Systems and methods for upregulation of exerkines

Abstract

Described herein are compositions, systems, and methods for modulating expression of exerkines (e.g., follistatin). Also described herein are systems and methods for improving muscle function or treating a disorder associated with loss of muscle function by activating expression of follistatin.

Description

Attorney Docket No. 232285-702601SYSTEMS AND METHODS FOR UPREGULATION OF EXERKINESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 790,595 filed April 17, 2025, and U.S. Provisional Application No. 63 / 790,637 filed April 17, 2025, both of which are incorporated by reference herein in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on April 6, 2026, is named 232285-70260 l_SL.xml and is 464,284 bytes in size.BACKGROUND

[0003] Exercise provides extensive health benefits across multiple organ systems, primarily driven by molecules known as exerkines. Exerkines are signaling proteins, peptides, or metabolites secreted into circulation during physical activity, influencing functions beyond their tissues of origin. These factors hold substantial therapeutic potential for cardiometabolic and muscle diseases. Several exerkines have been identified and characterized as promising therapeutic candidates, including follistatin (FST), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin (NRTN), brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), and sestrins. Collectively, these exerkines demonstrate the potential for enhancing metabolic function, improving cardiovascular health, increasing muscle mass, and reducing inflammation.

[0004] However, translating exerkines into therapeutic interventions presents challenges. Many exerkines exhibit a short circulating half-life, often on the order of minutes, significantly limiting their therapeutic efficacy and necessitating frequent administration. Additionally, there are inherent challenges associated with delivering multiple isoforms simultaneously, which may be necessary for comprehensive therapeutic effects. Finally, engineered proteins or exerkines modified for extended half-life may induce anti-drug antibodies, reducing therapeuticAtorney Docket No. 232285-702601effectiveness and increasing the risk of adverse reactions. Thus, there is a need to develop better compositions and systems for targeting exerkines, such as follistatin.SUMMARY

[0005] Provided herein are systems for upregulating expression of an exerkine in a cell, the system comprising: a polypeptide comprising: a DNA binding domain that comprises a nuclease that substantially lacks DNA cleavage activity, a transcriptional activation domain, and a nucleic acid comprising: a spacer region that is about 18 to 25 bases and has complementarity to a target sequence within a gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and a nuclease binding region that binds to the nuclease, wherein upon introduction of the system to the cell, the spacer region binds to the nuclease to form a complex with the polypeptide, thereby bringing the DNA binding domain and the transcriptional activation domain together to bind the target sequence or the regulatory region in the cell, initiating transcription of the gene, and consequently increasing an expression level of the exerkine in the cell as compared to the expression level of the exerkine in the cell prior to the introduction of the system to the cell. Further provided herein are systems, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids. Further provided herein are systems, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids. Further provided herein are systems, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids. Further provided herein are systems, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230. Further provided herein are systems, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230. Further provided herein are systems, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP128. Further provided herein are systems, wherein the nuclease is dCas 9 or dCas 12. Further provided herein are systems, wherein the nucleic acid is a guide RNA (gRNA). Further provided herein are systems, wherein the length of the spacer region is about 20 bases. Further provided herein are systems, wherein the length of the spacer region is about 23 bases. Further provided herein are systems, wherein the spacer comprises a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least aboutAttorney Docket No. 232285-70260195%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 252-313. Further provided herein are systems, wherein the spacer comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313. Further provided herein are systems, wherein the expression level of the exerkine in the cell is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold on day 6 following introduction of the system to the cell. Further provided herein are systems, wherein the expression level of the exerkine in the cell is increased by about 8-fold on day 6 following introduction of the system to the cell. Further provided herein are systems, wherein the expression level of the exerkine in the cell is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, or about 7-fold on day 12 following introduction of the system to the cell. Further provided herein are systems, wherein the expression level of the exerkine in the cell is increased by about 6-fold on day 12 following introduction of the system to the cell. Further provided herein are systems, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1 A (HSPA1 A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins. Further provided herein are systems, wherein the exerkine is FST. Further provided herein are systems, wherein the cell is a muscle cell or liver a cell. Further provided herein are systems, wherein the liver cell is a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal endothelial cell. Further provided herein are systems, wherein the system is in the form of a ribonucleoprotein complex (RNP-complex).

[0006] Provided herein are engineered proteins, comprising: a DNA binding domain; and a transcriptional activation domain, wherein the engineered protein binds a target gene encoding for an exerkine or a regulatory region of the target gene encoding for the exerkine. Further provided herein are engineered proteins, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1A (HSPA1A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2 A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin,Atorney Docket No. 232285-702601irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins. Further provided herein are engineered proteins, wherein the exerkine is FST. Further provided herein are engineered proteins, wherein the DNA binding domain is a zinc finger protein (ZFP). Further provided herein are engineered proteins, wherein the DNA binding domain is dCas 9 or dCas 12. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP128. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230. Further provided herein are engineered proteins, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230.

[0007] Provided herein are nucleic acids encoding for the engineered proteins provided herein.

[0008] Provided herein are compositions comprising the nucleic acids provided herein, wherein the nucleic acid is incorporated into a delivery vehicle. Further provided herein are compositions, wherein the delivery vehicle is a viral vector. Further provided herein are compositions, wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. Further provided herein are compositions, wherein the viral vector is an adeno-associated virus (AAV) vector. Further provided herein are compositions, wherein the delivery vehicle is a nanoparticle. Further provided herein are compositions, wherein the nanoparticle is a lipid nanoparticle.

[0009] Provided herein are compositions, comprising: an engineered protein provided herein; and a nucleic acid, wherein the nucleic acid nucleic acid is about 18 to 25 bases in length and comprises: a spacer region that has complementarity to an endogenous target sequence within a gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and a nuclease binding region, wherein the nuclease binding region comprises a secondaryAttorney Docket No. 232285-702601structure that binds to a nuclease. Further provided herein are compositions, wherein the nucleic acid is a guide RNA. Further provided herein are compositions, wherein the length of the spacer region is about 20 bases. Further provided herein are compositions, wherein the length of the spacer region is about 23 bases. Further provided herein are compositions, wherein the spacer region comprises a nucleic acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to any one of SEQ ID NOS: 252-313. Further provided herein are compositions, wherein the spacer region comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313. Further provided herein are compositions, wherein the engineered protein is complexed with the nucleic acid to form a ribonucleoprotein complex (RNP-complex).

[0010] Provided herein are compositions, comprising: a first nucleic acid encoding for a polypeptide comprising: a DNA binding domain, wherein the DNA binding domain comprises a nuclease that substantially lacks DNA cleavage activity; and a transcriptional activation domain; and a second nucleic acid, wherein the nucleic acid nucleic acid is about 18 to 25 bases in length and comprises: a spacer region that has complementarity to an endogenous target sequence within a gene encoding for an exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and a nuclease binding region, wherein the nuclease binding region comprises a secondary structure that binds to the nuclease. Further provided herein are compositions, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1A (HSPA1A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins. Further provided herein are compositions, wherein the exerkine is FST. Further provided herein are compositions, wherein the first nucleic acid is an mRNA. Further provided herein are compositions, wherein the second nucleic acid is a guide RNA. Further provided herein are compositions, wherein the first nucleic acid is an mRNA and the second nucleic acid is a guide RNA. Further provided herein are compositions, wherein the DNA binding domain is a zinc finger protein. Further provided herein are compositions, wherein the DNA binding domain is dCas 9 or dCas 12. Further provided herein are compositions, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP 128. Further provided herein are compositions, wherein the transcriptional activation domainAtorney Docket No. 232285-702601comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids. Further provided herein are compositions, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids. Further provided herein are compositions, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids. Further provided herein are compositions, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230. Further provided herein are compositions, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230. Further provided herein are compositions, wherein the length of the spacer region is about 20 bases. Further provided herein are compositions, wherein the length of the spacer region is about 23 bases. Further provided herein are compositions, wherein the spacer region comprises a nucleic acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to any one of SEQ ID NOS: 252-313. Further provided herein are compositions, wherein the spacer region comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313. Further provided herein are compositions, wherein the first nucleic acid and the second nucleic acid are incorporated into a delivery vehicle. Further provided herein are compositions, wherein the first nucleic acid and the second nucleic acid are incorporated into two separate delivery vehicles. Further provided herein are compositions, wherein each delivery vehicle is a viral vector. Further provided herein are compositions, wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. Further provided herein are compositions, wherein the viral vector is an adeno-associated virus (AAV) vector. Further provided herein are compositions, wherein each delivery vehicle is a nanoparticle. Further provided herein are compositions, wherein the nanoparticle is a lipid nanoparticle.

