety and forms part of the disclosure. 
     BACKGROUND OF THE DISCLOSURE 
     Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field. 
     The single copy gene Klotho plays important roles in ageing, cognition, anti-oxidative stress, neurological protection and development, and kidney health. Klotho is a Type I transmembrane protein which is mainly expressed in the brain, kidney and reproductive organs (Masuda et al., 2005 . Mech. Ageing Dev.  126(21): 1274-1283). It is also shed by proteolytic cleaving resulting in a soluble form that is detectable in serum and cerebrospinal fluid (CSF) (Bloch et al., 2009 . FEBS Lett.  583(19): 3221-3224; Chen et al., 2007 . Proc. Natl Acad. Sci. USA.  104(50): 19796-19801; Matsumura et al., 1998 . Biochem. Biophys. Res. Commun.  242(3): 626-630). A third form of Klotho, found mainly in the brain, results from differential mRNA splicing and is secreted from the cell into the blood and CSF (Masso et al., 2015 . PLoS One.  10(11): e0143623). Both the transmembrane and soluble forms of Klotho have important functions in many homeostatic processes. 
     Klotho promotes oligodendrocyte maturation, and it protects neurons from oxidative stress by increasing expression of antioxidant factors. It also induces re-myelination in vivo in the cuprizone-induced demyelination model of multiple sclerosis (Zeldich et al., 2015 . J. Mol. Neurosci.  57(2): 185-196). Studies have shown that Klotho overexpression reduces cognitive deficits in a mouse model of Alzheimer&#39;s disease, and that it enhances cognition in humans and mice (Dubal et al., 2014 . Cell Rep.  7(4): 1065-1076; Dubal et al., 2015 . Off. J. Socr. Neuroscience.  35(6): 2358-2371). 
     Despite efforts to modulate the activity of Klotho, or the activity of factors regulated by Klotho, there remains a need for compositions and methods for modulating Klotho activity, and preferably for increasing Klotho activity in humans. 
     SUMMARY OF THE DISCLOSURE 
     In a first aspect, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene, wherein the guide RNA associates with a transcriptional activation domain in the cell to thereby increase expression of the Klotho gene. 
     In certain examples, the method further comprises introducing into the cell an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to the transcriptional activation domain. 
     In some examples, the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. The adapter protein may bind to a tetra-loop and/or a stem loop 2 of the guide RNA. 
     The target sequence may be located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     The target sequence may be located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In certain examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     The target sequence may be located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In certain examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     The guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. In certain examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. The guide sequence may be at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. The guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In certain examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. The guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In certain examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. The guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. The guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In certain examples, the guide RNA is a single-molecule guide RNA (sgRNA). 
     The guide RNA may be between about 100 nucleotides and 200 nucleotides in length. 
     In certain examples, the method comprises introducing into the cell more than one guide RNA. For example, the method may comprise introducing into the cell two guide RNAs, wherein each guide RNA is independently as defined herein. 
     In some examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises or is attached to a second transcriptional activation domain. The second transcriptional activation domain may be selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. The CRISPR enzyme may be Cas9. For example, the CRISPR enzyme may be dead Cas9 (dCas9). 
     In certain examples, the cell is a neuronal cell or a kidney cell. The cell may be inside a human body. 
     In a second aspect, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In certain examples, the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     The target sequence may be located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In certain examples, the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In some examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. The guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In certain examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. In certain examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     Preferably, the guide RNA is a single-molecule guide RNA (sgRNA). 
     The guide RNA may be between about 100 nucleotides and about 200 nucleotides in length. 
     In certain examples, the method comprises introducing into the cell more than one guide RNA. For example, the method may comprise introducing into the cell two guide RNAs, wherein each guide RNA is independently as defined herein. 
     In some examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. The CRISPR enzyme may be Cas9. For example, the CRISPR enzyme may be dead Cas9 (dCas9). 
     In certain examples, the cell is a neuronal cell or a kidney cell. The cell may be inside a human body. 
     In a third aspect, the present disclosure provides a method of treating cancer in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In certain examples, the cancer is selected from the group consisting of colon cancer, prostate cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer and breast cancer. 
     In a fourth aspect, the present disclosure provides a method of treating a muscle disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In certain examples, the muscle disorder is selected from the group consisting of muscle atrophy and muscular dystrophy such as duchene muscular dystrophy. 
     In a fifth aspect, the present disclosure provides a method of treating a kidney disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In certain examples, the kidney disorder is selected from the group consisting of renal dysfunction, acute kidney injury and kidney disease such as chronic kidney disease. 
     In a sixth aspect, the present disclosure provides a method of enhancing cognition in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In an seventh aspect, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: 
     a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In certain examples, the neurological disorder is selected from the group consisting of memory loss, stress, biopolar disorder, epilepsy, dementia, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress and neurodegeneration. 
     In certain examples, the method further comprises administering to the subject an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to the transcriptional activation domain. 
     In some examples, the adapter protein is selected from the group consisting of MS2, PP7, Q8, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. The adapter protein may bind to a tetra-loop and/or a stem loop 2 of the guide RNA. 
     The target sequence may be located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     The target sequence may be located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In certain examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. In some examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     Preferably, the guide RNA is a single-molecule guide RNA (sgRNA). 
     In certain examples, the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In certain examples, the method comprises introducing into the cell more than one guide RNA. For example, the method may comprise introducing into the cell two guide RNAs, wherein each guide RNA is independently as defined herein. 
     In some examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In certain examples, the CRISPR enzyme comprises or is attached to a second transcriptional activation domain. The second transcriptional activation domain may be selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In certain examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. The CRISPR enzyme may be Cas9. For example, the CRISPR enzyme may be dead Cas9 (dCas9). 
     In an eighth aspect, the present disclosure provides a method of treating cancer in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In certain examples, the cancer is selected from the group consisting of colon cancer, prostate cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer and breast cancer. 
     In a ninth aspect, the present disclosure provides a method of treating a muscle disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In certain examples, the muscle disorder is selected from the group consisting of muscle atrophy and muscular dystrophy such as duchene muscular dystrophy. 
     In an tenth aspect, the present disclosure provides a method of treating a kidney disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In certain examples, the kidney disorder is selected from the group consisting of renal dysfunction, acute kidney injury and kidney disease such as chronic kidney disease. 
     In a eleventh aspect, the present disclosure provides a method of enhancing cognition in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In a twelfth aspect, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In some examples, the neurological disorder is selected from the group consisting of memory loss, stress, biopolar disorder, epilepsy, dementia, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress and neurodegeneration. 
     In some examples, the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     The target sequence may be located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     The target sequence may be located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In certain examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     Preferably, the guide RNA is a single-molecule guide RNA (sgRNA). 
     The guide RNA may be between about 100 nucleotides and about 200 nucleotides in length. 
     In certain examples, the method comprises introducing into the cell more than one guide RNA. For example, the method may comprise introducing into the cell two guide RNAs, wherein each guide RNA is independently as defined herein. 
     In some examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. The CRISPR enzyme may be Cas9. For example, the CRISPR enzyme may be dead Cas9 (dCas9). 
     In a thirteenth aspect, the present disclosure provides a guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a target sequence within or near a human Klotho gene. 
     The guide RNA may further comprise at least one protein binding sequence for binding to an adapter protein. The protein binding sequence may comprise an aptamer. 
     In certain examples, the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In some examples, the guide RNA comprises two protein binding sequences. 
     In some examples, the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     The target sequence may be located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     The guide sequence may be between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     Preferably, the guide RNA is a single-molecule guide RNA (sgRNA). 
     In certain examples, the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In a fourteenth aspect, the present disclosure provides an isolated or recombinant nucleic acid molecule encoding the guide RNA of the thirteenth aspect. 
     In a fifteenth aspect, the present disclosure provides a vector encoding the guide RNA of the thirteenth aspect. 
     The vector may be an adeno-associated viral (AAV) vector, an adenoviral vector (AdV) or a lentiviral (LV) vector. 
     In certain examples, the vector further encodes a CRISPR enzyme, an adapter protein or a transcriptional activation domain. 
     The CRISPR enzyme may comprise a mutation which abolishes or reduces its nuclease activity. In certain examples, the CRISPR enzyme is Cas9. For example, the CRISPR enzyme may be dead Cas9 (dCas9). 
     In certain examples, the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In certain examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In a sixteenth aspect, the present disclosure provides a ribonucleoprotein complex comprising: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a human Klotho gene, wherein the guide RNA comprises at least one protein binding sequence for binding to an adapter protein. 
     The protein binding sequence may comprise an aptamer. 
     In some examples, the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In some examples, the guide RNA comprises two protein binding sequences. 
     The target sequence may be located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     The guide sequence may be substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In some examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA is a single-molecule guide RNA (sgRNA). 
     In some examples, the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In some examples, the guide RNA is bound to the adapter protein. 
     In some examples, the adapter protein comprises or is attached to a transcriptional activation domain. The transcriptional activation domain may be selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In some examples, the CRISPR enzyme is Cas9. For example, the CRISPR enzyme may be dCas9. 
     In a seventeenth aspect, the present disclosure provides a ribonucleoprotein complex comprising: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a human Klotho gene; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In some examples, the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In some examples, the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. For example, the target sequence may be located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In some examples, the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In some examples, the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In some examples, the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may comprise at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In some examples, the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may be selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In some examples, the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may be at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. For example, the guide RNA may comprise a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In some examples, the guide RNA is a single-molecule guide RNA (sgRNA). 
     In some examples, the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. 
     In some examples, the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In some examples, the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In some examples, the CRISPR enzyme is Cas9. For example, the CRISPR enzyme may be dCas9. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1 . Representation of sgRNA3 (SEQ ID NO: 7) hybridised to a target sequence in the Klotho promoter. 
         FIG. 2 . Schematic view of the Klotho promoter region showing the location of the target sequences for sgRNA1-4. The 20 nt target sequence (solid box), and protospacer-adjacent motif (PAM) (empty box) are indicated. The number indicates the first nucleotide of the sgRNA relative to Klotho translation start site (“A” in ATG as number+1). 
         FIG. 3 . Klotho gene activation using a dual luciferase coincidence reporter system. (A) Schematic view of the firefly, NLuc coincidence reporter system under the control of Klotho 4 kb promoter. The P2A ribosome skipping sequence is indicated. (B) Fold increase in Klotho gene expression by SAM in stable HEK293 cells. Cells were analyzed by dual luciferase assay two days after transfection with dCas9-VP64, MS2-P65-HSF1 and the indicated sgRNA. Negative control: sgRNA cloning backbone empty vector. Positive control: Egr-1 transfected cells. Data are expressed as fold over negative control. Error bars show standard deviation among six replicates. (C) Fold change of the PGK promoter activities by SAM in stable HEK293 cells as in (B). (D) Validation of the PGK promoter system in stable HEK293 cells using Cilnidipine and Ataluren that were added to the cells 24 hours after plating and results assayed 24 hours later. Asterisks (*) indicate statistical significance of p&lt;0.05 by t-test. 
         FIG. 4 . Klotho gene activation using a CRISPR NLuc knock-in HEK293 cell line. (A) Schematic view of the 3′ end of Klotho including the 3′-UTR region. The stop codon, the sgRNA targets and polyA tail are shown. The P2A-NLuc sequence was inserted at the DSB site in the Klotho 3′-UTR. (B) PCR confirmation of positive clones using a forward primer located in intron 4 of Klotho and a NLuc reverse primer (NLrv) indicated in (A). Cell line numbers are indicated. (C) Fold increase in Klotho gene expression by SAM evaluated with a CRISPR NLuc knock-in HEK293 line. Cells were analyzed by NLuc luciferase assay two days after transfection with dCas9-VP64, MS2-P65-HSF1 and the indicated sgRNA. Negative control: sgRNA cloning backbone empty vector. Positive control: Egr-1 transfected cells. Data are expressed as fold over negative control. Error bars show standard deviation among six replicates. Asterisks (*) indicate statistical significance of p&lt;0.05 by t-test. 
         FIG. 5 . Klotho expression in kidney HK-2 (A, C, D) and neuronal SY5Y cells (B). (A) Fold change in Klotho mRNA evaluated in the HK-2 line by qPCR two days after transfection with SAM complex. Data are expressed as fold over negative control. Error bars show standard deviation among six replicates. (B) Fold change in Klotho mRNA evaluated in neuronal SY5Y line by qPCR two days after transfection with SAM complex. Data are expressed as fold over negative control. Error bars show standard deviation among six replicates. (C) Western blot analysis of Klotho gene expression by SAM evaluated in HK-2 cells. Two days after transfection with the SAM complex, cell lysates were analyzed by Western blot using an anti-Klotho antibody and an Actin control. (D) Statistical analysis of the results from (B). The intensities of the 130 kDa Klotho bands were analyzed and normalized to the Actin bands using the average intensity of the controls as 1 from 3 independent experiments. Error bar indicates standard deviation. Significance of results using student&#39;s t-test: *, p&lt;0.05. 
         FIG. 6 . Klotho gene activation using the dual luciferase coincidence reporter system. Graph illustrates fold increase in Klotho gene expression using different dCas9 systems in stable HEK293 cells. Cells were analyzed by dual luciferase assay two days after transfection with the indicated sgRNA, MS2-P65-HSF1 and various dCas9-activation systems (either SAM, Suntag, p300 or VPR). Negative control: sgRNA cloning backbone empty vector. Positive control: Egr-1 transfected cells. Data are expressed as fold over negative control. Error bars show standard deviation among six replicates. Asterisks (*) indicate statistical significance of p&lt;0.05 by t-test compared to negative control. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     In the context of this specification, the terms “a” and “an” are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
     The term “about” is understood to refer to a range of +/−10%, preferably +/−5% or +/−1% or, more preferably, +/−0.1%. 
     The terms “administration concurrently” or “administering concurrently” or “co-administering” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By “simultaneously” is meant that the active agents are administered at substantially the same time, and preferably together in the same formulation. 
     The terms “comprise”, “comprises”, “comprised” or “comprising”, “including” or “having” and the like in the present specification and claims are used in an inclusive sense, ie, to specify the presence of the stated features but not preclude the presence of additional or further features. 
     The term “substantially complementary” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to, and form a duplex structure with, an oligonucleotide or polynucleotide comprising the second nucleotide sequence. It will be understood that the sequence of a nucleic acid need not be 100% complementary to that of its target. Conditions under which hybridisation occurs may be stringent, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can also apply. Substantial complementarity allows the relevant function of the nucleic acid to proceed, eg, guide RNA hybridisation and CRISPR-mediated gene activation. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. 
     The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (ie, % homology=# of identical positions/total # of positions ×100), with optimal alignment determined taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. 
     The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. 
     The term “isolated” as used herein refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide” as used herein refers to a polynucleotide which has been purified from the sequences which flank it in a naturally-occurring state, eg, a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, ie, it is not associated with in vivo substances. 
     The term “pharmaceutically acceptable” as used herein refers to substances that do not cause substantial adverse allergic or immunological reactions when administered to a subject. A “pharmaceutically acceptable carrier” includes, but is not limited to, solvents, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and disintegrants. 
     The term “polynucleotide variant” refers to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions. The term also encompasses polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the term “polynucleotide variant” includes polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The term “polynucleotide variant” also includes naturally occurring allelic variants. The terms “peptide variant” and “polypeptide variant” and the like refer to peptides and polypeptides that are distinguished from a reference peptide or polypeptide by the addition, deletion or substitution of at least one amino acid residue. In certain examples, a peptide or polypeptide variant is distinguished from a reference peptide or polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain examples, the peptide or polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the peptide or polypeptide. Peptide and polypeptide variants also encompass peptides and polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues. 
     “Prevention” includes reduction of risk, incidence and/or severity of a condition or disorder. The terms “treatment” and “treat” include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms “treatment” and “treat” do not necessarily imply that a subject is treated until total recovery. The terms “treatment” and “treat” also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms “treatment” and “treat” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional. 
     The term “recombinant polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence. 
     The term “recombinant polypeptide” as used herein refers to a polypeptide made using recombinant techniques, ie, through the expression of a recombinant polynucleotide. 
     A “therapeutically effective amount” is at least the minimum concentration or amount required to effect a measurable improvement of a particular disease or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. 
     Klotho 
     Human Klotho plays important regulatory and protective roles in, inter alia, memory loss, stress, synaptic plasticity, biopolar disorder, epilepsy, Alzheimer&#39;s disease, Parkinson&#39;s disease, multiple sclerosis, myelin-related disease, neurogenic decline, neurodegeneration and kidney dysfunction (Vo et al., 2018 . Brain Plast.  3: 183-194). 
     The human Klotho gene is located on chromosome 13 and comprises five exons. The Klotho protein primarily exists in one of three forms. Transmembrane Klotho is an approximately 130 kDa, glyclosylated, Type I transmembrane protein. The transmembrane Klotho can be shed from the cell surface by ADAM10/17 metalloproteinases into a soluble form that is detectable in serum and cerebrospinal fluid (CSF) (Bloch et al., 2009 . FEBS Lett.  583(19): 3221-3224; Chen et al., 2007 . Proc. Natl Acad. Sci. USA.  104(50): 19796-19801; Matsumura et al., 1998 . Biochem. Biophys. Res. Commun.  242(3): 626-630). A third, secreted form of Klotho is generated by alternative splicing of exon 3 to produce a 70 kDa protein which is detectable in blood and CSF (Masso et al., 2015 . PLoS One.  10(11): e0143623). Both the transmembrane and soluble forms of Klotho have important functions in many homeostatic processes. 
     The present disclosure relates to methods and compositions for modulating Klotho expression. Table 1 lists various Klotho sequences relevant to the present disclosure. However, those skilled in the art will understand that several different Klotho alleles exist among humans, and all of those alleles are envisaged by the present disclosure. Skilled persons will also understand that greater levels of sequence variation may exist in genomic regions which do not directly encode amino acids. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 SEQ ID  
                   