[0011] Provided herein are uses of the composition provided herein in the manufacture of a medicament for treating or preventing a disease in a subject. Further provided are uses, wherein the disease is muscular dystrophy.Atorney Docket No. 232285-702601

[0012] Provided herein are methods of activating expression of FST in a cell, comprising administering to a cell, a tissue, or a subject the composition provided herein, wherein the second nucleic acid forms a complex with the DNA binding domain, wherein the complex binds to the endogenous target gene encoding for follistatin (FST) or the regulatory region of the gene encoding for FST, thereby activating expression of FST.

[0013] Provided herein are methods of improving muscle function, comprising administering to a cell, a tissue, or a subject the composition provided herein, wherein after administration, the level of FST in the cell, the tissue, or the subject is increased as compared to that before administration.

[0014] Provided herein are methods of treating muscular dystrophy, comprising administering to a cell, a tissue, or a subject the composition provided herein, wherein after administration, the level of FST in the cell, the tissue, or the subject is increased as compared to that before administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0016] FIGURES 1A-1B illustrates epigenetic upregulation of follistatin in liver using exemplary epigenetic editing systems described herein. FIG. 1A illustrates a system for screening epigenetic effectors for epigenetic upregulation of follistatin in liver. FIG. IB is a schematic depiction of several embodiments of the disclosed composition, namely a nucleic acid having a sequence encoding a DNA binding domain (DBD) including dCas9, dCasl2a, or zinc finger protein (ZFP), a sequence encoding an epigenetic effector that activates transcription of one or more target molecules, and associated structural elements, “a” depicts an embodiment for delivery of the composition in DNA plasmid format, including a promoter and mCherry-PuroR selection cassette separated by P2A cleavage peptide, “b”, “c”, and “d” depict embodiments for delivery of the composition as LNP-encapsulated mRNA.

[0017] FIGURE 2 illustrates the human FST targeted genomic region.

[0018] FIGURE 3 illustrates the specific binding loci of representative Casl2a crRNA guides. Numbered spacer labels indicate the experimentally observed (not “predicted”) relative on-target activity rankings of selected Casl2a crRNA guides (see FIGS. 4A-C, FIG. 5), when co-deliveredAtorney Docket No. 232285-702601effector is held constant, as measured by ELISA detection of human follistatin protein secreted by HepG2 cells at 6 days post-delivery. Guides are labeled such that “l_fst-5” denotes the highest activity, “3_fst-49” denotes third highest, etc., and guides designed using the alternative PAM range are denoted as “alt-”.

[0019] FIGURES 4A-4C are plots illustrating the observed relative on-target activity rankings of each Casl2a crRNA guide. FIG. 4A is a plot showing the fold changes in FST protein concentration (x-axis) as calculated with respect to non-targeting (NT) guide controls, with corresponding statistical significance values (y-axis) obtained by Ordinary One-Way ANOVA with Dunnett's multiple comparisons test. Dotted horizontal line indicates the significance threshold (p<0.05). FIG. 4B is a plot showing fold FST activation quantified by ELISA. FIG. 4C is a plot showing comparison of guide performance according to the designed spacer length. Y axis represents the score for 23 bp, whereas X axis represents the score for 20 bp. Each plotted dot represents the activation potency score of a 20-bp spacer (x-axis) relative to its 23-bp counterpart (y-axis), where both spacers target the same PAM locus within the targeted FST region. Dots above the diagonal line indicate outperformance by the 23-bp length spacer. Potency scores were calculated by normalization of observed fold FST protein (FIGS. 4A-4B) such that maximum activation = 100 (arbitrary units).

[0020] FIGURE 5 is a plot showing the ranked performance of 62 individual spacers, as tested in FIGURES 4A-4B, with targeting guides (dots) and non-targeting (NT) controls (boxes). Targeting guides having alternative PAM are grouped to the right, as labeled.

[0021] FIGURES 6A-6B illustrates FST activation fold changes at mRNA and protein levels as measured by RT-qPCR and ELISA, respectively, when targeted by the indicated crRNA guides.FIG. 6A is a plot showing the level of FST mRNA with different crRNA guides. FIG. 6B is a plot showing the fold increase of FST mRNA and protein with different crRNA guides.

[0022] FIGURE 7A is a bar plot illustrating the observed on-target activities of distinct dCasl2a-effector domains, when co-delivered crRNAs are held constant, as measured by ELISA detection of human follistatin protein secreted by HepG2 cells at 6 days and 12 days post-delivery.

[0023] FIGURE 7B is a plot illustrating fold activation over an experimental time series (0-12d) of FST in HEK293T cells. Y axis represents fold change of FST protein as measured by ELISA, whereas X axis represents time (days).

[0024] FIGURE 8 is a bar plot illustrating human fibroblast growth factor 21 (hFGF21) activation fold changes at mRNA level with different crRNA guides measured by RT-qPCR.Atorney Docket No. 232285-702601

[0025] FIGURE 9 is a bar plot illustrating apelin activation fold changes at mRNA level with different crRNA guides measured by RT-qPCR.

[0026] FIGURE 10 is a bar plot illustrating Fibronectin Type III Domain-Containing Protein 5 (FNDC5) activation fold changes at mRNA level with different crRNA guides measured by RT-qPCR.

[0027] FIGURE 11 is a bar plot illustrating Neurturin (NRTN) activation fold changes at mRNA level with different crRNA guides measured by RT-qPCR.

[0028] FIGURE 12 is a bar plot illustrating heat shock protein family A (Hsp70) Member 1A (HSPA1 A) activation fold changes at mRNA level with different crRNA guides measured by RT-qPCR.

[0029] FIGURES 13A-13B are plots illustrating co-upregulation of FST and apelin (FIG. 13A) and co-upregulation of FST and FNDC5 (FIG. 3B).

[0030] FIGURES 14A-14G illustrates the activation level fine-tuning capabilities conferred by adjustment of the crRNA spacer length, as observed following crRNA screens against six human gene targets (FST, NRTN, FGF21, HSPA1A, FNDC5, and APLN). In these experiments, the dCas-effector was kept constant across experiments to allow separate evaluation of crRNA spacer efficacies in arrayed screens in HEK293T and HepG2 cells. FIG. 14A illustrates the range of fold changes achieved by targeting guide hits (defined as at least 2SD above the mean of non-targeting controls) for each gene, relative to non-targeting controls, and the group mean differences (indicated as %) between guides of spacer length 20 nucleotides or 23 nucleotides. FIG. 14B compares the relative activity (normalized) observed on a PAM site-matched basis, as in specifically comparing the activation induced by 20-nt or 23-nt spacers targeting precisely the same PAM site within the FST promoter region. In the majority of cases, greater FST activation is achieved by the 23-nt version relative to the 20-nt version. FIG. 14C compares the relative activity observed on a PAM site-matched basis for the gene FGF21. FIG. 14D compares the relative activity observed on a PAM site-matched basis for the gene APLN. FIG. 14E compares the relative activity observed on a PAM site-matched basis for the gene HSPA1A. FIG. 14F compares the relative activity observed on a PAM site-matched basis for the gene NRTN. FIG.14G compares the relative activity observed on a PAM site-matched basis for the gene FNDC5.

[0031] FIGURES 15A-15C illustrate safety measures following delivery of compositions comprised of epigenetic effector domains sourced from human and viral proteins. FIG. 15A shows percentage of cell death in presence of epigenetic effector domains sourced from human, viral, or a combination of human and viral proteins. FIG. 15B shows the total cell count in presence ofAtorney Docket No. 232285-702601epigenetic effector domains sourced from human, viral, or a combination of human and viral proteins. FIG. 15C shows the safety metrics as measured by cell count versus cell death.

[0032] FIGURE 16A illustrates absolute activation folds at potency time points (3-6d) for benchmark effectors and selected engineered effector compositions. Y axis represents fold change of TRE3G-GFP in HEK293T cells relative to baseline, whereas X axis identifies the effectors.

[0033] FIGURE 16B is a bar plot illustrating absolute activation folds at Durability time points (9-18d) for benchmark effectors and selected engineered effector compositions. Y axis represents fold change of TRE3G-GFP in HEK293T cells relative to baseline, whereas X axis identifies the effectors.

[0034] FIGURES 17A-17D illustrate the functional effects on activation dynamics that result from tripartite effector fusions of various functionally discrete sub-domains of the endogenous human CTCF protein when fused C-terminally to a potent bipartite benchmark activator (vBi). Two of these sub-domains, when fused to vBi, consistently augment activation metrics relative to vBi alone, mildly for potency and roughly doubling its durability. FIG. 17A is a schematic depicting the rational partitioning of human CTCF protein into discrete sub-domains as defined by prior crystallography and DNA-binding assays. Independently synthesized fragments encoding the N-terminal domain (NTD), C-terminal domain (CTD), intrinsically disordered region (IDR), RNA-binding domain (RBD), Zinc Fingers 1-2 (NTD ZF1-2), and the Zinc Fingers 3-7 (DBD ZF3-7) DNA-binding domain were fused C-terminally to a potent bipartite benchmark activator (vBi) in order to determine their functional impact on activation dynamics of this benchmark activator. FIG. 17B illustrates absolute fold activation relative to baseline over a full experimental time series (0-18d) at TRE3G-GFP in HEK293T cells. The Y axis represents fold change of the fluorescent reporter (GFP), whereas the X axis represents time (days). Dark line indicates the benchmark effector vBi without fusion, and faint lines indicate individual vBi-CTCF domain fusion effectors. FIG. 17C illustrates Potency (3-6d) and Durability (9-18d) scores for vBi and each vBi-CTCF fusion effector. Dashed lines indicate the activation values achieved by the reference vBi effector alone. FIG. 17D illustrates the effect size over time of endogenous human CTCF protein with C-terminally fused IDR, NTD, NTD ZF1-2, RBD, CTD, and DBD ZF3-7.

[0035] FIGURE 18A illustrates 39 examples of engineered functional enhancements to activator potency, comparing activation by screen hit tiles (grey), their respective domains (black), and merged domains (white outlined in black) at a synthetic TRE3G-GFP promoter in HEK293T cells. Y axis represents absolute fold activation potency relative to non-targeting baseline levels (bars represent the mean of all 3-6d data), whereas X axis identifies the engineered effectors (either “tiles”, “domains”, or “merged domains”).Atorney Docket No. 232285-702601

[0036] FIGURE 18B is a bar plot showing fold activation with tiles, domains, or merged domains (potency of two adjacent full domains).

[0037] FIGURE 18C illustrates the design strategy for domains, merged, and bipartite domains based on initial high-throughput discovery of activator screen hit tiles.

[0038] FIGURES 19A-19C are bar plots showing in vivo activation of mouse FST with different effectors. FIG. 19A is a plot showing fold activation of FST protein as measured in mouse serum by ELISA, following mRNA-LNP dosing of effectors as compared to saline and non-targeting controls measured at day 1 post-dose. FIG. 19B is a plot showing fold activation of FST protein as measured in mouse serum by ELISA, in the presence following mRNA-LNP dosing of effectors as compared to saline and benchmark controls at day 4 post-dose. FIG. 19C shows the size (amino acid length) of effectors N06-C02, E1.057, E1.039, andEl.034, relative to benchmark VPH-SS 18.DETAILED DESCRIPTION OF THE INVENTION

[0039] The following description and examples illustrate embodiments of the invention in detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

[0040] Briefly, provided herein are (1) systems; (2) delivery vehicles and vectors; (3) cells; (4) pharmaceutical compositions, dosing, and administration; (5) kits; (6) methods for detecting epigenetic editing efficiency; and (7) use of the systems and compositions.Definitions

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Also, unless indicated otherwise, except within the claims, the use of “or” includes “and” and vice versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (in some aspects, “containing”, “including”, “having” and “comprising” typically indicate “including without limitation”). Examples of limiting terms include “consisting of’ and “consisting essentially of’. Singular forms including in the claims such as “a”, “an” and “the” include the plural reference unless expressly stated otherwise.

[0042] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to include small fluctuations, referring to the stated number and numbers + / — 10% thereof.Atorney Docket No. 232285-702601

[0043] The following standard one letter and three letter abbreviations for the amino acid residues used throughout the specification: A, Ala— alanine; R, Arg— Arginine; N, Asn— Asparagine; D, Asp— Aspartic acid; C, Cys— Cysteine; Q, Gin— Glutamine; E, Glu— Glutamic acid; G, Gly— Glycine; H, His— Histidine; I, He— Isoleucine; L, Leu— Leucine; K, Lys— Lysine; M, Met— Methionine; F, Phe— Phenyalanine; P, Pro— Proline; S, Ser— Serine; T, Thr— Threonine; W, Trp— Tryptophan; Y, Tyr— Tyrosine; and V, Vai — Valine.

[0044] The term “isolated peptide” or “isolated DNA” refers to a peptide or DNA molecule that has been produced and removed from the source that produced the peptide or DNA, such as recombinant cells or synthesis reactants. The term includes, without limitation, recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0045] The term “peptide” or “polypeptide” or “oligopeptide” as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term “peptide” is mutually inclusive of the terms “polypeptides”, “peptides” and “proteins”.

[0046] The term “sequence identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389- 3402 (1997). The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosureAtorney Docket No. 232285-702601encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0047] The term “mutation,” as used herein, refers to a deletion, an insertion of a heterologous nucleic acid, an inversion, or a substitution, including an open reading frame ablating mutations as commonly understood in the art.

[0048] The term “gene,” as used herein, refers to a segment of nucleic acid that encodes for an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory regions such as promoters, operators, terminators, and the like, which may be located upstream or downstream of the coding sequence.

[0049] A “promoter,” as used herein, refers a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. In some aspects, a promoter may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The terms “operatively positioned,” “operatively linked,” “under control” and “under transcriptional control” can mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and / or expression of that sequence. In some aspects, a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0050] The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product”. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated”, with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to theAtorney Docket No. 232285-702601transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0051] The terms “enhanced expression”, “increased expression”, or “upregulated expression” refer to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain e.g., a host cell). The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.

[0052] The terms “enhanced activity”, “increased activity”, or “upregulated activity” generally refer to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

[0053] The term “cell” refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, fems, clubmosses, homworts, liverworts, mosses), an algal cell, (e.g, Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sar gassum patens C. Agardh, and the like), seaweeds (e.g. kelp), a fungal cell (e.g, a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g. a cell can be a synthetically made, sometimes termed an artificial cell).Attorney Docket No. 232285-702601

[0054] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4' 5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRl 10]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein- 15 -dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR 770-9-dA TP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPYTMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5 -UTP, Texas Red-5 -dUTP, and Texas Red- 12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. SomeAtorney Docket No. 232285-702601non-limiting examples ofbiotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-dATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP e.g. biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).

[0055] The term "polynucleotide," "oligonucleotide," or "nucleic acid," as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, intrans, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0056] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Upregulated," with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state while "down-regulated" generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptideAtorney Docket No. 232285-702601sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression" the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0057] The term “engineered” and “modified” are used interchangeably herein. The terms “engineering” and "modifying" are used interchangeably herein. The terms "engineered cell" or "modified cell" are used interchangeably herein. The terms "engineered characteristic" and "modified characteristic" are used interchangeably herein.

[0058] The term "enhanced expression," "increased expression," or "upregulated expression" generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g, a host cell). The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.

[0059] The term "enhanced activity," "increased activity," or "upregulated activity" refers to activity of a moiety of interest (e.g, a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain.

[0060] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. In some embodiments, the terms “subject” and “patient” are used interchangeably herein in reference to a mammalian subject. In some embodiments, the subject is human.

[0061] The terms “treat,” “treating,” and “treatment” refers to alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition being treated.Overview

[0062] Provided herein are systems, compositions, and methods for enhancing muscle mass and function in a subject by upregulating the expression of an exerkine (e.g., follistatin (FST)) via epigenetic editing. “Exerkines” referred herein are signaling molecules released in response toAtorney Docket No. 232285-702601exercise that exert their effects through endocrine, paracrine and / or autocrine pathways. Exerkines can be polypeptides, nucleic acids, lipids, and microRNAs. In some embodiments, an exerkine provided herein is a polypeptide Exerkine. In some embodiments, an exerkine provided herein is a hepatokine, which is a hormone-like protein produced and secreted by liver cells (e.g., neurturin (NRTN)).