               
               
                 NO.  
                 Description 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 9  
                 4,000 nt genomic region extending upstream of Klotho  
               
               
                   
                 translation start site (−4,000 to +1) (sense)  
               
               
                 10  
                 4,000 nt genomic region extending upstream of Klotho  
               
               
                   
                 translation start site (−4,000 to +1) (antisense)  
               
               
                 11  
                 Genomic region between 200 nt and 4,000 nt upstream of  
               
               
                   
                 Klotho translation start site (−4,000 to −200) (sense)  
               
               
                 12  
                 Genomic region between 200 nt and 4,000 nt upstream of  
               
               
                   
                 Klotho translation start site (−4,000 to −200) (antisense)  
               
               
                 13  
                 Genomic region between 200 nt and 300 nt upstream of  
               
               
                   
                 Klotho translation start site (−300 to −200) (sense)  
               
               
                 14  
                 Genomic region between 200 nt and 300 nt upstream of  
               
               
                   
                 Klotho translation start site (−300 to −200) (antisense)  
               
               
                 15  
                 Klotho coding sequence  
               
               
                 16  
                 Klotho mRNA sequence  
               
               
                 17  
                 Genomic sequence of Klotho from translation start site to  
               
               
                   
                 translation stop site (sense)  
               
               
                 18  
                 Genomic sequence of Klotho from translation start site to  
               
               
                   
                 translation stop site (antisense)  
               
               
                 19  
                 Genomic region extending from 4,000 nt upstream of Klotho  
               
               
                   
                 translation start site to Klotho translation stop site (sense)  
               
               
                 20  
                 Genomic region extending from 4,000 nt upstream of Klotho  
               
               
                   
                 translation start site to Klotho translation stop site (antisense) 
               
               
                   
               
            
           
         
       
     
     Genome Editing 
     Genome editing generally refers to the process of modifying the nucleotide sequence of a genome in a precise or pre-determined manner. The genome of an organism may be edited using site-directed nucleases to cut DNA at precise target locations, thereby creating single-strand or double-strand DNA breaks. In some instances, the DNA breaks are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with the loss or addition of a nucleotide sequence. HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus. A third repair mechanism, referred to as microhomology-mediated end joining (MMEJ), or “Alternative NHEJ”, is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break. 
     A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes. In prokaryotes, the CRISPR locus encodes products that function as a type of immune system to help defend against foreign invaders, such as virus and phage. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acid. Five types of CRISPR systems (e.g., Type I, Type II, Type III, Type U, and Type V) have been identified. 
     A CRISPR locus includes a number of short repeating sequences referred to as “repeats.” When expressed, the repeats can form secondary structures (e.g., hairpins) and/or comprise unstructured single-stranded sequences. The repeats usually occur in clusters and frequently diverge between species. The repeats are regularly interspaced with unique intervening sequences referred to as “spacers” resulting in a repeat-spacer-repeat locus architecture. The spacers (sometimes referred to as “guide sequences”) are identical to, or have high homology with, foreign invader sequences. A spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequence that is involved in targeting a target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). A spacer sequence may be located at the 5′ or 3′ end of the crRNA. 
     A CRISPR locus also comprises polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and the interference stages of crRNA function in prokaryotes. Some Cas genes comprise homologous secondary and/or tertiary structures. 
     Type II CRISPR Systems 
     crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNaseIII, before hybridizing to a crRNA repeat in the pre-crRNA array. Endogenous RNaseIII can be recruited to cleave the pre-crRNA. Cleaved crRNAs can be subjected by exoribonuclease trimming to produce the mature crRNA form (e.g., 5′ trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The region of DNA targeted by a Type II CRISPR system includes a protospacer adjacent motif (PAM). In nature, the PAM facilitates binding of a CRISPR enzyme (e.g., Cas9) to the target nucleic acid. 
     Type II systems (also referred to as Nmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B (CASS4a). Jinek et al.,  Science,  337(6096):816-821 (2012) showed that the CRISPR/Cas9 system is useful for RNA-programmable genome editing, and international patent application publication number WO2013/176772 provides numerous examples and applications of the CRISPR/Cas endonuclease system for site-specific gene editing. 
     Type V CRISPR Systems 
     Type V CRISPR systems differ from Type II systems in many respects. For example, Cpf1 is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. Cpf1-associated CRISPR arrays can be processed into mature crRNAs without the requirement of an additional trans-activating tracrRNA. The Type V CRISPR array can be processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crRNA beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence. In contrast, mature crRNAs in Type II systems can start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacent motif such that Cpf1-crRNA complexes efficiently cleave target DNA preceded by a short T-rich PAM, which is in contrast to the G-rich PAM following the target DNA for Type II systems. Thus, Type V systems cleave at a point that is distant from the PAM, while Type II systems cleave at a point adjacent the PAM. In addition, in contrast to Type II systems, Cpf1 cleaves DNA via a staggered DNA double-stranded break with a 4- or 5-nucleotide 5′ overhang. Type II systems on the other hand cleave via a blunt double-stranded break. Similar to Type II systems, Cpf1 contains a predicted RuvC-like endonuclease domain, but lacks a second HNH endonuclease domain, which is in contrast to Type II systems. 
     CRISPR Enzymes 
     CRISPR enzymes are enzymes which can bind to a guide RNA and to a complementary target DNA sequence. CRISPR enzymes may have nuclease activity or they may be rendered nuclease inactive, for example, by mutation of their nuclease domain(s). The term “CRISPR enzyme” will be used herein in reference to both nuclease active and nuclease inactive enzymes. 
     Naturally-occurring wild-type Cas9 enzymes comprise two nuclease domains, a HNH nuclease domain and a RuvC domain. Herein, the term “Cas9” refers to both naturally occurring and recombinant Cas9. Cas9 enzymes contemplated herein can comprise a HNH or HNH-like nuclease domain, and/or a RuvC or RuvC-like nuclease domain. The Cas9 enzymes may also lack nuclease activity, or have reduced nuclease activity, due to mutations or modifications within one or both of its nuclease domains. HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-like domains comprise two antiparallel β-strands and an α-helix. HNH or HNH-like domains also comprise a metal binding site (e.g., a divalent cation binding site). HNH or HNH-like domains can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA targeted strand). RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold. The RNaseH domain comprises 5 β-strands surrounded by a plurality of α-helices. RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site (e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-like domains can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA). 
     Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to, Cas proteins identified in  Acaryochloris marina  MBIC11017 ; Acetohalobium arabaticum  DSM 5501 ; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans  ATCC 23270;  Alicyclobacillus acidocaldarius  LAA1;  Alicyclobacillus acidocaldarius  subsp.  acidocaldarius  DSM 446 ; Allochromatium vinosum  DSM 180 ; Ammonifex degensii  KC4;  Anabaena variabilis  ATCC 29413 ; Arthrospira maxima  CS-328 ; Arthrospira platensis  str.  Paraca; Arthrospira  sp. PCC 8005;  Bacillus pseudomycoides  DSM 12442;  Bacillus selenitireducens  MLS10 ; Burkholderiales bacterium  1_1_47 ; Caldicelulosiruptor becscii  DSM 6725 ; Candidatus Desulforudis audaxviator  MP104C;  Caldicellulosiruptor hydrothermalis _108;  Clostridium phage  c-st;  Clostridium botulinum  A3 str.  Loch Maree; Clostridium botulinum  Ba4 str. 657;  Clostridium difficile  QCD-63q42 ; Crocosphaera watsonii  WH 8501 ; Cyanothece  sp. ATCC 51142 ; Cyanothece  sp. CCY0110 ; Cyanothece  sp. PCC 7424 ; Cyanothece  sp. PCC 7822;  Exiguobacterium sibiricum  255-15;  Finegoldia magna  ATCC 29328 ; Ktedonobacter racemifer  DSM 44963;  Lactobacillus delbrueckii  subsp.  bulgaricus  PB2003/044-T3-4;  Lactobacillus salivarius  ATCC 11741;  Listeria innocua; Lyngbya  sp. PCC 8106;  Marinobacter  sp. ELB17 ; Methanohalobium evestigatum  Z-7303 ; Microcystis phage  Ma-LM M01 ; Microcystis aeruginosa  NI ES-843 ; Microscilla marina  ATCC 23134 ; Microcoleus chthonoplastes  PCC 7420;  Neisseria meningitidis; Nitrosococcus halophilus  Nc4 ; Nocardiopsis dassonvillei  subsp.  dassonvillei  DSM 43111 ; Nodularia spumigena  CCY9414;  Nostoc  sp. PCC 7120 ; Oscillatoria  sp. PCC 6506 ; Pelotomaculum thermopropionicum  SI;  Petrotoga mobilis  SJ95;  Polaromonas naphthalenivorans  CJ2;  Polaromonas  sp. JS666;  Pseudoalteromonas haloplanktis  TAC125;  Streptomyces pristinaespiralis  ATCC 25486;  Streptomyces pristinaespiralis  ATCC 25486;  Streptococcus thermophilus; Streptomyces viridochromogenes  DSM 40736 ; Streptosporangium roseum  DSM 43021 ; Synechococcus  sp. PCC 7335; and  Thermosipho africanus  TCF52B (Chylinski et al.,  RNA Biol.,  2013; 10(5): 726-737. 
     Other examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and homologues thereof. 
     CRISPR enzymes, if nuclease-active, can introduce double-strand breaks or single-strand breaks in genomic DNA. The double-strand break can stimulate a cell&#39;s endogenous DNA-repair pathways (e.g., HDR, NHEJ or MMEJ). In some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence at the target cleavage site. An exogenous polynucleotide sequence may be termed a donor polynucleotide (or donor or donor sequence). The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site. 
     The CRISPR enzyme may comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% amino acid sequence identity to a wild-type exemplary CRISPR enzyme [e.g., Cas9 from  S. pyogenes , US2014/0068797 Sequence ID No. 8 or Sapranauskas et al.,  Nucleic Acids Res,  39(21): 9275-9282 (2011)], and various other site-directed polypeptides. The CRISPR enzyme may comprise one or more mutations that reduces or abolishes its nucleic acid-cleaving activity. For example, the CRISPR enzyme may comprise a mutation such that it can induce a single-stranded break (SSB) on a target nucleic acid (e.g., by cutting only one of the sugar-phosphate backbones of a double-strand target nucleic acid). The mutation may result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the nucleic acid-cleaving domains of the wild-type CRISPR enzyme (e.g., Cas9 from  S. pyogenes , supra). The mutation may result in one or more of the nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation may result in one or more of the nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in the wild-type exemplary  S. pyogenes  Cas9 polypeptide, such as Asp10, His840, Asn854 and Asn856 may be mutated to inactivate one or more of the nucleic acid-cleaving domains. Non-limiting examples of mutations include D10A, H840A, N854A or N856A. N580A in Sa Cas9 may also be employed. Mutations other than alanine substitutions may also be suitable. Site-directed polypeptides that comprise one substantially inactive nuclease domain are referred to as “nickases”. 
     Nickase variants of CRISPR enzymes, for example Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild type Cas9 is typically guided by a single guide RNA designed to hybridize with an approximately 20 nucleotide target sequence. However, several mismatches can be tolerated between the guide sequence and the target locus, effectively reducing the length of required homology in the target site to, for example, as little as 13 nt of homology. This can increase the potential for binding and double-strand nucleic acid cleavage by the CRISPR/Cas9 complex elsewhere in the target genome—also known as “off-target” cleavage. Because nickase variants of Cas9 each only cut one strand, in order to create a double-strand break it is necessary for a pair of nickases to bind in close proximity and on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-strand break. This requires that two separate guide RNAs—one for each nickase—bind in close proximity and on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology needed for the double-strand break to occur, thereby reducing the likelihood that a double-strand cleavage event will occur elsewhere in the genome. 
     A Cas9 whose nuclease activity is abolished is often referred to as a “dead Cas9” or “dCas9”. A combination of D10A and H840A mutations or D10A and N863A mutations may render a Cas9 catalytically inactive. In some examples, the CRISPR enzyme of the present disclosure is dCas9. 
     A high fidelity CRISPR/Cas9 variant with reduced off-targeting was described by Kleinstiver et al. 2016. Nature. 529 (7587): 490-495. In certain examples, the CRISPR enzyme is a high fidelity Cas9 or a high fidelity dead Cas9 such as a Cas9 comprising mutations D10A, H840A, Y450A, N497A, R661A, Q695A and Q926A. 
     In certain examples, the CRISPR enzyme is truncated, for example to reduce the size of the CRISPR enzyme. In some examples, the CRISPR enzyme is a chimer, comprising amino acid stretches from more than one distinct CRISPR enzyme. 
     In addition to, or instead of, mutations which reduce or abolish the nuclease activity of a CRISPR enzyme, guide RNAs may be designed in such a way as to enable hybridisation between the guide sequence and the target sequence, but without enabling nuclease activity of the CRISPR enzyme. For example, a guide sequence which is 14 nucleotides or 15 nucleotides in length may be capable of guiding Cas9 to a target locus without enabling cleavage of the target locus, even if the Cas9 comprises functional nuclease domains. Such guide RNAs may be referred to as “dead guide RNAs” (dgRNAs). In certain examples, the guide RNA of the present disclosure is a dead guide RNA. The guide RNA may comprise a guide sequence that is no greater than 15 nucleotides in length, or no greater than 14 nucleotides in length. 
     The CRISPR enzyme may also be modified to comprise, or associate with, a transcriptional activation domain. For example, the CRISPR enzyme may be fused to a transcriptional activation domain as a single polypeptide. In such examples, a flexible linker may fuse the CRISPR enzyme to the transcriptional activation domain. For example, GlySer linkers (eg, GGGGS) may be used. They may be repeats of 3 ((GGGGS) 3 ), 6, 9, 12 or more, to provide suitable lengths as desired. The linker may also be rigid such as an Ala(GluAlaAlaAlaLys)Ala linker. Alternatively, the CRISPR enzyme may bind to a separate protein which comprises a transcriptional activation domain. The transcriptional activation domain may enhance transcription of nearby genes, for example, by recruiting transcription factors and or chromatin remodelling complexes. Indeed, the transcriptional activation domain may itself contribute to chromatin remodelling. Suitable transcriptional activation domains may include VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, SET7/9 as well as domains from chromatin-remodelling enzymes such as DNA demethylases and histone acetyltransferases. For example, members of the ten-eleven translocation (TET) family of zinc finger proteins catalyze the oxidation of methylated cytosine residues which leads to the replaced of methylated cytosine residues with naked cytosines and ultimately an increase in transcriptional activity. In certain examples, the transcriptional activation domain of the present disclosure is a TET catalytic domain (CD) such as the catalytic domain of TET1, TET2 or TET3. In some examples, the transcriptional activation domain is an acetyltransferase such as p300. 
     A CRISPR enzyme such as Cas9, and preferably dCas9, may comprise or associate with a transcriptional activation domain in many different ways. For example, dCas9-VP64 comprises dCas9 and four copies of the VP16 transcriptional activation domain fused to the C-terminus of the dCas9. dCas9-VPR comprises dCas9 fused to the activation domains VP64, p65 and RTA, with each activation domain optionally separated by a short amino acid linker, thereby resulting in six transcriptional activation domains fused to the C-terminus of dCas9. Suntag comprises a dCas9 and a chain of 10 peptide epitopes called GCN4 fused repetitively to the C-terminus of the dCas9. Single chain antibodies with specificity to the GCN4 epitope and fused to VP64 can then be co-administered (eg, co-expressed) with the dCas9 fusion. P300 is the catalytic core of the human acetyltransferase p300 protein directly fused to dCas9. VP160 comprises 10 repeats of VP16 fused to the C-terminus of dCas9. VP64-dCas9-BFP-VP64 is dCas9 with VP64 fused to the N-terminus and BFP-VP64 fused to the C-terminus resulting in eight transcriptional activation domains. Vectors encoding such fusions are commercially available, for example, from Addgene. 
     In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10, and preferably to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, such as to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain, wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a CRISPR enzyme, wherein the CRISPR enzyme is fused to VP64, and wherein the CRISPR enzyme is dead Cas9 (dCas9). In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a CRISPR enzyme, wherein the CRISPR enzyme is attached to VP64 via a GCN4 fusion, and wherein the CRISPR enzyme is dead Cas9 (dCas9). In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a CRISPR enzyme, wherein the CRISPR enzyme is fused to VP64, p65 and RTA, and wherein the CRISPR enzyme is dead Cas9 (dCas9). 
     Table 2 provides exemplary sequences of a dCas9 fused to an NLS and a transcriptional activation domain. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Descrip- 
                   