[0063] Unlike traditional gene therapies, the systems, compositions, and methods provided herein regulate expression levels of an exerkine in a cell by targeting the regulatory elements of an exerkine, leaving the underlying DNA sequence unchanged. In some embodiments, using programmable DNA-binding domains fused to transcriptional activation domains, and in complex with target-specific CRISPR RNA (crRNA) guides, the systems, compositions, and methods provided herein selectively modify chromatin states at the exerkine locus to increase gene transcription. In some embodiments, a composition described herein is encoded by a synthetic messenger RNA (mRNA) molecule which is encapsulated into or in complex with a lipid nanoparticle (LNP) for systemic or local delivery of the mRNA-LNP complex. By utilizing epigenetic mechanisms, the systems, compositions, and methods described herein offer a nonpermanent yet durable therapeutic effect to elevate exerkine levels within a physiological range by closely mimicking natural regulatory processes.Systems

[0064] Provided herein are systems for upregulating expression of an exerkine in a cell, wherein the systems comprise: an engineered protein or a polynucleotide encoding the engineered protein and a guide nucleic acid. In some embodiments, a system described herein comprises: (a) a polypeptide comprising a DNA binding domain and a transcriptional activation domain and (b) a guide nucleic acid comprising a spacer region that is about 20 to 25 bases and a nuclease binding region that binds to the DNA binding domain. In some embodiments, a system described herein comprises (a) a polypeptide comprising (i) a DNA binding domain that comprises a nuclease and (ii) a transcriptional activation domain and (b) a guide nucleic acid comprising: (i) a spacer region that is about 20 to 25 bases and has complementarity to a target sequence within the gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and (ii) a nuclease binding region that binds to the nuclease. In some embodiments, a system described herein is in the form of a ribonucleoprotein complex (RNP-complex)

[0065] In some embodiments, upon introduction of the system to the cell, the spacer region binds to the nuclease to form a complex with the polypeptide, thereby bringing in the DNA bindingAttorney Docket No. 232285-702601domain and the transcriptional activation domain together to bind the target sequence of the gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in the cell, initiating transcription of the gene, and consequently increasing an expression level of the exerkine in the cell as compared to the expression level of the exerkine in the cell prior to the introduction of the system. In some embodiments, an expression level is an RNA expression level can be measured by, for example, RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, an expression level is a protein expression level can be measured by, for example, Western Blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, etc.

[0066] In some embodiments, the system increases the expression of the gene encoding for an exerkine by at least about 1.01 fold to about 5,000 fold (e.g., as compared to a control cell lacking the polypeptide and / or the guide nucleic acid). In some embodiments, the system increases the expression of an exerkine by at least about 1.01 fold to about 1.05 fold, about 1.01 fold to about 1.1 fold, about 1.01 fold to about 1.5 fold, about 1.01 fold to about 2 fold, about 1.01 fold to about 5 fold, about 1.01 fold to about 10 fold, about 1.01 fold to about 50 fold, about 1.01 fold to about 100 fold, about 1.01 fold to about 500 fold, about 1.01 fold to about 1,000 fold, about 1.01 fold to about 5,000 fold, about 1.05 fold to about 1.1 fold, about 1.05 fold to about 1.5 fold, about 1.05 fold to about 2 fold, about 1.05 fold to about 5 fold, about 1.05 fold to about 10 fold, about 1.05 fold to about 50 fold, about 1.05 fold to about 100 fold, about 1.05 fold to about 500 fold, about 1.05 fold to about 1,000 fold, about 1.05 fold to about 5,000 fold, about 1.1 fold to about 1.5 fold, about 1.1 fold to about 2 fold, about 1.1 fold to about 5 fold, about 1.1 fold to about 10 fold, about 1.1 fold to about 50 fold, about 1.1 fold to about 100 fold, about 1.1 fold to about 500 fold, about 1.1 fold to about 1,000 fold, about 1.1 fold to about 5,000 fold, about 1.5 fold to about 1 fold, about 1.5 fold to about 5 fold, about 1.5 fold to about 10 fold, about 1.5 fold to about 50 fold, about 1.5 fold to about 100 fold, about 1.5 fold to about 500 fold, about 1.5 fold to about 1,000 fold, about 1.5 fold to about 5,000 fold, about 1 fold to about 5 fold, about 1 fold to about 10 fold, about 1 fold to about 50 fold, about 1 fold to about 100 fold, about 1 fold to about 500 fold, about 1 fold to about 1,000 fold, about 1 fold to about 5,000 fold, about 5 fold to about 10 fold, about 5 fold to about 50 fold, about 5 fold to about 100 fold, about 5 fold to about 500 fold, about 5 fold to about 1,000 fold, about 5 fold to about 5,000 fold, about 10 fold to about 50 fold, about 10 fold to about 100 fold, about 10 fold to about 500 fold, about 10 fold to about 1,000 fold, about 10 fold to about 5,000 fold, about 50 fold to about 100 fold, about 50 fold to about 500 fold, about 50 fold to about 1,000 fold, about 50 fold to about 5,000 fold, about 100 fold to about 500 fold, about 100 fold to about 1,000 fold, about 100 fold to about 5,000 fold, about 500 fold to about 1,000 fold, about 500 fold to about 5,000 fold, or about 1,000 fold to about 5,000 fold (e.g., as compared to a control cellAttorney Docket No. 232285-702601lacking the heterologous polypeptide and / or the guide nucleic acid). In some embodiments, the system increases the expression of an exerkine by at least about 1.01 fold, about 1.05 fold, about 1.1 fold, about 1.5 fold, about 1 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold (e.g., as compared to a control cell lacking the polypeptide and / or the guide nucleic acid). In some embodiment, the gene encoding for an exerkine is endogenous. In some embodiments, the system increases the expression of the endogenous gene encoding for the exerkine by at least at least about 1.01 fold, about 1.05 fold, about 1.1 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, or about 1,000 fold (e.g, as compared to a control cell lacking the heterologous polypeptide and / or the guide nucleic acid). In some embodiments, the system increases the expression of an exerkine by at least at most about 1.05 fold, about 1.1 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold (e.g, as compared to a control cell lacking the polypeptide and / or the guide nucleic acid).

[0067] In some embodiments, the increase in the expression level of an exerkine (e.g., FST) in a subject after administration of a composition as provided herein results in enhanced (or increased) muscle mass, as compared to that in the subject prior to the administration. In some embodiments, the muscle mass of a subject can be assessed by measuring the muscle weight based on the weight of specific muscles (e.g., tibialis anterior, gastrocnemius, quadriceps) normalized to body weight or tibial length. In some embodiments, the muscle mass of a subject can be assessed based on the lean mass measurement using DEXA (Dual-Energy X-ray Absorptiometry) or EchoMRI for whole-body lean mass assessment. The increase in the muscle mass (e.g., average muscle mass) can be at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%, as compared to such control subject. The increase in the muscle mass (e.g., average muscle mass) can be at least or up to about 1.1 -fold, at least or up to about 1.2-fold, at least or up to about 1.5- fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, atAtorney Docket No. 232285-702601least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 150-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold, as compared to that in the subject prior to administration.

[0068] In some embodiments, the increase in the expression level of an exerkine (e.g., FST) in a subject after administration of a composition as provided herein results in enhanced (or increased) muscle function, as compared to the subject prior to the administration. In some embodiments, muscle function is assessed by grip strength test (e.g., measurement of forelimb / hindlimb grip strength using a grip strength meter), in vivo force measurement (recording contractile force using electrical stimulation of isolated muscles (e.g., EDL, soleus), ultrasound, or MRI (imaging muscle volume and structure non-invasively). The increase in the muscle function can be at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%, as compared to such control subject. The increase in the muscle function can be at least or up to about 1.1 -fold, at least or up to about 1.2-fold, at least or up to about 1.5- fold, at least or up to about 1.8-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 150-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold, as compared to that in the subject prior to administration.a. Polypeptide

[0069] Provided herein are polypeptides that specifically bind to a target polynucleotide using a guide nucleic acid described herein to hybridize to the target sequence. In some embodiments, a polypeptide provided herein is an engineered protein comprising a DNA binding domain and aAtorney Docket No. 232285-702601transcriptional activation domain. In some embodiments, the DNA binding domain is a nuclease, wherein the nuclease substantially lacks DNA cleavage activity.(1) DNA-binding domain

[0070] In some embodiments, the DNA-binding domain is a zinc finger protein or a Cas protein. In some embodiments, the DNA-binding domain comprises a nuclease that substantially lacks DNA cleavage activity. In some embodiments, the nuclease is a Cas protein or a derivative thereof. In some embodiments, a dCas protein or derivative thereof is a catalytically dead Cas or a partially dead Cas.

[0071] In some embodiments, the DNA binding domain is a zinc finger protein. A “zinc finger protein” (ZFP) is a protein with a zinc-stabilized DNA-binding domain. ZFPs bind to DNA in a sequence-specific manner. Each binding unit is referred to as a “zinc finger.” A typical zinc finger is approximately 30 amino acids long and chelates zinc. Each zinc finger contains a recognition helix of about seven amino acids that determines binding specificity. In some embodiments, each zinc finger binds two to four base pairs of DNA. In some embodiments, each zinc finger binds three or four base pairs of DNA. In some embodiments, a ZFP domain provided herein comprises at least one zinc finger. In some embodiments, a ZFP comprises at least two, three, four, five or six zinc fingers. In some embodiments, a ZFP comprises three zinc fingers (“three-finger ZFP”). In some embodiments, a ZFP comprises four zinc fingers (“four-finger ZFP”). In some embodiments, a ZFP comprises five zinc fingers (“five-finger ZFP”). In some embodiments, a ZFP comprises six zinc fingers (“six-finger ZFP”). In some embodiments, a ZFP recognizes 6 to 25 nucleotides at a target site. In some embodiments, a three-finger ZFP recognizes 9 or 10 nucleotides at a target site. In some embodiments, a four-finger ZFP recognizes 12 to 14 nucleotides at a target site. In some embodiments, a six-finger ZFPs typically recognize target sites of 18 to 21 nucleotides.

[0072] In some embodiments, a ZFP described herein comprise a C2H2 motif, a Zn2 / Cys6 zinc finger, a Gag-knuckle, a zinc ribbon, a treble clef, or a TAZ zinc finger (transcription adaptor putative zinc finger). In some embodiments, a ZFP described herein comprises one or more modifications. In some embodiments, a ZFP comprises at least one flexible linker between its individual zinc fingers. In some embodiments, a ZFP comprises a hinge domain. In some embodiments, a hinge domain provided herein improves conformational flexibility. In some embodiments, a ZFP comprises a motif that facilitates dimerization. In some embodiments, the motif that facilitates dimerization is a coiled-coil motif. In some embodiments, the motif that facilitates dimerization is near the C-terminus of a ZFP provided herein.1Attorney Docket No. 232285-702601

[0073] In some embodiments, a system or a composition described herein comprises a ZFP comprising an amino acid sequence that is at least 75% identical to any one of SEQ ID NOS: 1- 35. In some embodiments, a system or a composition described herein comprises a ZFP comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 1-35. In some embodiments, a system or a composition described herein comprises a ZFP comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1-35. In some embodiments, a system or a composition provided herein comprises a ZFP comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 1-35. In some embodiments, a system or a composition provided herein comprises a ZFP comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1-35. In some embodiments, a system or a composition provided herein comprises a ZFP comprising an amino acid sequence that is at least 99% identical to any one of SEQ ID NOS: 1-35. In some embodiments, a system or a composition provided herein comprises any one of SEQ ID NOS: 1- 35.Table 1. ZFP Sequences and Binding SiteSEQ ZF Sequence SEQ ZF Binding Site ID NO ID NO1 HVC AECGKAFLES SKLRRHQLVHTGERPYECNECG 36 GGTGGGGGCG KAF VVFQHFIRHRKIHTGERPHVCAECGKAFLES SK CTGGAGGCGG LRRHQLVHTGERPHKCLECGKCF SQNTHLTRHQRT TGGC HTGERPHFCPVCLRAFPYLSDLERHSISHTGERPHV C AECGKAFLES SKLRRHQLVHTGERPYNCNECGKA FTRIFHLTRHQKIHTGERPYECNECGKAFVVFQHFIR HR.KIH2 YVCRECGKAFTQPANFVRHNRIHTGERPFFCELQG 37 GAAGAAATA CKYEFVTREALLMHYLKKHTGERPHFCPVCLRAFP AATTCAGCTT YLSDLERHSISHTGERPYKCDMCEKTFVQSSDLTSH TGGAA QRIHTGERP YKCPQC S YAS A IK AN LN VHLRKHTGE RPYHCKECGKVFSQSAGLIQHQRIHTGERPYVCREC GKAFTQPANFVRHNRIHTGERPYVCRECGKAFTQP ANFVRHNRIHAtorney Docket No. 232285-702601HVC AECGKAFLES SKLRRHQLVHTGERPYECNECG 38 GGAGCCGGCG KAFVVFQHFIRHRKIHTGERPFPCELCGKKFKRKKD CGGCGGCGGG VKRHVLQVHTGERPFPCELCGKKFKRKKDVKRHV TGGC LQVHTGERPFPCELCGKKFKRKKDVKRHVLQVHT GERPHVCAECGKAFLESSKLRRHQLVHTGERPYEC NECGKSF AES SDLTRHRRIHTGERPHKCLECGKCF S QNTHLTRHQRTHHFCPVCLRAFPYLSDLERHSISHTGERPYKCDMCEK 39 AATAAAGAA TFVQSSDLTSHQRIHTGERPYKCPQCSYASAIKANL GAAATAAATT NVHLRKHTGERPYHCKECGKVFSQSAGLIQHQRIH CAGCT TGERPYVCRECGKAFTQPANFVRHNRIHTGERPYV CRECGKAFTQPANFVRHNRIHTGERPYECSECGKSF SQ S SNLTNHQRIHTGERP YKCPQC S Y AS AIK ANLNV HLRKHFFCELQGCKYEFVTREALLMHYLKKHTGERPHFCP 40 AAAGAAGAA VCLRAFPYLSDLERHSISHTGERPYKCDMCEKTFVQ ATAAATTCAG SSDLTSHQRIHTGERPYKCPQCSYASAIKANLNVHL CTTTG RKHTGERPYHCKECGKVFSQSAGLIQHQRIHTGERP YVCRECGKAFTQPANFVRHNRIHTGERPYVCRECG KAFTQPANFVRHNRIHTGERPYECSECGKSFSQSSN LTNHQRIHYNCNECGKAFTRIFHLTRHQKIHTGERPYNCNECG 41 GGCGCTGGAG KAFTRIFHLTRHQKIHTGERPHVC AECGKAFLES SK GCGGTGGCGG LRRHQLVHTGERPYECNECGKAFVVFQHFIRHRKI GGGG HTGERPHVC AECGKAFLES SKLRRHQLVHTGERPH KCLECGKCFSQNTHLTRHQRTHTGERPHFCPVCLR AFP YLSDLERHSISHTGERPHVC AECGKAFLES SKL RRHQLVHYNCNECGKAFTRIFHLTRHQKIHTGERPHVCAECG 42 GGGGGCGCTG K AF LES SKLRRHQLVHTGERPYECNECGKAF VVFQ GAGGCGGTGG HFIRHRKIHTGERPHVC AECGKAFLES SKLRRHQL V CGGG HTGERPHKCLECGKCF SQNTHLTRHQRTHTGERPHAtorney Docket No. 232285-702601 FCPVCLRAFPYLSDLERHSISHTGERPHVCAECGKA FLES SKLRRHQLVHTGERPYNCNECGKAFTRIFHLT RHQKIHYNCNECGKAFTRIFHLTRHQKIHTGERPFPCELCGK 43 GGTGCAGGTG KFKRKKDVKRHVLQVHTGERPYECNECGKSFAESS CGGCGGCCGC DLTRHRRIHTGERPFPCELCGKKFKRKKDVKRHVL GGGG QVHTGERPFPCELCGKKFKRKKDVKRHVLQVHTG ERPYECNECGKAFVVFQHFIRHRKIHTGERPFSCNE CKKTFAQRTDLARHQQIHTGERPYECNECGKAFVV FQHFIRHRKIHYECNECGKSFAESSDLTRHRRIHTGERPYECSECGK 44 GCAGCAGCAG SFSQSSNLTNHQRIHTGERPHVCAECGKAFLESSKL CAGGAGGCA RRHQLVHTGERPHKCLECGKCF SQNTHLTRHQRTH AAGCC TGERPFSCNECKKTFAQRTDLARHQQIHTGERPFSC NECKKTF AQRTDLARHQQIHTGERPF SCNECKKTF AQRTDLARHQQIHTGERPFSCNECKKTFAQRTDLA RHQQIH0 YACKECGRCFRQRTTLVNHQRTHTGERPYICEYCN 45 TCAGACGGCT RACAKPSVLLKHIRSHTGERPHVCAECGKAFLESSK GTGGGGGCTC LRRHQLVHTGERPYNCNECGKAFTRIFHLTRHQKIH CACA TGERPFICGQCGLAFKWSSHYQYHLRQHTGERPHV C AECGKAFLES SKLRRHQLVHTGERPYECENC AKV FTDPSNLQRHIRSQHTGERPYKCDMCEKTFVQSSDL TSHQRIH1 FICGQCGLAFKWSSHYQYHLRQHTGERPYTCNECG 46 GGTGGCGGGT KVF SRRENLARHHRLHTGERPFICGQCGLAFKWS S GTGTGTGTGA HYQYHLRQHTGERPYECVECGKAFNRRSPLTRHQR GTGT IHTGERPFICGQCGLAFKWSSHYQYHLRQHTGERP YNCNECGKAFTRIFHLTRHQKIHTGERPHVCAECG RAF LES SI< LRRHQLVHTGERP YECNECGK AF VVFQHFIRHRKIHAtorney Docket No. 232285-7026012 YECVECGKAFNRRSPLTRHQRIHTGERPFICGQCGL 47 TGTGAGTGTG AFKWS SHYQYHLRQHTGERP YTCNECGKVF SRRE TGTGTGAGTG NLARHHRLHTGERPFICGQCGLAFKWS SHYQYHLR TGTG QHTGERP YECVECGKAFNRRSPLTRHQRIHTGERPF ICGQCGLAFKWS SHYQYHLRQHTGERP YTCNECGK VFSRRENLARHHRLHTGERPFICGQCGLAFKWSSH YQYHLRQH3 FICGQCGLAFKWS SHYQYHLRQHTGERP YTCNECG 48 GTGTGTGAGT KVF SRRENLARHHRLHTGERPFICGQCGLAFKWS S GTGTGTGTGA HYQYHLRQHTGERPYECVECGKAFNRRSPLTRHQR GTGT IHTGERPFICGQCGLAFKWSSHYQYHLRQHTGERP YTCNECGKVF SRRENLARHHRLHTGERPFICGQCG L AFKWS SHYQYHLRQHTGERPYECVECGKAFNRR SPLTRHQRIH4 YTCNECGKVF SRRENLARHHRLHTGERPFICGQCG 49 TGTGTGTGTG L AFKWS SHYQYHLRQHTGERPYECVECGKAFNRR AGTGTGTGTG SPLTRHQRIHTGERPFICGQCGLAFKWS SHYQYHLR TGAG QHTGERP YTCNECGKVF S RREN LA RHH RLHTGE RP FICGQCGLAFKWS SHYQYHLRQHTGERP YECVECG KAFNRRSPLTRHQRIHTGERPFICGQCGLAFKWSSH