               
               
                 SEQ 
                 tion 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 21 
                 NLS- 
                 ATGAGCcccaagaagaagagaaaggtgGAGGCCAGC GACAAGAAGTACAGCATCGGCCTGGCCATCGGCA   
               
               
                   
                 dCas9 
                 
                   CCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGG 
                 
               
               
                   
                 (D10A, 
                 
                   CAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC 
                 
               
               
                   
                 H840A)- 
                 
                   GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGC 
                 
               
               
                   
                 NLS- 
                 
                   AAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT 
                 
               
               
                   
                 VP64 
                 
                   GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCAC 
                 
               
               
                   
                 dCas9 
                 
                   GAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGC 
                 
               
               
                   
                 under- 
                 
                   TGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCC 
                 
               
               
                   
                 lined 
                 
                   CGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC 
                 
               
               
                   
                 NLS 
                 
                   CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGG 
                 
               
               
                   
                 lower- 
                 
                   AAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCT 
                 
               
               
                   
                 case 
                 
                   GGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGAC 
                 
               
               
                   
                 VP64 
                 
                   ACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCG 
                 
               
               
                   
                 high- 
                 
                   CCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCC 
                 
               
               
                   
                 lighted 
                 
                   CCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTG 
                 
               
               
                   
                   
                 
                   CGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACA 
                 
               
               
                   
                   
                 
                   TTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCOTGGAAAAGATGGACGGCAC 
                 
               
               
                   
                   
                 
                   CGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGC 
                 
               
               
                   
                   
                 
                   ATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCC 
                 
               
               
                   
                   
                 
                   TGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGC 
                 
               
               
                   
                   
                 
                   CAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAG 
                 
               
               
                   
                   
                 
                   GAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGC 
                 
               
               
                   
                   
                 
                   CCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAA 
                 
               
               
                   
                   
                 
                   AGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTG 
                 
               
               
                   
                   
                 
                   GACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCG 
                 
               
               
                   
                   
                 
                   AGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGA 
                 
               
               
                   
                   
                 
                   TCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATC 
                 
               
               
                   
                   
                 
                   GTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT 
                 
               
               
                   
                   
                 
                   TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCT 
                 
               
               
                   
                   
                 
                   GATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCC 
                 
               
               
                   
                   
                 
                   AACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGG 
                 
               
               
                   
                   
                 
                   TGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAPAAGGG 
                 
               
               
                   
                   
                 
                   CATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATC 
                 
               
               
                   
                   
                 
                   GTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGC 
                 
               
               
                   
                   
                 
                   GGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCT 
                 
               
               
                   
                   
                 
                   GCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGAC 
                 
               
               
                   
                   
                 
                   ATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCG 
                 
               
               
                   
                   
                 
                   ACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGT 
                 
               
               
                   
                   
                 
                   GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAAT 
                 
               
               
                   
                   
                 
                   CTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGG 
                 
               
               
                   
                   
                 
                   AAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAA 
                 
               
               
                   
                   
                 
                   TGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGAT 
                 
               
               
                   
                   
                 
                   TTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCG 
                 
               
               
                   
                   
                 
                   TGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTA 
                 
               
               
                   
                   
                 
                   CGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTAC 
                 
               
               
                   
                   
                 
                   AGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGA 
                 
               
               
                   
                   
                 
                   TCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGT 
                 
               
               
                   
                   
                 
                   GCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCT 
                 
               
               
                   
                   
                 
                   ATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCG 
                 
               
               
                   
                   
                 
                   GCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAA 
                 
               
               
                   
                   
                 
                   ACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATC 
                 
               
               
                   
                   
                 
                   GACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCC 
                 
               
               
                   
                   
                 
                   TGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACT 
                 
               
               
                   
                   
                 
                   GGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCC 
                 
               
               
                   
                   
                 
                   GAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGA 
                 
               
               
                   
                   
                 
                   TCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA 
                 
               
               
                   
                   
                 
                   GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGA 
                 
               
               
                   
                   
                 
                   GCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGC 
                 
               
               
                   
                   
                 
                   TGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGG 
                 
               
               
                   
                   
                   AGGCGAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCggacctaagaaa 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 22 
                 dCas9 
                 
                   GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGT 
                 
               
               
                   
                 (D10A, 
                 
                   ACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGAT 
                 
               
               
                   
                 H840A)- 
                 
                   CGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGA 
                 
               
               
                   
                 NLS- 
                 
                   TACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACG 
                 
               
               
                   
                 p-65 
                 
                   ACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCAT 
                 
               
               
                   
                 dCas9 
                 
                   CTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAA 
                 
               
               
                   
                 under- 
                 
                   CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCC 
                 
               
               
                   
                 lined 
                 
                   GGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT 
                 
               
               
                   
                 NLS 
                 
                   GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATC 
                 
               
               
                   
                 lower- 
                 
                   CTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGA 
                 
               
               
                   
                 case 
                 
                   ATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCT 
                 
               
               
                   
                 p65 
                 
                   GGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAG 
                 
               
               
                   
                 high- 
                 
                   ATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACA 
                 
               
               
                   
                 lighted 
                 
                   TCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCA 
                 
               
               
                   
                   
                 
                   CCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTC 
                 
               
               
                   
                   
                 
                   TTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGT 
                 
               
               
                   
                   
                 
                   TCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCT 
                 
               
               
                   
                   
                 
                   GCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCC 
                 
               
               
                   
                   
                 
                   ATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGA 
                 
               
               
                   
                   
                 
                   CCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAA 
                 
               
               
                   
                   
                 
                   GAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTC 
                 
               
               
                   
                   
                 
                   ATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGT 
                 
               
               
                   
                   
                 
                   ACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGC 
                 
               
               
                   
                   
                 
                   CTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTG 
                 
               
               
                   
                   
                 
                   AAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAG 
                 
               
               
                   
                   
                 
                   ATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGA 
                 
               
               
                   
                   
                 
                   CAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATG 
                 
               
               
                   
                   
                 
                   ATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGA 
                 
               
               
                   
                   
                 
                   GATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGAC 
                 
               
               
                   
                   
                 
                   AATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC 
                 
               
               
                   
                   
                 
                   CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTG 
                 
               
               
                   
                   
                 
                   CCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGT 
                 
               
               
                   
                   
                 
                   GAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAG 
                 
               
               
                   
                   
                 
                   AAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGA 
                 
               
               
                   
                   
                 
                   TCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAA 
                 
               
               
                   
                   
                 
                   TGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATC 
                 
               
               
                   
                   
                 
                   GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGG 
                 
               
               
                   
                   
                 
                   GCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAA 
                 
               
               
                   
                   
                 
                   CGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG 
                 
               
               
                   
                   
                 
                   GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCC 
                 
               
               
                   
                   
                 
                   TGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCT 
                 
               
               
                   
                   
                 
                   GAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTAC 
                 
               
               
                   
                   
                 
                   CACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGG 
                 
               
               
                   
                   
                 
                   AAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGA 
                 
               
               
                   
                   
                 
                   AATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACC 
                 
               
               
                   
                   
                 
                   CTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGG 
                 
               
               
                   
                   
                 
                   ATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC 
                 
               
               
                   
                   
                 
                   CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCC 
                 
               
               
                   
                   
                 
                   AGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGG 
                 
               
               
                   
                   
                 
                   TGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCAT 
                 
               
               
                   
                   
                 
                   CATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAGA 
                 
               
               
                   
                   
                 
                   AAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGG 
                 
               
               
                   
                   
                 
                   CCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCT 
                 
               
               
                   
                   
                 
                   GGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG 
                 
               
               
                   
                   
                 
                   CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACG 
                 
               
               
                   
                   
                 
                   CTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAA 
                 
               
               
                   
                   
                 
                   TATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATC 
                 
               
               
                   
                   
                 
                   GACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCC 
                 
               
               
                   
                   
                   TGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGG 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     It will be understood that in examples where the CRISPR enzyme comprises or associates with a transcriptional activation domain, expression of the Klotho gene may be enhanced in the absence of an adapter protein. Accordingly, the present disclosure provides methods of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In certain examples, the CRISPR enzyme is modified to include a nuclear localisation signal (NLS). For example, the CRISPR enzyme may be flanked at its N-terminus, its C-terminus, or both the N-terminus and C-terminus by one or more NLSs. For example, a Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as a SV40 NLS. A non-limiting example of an SV40 NLS is set forth in SEQ ID NO. 27. 
     Guide RNA, Adapter Proteins and Transcriptional Activation Domains 
     A guide RNA (gRNA) can comprise at least a guide (also called a “spacer”) sequence, which is the sequence of the guide RNA that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. The term guide sequence is not to be construed as referring to the sequence of the entire guide RNA, but rather, the portion of the guide RNA that hybridizes to the target sequence. In wild type Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex can bind a CRISPR enzyme, such that the guide RNA and the CRISPR enzyme form a complex. The guide RNA provides target specificity to the complex by virtue of its association with the CRISPR enzyme. 
     A double-molecule guide RNA can comprise two strands of RNA. The first strand comprises in the 5′ to 3′ direction, an optional spacer extension sequence, a guide sequence and a minimum CRISPR repeat sequence. The second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and an optional tracrRNA extension sequence. 
     A single-molecule guide RNA (sgRNA) comprises only one strand of RNA. The sgRNA may comprise a guide sequence fused to a tracr sequence. Without wishing to be bound to any particular arrangement of sequences or structure, a sgRNA in a Type II system may comprise, in a 5′ to 3′ direction, an optional spacer extension sequence, a guide sequence (also referred to as a spacer sequence), a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. 
     The length of the guide sequence may vary from between 10 nucleotides to 40 nucleotides and is generally located at the 5′ end of the guide RNA. In certain examples, the guide sequence is less than 40 nucleotides in length such as less than 25 nucleotides. The guide sequence may be 24 nucleotides, 23 nucleotides, 22 nucleotides, 21 nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, 14 nucleotides, 13 nucleotides, 12, nucleotides, 11 nucleotides or 10 nucleotides in length. 
     The length of the guide RNA may vary from 50 nucleotides to more than 300 nucleotides. For example, the guide RNA of the present disclosure may be between about 50 nucleotides and 300 nucleotides in length, such as between about 70 nucleotides and 250 nucleotides in length, or between about 80 nucleotides and 225 nucleotides in length, or between about 90 nucleotides and 215 nucleotides in length, or between about 100 nucleotides and 200 nucleotides in length, or between about 110 nucleotides and 190 nucleotides in length, or between about 125 nucleotides and 170 nucleotides in length, or between about 140 nucleotides and 165 nucleotides in length. 
     In a CRISPR/Cas system, the guide sequence can be designed to hybridize to a target nucleic acid that is located immediately upstream (5′) of a PAM of the Cas9 enzyme used in the system. The guide sequence can be perfectly complementary with the target sequence or it can have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example,  S. pyogenes  recognizes in a target nucleic acid a PAM that comprises the sequence 5′-NRG-3′, where R comprises either A or G, where N is any nucleotide and N is immediately 3′ of the target nucleic acid sequence targeted by the spacer sequence. 
     In certain examples, the guide RNA of the present disclosure comprises a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene. A target sequence will be considered “near” the Klotho gene if, despite not being located within the Klotho gene, the target sequence is sufficiently close to the Klotho gene such that when the target sequence is bound by a guide RNA of the present disclosure along with a CRISPR enzyme and a transcriptional activation domain (optionally via an adapter protein), expression of the Klotho gene is increased relative to an absence of such binding. 
     In some examples, the percent complementarity between the guide sequence and the target sequence is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 100%. In some examples, the percent complementarity between the guide sequence and the target nucleic acid is 100% over the six contiguous 5-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the guide sequence and the target nucleic acid can be at least 60%, such as at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% over about 20 contiguous nucleotides. The length of the guide sequence and the target nucleic acid can differ by 1 to 6 nucleotides, the difference in length may result in a bulge or bulges. In certain examples, the guide sequence is at least about 80% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, such as at least about 85%, at least about 90%, at least about 95% or about 100% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. In certain examples, the guide sequence comprises at least 10 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. For example, the guide sequence may comprises at least 11 contiguous nucleotides, or at least 12 contiguous nucleotides, or at least 13 contiguous nucleotides, or at least 14 contiguous nucleotides, or at least 15 contiguous nucleotides, or at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides or at least 20 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     The target sequence may be located anywhere within or near the Klotho gene such that binding of a guide RNA of the present disclosure to the target sequence, along with a CRISPR enzyme and a transcriptional activation domain (optionally via an adapter protein), enhances transcription of Klotho. In certain examples, the target sequence is located within a region between the Klotho gene translation start site and about 5 kb upstream of the Klotho translation start site. In other words, the target sequence may be located within a window starting from the translation initiation site and extending 5 kb upstream. In some examples, the target sequence may be located within a region extending about 4.5 kb upstream of the Klotho translation start site, or about 4 kb upstream of the Klotho translation start site, or about 3.5 kb upstream of the Klotho translation start site, or about 3 kb upstream of the Klotho translation start site, or about 2.5 kb upstream of the Klotho translation start site, or about 2 kb upstream of the Klotho translation start site, or about 1.5 kb upstream of the Klotho translation start site, or about 1 kb upstream of the Klotho translation start site, or about 0.5 kb upstream of the Klotho translation start site. In certain examples, the target sequence is located within a region between about 100 nucleotides and 5000 nucleotides upstream of the Klotho translation start site, such as within a region between about 150 nucleotides and 5000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 5000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 4500 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 4000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 3500 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 3000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 2500 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 2000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 1500 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 1000 nucleotides upstream of the Klotho translation start site, or within a region between about 200 nucleotides and 500 nucleotides upstream of the Klotho translation start site, within a region between about 200 nucleotides and 300 nucleotides upstream of the Klotho translation start site, within a region between about 210 nucleotides and 300 nucleotides upstream of the Klotho translation start site, within a region between about 220 nucleotides and 300 nucleotides upstream of the Klotho translation start site, within a region between about 230 nucleotides and 300 nucleotides upstream of the Klotho translation start site, or within a region between about 240 nucleotides and 300 nucleotides upstream of the Klotho translation start site. 
     In certain examples, the target sequence is located within a regulatory element of the Klotho gene such as a promoter or an enhancer. Expression of eukaryotic protein-coding genes generally is regulated through multiple cis-acting transcription-control regions. Some control elements are located close to the start site (promoter-proximal elements), whereas others lie more distal (enhancers and silencers). Promoters determine the site of transcription initiation and direct binding of RNA polymerase II. They may extend a few hundred base pairs to several kilobases upstream of a transcription start site. Enhancers may be 100 to 200 base pairs in length and may be located a hundred base pairs up to tens of kilobases upstream or down stream of a promoter. Promoters of highly expressed genes often comprise a TATA box. CpG islands are characteristic of transcribed genes. 
     For the purposes of the present disclosure, a target sequence will be considered “within” a region if it is entirely within that region or partially within that region. For example, the target sequence may be located entirely within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site, or alternatively, only a part of the target sequence may be located with that region. In either case, the target sequence is deemed to be “within” a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     The sgRNA of the present disclosure may comprise no uracil at the 3′ end of the sgRNA. Alternatively, the sgRNA may comprise 1 uracil (U) at the 3′ end of the sgRNA. The sgRNA may comprise 2 uracil (UU) at the 3′ end of the sgRNA. The sgRNA may comprise 3 uracil (UUU) at the 3′ end of the sgRNA. The sgRNA may comprise 4 uracil (UUUU) at the 3′ end of the sgRNA. The sgRNA may comprise 5 uracil (UUUUU) at the 3′ end of the sgRNA. The sgRNA may comprise 6 uracil (UUUUUU) at the 3′ end of the sgRNA. The sgRNA may comprise 7 uracil (UUUUUUU) at the 3′ end of the sgRNA. The sgRNA may comprise 8 uracil (UUUUUUUU) at the 3′ end of the sgRNA. 
     The guide sequence may be designed or chosen using a computer program. The computer program may use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more positions as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like. 
     The guide RNA of the present disclosure preferably associates with a transcriptional activation domain. The guide RNA may associate with a transcriptional activation domain via the CRISPR enzyme which may be modified to comprise, or bind to, a transcriptional activation domain. Alternatively, or additionally, the guide RNA may associate with a transcriptional activation domain by way of an adapter protein which is capable of binding to the guide RNA and which comprises, or binds to, a transcriptional activation domain. 
     The sgRNA may comprise multiple loop structures owing to self-complementarity within the sgRNA molecule. Two of those loops, namely the tetra-loop and step loop 2 have been shown to protrude outside of the Cas9-sgRNA ribonucleoprotein complex, with the distal four base pairs of each stem free of interactions with Cas9 amino acid side chains (Nishimasu et al. (2014)  Cell  156: 935-949). Those loops may be engineered to include a protein-binding sequence, such as an RNA aptamer, thus enabling the guide RNA to bind to one or more adapter proteins as well as to the CRISPR enzyme. The adapter protein may comprise or attach to a transcriptional activation domain. Those skilled in the art will be able to identify suitable locations within a particular guide RNA for insertion of a protein-binding sequence, such as at the tetra-loop and/or stem loop 2 which may be modified, altered or replaced to include the protein-binding sequence. Suitable guide RNAs comprising protein-binding sequences are described herein. Konermann et al. ( Nature  (2015) 517: 583-588) have also described guide RNAs comprising protein-binding sequences. 
     The exposed or extraneous portion of the guide (when the guide-Cas9-DNA complex is formed) is preferably a 4 (four) nucleotide stretch. In some examples, the stretch may be in the tetra-loop as described. In some examples, the stretch may be in the stem loop 2 as described. In some examples, stretches in both the tetra-loop and the stem loop 2 are envisaged. This stretch may be modified, altered or entirely replaced. It is not generally preferred to reduce the number of nucleotides in the exposed stretch to less than 4 for stearic reasons as this could affect the secondary structure of the rest of the guide RNA and thus affect formation of the Cas9-guide-DNA complex or the exposure of the stretch. It may be modified or altered in that all four of the original 4 nucleotides in the stretch are retained and additions (or further nucleotides) are made between 1 and 2, 2 and 3, or 3 and 4. It is also envisaged that additions may be made immediately 5′ to 1 or 3′ immediately to 4. The stem may be flexible, but it is preferred that it is largely self-complementary throughout. 
     Unafold is a software tool that can be used to help predict RNA secondary structure in the guide and so assist the skilled person to determine what changes to the guide RNA may be acceptable within the framework discussed herein. 
     In some examples, one or more GC tracts may replace the stem portion of stem loop 2 and/or the tetra-loop. Preferably, the loop feature should be retained but protein binding section of the distinct RNA added to the guide may determine this. The non-loop ends abutting the edge of the enzyme should preferably be retained in the sense that they should be present, but the primary sequence of the original guide can be changed, for example by insertion of one or more GC tract(s). Preferably, this should be done at the non-loop (non-protein-binding end) of the distinct RNA added, which may be extended. The secondary structure of the non-protein-binding region of the distinct RNA preferably forms a stem. 
       FIG. 1  shows an exemplary guide RNA of the present disclosure (sgRNA3; SEQ ID NO: 3) hybridised to a target sequence in the Klotho promoter. The secondary structure of the guide RNA is also indicated including the tetra-loop and the stem loop 2 which have been engineered to include a MS2 binding sequence. 
     Synergistic activation mediator (SAM) comprises dCas9-VP64 with a guide RNA that associates with the fusion polypeptide MS2-p65-HSF1 (sometimes referred to as MCP-p65-HSF1 where MCP is an abbreviation for MS2 coat protein). The guide RNA of the SAM system typically comprises two MS2 binding sequences at the tetraloop and stem loop 2, and are used to recruit MS2-p65-HSF1. MS2-p65-HSF1 binds to the MS2 binding loops as a dimer, thereby resulting in four sets of the activation domains p65 and HSF1 being recruited. Together with the VP64 component, 12 transcriptional activation domains may be involved in a SAM complex. 
     Scaffold comprises a dCas9 component and a guide RNA wherein the guide RNA comprises an MS2 binding loop, which associates with a dimer of MS2-VP4 (sometimes referred to as MCP-VP64), and an F6 aptamer, which associates with two MS2-VP64 fusions resulting in the recruitment of 16 transcriptional activation domains. Vectors encoding such fusions are commercially available, for example, from Addgene. 
     Suitable adapter proteins for binding to guide RNAs of the present disclosure may include bacteriophage coat proteins such as Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In preferred examples, the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. A suitable protein binding sequence may include the MS2-binding loop ggccAACATGAGGATCACCCATGTCTGCAGggcc (SEQ ID NO: 28) or ggccAGCATGAGGATCACCCATGCCTGCAGggcc (SEQ ID NO: 29), or the F6 aptamer. Accordingly, in certain examples, the guide RNA of the present disclosure is a sgRNA which is capable of binding to an adapter protein. 
     The guide RNA may comprise more than one protein binding sequence, and each protein binding sequence may bind the same or different adapter proteins. The adapter protein may comprise a nuclear localisation signal (NLS) such as that from SV40. 
     A PP7 variant may be used in some examples. For example, the PP7 Pseudomonas bacteriophage coat protein (with amino acids 68-69 mutated to SG and amino acids 70-75 deleted from the wild type protein as described in Wu et al.  Biophysical Journal.  102.1 (2012): 2936-2944 and Chao and Singer,  Nature Structural  &amp;  Molecular Biology  15.1 (2007): 103-105 may be desirable. 
     An MS2 variant may also be used, such as the N55 mutant, particularly the N55K mutant. This is the N55K mutant of the MS2 bacteriophage coat protein (shown to have higher binding affinity than wild type MS2 in Lim et al.  Journal of Biological Chemistry  269.12 (1994): 9006-9010). 
     The adapter protein may itself comprise a transcriptional activation domain or it may be fused to the transcriptional activation domain of another protein. For example, GlySer linkers (eg, GGGGS) may be used. They may be repeats of 3 ((GGGGS) 3 ), 6, 9, 12 or more, to provide suitable lengths as desired. Alternatively, the adapter protein may bind to another protein which comprises a transcriptional activation domain. In further examples, the adapter protein may bind to a further adapter such as an antibody which then binds to a protein which comprises a transcriptional activation domain. The transcriptional activation domain may enhance transcription of nearby genes, for example, by recruiting transcription factors and or chromatin remodelling complexes. Indeed, the transcriptional activation domain may itself contribute to chromatin remodelling. Suitable transcriptional activation domains may include VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, SET7/9 as well as domains from chromatin-remodelling enzymes such as DNA demethylases and histone acetyltransferases. In certain examples, the transcriptional activation domain is a TET catalytic domain such as the catalytic domain of TET1, TET2 or TET3. In other examples, the transcriptional activation domain is an acetyltransferase such as p300. 
     Table 3 provides exemplary sequences of an adapter protein (MS2) fused to an NLS and one or more transcriptional activation domains. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 SEQ 
                 Description 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 23 
                 MS2-NLS-VP64 
                 
                   ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGAT 
                 
               
               
                   
                 MS2 underlined 
                 
                   GTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCC 
                 
               
               
                   
                 NLS lowercase 
                 
                   AACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCC 
                 
               
               
                   
                 VP64 
                 
                   CAGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACA 
                 
               
               
                   
                 highlighted 
                 
                   GTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAG 
                 
               
               
                   
                   
                 
                   CTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCA 
                 
               
               
                   
                   
                 
                   ATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAAC 
                 
               
               
                   
                   
                   TCAGGTATCTAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGA 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 24 
                 MS2-NLS-P65 
                 
                   ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGAT 
                 
               
               
                   
                 MS2 underlined 
                 
                   GTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCC 
                 
               
               
                   
                 NLS lowercase 
                 
                   AACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCC 
                 
               
               
                   
                 P65 
                 
                   CAGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACA 
                 
               
               
                   
                 highlighted 
                 
                   GTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAG 
                 
               
               
                   
                   
                 
                   CTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCA 
                 
               
               
                   
                   
                 
                   ATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAAC 
                 
               
               
                   
                   
                   TCAGGTATCTAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGA 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 25 
                 MS2-NLS-P65- 
                 
                   ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGAT 
                 
               
               
                   
                 HSF1 
                 
                   GTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCC 
                 
               
               
                   
                 MS2 underlined 
                 
                   AACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCC 
                 
               
               
                   
                 NLS lowercase 
                 
                   CAGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACA 
                 
               
               
                   
                 P65 
                 
                   GTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAG 
                 
               
               
                   
                 highlighted 
                 
                   CTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCA 
                 
               
               
                   
                 HSF1 bold 
                 
                   ATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAAC 
                 
               
               
                   
                   
                   TCAGGTATCTAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGA 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   CTGTTCAGCCCCTCGGTGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGC 
                 
               
               
                   
                   
                 
                   AGCCTGGCCAGTATCCAAGAGCTCCTGTCTCCCCAGGAGCCCCCCAGGCCTCCC 
                 
               
               
                   
                   
                 
                   GAGGCAGAGAACAGCAGCCCGGATTCAGGGAAGCAGCTGGTGCACTACACAGCG 
                 
               
               
                   
                   
                 
                   CAGCCGCTGTTCCTGCTGGACCCCGGCTCCGTGGACACCGGGAGCAACGACCTG 
                 
               
               
                   
                   
                 
                   CCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACTTCTCCGAAGGGGACGGCTTC 
                 
               
               
                   
                   
                 
                   GCCGAGGACCCCACCATCTCCCTGCTGACAGGCTCGGAGCCTCCCAAAGCCAAG 
                 
               
               
                   
                   
                 
                   GACCCCACTGTCTCC 
                 
               
               
                   
               
               
                 26 
                 MS2-NLS-P65- 
                 
                   ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGAT 
                 
               
               
                   
                 MyoD1 
                 
                   GTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCC 
                 
               
               
                   
                 MS2 underlined 
                 
                   AACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCC 
                 
               
               
                   
                 NLS lowercase 
                 
                   CAGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACA 
                 
               
               
                   
                 P65 
                 
                   GTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAG 
                 
               
               
                   
                 highlighted 
                 
                   CTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCA 
                 
               
               
                   
                 MyoD1 bold 
                 
                   ATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAAC 
                 
               
               
                   
                   
                   TCAGGTATCTAC AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGA 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
                 
                   GACCTGACTGCGCCCGACGGCTCTCTTTGCTCCTTCGCCACAACCGACGACTTC 
                 
               
               
                   
                   
                 
                   TACGATGATCCATGTTTTGACAGCCCCGATCTCAGGTTCTTTGAGGATCTCGAT 
                 
               
               
                   
                   
                 
                   CCTAGACTGATGCACGTGGGCGCACTGCTCAAACCTGAGGAACATAGC 
                 
               
               
                   
               
            
           
         
       