YQYHLRQH5 FICGQCGLAFKWS SHYQYHLRQHTGERP YECVECG 50 GCGGGTGTGT KAFNRRSPLTRHQRIHTGERPYKCEECGKAF S VPSK GTGTGAGTGT LNNHKRIHTGERPYECVECGKAFNRRSPLTRHQRIH GTGT TGERPFICGQCGLAFKWSSHYQYHLRQHTGERPYE CVECGKAFNRRSPLTRHQRIHTGERPYECNECGKA FVVFQHFIRHRKIHTGERPFPCELCGKKFKRKKDVK RHVLQVH6 YECVECGKAFNRRSPLTRHQRIHTGERPFICGQCGL 51 GGCGGGTGTG AFKWS SHYQYHLRQHTGERP YTCNECGKVF SRRE TGTGTGAGTG NLARHHRLHTGERPFICGQCGLAFKWS SHYQYHLR TGTG QHTGERP YECVECGKAFNRRSPLTRHQRIHTGERPFAtorney Docket No. 232285-702601ICGQCGLAFKWSSHYQYHLRQHTGERPYNCNECG KAFTRIFHLTRHQKIHTGERPHVCAECGKAFLES SK LRRHQLVH7 YECVECGKAFNRRSPLTRHQRIHTGERPYKCEECG 52 GTGGCGGGTG KAFSVPSKLNNHKRIHTGERPYECVECGKAFNRRSP TGTGTGTGAG LTRHQRIHTGERPFICGQCGLAFKWS SHYQYHLRQ TGTG HTGERPYECVECGKAFNRRSPLTRHQRIHTGERPYE CNECGKAFVVFQHFIRHRKIHTGERPFPCELCGKKF KRKKDVKRHVLQVHTGERPYECVECGKAFNRRSP LTRHQRIH8 YICEYCNRACAKPSVLLKHIRSHTGERPFNCPVCDY 53 TTTTTGTTTGC AASKKCNLQYHFKSKHPTCTGERPYECENCAKVFT TTCCGACTAG DPSNLQRHIRSQHTGERPYICEYCNRACAKPSVLLK TCC HIRSHTGERPHFCPVCLRAFPYLSDLERHSISHTGER PNSCSQCGKLFRSPKSLSYHRRMHTGERPFFCELQG CKYEF VTREALLMHYLKKHTGERPNSC SQCGKLFR SPKSLSYHRRMH9 YECVECGKAFNRRSPLTRHQRIHTGERPHFCPVCLR 54 AGAAAGGGA AFPYLSDLERHSISHTGERPYTCNECGKVFSRRENL GAGGGCGAG ARHHRLHTGERPHVCAECGKAFLESSKLRRHQLVH GCTGTG TGERPYTCNECGKVFSRRENLARHHRLHTGERPHK CLECGKCFSQNTHLTRHQRTHTGERPYICTTCKQPF NSAWFLLQHAQNTHTGERPYKCNECGKSFNQNSH LIIHQRIH0 YKCDECPFTCKSSLKLGAHKQCHTGERPYECNECG 55 GCGGCGTCTG KSFAESSDLTRHRRIHTGERPHKCLECGKCFSQNTH GAGTCGGAGC LTRHQRTHTGERP YSCGICGKSF SDS S AKRRHCILH CCGG TGERPHKCLECGKCF SQNTHLTRHQRTHTGERPHK CEECGKAFGWSTDLNKHKIIHTGERPFPCELCGKKF KRKKDVKRHVLQVHTGERPFPCELCGKKFKRKKD VKRHVLQVHAtorney Docket No. 232285-7026011 HFCPVCLRAFPYLSDLERHSISHTGERPYTCNECGK 56 AAAAGAAAG VF SRRENL ARHHRLHTGERPHVC AECGKAFLES SK GGAGAGGGC LRRHQLVHTGERPYTCNECGKVFSRRENLARHHRL GAGGCT HTGERPHKCLECGKCF SQNTHLTRHQRTHTGERP YI CTTCKQPFNSAWFLLQHAQNTHTGERPYKCNECG KSFNQNSHLIIHQRIHTGERPYECSECGKSFSQSSNL TNHQRIH2 YECKDCGKTFRQCTHLTRHQRLHTGERPFECSKCG 57 AAAGAAAGA KACTRRCNLIQHQKVHTGERPFICEVCHKS YTQF SN GAGGGAGAA LCRHKRMHADCTGERPYECKECGKTFRQCSHLKR AAGGGA HQRIHTGERPHQCIECGKSFNRHCNLIRHQKIHTGE RPYACDLCSKRFAQWSHLAQHQLLHTGERPYQCK ECGKSFSQLCNLTRHQRIHTGERPYKCGECGKGFSQ SSNLHIHRCIH3 YKCNDCGKAFIHQSSLARHHRLHTGERPHGCHLCG 58 GCAGTTGGTG KAFTHCSDLRKHERTHTGERPYQCGQCGKSFRQSS GAAAAAAATC NLHQHHRLHHTGERPYQCGQCGKSFRQSSNLHQH TGTT HRLHHTGERPYACHLCRKAFTQCSHLRRHEKTHTG ERPYKCNECGKTFNVQSHLSRHHRLHTGERPYKCT ICGKAFYFLNSVERHQRTHTGERPYECGQCGRYFIQ MADFHRHEKCH4 FICEVCHKSYTQFSNLCRHKRMHADCTGERPYECK 59 ACTGGAAAAG ECGKTFRQCSHLKRHQRIHTGERPHQCIECGKSFNR AAAGAGAGG HCNLIRHQKIHTGERPYACDLCSKRFAQWSHLAQH GAGAA QLLHTGERPYQCKECGKSFSQLCNLTRHQRIHTGER PYQCGQCGKSFRQSSNLHQHHRLHHTGERPYECKE CGKTFRQCSHLKRHQRIHTGERPYHCKECGKSFTV GSTLLQHQQIH5 YECKDCGKTFRQCTHLTRHQRLHTGERPHQCIECG 60 GAGACTGGAA KSFNRHCNLIRHQKIHTGERPYACDLCSKRFAQWS AAGAAAGAG HL AQHQLLHTGERP YQCKECGKSF SQLCNLTRHQR AGGGA IHTGERPYQCGQCGKSFRQSSNLHQHHRLHHTGERAtorney Docket No. 232285-702601 PYECKECGKTFRQCSHLKRHQRIHTGERPYECKQC GKTFTWSSTLHNHVRMHTGERPFQCELCSYTCPRR SNLDRHMKSH6 FEC SKCGK ACTRRCNLIQHQKVHTGERPFICE VCHK 61 GGAAAAGAA S YTQF SNLCRHKRMHADCTGERPYECKECGKTFRQ AGAGAGGGA C SHLK RHQRI HTGERPHQC IECGK SFN RHC N LI RHQ GAAAAG KIHTGERPYACDLCSKRFAQWSHLAQHQLLHTGER PYQCKECGKSFSQLCNLTRHQRIHTGERPYQCGQC GKSFRQSSNLHQHHRLHHTGERPYECKECGKTFRQ C SHLK RHQ RIH7 HECRDCGKAFKTRNRLCMHQLIHTGERPYKCNECG 62 CGAGCAGGAC KTFNVQSHLSRHHRLHTGERPYTCEICNKCFTRSAV TTGAAGTGGG LRRHKKMHCTGERPFICEVCHKS YTQF SNLCRHKR TGTT MHADCTGERPHKCAFCPMAFKTASSTADHSATQH TGERPYECKECGKTFRQC SHLK RHQRI HTGERPYEC GQCGRYFIQMADFHRHEKCHTGERPYNCEECGRAF SQASHLQDHQRLH8 YACGECGEAFAWLSHLMEHHSSHTGERPYTCEICN 63 GCGGGTGTGT KCFTRS AVLRRHI<I<MHCTGERPYECHLCGI<AF SHC GTGTGAGTGT SHLRQHERSHTGERPYTCEICNKCFTRSAVLRRHKK GTGT MHCTGERPYACGECGEAFAWLSHLMEHHSSHTGE RPYTCEICNKCFTRSAVLRRHKKMHCTGERPYKCN ECGKTFNVQSHLSRHHRLHTGERPYSCEQCGKVFR RNCDLRRHSLTH9 YKCNECGKAFNEQSHLSRHHRIHTGERPWQCRICE 64 TCCAAAGAGA DMFDSQEYVKQHCMSLASHTGERPYECGQCGRYFI CTTTTGCAAA QMADFHRHEKCHTGERPFVCKECGMAFRYHYQLI CGGC EHCQIHTGERPYECKQCGKTFTWSSTLHNHVRMHT GERPHQCIECGKSFNRHCNLIRHQKIHTGERPYQCG QCGKSFRQSSNLHQHHRLHHTGERPYLCQHPGCQK AF SNS SDRAKHQRTHAtorney Docket No. 232285-7026010 YTCEICNKCFTRSAVLRRHKKMHCTGERPYACGEC 65 GGTGTGTGTG GEAFAWLSHLMEHHSSHTGERP YTCEICNKCFTRS TGAGTGTGTG AVLRRHI<I<MHCTGERPYECHLCGI<AFSHCSHLRQ TGTG HERSHTGERPYTCEICNKCFTRSAVLRRHKKMHCT GERPYACGECGEAFAWLSHLMEHHSSHTGERPYTC EICNKCFTRSAVLRRHKKMHCTGERPYKCNECGKT FNVQSHLSRHHRLH1 YGCKKCGRRFGRLSNCTRHEKTHSACTGERPYACG 66 TGTGTGTGTG ECGEAF AWLSHLMEHHS SHTGERP YTCEICNKCFT AGTGTGTGTG RSAVLRRHKKMHCTGERPYACGECGEAFAWLSHL TGAG MEHHS SHTGERPHQCIECGKSFNRHCNLIRHQKIHT GERPYACGECGEAFAWLSHLMEHHSSHTGERPYTC EICNKCFTRSAVLRRHKKMHCTGERPYACGECGEA FAWLSHLMEHHSSH2 YACGECGEAF AWLSHLMEHHS SHTGERPHQCIECG 67 GTGTGTGAGT KSFNRHCNLIRHQKIHTGERPYACGECGEAFAWLS GTGTGTGTGA HLMEHHS SHTGERP YTCEICNKCFTRS AVLRRHKK GTGT MHCTGERPYACGECGEAFAWLSHLMEHHSSHTGE RPHQCffiCGKSFNRHCNLIRHQKIHTGERPYACGEC GEAFAWLSHLMEHHSSHTGERP YKCNECGKTFSRK SSLTRHCRLH3 YTCEICNKCFTRSAVLRRHKKMHCTGERPYACGEC 68 TGTGAGTGTG GEAFAWLSHLMEHHSSHTGERPHQCIECGKSFNRH TGTGTGAGTG CNLIRHQKIHTGERPYACGECGEAFAWLSHLMEHH TGTG S SHTGERP YTCEICNKCFTRS AVLRRHKKMHCTGE RP YACGECGEAF AWLSHLMEHHS SHTGERPHQCIE CGKSFNRHCNLIRHQKIHTGERP YACGECGEAF AW LSHLMEHHSSH4 YQCGQCGKSFRQSSNLHQHHRLHHTGERPFECSKC 69 CGAAGTGAAA GKACTRRCNLIQHQKVHTGERPFICEVCHKSYTQFS GGGGAGAAA NLCRHKRMHADCTGERP YECKECGKTFRQC SHLK AGAAA RHQRIHTGERPFECAVCGESFVNPAELADHVTVHTAtorney Docket No. 232285-702601 GERPFICEVCHKSYTQFSNLCRHKRMHADCTGERP YECHLCGKAFSHCSHLRQHERSHTGERPYNCEECG RAFSQASHLQDHQRLH5 YGCKKCGRRFGRLSNCTRHEKTHSACTGERPYACH 70 AAAGGGGAG LCRKAFTQCSHLRRHEKTHTGERPFECPKCGKCYF AAAAGAAAG RKENLLEHEARNCTGERPYACDLCSKRFAQWSHLA GGAGAG QHQLLHTGERPYQCGQCGKSFRQSSNLHQHHRLH HTGERPHQCIECGKSFNRHCNLIRHQKIHTGERPYE CQECQKTF SRS SHLLRHQS VHCTGERPYKCGECGK GFSQSSNLHIHRCIH