     
     In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, preferably at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In some examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, preferably at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1, and wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein is MS2 fused to p65 and HSF1. In certain examples, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein is MS2 fused to p65 and HSF1, and wherein the CRISPR enzyme is fused to VP64, and wherein the CRISPR enzyme is dead Cas9 (dCas9). 
     In certain examples, the present disclosure provides a guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site, and wherein the guide RNA is between about 100 nt and 200 nt in length. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14, wherein the guide RNA is between about 100 nt and 200 nt in length. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, preferably to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. In some examples, the present disclosure provides a single-molecule guide RNA comprising a guide sequence wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In certain examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is substantially complementary to a target sequence within or near a human Klotho gene; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1, preferably MS2. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, preferably to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4; and at least one protein binding sequence for binding to an adapter protein, wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1, wherein the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. In some examples, the present disclosure provides a single-molecule guide RNA comprising: a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4, preferably to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4; and at least one protein binding sequence for binding to MS2, wherein the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. 
     Nucleic Acid Modifications 
     In some cases, polynucleotides introduced into cells can comprise one or more modifications that can be used individually or in combination, for example, to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or for other enhancements, as further described herein and known in the art. 
     Modifications of guide RNAs may be used to enhance the initiation, stability or kinetics of interactions between the CRISPR enzyme and the target sequence in the genome, and can be used, for example, to enhance on-target activity. Modifications of guide RNAs can also or alternatively be used to enhance specificity, e.g., the relative rates of on-targeting as compared to off-targeting. Modifications may also or alternatively be used to increase the stability of a guide RNA, e.g., by increasing its resistance to degradation by ribonucleases (RNases) present in a cell, thereby causing its half-life in the cell to be increased. Modifications enhancing guide RNA half-life can be particularly useful in aspects in which a CRISPR enzyme is introduced into a cell via an RNA that needs to be translated in order to generate the CRISPR enzyme, because increasing the half-life of guide RNAs introduced at the same time as the RNA encoding the CRISPR enzyme can be used to increase the time that the guide RNAs and the encoded CRISPR enzyme co-exist in the cell. 
     Modifications may also or alternatively be used to decrease the likelihood or degree to which RNAs introduced into cells elicit innate immune responses. Such responses tend to be associated with reduced half-life of the RNA and/or the elicitation of cytokines or other factors associated with immune responses. 
     One or more types of modifications can also be made to RNAs encoding a 
     CRISPR enzyme that are introduced into a cell, including, without limitation, modifications that enhance the stability of the RNA, modifications that enhance translation of the resulting product (i.e. the CRISPR enzyme), and/or modifications that decrease the likelihood or degree to which the RNAs introduced into cells elicit innate immune responses. 
     By way of illustration of various types of modifications, especially those used frequently with smaller chemically synthesized RNAs, modifications can comprise one or more nucleotides modified at the 2′ position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl, or 2′-fluoro-modified nucleotide. In some examples, RNA modifications can comprise 2′-fluoro, 2′-amino or 2′-O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3′ end of the RNA. Such modifications can be routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2′-deoxyoligonucleotides against a given target. 
     A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligonucleotide. These modified oligos survive intact for a longer period of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH 2 —NH—O—CH 2 , CH, ˜N(CH 3 )—O—CH 2  (known as a methylene(methylimino) or MMI backbone), CH 2 —O—N (CH 3 )—CH 2 , CH 2 —N(CH 3 )—N(CH 3 )—CH 2  and O-N(CH 3 )—CH 2 —CH 2  backbones, wherein the native phosphodiester backbone is represented as O— P— O— CH); amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3′alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5-3′ or 2′-5′ to 5-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050. 
     Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. 
     Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122: 8595-8602 (2000). 
     Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH 2  component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. 
     One or more substituted sugar moieties can also be included, e.g., one of the following at the 2′ position: OH, SH, SCH 3 , F, OCN, OCH 3  OCH 3 , OCH 3  O(CH 2 )n CH 3 , O(CH 2 )n NH 2 , or O(CH 2 )n CH 3 , where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2  CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. In some aspects, a modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2′-methoxy (2′-O—CH 3 ), 2′-propoxy (2′-OCH 2  CH 2 CH 3 ) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group. 
     In some examples, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991). 
     Guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are not found, or found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino) adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino) adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman &amp; Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions. 
     Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. 
     Further, nucleobases can comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in ‘The Concise Encyclopedia of Polymer Science and Engineering’, pages 858-859, Kroschwitz, J. I., ed. John Wley &amp; Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition’, 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications&#39;, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research and Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication 2003/0158403. 
     Thus, the term “modified” refers to a non-natural sugar, phosphate, or base that is incorporated into a nucleic acid such as a guide RNA or a nucleic acid encoding a CRISPR enzyme. It is not necessary for all positions in a given nucleic acid molecule to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a nucleic acid molecule, or even in a single nucleoside within an oligonucleotide. 
     The guide RNAs and/or the RNA or DNA encoding a CRISPR enzyme may be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake. Such moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety [Letsinger et al., Proc. Natl. Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g., hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660: 306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3: 2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol or undecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) and Svinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain [Mancharan et al., Nucleosides &amp; Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941. 
     Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites. For example, hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and/or complexes thereof to particular target cells of interest. These targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups. Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this present disclosure, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992 (published as WO1993007883), and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium I,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941. 
     Longer polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can also be modified by various means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of particular sequences or other moieties at the 5′ or 3′ ends of molecules, and other modifications. By way of illustration, the mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied to, e.g., increase its translation or stability (such as by increasing its resistance to degradation with a cell), or to reduce the tendency of the RNA to elicit an innate immune response that is often observed in cells following introduction of exogenous RNAs, particularly longer RNAs such as that encoding Cas9. Numerous such modifications have been described in the art, such as polyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) or m7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs), use of modified bases (such as Pseudo-UTP, 2-Thio-UTP, 5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), or treatment with phosphatase to remove 5′ terminal phosphates. These and other modifications are known in the art, and new modifications of RNAs are regularly being developed. 
     There are numerous commercial suppliers of modified RNAs, including for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon and many others. As described by TriLink, for example, 5-Methyl-CTP can be used to impart desirable characteristics, such as increased nuclease stability, increased translation or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as well as Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as illustrated in publications by Kormann et al. and Warren et al. referred to below. 
     It has been shown that chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29, 154-157 (2011). Such modifications can be used, for example, to increase the stability of the RNA molecule and/or reduce its immunogenicity. Using chemical modifications such as Pseudo-U, N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substituting just one quarter of the uridine and cytidine residues with 2-Thio-U and 5-Methyl-C respectively resulted in a significant decrease in toll-like receptor (TLR) mediated recognition of the mRNA in mice. By reducing the activation of the innate immune system, these modifications can be used to effectively increase the stability and longevity of the mRNA in vivo; see, e.g., Kormann et al., supra. 
     It has also been shown that repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass innate anti-viral responses can reprogram differentiated human cells to pluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs that act as primary reprogramming proteins can be an efficient means of reprogramming multiple human cell types. Such cells are referred to as induced pluripotency stem cells (iPSCs), and it was found that enzymatically synthesized RNA incorporating 5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could be used to effectively evade the cell&#39;s antiviral response; see, e.g., Warren et al., supra. 
     Other modifications of polynucleotides described in the art include, for example, the use of polyA tails, the addition of 5′ cap analogs such as (m7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions (UTRs), or treatment with phosphatase to remove 5 terminal phosphates—and new approaches are regularly being developed. 
     A number of compositions and techniques applicable to the generation of modified RNAs for use herein have been developed in connection with the modification of RNAs used in RNA interference (RNAi), including small-interfering RNAs (siRNAs). siRNAs present particular challenges in vivo because their effects on gene silencing via RNA interference are generally transient, which can require repeat administration. In addition, siRNAs are double-stranded RNAs (dsRNA) and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase), and potentially retinoic acid-inducible gene I (RIG-I), that can mediate cellular responses to dsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) that can trigger the induction of cytokines in response to such molecules; see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4): 440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012); Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan, Hum Gene Ther 19(2):111-24 (2008). 
     A large variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and/or achieve other benefits that can be useful in connection with the introduction of polynucleotides into human cells, as described herein; see, e.g., the reviews by Whitehead K A et al., Annual Review of Chemical and Biomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin Mol Ther., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsen et al., Front Genet 3:154 (2012). 
     There are a number of commercial suppliers of modified RNAs, many of which have specialized in modifications designed to improve the effectiveness of siRNAs. A variety of approaches are offered based on various findings reported in the literature. For example, Dharmacon notes that replacement of a non-bridging oxygen with sulfur (phosphorothioate, PS) has been extensively used to improve nuclease resistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery 11:125-140 (2012). Modifications of the 2′-position of the ribose have been reported to improve nuclease resistance of the internucleotide phosphate bond while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. A combination of moderate PS backbone modifications with small, well-tolerated 2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associated with highly stable siRNAs for applications in vivo, as reported by Soutschek et al. Nature 432:173-178 (2004); and 2′-O-Methyl modifications have been reported to be effective in improving stability as reported by Volkov, Oligonucleotides 19:191-202 (2009). With respect to decreasing the induction of innate immune responses, modifying specific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have been reported to reduce TLR7/TLR8 interaction while generally preserving silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, K. et al., Immunity 23:165-175 (2005). 
     As is also known in the art, and commercially available, a number of conjugates can be applied to polynucleotides, such as RNAs, for use herein that can enhance their delivery and/or uptake by cells, including for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther. Deliv. 4:791-809 (2013). 
     Target Sequence Selection 
     Many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as the requirement of a PAM sequence in a particular position adjacent the DNA cleavage site in the case of CRISPR Type II or Type V enzymes. 
     The frequency of off-target activity can be assessed relative to the frequency of on-target activity. In some cases, cells that have been correctly targeted at the desired locus can have a selective advantage relative to other cells. Illustrative, but non-limiting, examples of a selective advantage include the acquisition of attributes such as enhanced rates of replication, persistence, resistance to certain conditions, enhanced rates of successful engraftment or persistence in vivo following introduction into a patient, and other attributes associated with the maintenance or increased numbers or viability of such cells. In other cases, cells that have been correctly targeted at the desired locus can be positively selected for by one or more screening methods used to identify, sort or otherwise select for cells that have been correctly targeted. In some cases, cells can be targeted two or more times in order to select or purify the intended population of cells. Such a second targeting could be created by adding a second gRNA for a selectable or screenable marker. DNA or RNA sequencing such as whole genome sequencing can be used to detect off-target activity. 
     The occurrence of off-target activity can be influenced by a number of factors including similarities and dissimilarities between the target site and various off-target sites, as well as the particular CRISPR enzyme used. Bioinformatics tools such as MIT&#39;s CRISPR design tool (http://crispr.mit.edu; Ran et al. 2013 . Nat. Protoc.  8(11): 2281-2308) are available to assist in the prediction of off-target activity, and frequently such tools can also be used to identify the most likely sites of off-target activity, which can then be assessed in experimental settings to evaluate relative frequencies of off-target to on-target activity, thereby allowing the selection of sequences that have higher relative on-target activities. 
     Nucleic Acids Encoding System Components 
     Those skilled in the art will understand that RNA molecules of the present disclosure (eg, guide RNA) may be introduced into a cell directly or via a DNA molecule which is transcribed to produce the RNA. In either case, the RNA is considered to be introduced into the cell. Similarly, proteins such as a CRISPR enzyme or an adapter protein may be delivered to a cell directly or as an RNA molecule which is translated or as a DNA molecule which is transcribed to produce an RNA molecule which is subsequently translated. 
     The nucleic acids of the present disclosure may include a vector (eg, a recombinant expression vector). The term “vector” refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector; wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. 
     In some examples, vectors can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors”, or more simply “expression vectors”, which serve equivalent functions. 
     The term “operably linked” means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term “regulatory sequence” is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like. 
     Expression vectors contemplated include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell. 
     In some examples, a vector can comprise one or more transcription and/or translation control elements. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. The vector can be a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements. 
     Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EF1), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I. 
     For expressing small RNAs, including guide RNAs, various promoters such as RNA polymerase III promoters, including for example U6 and H1, can be advantageous. Descriptions of and parameters for enhancing the use of such promoters are known in art, and additional information and approaches are regularly being described; see, e.g., Ma, H. et al.,  Molecular Therapy—Nucleic Acids  3 , el  61 (2014) doi:10.1038/mtna.2014.12. 
     The expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also comprise appropriate sequences for amplifying expression. The expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein. 
     A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.). 
     A polynucleotide encoding a CRISPR enzyme of the disclosure, an adapter protein of the disclosure, a transcriptional activation domain of the disclosure (or a protein comprising a transcriptional activation domain) and/or any proteinaceous molecule necessary to carry out the aspects of the methods of the disclosure can be codon-optimized according to methods standard in the art for expression in the cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding Cas9 is contemplated for use for producing the Cas9 polypeptide. 
     Introduction of the complexes, polypeptides, and nucleic acids of the disclosure into cells can occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like. 
     Delivery and Treatments 
     The present disclosure provides methods for increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene, wherein the guide RNA associates with a transcriptional activation domain in the cell to thereby increase expression of the Klotho gene. The methods may comprise introducing into the cell an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to the transcriptional activation domain. It will be understood that the guide RNA may be delivered directly into the cell, or as a DNA vector which is transcribed in the cell to thereby produce the guide RNA. Likewise, the CRISPR enzyme and the adapter protein may be introduced into the cell directly, or one or both may be introduced as a DNA or RNA molecule which is transcribed (in the case of a DNA molecule) and translated to thereby produce the CRISPR enzyme and/or the adapter protein. 
     Guide RNA polynucleotides (RNA or DNA) and/or polynucleotides encoding a CRISPR enzyme of the disclosure, an adapter protein of the disclosure, a transcriptional activation domain of the disclosure (or a protein comprising a transcriptional activation domain) and/or any proteinaceous molecule necessary to carry out the aspects of the methods of the disclosure can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, CRISPR enzyme polypeptides, adapter proteins and activation domain polypeptides can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In further alternative aspects, the CRISPR enzyme polypeptides, adapter proteins and activation domain polypeptides can be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs. 
     Polynucleotides can be delivered by non-viral delivery vehicles including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vehicles for siRNA that are also useful for delivery of other polynucleotides). 
     Polynucleotides, such as guide RNA, sgRNA, and mRNA, can be delivered to a cell or a patient by a lipid nanoparticle (LNP). A LNP has a diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. Alternatively, a nanoparticle can range in size from about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm. LNPs can be made from cationic, anionic or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as ‘helper lipids’ to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. 
     Any lipid or combination of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerC14, and PEG-CerC20. The lipids can be combined in any number of molar ratios to produce a LNP. In addition, the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP. 
     The CRISPR enzyme, the adapter protein and guide RNA can each be administered separately to a cell or a patient. On the other hand, the CRISPR enzyme and/or the adapter protein can be pre-complexed with one or more guide RNAs. The pre-complexed material can then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP). RNPs can provide the advantage of reducing undesirable nucleic acid interactions, and protecting the RNA from degradation. 
     Adeno-Associated Virus (AAV) 
     A recombinant adeno-associated virus (AAV) vector can be used for delivery. Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are known in the art. Production of rAAV typically prefers that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be from any AAV serotype for which recombinant virus can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes described herein. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01/83692. 
     AAV particles packaging polynucleotides encoding compositions of the present disclosure, e.g., CRISPR enzyme, adapter protein or RNA guide molecules can comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, the AAV particles can utilize or be based on a serotype selected from any of the following serotypes, and variants thereof including but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60, AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5T, AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3, AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-1 b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r11.64, AAV4-8/rh.64, AAV4-9/rh.54, AAV5, AAV52.1/hu.20, AAV52/hu.19, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-1/hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14, AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5, AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprine AAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8. 
     In some examples, the AAV serotype can be, or have, a mutation in the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), such as but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84. 
     In some examples, the AAV serotype can be, or have, a sequence as described in U.S. Pat. No. 6,156,303, such as, but not limited to, AAV3B (SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NO: 2, 7 and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NO: 3 and 8 of U.S. Pat. No. 6,156,303), AAV3A (SEQ ID NO: 4 and 9, of U.S. Pat. No. 6,156,303), or derivatives thereof. 
     In some examples, the serotype can be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8 can comprise two or more mutations in order to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, can comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, can comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). 
     In some examples, the AAV serotype can be, or have, a sequence as described in International Publication No. WO2015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), “UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ ID NO: 9 of WO2015121501), or variants thereof. 
     According to the present disclosure, AAV capsid serotype selection or use can be from a variety of species. In one example, the AAV can be an avian AAV (AAAV). The AAAV serotype can be, or have, a sequence as described in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800), or variants thereof. 
     In some examples, the AAV can be a bovine AAV (BAAV). The BAAV serotype can be, or have, a sequence as described in U.S. Pat. No. 9,193,769, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No. 9,193,769), or variants thereof. The BAAV serotype can be or have a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), or variants thereof. 
     In some examples, the AAV can be a caprine AAV. The caprine AAV serotype can be, or have, a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No. 7,427,396), or variants thereof. 
     In other examples the AAV can be engineered as a hybrid AAV from two or more parental serotypes. In one example, the AAV can be AAV2G9 which comprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype can be, or have, a sequence as described in United States Patent Publication No. US20160017005. 
     In some examples, the AAV can be a serotype generated by the AAV9 capsid library with mutations in amino acids 390-627 (VP1 numbering) as described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011). The serotype and corresponding nucleotide and amino acid substitutions can be, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T12500 and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, 01760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F4111), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V6061), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T4921, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N4981), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A, G481R), AAV9.83 (C1402A, A1500T; P4681, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A16751; M559L) and AAV9.95 (T1605A; F535L). 
     In some examples, the AAV can be a serotype comprising at least one AAV capsid CD8+ T-cell epitope. As a non-limiting example, the serotype can be AAV1, AAV2 or AAV8. 
     In some examples, the AAV can be a variant, such as PHP.A or PHP.B as described in Deverman. 2016. Nature Biotechnology. 34(2): 204-209. 
     A method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy &amp; Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line can then be infected with a helper virus, such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells. 
     General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Viral., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595. 
     AAV vector serotypes can be matched to target cell types. For example, the following exemplary cell types can be transduced by the indicated AAV serotypes among others: 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Tissue/Cell Type  
                 Serotype 
               
               
                   
               
             
            
               
                 Liver  
                 AAV3, AA5, AAV8, AAV9  
               
               
                 Skeletal muscle  
                 AAV1, AAV7, AAV6, AAV8, AAV9  
               
               
                 Central nervous system  
                 AAV1, AAV4, AAV5, AAV8, AAV9  
               
               
                 RPE  
                 AAV5, AAV4, AAV2, AAV8, AAV9  
               
               
                   
                 AAVrh8r  
               
               
                 Photoreceptor cells  
                 AAV5, AAV8, AAV9, AAVrh8R  
               
               
                 Lung  
                 AAV9, AAV5  
               
               
                 Heart  
                 AAV8  
               
               
                 Pancreas  
                 AAV8  
               
               
                 Kidney  
                 AAV2, AAV8 
               
               
                   
               
            
           
         
       
     
     In addition to adeno-associated viral vectors, other viral vectors can be used. Such viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplex virus. 
     In some cases, the components of the present disclosure (eg, gRNA, CRISPR enzyme and adapter protein) are each separately formulated into lipid nanoparticles, or are all co-formulated into one lipid nanoparticle. In some cases, the CRISPR enzyme is formulated in a lipid nanoparticle, while the gRNA is delivered in a viral vector such as an AAV vector. A range of non-viral delivery methods also exist that can deliver each of these components, or non-viral and viral methods can be employed in tandem. 
     Lentivirus 
     In some examples, lentiviral vectors or particles can be used as delivery vehicles. Lentiviruses are subgroup of the Retroviridae family of viruses. Lentiviral particles are able to integrate their genetic material into the genome of a target/host cell. Examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV), the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV). LV&#39;s are capable of infecting both dividing and non-dividing cells due to their unique ability to pass through a target cell&#39;s intact nuclear membrane Greenberg et al., University of Berkeley, Calif.; 2006). Lentiviral particles that form the gene delivery vehicle are replication defective and are generated by attenuating the HIV virulence genes. For example, the genes Vpu, Vpr, Nef, Env, and Tat are excised making the vector biologically safe. Lentiviral vehicles, for example, derived from HIV-1/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non-dividing cells. 
     In order to produce a lentivirus that is capable of infecting host cells, three types of vectors should be co-expressed in virus producing cells: a backbone vector containing the transgene of interests and self-inactivating 3′-LTR regions, one construct expressing viral structure proteins, and one vector encoding vesicular stomatitis virus glycoprotein (VSVG) for encapsulation (Naldini, L. et al., Science 1996; 272, 263-267). Separation of the Rev gene from other structural genes further increases the biosafety by reducing the possibility of reverse recombination. Cell lines that can be used to produce high-titer lentiviral particles may include, but are not limited to 293T cells, 293FT cells, and 293SF-3F6 cells (Witting et al., Human Gene Therapy, 2012; 23: 243-249; Ansorge et al., Journal of Genetic Medicine, 2009; 11: 868-876). 
     Methods for generating recombinant lentiviral particles are discussed in the art, for example, WO 2013076309 (PCT/EP2012/073645); WO 2009153563 (PCT/GB2009/001527); U.S. Pat. Nos. 7,629,153; and 6,808,905. 
     Guide RNA Formulation 
     Guide RNAs of the present disclosure can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. Guide RNA compositions can be formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, for example, from about pH 3 to about pH 7, depending on the formulation and route of administration. In some cases, the pH can be adjusted to a range from about pH 5 to about pH 8. In some cases, the compositions can comprise a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients. 
     Suitable excipients include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients can include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like. 
     Administration and Efficacy 
     In some examples, the compositions of the present disclosure are administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration, which is then covered by a dressing that occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis and spinal. 
     Modes of administration include injection, infusion, instillation, and/or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some examples, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made. 
     In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). The release system material can be selected so that components having different molecular weights are released by diffusion or through degradation of the material. Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Poly(lactide-co-glycolide) microspheres can also be used. 
     Dosages may vary with the type and severity of the condition to be treated, and may include single or multiple dosses. Specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the practitioner administering the composition. When administered to a human subject, the dosage regimen may vary depending on a variety of factors including the type and severity of the condition, the age, sex, weight or medical condition of the subject and the route of administration. 
     The compositions described herein may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the condition being treated, whether a recurrence is considered likely, etc. The administration may be constant, eg, constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, eg, once per day over a period of days, once per hour over a period of hours, or any other such schedule as deemed suitable. 
     Treatments 
     The present disclosure provides methods of treating a neurological disorder in a subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. The neurological disorder may be associated with memory loss, psychological dysfunction, stress, biopolar disorder, epilepsy, dementia (eg, post stroke dementia, post-traumatic dementia, senile dementia), Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress, neurogenic decline or neurodegeneration. Symptoms of neurological disorders may include memory loss, anxiety, depression, insomnia, disorientation, irrational fear, decline of motor skills or locomotor activity, neophobia, apathy, agitation, tremors, loss of balance, irritability or agoraphobia. 
     In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10, such as to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising increasing expression of a Klotho gene of the subject by administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to a transcriptional activation domain, wherein the neurological disorder is selected from the group consisting of memory loss, stress, biopolar disorder, epilepsy, dementia, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress and neurodegeneration. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising increasing expression of a Klotho gene of the subject by administering to the subject: a CRISPR enzyme; a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and an adapter protein capable of binding to the guide RNA wherein the adapter protein is MS2 fused to p65 and HSF1, and wherein the CRISPR enzyme is fused to VP64, and wherein the CRISPR enzyme is dead Cas9 (dCas9). 
     In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a dead Cas9 (dCas9), wherein the dCas9 is attached to VP64 via a GCN4 fusion. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a dead Cas9 (dCas9), wherein the dCas9 is fused to VP64. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a target sequence located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site; and a dead Cas9 (dCas9), wherein the dCas9 is fused to VP64, p65 and RTA. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10, such as to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. In certain examples, the present disclosure provides a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a single-molecule guide RNA comprising a guide sequence that is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     The method may further comprise administering to the subject an active agent suitable for the treatment of a neurological disorder such as donepezil hydrochloride, memantine, rivastigmine, ligustilide, aripiprazole, asenapine, cariprazine, clozapine, lurasidone, olanzapine, quetiapine, risperidone, ziprasidone, xenazine, tetrabenazine, baclofen, lioresal, kemstro, deutetrabenazine, austedo, cannabis extract, a cannabinoid or cannabinol, an antidepressant, memantine, a cholinesterase inhibitor, an antipsychotic, antioxidants, levodopa, carbidopa, trazodone or dibenzoylmethane. Those skilled in the art will be aware of other active agents that may be suitable for treatment of neurological disorders. 
     The present disclosure also provides a method of enhancing cognitive ability in a subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. For example, the method may enhance memory or learning in the subject. 
     Klotho also plays important regulatory and protective roles in the kidney. In that regard, the present disclosure provides a method of treating renal dysfunction in a subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. In certain examples, the renal dysfunction is associated with renal fibrosis. The present disclosure also provides a method of treating acute kidney injury or a kidney disease such as chronic kidney disease in a subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     Studies have also shown that Klotho plays important roles in regulating fertility. In that regard, the present disclosure provides a method of treating infertility in a subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     Klotho protein has been identified as a regulator of various tumorigenesis and cancer signalling pathways. In that regard, the present disclosure provides methods for treating cancer in a subject comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. In certain examples the cancer is mediated by IGF-1, WNT, bFGF or TGF-β. The cancer may be colon cancer, prostate cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer or breast cancer. Further, non-limiting examples of cancer include leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyclocytic leukemia, acute myclomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin&#39;s disease, non-Hodgkin&#39;s disease), Waldenstrom&#39;s macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing&#39;s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, Sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm&#39;s tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, Small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, Schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the cancer is metastatic cancer. 
     The present disclosure also provides methods of suppressing tumorigenesis, such as breast tumorigenesis and pancreatic tumorigenesis in a subject comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     The present disclosure also provides methods for treating an age-related condition in a subject comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. The age-related condition may be sarcopenia, skin atrophy, muscle wasting, brain atrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis, immunologic incompetence, high blood pressure, dementia, Huntington&#39;s disease, Alzheimer&#39;s disease, cataracts, age-related macular degeneration, prostate cancer, stroke, memory loss, wrinkles, impaired kidney function or hearing loss. 
     The present disclosure also provides methods for treating a muscular disorder such as muscle atrophy and muscular dystrophy (eg, duchene muscular dystrophy) in a subject comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. Muscle atrophy is associated with numerous neuromuscular, metabolic, immunological and neurological disorders and diseases as well as starvation, nutritional deficiency, metabolic stress, diabetes, aging, muscular dystrophy or myopathy. Symptoms include a decline in skeletal muscle tissue mass. In human males, muscle mass declines by one-third between the ages of 50 and 80. Some molecular features of muscle atrophy include the upregulation of ubiquitin ligases, and the loss of myofibrillar proteins (Furuno et al. 1990. J. Biol. Chem. 265: 8550-8557). The degradation of these proteins can be detected, eg, by measuring 3-methyl-histidine production, which is a specific component of actin, and in certain muscles of myosin. Release of creatine kinase can also be indicative. 
     The present disclosure also provides methods for treating a metabolic disorder in a subject comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. In certain examples, the metabolic disorder is selected from Type II Diabetes, Metabolic Syndrome, hyperglycemia and obesity. 
     Methods and Compositions of the Disclosure 
     In a first method, Method 1, the present disclosure provides a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene, wherein the guide RNA associates with a transcriptional activation domain in the cell to thereby increase expression of the Klotho gene. 
     In another method, Method 2, the present disclosure provides the method of Method 1 further comprising introducing into the cell an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to the transcriptional activation domain. 
     In another method, Method 3, the present disclosure provides the method of Method 2 wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, 102, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In another method, Method 4, the present disclosure provides the Method of Method 2 or Method 3 wherein the adapter protein binds to a tetra-loop and/or a stem loop 2 of the guide RNA. 
     In another method, Method 5, there is provided the method of any one of Methods 1 to 4 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 6, there is provided the method of any one of Methods 1 to 5 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another method, Method 7, there is provided the method of any one of Methods 1 to 6 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 8, there is provided the method of any one of Methods 1 to 7 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another method, Method 9, there is provided the method of any one of Methods 1 to 8 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 10, there is provided the method of any one of Methods 1 to 9 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 11, there is provided the method of any one of Methods 1 to 10 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another method, Method 12, there is provided the method of any one of Methods 1 to 11 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another method, Method 13, there is provided the method of any one of Methods 1 to 6 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 14, there is provided the method of any one of Methods 1 to 13 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 15, there is provided the method of any one of Methods 1 to 6 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 16, there is provided the method of any one of Methods 1 to 15 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 17, there is provided the method of any one of Methods 1 to 6 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 18, there is provided the method of any one of Methods 1 to 17 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another method, Method 19, there is provided the method of any one of Methods 1 to 6 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 20, there is provided the method of any one of Methods 1 to 19 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 21, there is provided the method of any one of Methods 1 to 6 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 22, there is provided the method of any one of Methods 1 to 21 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 23, there is provided the method of any one of Methods 1 to 22 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another method, Method 24, there is provided the method of any one of Methods 1 to 23 wherein the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In another method, Method 25, there is provided the method of any one of Methods 1 to 24 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 26, there is provided the method of any one of Methods 1 to 25 wherein the CRISPR enzyme comprises or is attached to a second transcriptional activation domain. 
     In another method, Method 27, there is provided the method of Method 26 wherein the second transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 28, there is provided the method of any one of Methods 1 to 27 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another method, Method 29, there is provided the method of any one of Methods 1 to 28 wherein the CRISPR enzyme is Cas9. 
     In another method, Method 30, there is provided the method of any one of Methods 1 to 29 wherein the cell is a neuronal cell or a kidney cell. 
     In another method, Method 31, there is provided the method of any one of Methods 1 to 30 wherein the cell is inside a human body. 
     In another method, Method 32, there is provided a method of increasing expression of a Klotho gene in a human cell the method comprising introducing into the cell: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 33, there is provided the method of Method 32 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 34, there is provided the method of Method 32 or Method 33 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another method, Method 35, there is provided the method of any one of Methods 32 to 34 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 36, there is provided the method of any one of Methods 32 to 35 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another method, Method 37, there is provided the method of any one of Methods 32 to 36 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 38, there is provided the method of any one of Methods 32 to 37 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 39, there is provided the method of any one of Methods 32 to 38 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another method, Method 40, there is provided the method of any one of Methods 32 to 39 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another method, Method 41, there is provided the method of any one of Methods 32 to 34 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 42, there is provided the method of any one of Methods 32 to 41 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 43, there is provided the method of any one of Methods 32 to 34 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 44, there is provided the method of any one of Methods 32 to 43 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 45, there is provided the method of any one of Methods 32 to 34 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 46, there is provided the method of any one of Methods 32 to 45 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another method, Method 47, there is provided the method of any one of Methods 32 to 34 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 48, there is provided the method of any one of Methods 32 to 47 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 49, there is provided the method of any one of Methods 32 to 34 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 50, there is provided the method of any one of Methods 32 to 49 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 51, there is provided the method of any one of Methods 32 to 50 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another method, Method 52, there is provided the method of any one of Methods 32 to 51 wherein the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. 
     In another method, Method 53, there is provided the method of any one of Methods 32 to 52 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 54, there is provided the method of any one of Methods 32 to 53 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another method, Method 55, there is provided the method of any one of Methods 32 to 54 wherein the CRISPR enzyme is Cas9. 
     In another method, Method 56, there is provided the method of any one of Methods 32 to 55 wherein the cell is a neuronal cell or a kidney cell. 
     In another method, Method 57, there is provided the method of any one of Methods 32 to 56 wherein the cell is inside a human body. 
     In another method, Method 58, there is provided a method of treating cancer in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In another method, Method 59, there is provided the method of Method 58 wherein the cancer is selected from the group consisting of colon cancer, prostate cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer and breast cancer. 
     In another method, Method 60, there is provided a method of treating a muscle disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In another method, Method 61, there is provided the method of Method 60 wherein the muscle disorder is selected from the group consisting of muscle atrophy and muscular dystrophy such as duchene muscular dystrophy. 
     In another method, Method 62, there is provided a method of treating a kidney disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In another method, Method 63, there is provided the method of Method 62 wherein the kidney disorder is selected from the group consisting of renal dysfunction, acute kidney injury and kidney disease such as chronic kidney disease. 
     In another method, Method 64, there is provided a method of enhancing cognition in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In another method, Method 65, there is provided a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject, wherein the guide RNA associates with a transcriptional activation domain in a cell of the subject and thereby increases expression of the Klotho gene. 
     In another method, Method 66, there is provided the method of Method 65 wherein the neurological disorder is selected from the group consisting of memory loss, stress, biopolar disorder, epilepsy, dementia, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress and neurodegeneration. 
     In another method, Method 67, there is provided the method of any one of Methods 58 to 66 further comprising administering to the subject an adapter protein capable of binding to the guide RNA wherein the adapter protein comprises or is attached to the transcriptional activation domain. 
     In another method, Method 68, there is provided the method of Method 67 wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In another method, Method 69, there is provided the method of Method 67 or Method 68 wherein the adapter protein binds to a tetra-loop and/or a stem loop 2 of the guide RNA. 
     In another method, Method 70, there is provided the method of any one of Methods 58 to 69 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 71, there is provided the method of any one of Methods 58 to 70 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another method, Method 72, there is provided the method of any one of Methods 58 to 71 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 73, there is provided the method of any one of Methods 58 to 72 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another method, Method 74, there is provided the method of any one of Methods 58 to 73 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 75, there is provided the method of any one of Methods 58 to 74 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 76, there is provided the method of any one of Methods 58 to 75 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another method, Method 77, there is provided the method of any one of Methods 58 to 76 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another method, Method 78, there is provided the method of any one of Methods 58 to 71 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 79, there is provided the method of any one of Methods 58 to 78 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 80, there is provided the method of any one of Methods 58 to 71 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 81, there is provided the method of any one of Methods 58 to 80 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 82, there is provided the method of any one of Methods 58 to 71 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 83, there is provided the method of any one of Methods 58 to 82 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another method, Method 84, there is provided the method of any one of Methods 58 to 71 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 85, there is provided the method of any one of Methods 58 to 84 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 86, there is provided the method of any one of Methods 58 to 71 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 87, there is provided the method of any one of Methods 58 to 86 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 88, there is provided the method of any one of Methods 58 to 87 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another method, Method 89, there is provided the method of any one of Methods 58 to 88 wherein the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In another method, Method 90, there is provided the method of any one of Methods 58 to 89 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 91, there is provided the method of any one of Methods 58 to 90 wherein the CRISPR enzyme comprises or is attached to a second transcriptional activation domain. 
     In another method, Method 92, there is provided the method of Method 91 wherein the second transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 93, there is provided the method of any one of Methods 58 to 92 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another method, Method 94, there is provided the method of any one of Methods 58 to 93 wherein the CRISPR enzyme is Cas9. 
     In another method, Method 95, there is provided a method of treating cancer in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 96, there is provided the method of Method 95 wherein the cancer is selected from the group consisting of colon cancer, prostate cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer and breast cancer. 
     In another method, Method 97, there is provided a method of treating a muscle disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 98, there is provided the method of Method 97 wherein the muscle disorder is selected from the group consisting of muscle atrophy and muscular dystrophy such as duchene muscular dystrophy. 
     In another method, Method 99, there is provided a method of treating a kidney disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 100, there is provided the method of Method 99 wherein the kidney disorder is selected from the group consisting of renal dysfunction, acute kidney injury and kidney disease such as chronic kidney disease. 
     In another method, Method 101, there is provided a method of enhancing cognition in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 102, there is provided a method of treating a neurological disorder in a human subject the method comprising administering to the subject: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a Klotho gene of the subject; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another method, Method 103, there is provided the method of Method 102 wherein the neurological disorder is selected from the group consisting of memory loss, stress, biopolar disorder, epilepsy, dementia, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, ataxia telangiectasia, craniocerebral trauma, amyotrophic lateral sclerosis, depression, schizophrenia, multiple sclerosis, myelin-related disease, oxidative stress and neurodegeneration. 
     In another method, Method 104, there is provided the method of any one of Methods 95 to 103 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 105, there is provided the method of any one of Methods 95 to 104 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another method, Method 106, there is provided the method of any one of Methods 95 to 105 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 107, there is provided the method of any one of Methods 95 to 106 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another method, Method 108, there is provided the method of any one of Methods 95 to 107 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 109, there is provided the method of any one of Methods 95 to 108 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another method, Method 110, there is provided the method of any one of Methods 95 to 109 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another method, Method 111, there is provided the method of any one of Methods 95 to 110 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another method, Method 112, there is provided the method of any one of Methods 95 to 105 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 113, there is provided the method of any one of Methods 95 to 112 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 114, there is provided the method of any one of Methods 95 to 105 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 115, there is provided the method of any one of Methods 95 to 114 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another method, Method 116, there is provided the method of any one of Methods 95 to 105 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another method, Method 117, there is provided the method of any one of Methods 95 to 116 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another method, Method 118, there is provided the method of any one of Methods 95 to 105 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 119, there is provided the method of any one of Methods 95 to 118 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 120, there is provided the method of any one of Methods 95 to 105 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another method, Method 121, there is provided the method of any one of Methods 95 to 120 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another method, Method 122, there is provided the method of any one of Methods 95 to 121 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another method, Method 123, there is provided the method of any one of Methods 95 to 122 wherein the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. 
     In another method, Method 124, there is provided the method of any one of Methods 95 to 123 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another method, Method 125, there is provided the method of any one of Methods 95 to 124 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another method, Method 126, there is provided the method of any one of Methods 95 to 125 wherein the CRISPR enzyme is Cas9. 
     The present disclosure also provides a composition, Composition 1 which comprises a guide RNA comprising a guide sequence wherein the guide sequence is substantially complementary to a target sequence within or near a human Klotho gene. 
     In another composition, Composition 2, there is provided the composition of Composition 1 wherein the guide RNA further comprises at least one protein binding sequence for binding to an adapter protein. 
     In another composition, Composition 3, there is provided the composition of Composition 2 wherein the protein binding sequence comprises an aptamer. 
     In another composition, Composition 4, there is provided the composition of Composition 2 or Composition 3 wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In another composition, Composition 5, there is provided the composition of any one of Compositions 2 to 4 wherein the guide RNA comprises two protein binding sequences. 
     In another composition, Composition 6, there is provided the composition of any one of Compositions 1 to 5 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 7, there is provided the composition of any one of Compositions 1 to 6 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another composition, Composition 8, there is provided the composition of any one of Compositions 1 to 7 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 9, there is provided the composition of any one of Compositions 1 to 8 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another composition, Composition 10, there is provided the composition of any one of Compositions 1 to 9 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 11, there is provided the composition of any one of Compositions 1 to 10 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 12, there is provided the composition of any one of Compositions 1 to 11 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another composition, Composition 13, there is provided the composition of any one of Compositions 1 to 12 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another composition, Composition 14, there is provided the composition of any one of Compositions 1 to 7 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 15, there is provided the composition of any one of Compositions 1 to 14 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 16, there is provided the composition of any one of Compositions 1 to 7 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 17, there is provided the composition of any one of Compositions 1 to 16 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 18, there is provided the composition of any one of Compositions 1 to 7 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 19, there is provided the composition of any one of Compositions 1 to 18 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another composition, Composition 20, there is provided the composition of any one of Compositions 1 to 7 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 21, there is provided the composition of any one of Compositions 1 to 20 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 22, there is provided the composition of any one of Compositions 1 to 7 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 23, there is provided the composition of any one of Compositions 1 to 22 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 24, there is provided the composition of any one of Compositions 1 to 23 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another composition, Composition 25, there is provided the composition of any one of Compositions 1 to 24 wherein the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In another composition, Composition 26, there is provided an isolated or recombinant nucleic acid molecule encoding the guide RNA of any one of Compositions 1 to 25. 
     In another composition, Composition 27, there is provided a vector encoding the guide RNA of any one of Compositions 1 to 25. 
     In another composition, Composition 28, there is provided the composition of Composition 27 wherein the vector is an adeno-associated viral (AAV) vector, an adenoviral vector (AdV) or a lentiviral (LV) vector. 
     In another composition, Composition 29, there is provided the composition of Composition 27 or Composition 28 wherein the vector further encodes a CRISPR enzyme, an adapter protein or a transcriptional activation domain. 
     In another composition, Composition 30, there is provided the composition of Composition 29 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another composition, Composition 31, there is provided the composition of Composition 29 or Composition 30 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another composition, Composition 32, there is provided the composition of any one of Compositions 29 to 31 wherein the CRISPR enzyme is Cas9. 
     In another composition, Composition 33, there is provided the composition of any one of Compositions 29 to 32 wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In another composition, Composition 34, there is provided a ribonucleoprotein complex comprising: a CRISPR enzyme; and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a human Klotho gene, wherein the guide RNA comprises at least one protein binding sequence for binding to an adapter protein. 
     In another composition, Composition 35, there is provided the composition of Composition 34 wherein the protein binding sequence comprises an aptamer. 
     In another composition, Composition 36, there is provided the composition of Composition 34 or Composition 35 wherein the adapter protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. 
     In another composition, Composition 37, there is provided the composition of any one of Compositions 34 to 36 wherein the guide RNA comprises two protein binding sequences. 
     In another composition, Composition 38, there is provided the composition of any one of Compositions 34 to 37 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 39, there is provided the composition of any one of Compositions 34 to 38 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another composition, Composition 40, there is provided the composition of any one of Compositions 34 to 39 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 41, there is provided the composition of any one of Compositions 34 to 40 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another composition, Composition 42, there is provided the composition of any one of Compositions 34 to 41 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 43, there is provided the composition of any one of Compositions 34 to 42 wherein the target sequence is located within a region between 240 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 44, there is provided the composition of any one of Compositions 34 to 43 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another composition, Composition 45, there is provided the composition of any one of Compositions 34 to 44 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another composition, Composition 46, there is provided the composition of any one of Compositions 34 to 39 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 47, there is provided the composition of any one of Compositions 34 to 46 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 48, there is provided the composition of any one of Compositions 34 to 39 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 49, there is provided the composition of any one of Compositions 34 to 48 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 50, there is provided the composition of any one of Compositions 34 to 39 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 51, there is provided the composition of any one of Compositions 34 to 50 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another composition, Composition 52, there is provided the composition of any one of Compositions 34 to 39 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 53, there is provided the composition of any one of Compositions 34 to 52 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 54, there is provided the composition of any one of Compositions 34 to 39 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 55, there is provided the composition of any one of Compositions 34 to 54 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 56, there is provided the composition of any one of Compositions 34 to 55 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another composition, Composition 57, there is provided the composition of any one of Compositions 34 to 56 wherein the guide RNA is between about 100 nucleotides and 200 nucleotides in length. 
     In another composition, Composition 58, there is provided the composition of any one of Compositions 34 to 57 wherein the guide RNA is bound to the adapter protein. 
     In another composition, Composition 59, there is provided the composition of any one of Compositions 34 to 58 wherein the adapter protein comprises or is attached to a transcriptional activation domain. 
     In another composition, Composition 60, there is provided the composition of Composition 59 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another composition, Composition 61, there is provided a ribonucleoprotein complex comprising: a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near a human Klotho gene; and a CRISPR enzyme, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain. 
     In another composition, Composition 62, there is provided the composition of Composition 61 wherein the target sequence is located within a region between the Klotho gene translation start site and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 63, there is provided the composition of Composition 61 or Composition 62 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. 
     In another composition, Composition 64, there is provided the composition of any one of Compositions 61 to 63 wherein the target sequence is located within a region between 200 nucleotides and 4000 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 65, there is provided the composition of any one of Compositions 61 to 64 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. 
     In another composition, Composition 66, there is provided the composition of any one of Compositions 61 to 65 wherein the target sequence is located within a region between 200 nucleotides and 300 nucleotides upstream of the Klotho gene translation start site. 
     In another composition, Composition 67, there is provided the composition of any one of Compositions 61 to 66 wherein the guide sequence is substantially complementary to a nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14. 
     In another composition, Composition 68, there is provided the composition of any one of Compositions 61 to 67 wherein the guide sequence is between 14 nucleotides and 25 nucleotides in length. 
     In another composition, Composition 69, there is provided the composition of any one of Compositions 61 to 63 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 70, there is provided the composition of any one of Compositions 61 to 69 wherein the guide sequence comprises at least 14 contiguous nucleotides which are identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 71, there is provided the composition of any one of Compositions 61 to 63 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 72, there is provided the composition of any one of Compositions 61 to 71 wherein the guide sequence is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 4. 
     In another composition, Composition 73, there is provided the composition of any one of Compositions 61 to 63 wherein the guide sequence is selected from a nucleotide sequence set forth in any one of SEQ ID NOs 1 to 4. 
     In another composition, Composition 74, there is provided the composition of any one of Compositions 61 to 73 wherein the guide sequence is selected from a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4. 
     In another composition, Composition 75, there is provided the composition of any one of Compositions 61 to 63 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 76, there is provided the composition of any one of Compositions 61 to 75 wherein the guide RNA is at least 90% identical to a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 77, there is provided the composition of any one of Compositions 61 to 63 wherein the guide RNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 5 to 8. 
     In another composition, Composition 78, there is provided the composition of any one of Compositions 61 to 77 wherein the guide RNA comprises a nucleotide sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8. 
     In another composition, Composition 79, there is provided the composition of any one of Compositions 61 to 78 wherein the guide RNA is a single-molecule guide RNA (sgRNA). 
     In another composition, Composition 80, there is provided the composition of any one of Compositions 61 to 79 wherein the guide RNA is between about 100 nucleotides and about 200 nucleotides in length. 
     In another composition, Composition 81, there is provided the composition of any one of Compositions 61 to 80 wherein the transcriptional activation domain is selected from the group consisting of VP16, or a plurality thereof, VP64, VP160, p65, MyoD1, HSF1, RTA, TET3CD, p300 and SET7/9. 
     In another composition, Composition 82, there is provided the composition of any one of Compositions 34 to 81 wherein the CRISPR enzyme comprises a mutation which abolishes or reduces its nuclease activity. 
     In another composition, Composition 83, there is provided the composition of any one of Compositions 34 to 82 wherein the CRISPR enzyme is Cas9. 
     EXAMPLES 
     Guide RNAs 
     Guide RNA sequences relevant to the present examples are listed in Table 5. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 SEQ ID 
                   