[0074] In some embodiments, a DNA binding domain described herein is a Cas protein that can be used in a CRISPR / Cas system. In some embodiments, a CRISPR / Cas system can be a type I, a type II, a type III, a type IV, a type V, a type VI system, or any other suitable CRISPR / Cas system. A CRISPR / Cas system as used herein can be a Class 1, Class 2, or any other suitably classified CRISPR / Cas system. Class 1 or Class 2 determination can be based upon the genes encoding the transcriptional activation domain. Class 1 systems generally have a multi-subunit sgRNA-protein complex, whereas Class 2 systems generally have a single protein, such as Cas9, Cpfl, C2cl, C2c2, C2c3 or a sgRNA-protein complex. A Class I CRISPR / Cas system can use a complex of multiple Cas proteins to effect regulation. A Class 1 CRISPR / Cas system can comprise, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., Ill, IIIA, IIIB, IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR / Cas type. A Class 2 CRISPR / Cas system can use a single large Cas protein to effect regulation. A Class 2 CRISPR / Cas systems can comprise, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas type. CRISPR systems can be complementary to each other, and / or can lend functional units in trans to facilitate CRISPR locus targeting.

[0075] In some embodiments, a Cas protein is a type I, II, type III, type IV, type V Cas protein, or type VI Cas protein. A Cas protein comprises one or more domains. Non-limiting examples of domains include, guide nucleic acid recognition and / or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. In some embodiments, a guide nucleic acid recognition and / or binding domain can interact with a guide nucleic acid. A nuclease domain can comprise catalytic activity for nucleic acid cleavage. In some embodiments, a nuclease domain lacks catalytic activity to prevent nucleic acid cleavage. In some embodiments,Atorney Docket No. 232285-702601a Cas protein is a chimeric Cas protein or fragment thereof that is fused to other proteins or polypeptides. In some embodiments, a Cas protein is a chimera of various Cas proteins, for example, comprising domains from different Cas proteins.

[0076] In some embodiments, Non -limiting examples of Cas proteins include c2cl, C2c2, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Casl2 CaslO, CaslOd, CaslO, CaslOd, CasF, CasG, CasH, Cpfl, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, Casl3a, Casl3b, Casl3c, Casl3d, Casl3X, Casl3Y, Casl4 (e.g., Casl4 variants, such as Casl4a, Casl4b, Casl4c, etc.) and homologs or modified versions thereof.

[0077] A Cas protein or fragment or derivative thereof can be from any suitable organism. Nonlimiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polar omonas nap hthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalter omonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus).

[0078] In some embodiments, a Cas protein is derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii,Atorney Docket No. 232285-702601Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudinlermedius. Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinellasuccinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0079] In some embodiments, preferably, a Cas protein described herein is a dead Cas protein. A dead Cas protein (dCas) can comprise one or more mutations relative to a wild-type version of the protein. In some embodiments, a dCas protein is Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2f, Casl2k, or a derivative thereof. In some embodiments, a dCas is an ortholog of Casl2a. In some embodiments, the ortholog of Casl2a is AsCasl2a, LbCasl2a, FnCasl2a. In some embodiments, a dCas protein described herein is a Cas9 protein. In some embodiments, a dCas protein described herein is an ortholog of Cas 9. In some embodiments, the ortholog of Cas 9 is SpCas9, SaCas9, or NmCas9. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to bind to the target nucleic acid but losing its ability to cleave the double strands of the target nucleic acid. A dCas protein can comprise an amino acid sequence having at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, identity or sequence similarity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wildtype Cas protein.Attorney Docket No. 232285-702601