                   
               
               
                 NO. 
                 Description 
                 Sequence 
               
               
                   
               
             
            
               
                 1 
                 sgRNA1 guide 
                 GGCAUAAAGGGGCGCGGCGC 
               
               
                   
                 sequence 
                   
               
               
                   
               
               
                 2 
                 sgRNA2 guide 
                 CGGCGGGGCGCGGGCAUAAA 
               
               
                   
                 sequence 
                   
               
               
                   
               
               
                 3 
                 sgRNA3 guide 
                 GUGCCUUUCUCCGACGUCCG 
               
               
                   
                 sequence 
                   
               
               
                   
               
               
                 4 
                 sgRNA4 guide 
                 GAAACGUCCUGCACGGCUCC 
               
               
                   
                 sequence 
                   
               
               
                   
               
               
                 5 
                 sgRNA1 
                   GGCAUAAAGGGGCGCGGCGC GUUUUAGAGCUA GGCCAACAUGAGGAUC   
               
               
                   
                 guide sequence 
                   ACCCAUGUCUGCAGGGCC UAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
               
               
                   
                 underlined 
                 CAACUU GGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCC AAGUGGCA 
               
               
                   
                 MS2 stem loop 
                 CCGAGUCGGUGCUUUUU 
               
               
                   
                 in bold 
                   
               
               
                   
               
               
                 6 
                 sgRNA2 
                   GGCGGGGCGCGGGCAUAAA GUUUUAGAGCUA GGCCAACAUGAGGAUCA   
               
               
                   
                 guide sequence 
                   CCCAUGUCUGCAGGGCC UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
                 underlined 
                 AACUU GGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCC AAGUGGCAC 
               
               
                   
                 MS2 stem loop 
                 CGAGUCGGUGCUUUUU 
               
               
                   
                 in bold 
                   
               
               
                   
               
               
                 7 
                 sgRNA3 
                   GUGCCUUUCUCCGACGUCCG GUUUUAGAGCUA GGCCAACAUGAGGAUC   
               
               
                   
                 guide sequence 
                   ACCCAUGUCUGCAGGGCC UAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
               
               
                   
                 underlined 
                 CAACUU GGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCC AAGUGGCA 
               
               
                   
                 MS2 stem loop 
                 CCGAGUCGGUGCUUUUU 
               
               
                   
                 in bold 
                   
               
               
                   
               
               
                 8 
                 sgRNA4 
                   GAAACGUCCUGCACGGCUCC GUUUUAGAGCUA GGCCAACAUGAGGAUC   
               
               
                   
                 guide sequence 
                   ACCCAUGUCUGCAGGGCC UAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
               
               
                   
                 underlined 
                 CAACUU GGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCC AAGUGGCA 
               
               
                   
                 MS2 stem loop 
                 CCGAGUCGGUGCUUUUU 
               
               
                   
                 in bold 
               
               
                   
               
            
           
         
       
     
       FIG. 1  provides a representation of sgRNA3 (SEQ ID NO: 3) hybridised to a target sequence in the Klotho promoter. The secondary structure of the guide RNA is also indicated including the tetra-loop and the stem loop 2 which have been engineered to include a MS2 binding sequence. 
     Designing and Cloning of sgRNA Plasmid 
     Guide RNAs were designed to target loci within the first 300 bp upstream of the Klotho translation initiation site (“A” in ATG as number+1). Target sequences were selected according to predicted off-target scores using an online CRISPR design tool (http://crispr.mit.edu (Ran et al. 2013 . Nat. Protoc.  8(11): 2291-2308) and subsequently filtered for a maximum GC content of 25%, and minimal overlap of the target sequence. After filtering, four guide RNAs on the sense strand with the best off-target scores were selected, and then cloned into sgRNA (MS2) backbone (Addgene #61424) at the BbsI site. Final plasmid constructs were confirmed by DNA sequencing. The following primers were used for cloning: 
     
       
         
           
               
               
            
               
                   
                 sgRNA1 sense: 
               
               
                   
                 (SEQ ID NO. 30) 
               
               
                   
                 5′-CACCGGGCATAAAGGGGCGCGGCGC-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA1 anti-sense: 
               
               
                   
                 (SEQ ID NO. 31) 
               
               
                   
                 5′-AAACGCGCCGCGCCCCTTTATGCCC-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA2 sense: 
               
               
                   
                 (SEQ ID NO. 32) 
               
               
                   
                 5′-CACCGCGGCGGGGCGCGGGCATAAA-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA2 anti-sense: 
               
               
                   
                 (SEQ ID NO. 33) 
               
               
                   
                 5′-AAACTTTATGCCCGCGCCCCGCCGC-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA3 sense: 
               
               
                   
                 (SEQ ID NO. 34) 
               
               
                   
                 5′-CACCGGTGCCTTTCTCCGACGTCCG-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA3 anti-sense: 
               
               
                   
                 (SEQ ID NO. 35) 
               
               
                   
                 5′-AAACCGGACGTCGGAGAAAGGCACC-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA4 sense: 
               
               
                   
                 (SEQ ID NO. 36) 
               
               
                   
                 5′-CACCGGAAACGTCCTGCACGGCTCC-3′ 
               
               
                   
                   
               
               
                   
                 sgRNA4 anti-sense: 
               
               
                   
                 (SEQ ID NO. 37) 
               
               
                   
                 5′-AAACGGAGCCGTGCAGGACGTTTCC-3′ 
               
            
           
         
       
     
     Cell Culture 
     Cell lines were maintained under standard growth conditions and propagated in DMEM (Dulbecco&#39;s modified Eagle&#39;s medium) (4.5 g/ml glucose) containing 10% FBS (fetal bovine serum) (Atlanta Biologicals) and 1% penicillin/streptomycin (100 units/ml). For HK-2 kidney cells, DMEM:F12 (1:1) medium was used. Cell culture solutions were obtained from Cellgro unless otherwise noted in the examples. 
     Cloning of Klotho (KL) 4 kb Promoter into FLuc and NLuc Luciferase Coincidence Reporter and Stable Cell Generation 
     To clone the SV40 enhancer into pNLCol1[luc2-P2A-NlucP/Hygro] Vector (Promega), the SV40 enhancer was amplified from pGL3 using the following primers and Clontech HiFi according to the manufacturer&#39;s protocol: 
     
       
         
           
               
               
            
               
                   
                 Forward primer: 
               
               
                   
                 (SEQ ID NO. 38) 
               
               
                   
                 5′-AAATCGATAAGGATCCGATGGAGCGGAGAATGGGCGG-3′ 
               
               
                   
                   
               
               
                   
                 Reverse primer: 
               
               
                   
                 (SEQ ID NO. 39) 
               
               
                   
                 5′-ATACGCAAACGGATCCGCTGTGGAATGTGTGTCAG-3′ 
               
            
           
         
       
     
     The PCR product was isolated and ligated to the pNL vector by digestion with BamHI to generate the pNLCol1-SV40 vector. Approximately 1.8 kb of the Klotho promoter was digested from pGL3-KL1800 (King et al. 2012 . Biochem. J.  441(1): 453-461) with HindIII and XhoI, and subcloned into pNLCol1-SV40 vector using the same restriction sites to generate pNLCol1-SV40-KL1800. 
     To clone an approximately 4 kb region of the Klotho promoter, the additional approximately 2.2 Kb (about −1800 to −4000) and the vector were amplified from HEK293 genomic DNA and pNLCol1-SV40-KL1800, respectively, using the following primers and ClonAmp HiFi polymerase according to the manufacturer&#39;s protocol: 
     
       
         
           
               
               
            
               
                   
                 Forward primer for 2.2kb KL promoter insert: 
               
               
                   
                 (SEQ ID NO. 40) 
               
               
                   
                 5′-CTCGCTAGCCTCGAGATCTATAGTGCCACATGGTGAC-3′ 
               
               
                   
                   
               
               
                   
                 Reverse primer for 2.2kb KL promoter insert: 
               
               
                   
                 (SEQ ID NO. 41) 
               
               
                   
                 5′-AGTATCACATTTCCCTTCTAGAAGTGAAGATTGGAGTG-3′ 
               
               
                   
                   
               
               
                   
                 Forward primer for vector: 
               
               
                   
                 (SEQ ID NO. 42) 
               
               
                   
                 5′-GGGAAATGTGATACTCCATGTAG-3′ 
               
               
                   
                   
               
               
                   
                 Reverse primer for vector: 
               
               
                   
                 (SEQ ID NO. 43) 
               
               
                   
                 5′-CTCGAGGCTAGCGAGCTCAGGTACC-3′ 
               
            
           
         
       
     
     The insert and vector bands (100 ng each) were ligated together using In Fusion kit (Clontech) to generate pNLCol1-SV40-KL4000. Stable HEK-293 cells expressing 4 kb of the KL promoter or the PGK promoter with the coincidence reporter were generated from single clones and selected using Hygromycin (Invivogen, USA) at a concentration of 75 μg/ml for two weeks following Hygromycin 25 μg/ml for maintenance. 
     Generation of a NLuc Knock-in HEK293 Cell Line by CRISPR Genomic Editing 
     A double nicking strategy with Cas9n was used to introduce double-stranded breaks (DSB) by a pair of appropriately spaced and oriented sgRNAs at the Klotho 3′-UTR. Guide RNAs were designed to target the Klotho 3′-UTR using the online CRISPR design tools as described above. The best off-target scoring (with a score of 100, and 0 off-target) gRNA pair was selected, and then cloned into pSpCase9n(BB)-2A-Puro (PX462) V2.0 plasmid (Addgene #62987) digested with BbsI, and confirmed by DNA sequencing. The following primers were used for cloning: 
     
       
         
           
               
               
            
               
                   
                 gRNA pair 1 sense: 
               
               
                   
                 (SEQ ID NO. 44) 
               
               
                   
                 5′-CACCGGTCTCACTGGCATCTTGTTG-3′ 
               
               
                   
                   
               
               
                   
                 gRNA pair 1 anti-sense: 
               
               
                   
                 (SEQ ID NO. 45) 
               
               
                   
                 5′-AAACCAACAAGATGCCAGTGAGACC-3′ 
               
               
                   
                   
               
               
                   
                 gRNA pair 2 sense: 
               
               
                   
                 (SEQ ID NO. 46) 
               
               
                   
                 5′-CACCGCAGGGACACAGGGTTTAGAC-3′ 
               
               
                   
                   
               
               
                   
                 gRNA pair 2 anti-sense: 
               
               
                   
                 (SEQ ID NO. 47) 
               
               
                   
                 5′-AAACGTCTAAACCCTGTGTCCCTGC-3′ 
               
            
           
         
       
     
     The P2A-NLuc sequence was inserted at the DSB site of the Klotho 3′-UTR by co-transfection of a template DNA with 1 kb homology arms to each side of the DSB site of the Klotho genomic DNA sequence. The template DNA containing the homology arms flanking the P2A-NLuc sequence was amplified from HEK cell genomic DNA or pNLCol1[luc2-P2A-NlucP/Hygro] and constructed into pcDNA3.1 plasmid without the CMV promoter region using ClonAmp HiFi and In Fusion cloning kit (Clontech). The following primers were used: 
     
       
         
           
               
               
            
               
                   
                 pcDNA3.1 forward: 
               
               
                   
                 (SEQ ID NO. 48) 
               
               
                   
                 5′-CTCGAGTCTAGAGGGCCCGCGGTTC-3′ 
               
               
                   
                   
               
               
                   
                 pcDNA3.1 reverse: 
               
               
                   
                 (SEQ ID NO. 49) 
               
               
                   
                 5′-AAGCTTCGTATATCTGGCCCGTACATCGCG-3′ 
               
               
                   
                   
               
               
                   
                 KL 2710 forward: 
               
               
                   
                 (SEQ ID NO. 50) 
               
               
                   
                 5′-AGATATACGAAGCTTCCCACATACTGGATGGTATCAATC-3′ 
               
               
                   
                   
               
               
                   
                 KL 3700 reverse: 
               
               
                   
                 (SEQ ID NO. 51) 
               
               
                   
                 5′-AGTAGCTCCGCTTCCGACAGGACCTCAAAAATCATATAA-3′ 
               
               
                   
                   
               
               
                   
                 P2A NLuc forward: 
               
               
                   
                 (SEQ ID NO. 52) 
               
               
                   
                 5′-GGAAGCGGAGCTACTAACTTCAGCC-3′ 
               
               
                   
                   
               
               
                   
                 P2A NLuc reverse: 
               
               
                   
                 (SEQ ID NO. 53) 
               
               
                   
                 5′-TTAGACGTTGATGCGAGCTGAAGC-3′ 
               
               
                   
                   
               
               
                   
                 KL 3743 forward: 
               
               
                   
                 (SEQ ID NO. 54) 
               
               
                   
                 5′-CGCATCAACGTCTAATTGAGGGCCTTGCACATAGGAAAC-3′ 
               
               
                   
                   
               
               
                   
                 KL 4978 reverse: 
               
               
                   
                 (SEQ ID NO. 55) 
               
               
                   
                 5′-CCCTCTAGACTCGAGATTATGAAAGAAGGCAAAAAGTTGC-3′ 
               
            
           
         
       
     
     Isolation of clonal cell lines comprising P2A-NLuc insertion was achieved after co-transfection of the two pairs of gRNA and template plasmids into HEK293 cells by isolating single cells through serial dilutions in 96 well plates, followed by an expansion period to establish a new clonal cell line. NLuc positive lines were confirmed by PCR using Terra PCR direct kit (Clontech) and the following primers: 
     
       
         
           
               
               
            
               
                   
                 Klotho intron 4 forward: 
               
               
                   
                 (SEQ ID NO. 56) 
               
               
                   
                 5′- GTGTTGTGTGCAAAATACGTAATAA-3′ 
               
               
                   
                   
               
               
                   
                 NLuc reverse: 
               
               
                   
                 (SEQ ID NO. 57) 
               
               
                   
                 5′- TGACATGGATGTCGATCTTCAG-3′ 
               
            
           
         
       
     
     The forward primer is located in intron 4 of Klotho upstream to the left homology arm, and can therefore avoid false positive stable colonies with random insertion into genomic DNA. As a control for a specific activation of the Klotho promoter in a coincidence reporter vector, the pNLCol4[luc2-P2A-NlucP/PGK/Hygro] Vector (cat. 1492, Promega, USA) was used. Ataluren (PTC124) (cat. S6003, SelleckChem, USA) for FLuc and Cilnidipine (cat. S1293, SelleckChem, USA) for NLuc luciferase were used as positive controls for validation of the dual luciferase system. 
     Transfection 
     Cells were grown on poly-D-lysine-coated plates in 96-, 12- or 6-well formats. Twenty-four hours after plating, cells reached 70-80% confluency and were transfected with a 1:1:1 ratio of Klotho-specific sgRNA plasmid or control sgRNA cloning backbone plasmid, MS2-P65-HSF1effector plasmid (Addgene, #61423) and dCas9-VP64 effector plasmid (Addgene, #61422). For positive controls, cells were transfected with a 1:2 ratio of Egr1 (a transcription factor which activates Klotho transcription) or pcDNA3.1 empty vector. Transfections were carried out using Mirus TranslT-X2 with 100 ng, 1 μg or 2 μg of total plasmid DNA per well in 96-, 12- or 6-well plates, respectively. Transfection medium was removed and replaced with fresh medium after 5 hours. 
     Luciferase Assays 
     For measurement of FLuc and NLuc expression, the coincidence reporter vector under Klotho promoter, Nano-Glo® Dual-Luciferase® Reporter Assay System (cat. N1620, Promega) was used according to the manufacturer&#39;s instructions. Briefly, 24 hours after transfection in white 96-well culture plates, the medium was replaced with 70 μL of the fresh medium and assay was performed after an additional 24 hours. The 96-well plates and the reagents were equilibrated to room temperature and 70 μL ONE-Glo™ EX was added to the culture medium. The samples were incubated for 10 minutes and firefly luminescence was measured with a plate reader (GloMax® Discover System, Promega). To measure NLuc luciferase activity, 70 μL of NanoDLR™ Stop&amp; Glo® Reagent was added to each well and the luminescence was measured after 20 minutes. 
     Klotho promoter-induced NLuc expression was measured in a NLuc knock-in HEK293 cell line using the Nano-Glo Luciferase Assay System (cat. N1110, Promega) according to the manufacturer&#39;s instructions. Briefly, 24 hours after transfection in white 96-well culture plates, the medium was replaced with 70 μL of the fresh medium and assayed after an additional 24 hours. The 96-well plates and reagents were equilibrated to room temperature and 70 μL of Nano-Glo® Luciferase Assay Reagent was added to the culture medium. The samples were incubated for 10 minutes and the luminescence was measured using a plate reader (GloMax® Discover System, Promega). 
     qPCR Experiments and Analysis 
     48 hours after transfection, total RNA was isolated using the RNeasy mini plus Kit (QIAGEN) and 1 μg of total RNA was reverse transcribed using the SuperScript™ VILO™ cDNA Synthesis Kit according to the manufacturer&#39;s instructions (cat. 11754050, ThermoFisher scientific). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was carried out using human TaqMan Gene Expression Assays (Life Technologies): Klotho (Assay ID Hs00934627_m1 FAM); Peptidylprolyl isomerase A (PPIA) (Assay ID Hs04194521_s1; FAM) and beta actin (ActinB) (Assay ID Hs01060665_g1; VIC), on a BioRad 7900HT Real-Time PCR system using Fast Advanced Master Mix (Life Technologies), according to the manufacturer&#39;s protocol. The Klotho transcript was normalized to PPIA and ActinB which were used as endogenous controls. Samples were run in triplicates at 1 μg of cDNA per reaction. The presence or absence of transcripts was assessed by whether a critical threshold (CT) value was determined or undetermined, respectively, at the threshold chosen by BioRad software v2.4. To normalize sample input, ΔCT values were calculated for each gene. Data were analyzed further by the ΔΔCt method and fold changes in Klotho gene expression were determined by Gene Expression Module of CSX Manager software (BioRad), and a p-value ≤0.05 was considered significant. 
     Western Blotting 
     Protein Western blotting was performed as described (Chen et al. 2007 . Proc. Natl. Acad. Sci. USA.  104(50): 19796-19801). Protein expression based on Western blots was assessed and normalized by densitometry using ImageJ. 
     Anti-Klotho (K0603, clone number KM2076, 1:500, Transgenic) and anti-Actin (1:1000, Cell Signaling, Danvers, Mass.) antibodies were used. 
     Statistical Analysis 
     For Western blotting and luciferase assay, the significance was calculated using the traditional Student&#39;s t-test. Quantitative data are expressed as the means±SD. 
     Statistical comparisons between experimental groups were made using the two-tailed, unpaired Student&#39;s t-test. P-values of p&lt;0.05 were considered significant. 
     Example 1: sgRNA Target Site Selection 
     To increase endogenous Klotho gene transcription, a Synergistic Activation Mediator (SAM) system comprising NLS-dCas9-NLS-VP64, MS2-NLS-p65-HSF1 and a sgRNA, was employed. sgRNAs targeting the human Klotho promoter region were designed using the online CRISPR design tool http://crispr.mit.edu. Specifically, sgRNA targeting −300 to +1 of the Klotho promoter region were designed ( FIG. 2 ). Four guide RNAs on the sense strand with the best off-target scores were selected for gene activation experiments. 
     Example 2: Evaluation of Klotho Gene Expression Using a Dual Luciferase Coincidence Reporter System 
     4,000 bp of the Klotho promoter drove the stoichiometric expression of two orthologous reporters, firefly luciferase (FLuc) and PEST-destabilized NLuc luciferase (NLuc). The reporters were expressed from the same Klotho promoter using ribosome skipping mediated by the P2A peptide ( FIG. 3A ). To examine the efficiency of the sgRNA on Klotho gene activation, the four guide RNA sequences were introduced into sgRNA expression plasmids and co-transfected with the dCas9-VP64 and MS2-P65-HSF1 expression plasmids into stable FLuc NLuc coincidence reporter HEK293 cells. The results showed that all sgRNAs increased Klotho transcription, with sgRNA3 and 4, surprisingly, having a more substantial effect on expression than sgRNA1 or 2 ( FIG. 3B ). 
     In the 4 kb Klotho promoter reporter system, the positive control transcription factor Egr-1 (Choi et al. 2010 . Gene.  450(1-2): 121-127) resulted in a 3- to 4-fold increase in Klotho expression, comparable to sgRNA-mediated activation ( FIG. 3B ). The specificity of Klotho gene activation was tested in HEK293 cells stably transfected with a control promoter of the yeast gene encoding phosphoglycerate kinase (PGK) using the FLuc NLuc coincidence reporter vector. The results showed that none of the sgRNAs increased PGK promoter activity. sgRNA3 slightly reduced PGK-1 promoter activity ( FIG. 3C ). The PGK promoter system was validated using two known luciferase inhibitors: Cilnidipine, a Nluc luciferase inhibitor; and Ataluren (PTC124), a FLuc inhibitor. Cilnidipine treatment elicited a NLuc-specific increase that fit the 7-parameters bell-shape, while treatment with Ataluren led to stabilization of the FLuc reporter, manifested as an enhanced signal at intermediate concentrations followed by a signal decrease at high concentrations ( FIG. 3D ). The validation results using Cilnidipine and Ataluren are in line with the results obtained with the Klotho 4 kb promoter in the coincidence reporter vector (data not shown) and with the previously published protocol (Schuck et al. 2017.  Curr. Protoc. Neurosci.  79:5 32 31-35 32 27). These results indicate that the sgRNAs specifically activated Klotho gene expression but not the PGK promoter. 
     Example 3: Evaluation of Klotho Activation Using CRISPR NLuc Knock-in HEK293 Cell Line 
     To monitor endogenous Klotho gene expression, a CRISPR system was used to generate a HEK knock-in line with NLuc inserted into 3′-UTR of the Klotho gene ( FIG. 4A ). In this system, Klotho and NLuc are expressed from the same Klotho promoter by the P2A sequence. Thus, Klotho transcription can be monitored based on NLuc activity. The positive lines were selected by NLuc assay. Further confirmation of successful CRISPR genomic editing was performed by direct PCR amplification from NLuc positive cells using two primer pairs, the forward primer located in intron 4 of Klotho, upstream of the left homology arm, and the NL reverse primer located in the NLuc sequence ( FIG. 4B ). To examine the efficiency of the sgRNA on endogenous Klotho expression, sgRNA/dCas9-VP64/MS2-P65-HSF1 plasmids were introduced into the NLuc knock-in HEK293 cell line. The results showed that all sgRNAs increased Klotho transcription, with sgRNA3 and 4 directing the highest level of Klotho expression ( FIG. 4C ). Egr-1 was used as positive control ( FIG. 4C ). 
     Example 4: Evaluation of Klotho Activation in HK-2 and SY5Y Cells 
     Klotho is largely expressed in the brain and kidney (Masuda et al. 2005 . Mech. Ageing Dev.  126(12): 1274-1283). Experiments were therefore conducted to investigate whether the sgRNA targeting the Klotho promoter region can enhance Klotho expression in cell lines from these organs. The SAM complex was transfected into HK-2 cells, and Klotho mRNA and protein levels were measured. qPCR showed that sgRNA3 and 4 enhanced Klotho mRNA levels by 2- to 4-fold ( FIG. 5A ). Klotho protein levels were evaluated by Western blot. The results showed that sgRNA increased Klotho protein levels by about 4- to 6-fold ( FIGS. 5B and 5C ). Egr-1 overexpression also enhanced Klotho gene expression ( FIG. 5C ). 
     Klotho expression was then tested in neuronal SY5Y cells. In this system, Klotho mRNA level is detectable by qPCR, however the protein level is undetectable by Western blot. The SAM complex was transfected into SY5Y cells, and Klotho mRNA levels were measured by qPCR. sgRNA3 and the positive control Egr-1 enhanced Klotho expression by 15- to 20-fold ( FIG. 5B ). sgRNA4 enhanced Klotho gene expression by about 4-fold ( FIG. 5B ). In both HK-2 and SY5Y cells, sgRNA3 increased mRNA expression more than sgRNA4. However, in terms of protein level enhancement, sgRNA4 had a stronger effect than sgRNA3 in HK-2 cells ( FIGS. 5C and 5D ). 
     Example 5: Increasing Klotho Expression Using a Variety of CRISPR Complexes 
     Using the FLuc and NLuc reporters, the SAM system was compared to other CRISPR-mediated systems of increasing Klotho expression. In that regard, sgRNA3 or sgRNA4 were co-transfected with MS2-P65-HSF1, and either dCas9-VP64, dCas9-Suntag, dCas9-p300 or dCas9-VPR into stable FLuc NLuc coincidence reporter HEK293 cells. As shown in  FIG. 6 , all of the systems tested increased Klotho expression, with dCas9-VPR and dCas9-VP64 (SAM) providing the greatest increase.