[0080] In some embodiments, a system or a composition described herein comprises a Cas protein comprising an amino acid sequence that is at least 75% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition described herein comprises a Cas protein comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition described herein comprises a Cas protein comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition provided herein comprises a Cas protein comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition provided herein comprises a Cas protein comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition provided herein comprises a Cas protein comprising an amino acid sequence that is at least 99% identical to any one of SEQ ID NOS: 71- 73. In some embodiments, a system or a composition provided herein comprises any one of SEQ ID NOS: 71-73Table 2. Cas proteins.SEQ ID Name SequenceNO71 dHyperCasl MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRA 2a EDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTE KENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLD DKDEIALVNSFNGFTTAFTGFFRNRENMFSEEAKSTSIAFRCINE NLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE FFNFVLTQEGIRVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQ KLPKFKPLYKQVLSDRESLSFYGRGYTSDEEVLEVFRNTLNKNS EIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKRIFGEWNVIR DKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQE YADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSL KKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGD FVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMG GWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDV NGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYK NGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSET EKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQAtorney Docket No. 232285-702601IYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFM RRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRF SEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIARGE RNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKE KERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIAL EDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCA TGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFV NLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTD ADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELF NKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSIT GRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAY NIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH2 dLbCasl2a MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRA EDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTE KENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLD DKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINE NLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE FFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQ KLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNS EIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIR DKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQE YADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSL KKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGD FVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMG GWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDV NGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYK NGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSET EKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQ IYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFM RRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRF SEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIARGE RNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKE KERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIAL EDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCAAtorney Docket No. 232285-702601TGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFV NLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTD ADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELF NKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSIT GRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAY NIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH3 SpdCas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHE KYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGF IKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSI<LVSDFRI<DFQFYI<VREINNYHHAHDAYLNAVVGTALII<I<YP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNF FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELAtorney Docket No. 232285-702601QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGD

[0081] A Cas protein, variant or derivative thereof can be modified to enhance regulation of gene expression by compositions and methods of the disclosure, e.g., as part of a complex disclosed herein. A Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzymatic activity, and / or binding to other factors, such as heterodimerization or oligomerization domains and induce ligands. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the desired function of the protein or complex. A Cas protein can be modified to modulate (e.g., enhance or reduce) the activity of the Cas protein for regulating gene expression by a complex of the disclosure that comprises a heterologous gene effector.(2) Transcriptional activation domain

[0082] In some embodiments, a system or a polypeptide described herein comprises a transcriptional activation domain, wherein the transcriptional activation domain comprises one or more transcriptional activation effector fragments that activate the expression of an exerkine. In some embodiments, a transcriptional activation domain ranges in size from about 80 amino acids to about 800 amino acids. In some embodiments, a transcriptional activation domain described herein is derived from gene activating modulators such as VP 16, VP64, VP48, VP 160, p65 subdomain (e.g, from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g, for activity in plants); and histone epigenetic modifier such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, IHDM2a / b, UTX, JMID3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, M0ZMYST3, M0RFMYST4, SRC1, ACTR, PI 60, CLOCK, Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like. Additional examples of such gene activating modulators include, but are not limited to, VP64-p65-Rta fusion polypeptide (VPR), DOT1L, SMYD3, MRTF-A, STAT1, eNRF2, HSF1, MCP, GADD45A, Rta, BAF, dMSK, PRDM9, GCN5, and VP 192.

[0083] In some embodiments, a transcriptional activation domain comprises an amino acid sequence having at least or up to about 50%, at least or up to about 55%, at least or up to aboutAttorney Docket No. 232285-70260160%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to the polypeptide sequence of any one of SEQ ID NOS: 74-230.

[0084] In some embodiments, a transcriptional activation domain disclosed herein (e.g., engineered gene activator) can comprise an amino acid sequence (e.g., a consecutive polypeptide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to the polypeptide sequence of a member selected from SEQ ID NOs: 74-230. Table 3. Sequences of Transcriptional ActivatorsSEQ Sources Effector Leng SequenceID ID thNO74 CTCF CTCF 727 MEGDAVEAIVEESETFIKGKERKTYQRRREGG QEEDACHLPQNQTDGGEWQDVNSSVQMVM MEQLDPTLLQMKTEVMEGTVAPEAEAAVDDT QIITLQWNMEEQPINIGELQLVQVPVPVTVPVA TTSVEELQGAYENEVSKEGLAESEPMICHTLPL PEGFQWKVGANGEVETLEQGELPPQEDPSWQ KDPDYQPPAKKTKKTKKSKLRYTEEGKDVDVS VYDFEEEQQEGLLSEVNAEKWGNMKPPKPTK IKKKGVKKTFQCELCSYTCPRRSNLDRHMKSH TDERPHKCHLCGRAFRTVTLLRNHLNTHTGTR PHKCPDCDMAFVTSGELVRHRRYKHTHEKPFK CSMCDYASVEVSKLKRHIRSHTGERPFQCSLCS YASRDTYKLKRHMRTHSGEKPYECYICHARFT QSGTMI<MHILQI<HTENVAI<FHCPHCDTVIARI< SDLGVHLRKQHSYIEQGKKCRYCDAVFHERYA LIQHQKSHKNEKRFKCDQCDYACRQERHMIM HKRTHTGEKPYACSHCDKTFRQKQLLDMHFKRYHDPNFVPAAFVCSKCGKTFTRRNTMARHADAtorney Docket No. 232285-702601NCAGPDGVEGENGGETKKSKRGRKRKMRSKK EDSSDSENAEPDLDDNEDEEEPAVEIEPEPEPQP VTPAPPPAKKRRGRPPGRTNQPKQNQPTAIIQV EDQNTGAIENIIVEVKKEPDAEPAEGEEEEAQP AATDAPNGDLTPEMILSMMDR75 SS18 SS18 417 SVAFAAPRQRGKGEITPAAIQKMLDENNHLIQC IMDYQNKGKASECSQYQQILHTNLVYL ATIAD S NQNMQSLLPAPPTQTMPMGPGGMSQSGPPPPP RSHNMPSDGMVGGGPPAPHMQNQMNGQMPG PNHMPMQGPGPSQLSMTNSSMNMPSSSHGSM GGYNHSVPSSQSMPVQNQMTMSQGQPMGNYG PRPNMNMQPNQGPMMHQQPPSQQYNMPPGG AQHYQGQQAPMGLMGQVNQGSHMMGQRQM PPYRPPQQGPPQQYSGQEDYYGDQYSHGGQGP PEGMNQQYYPDGHNDYGYQQPSYPEQGYDRP YEDSSQHYYEGGNSQYGQQQDAYQGPPPQQG YPPQQQQYPGQQGYPGQQQSYGPSQGGPGPQY PNYPQGQGQQYGGYRPTQPGPPQPPQQRPYGY DQGQYGNYQQ76 NCOA3, NFZ 145 EGQSDERALLDQLHTLLSNTDATGLEEIDRALG FOXO3, IPELVNQGQALEPKQGGGGSGHEKFPSDLDLD ZNF473 MFNGSLECDMESIIRSELMDADGLDFNFDSGGG GSGFVTLKDVGMDFTLGDWEQLGLEQGDTFW DTALDNCQDLFLLDPP77 HSV1 VP16 VP128 104 GSDALDDFDLDMLGSDALDDFDLDMLGSDAL (P06492) DDFDLDMLGSDALDDFDLDMLGSDALDDFDL DMLGSDALDDFDLDMLGSDALDDFDLDMLGS DALDDFDLDML78 HSV1 VP16 mVPR 295 DALDDFDLDMLGSDALDDFDLDMLGSDALDD (P06492), P65, FDLDMLGSDALDDFDLDMLGSPKKKRKVGSV Rta LPQAPAPAPAPAMVSALAQAPAPVPVLAPGPP QAVAPPAPKPTQAGEGTLSEALLQLQFDDEDL GALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPA PLGAPGLPNGLLSGDEDFSSIADMDFSALLSGS GSGSDLSHPPPRGHLDELTTTLESMTEDLNLDS PLTPELNEILDTFLNDECLLHAMHISTGLSIFDTS LF79 KSHV VIRF2 vBi 85 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDML(P06492)80 KSHV VIRF2 vTri 108 GGGSLDDCLPMVDHIEGCLLDLLSDVGQELPD (Q2HR71) LGDLGGGSLDDCLPMVDHIEGCLLDLLSDVGQ ELPDLGDLGGGSLDDCLPMVDHIEGCLLDLLSD VGQELPDLGDL81 KSHV VIRF2 vQuad 98 GGSDALDDFDLDMLGGSLDDCLPMVDHIEGCL (Q2HR71), LDLLSDVGQELPDLGDLGGSDALDDFDLDMLG HSV1 VP16 GSLDDCLPMVDHIEGCLLDLLSDVGQELPDLG (P06492) DL82 KSHV VIRF2 vTile 85 PPAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHI (Q2HR71) EGCLLDLLSDVGQELPDLGDLGELLCETASPQG PMQSEGGEEGSTESVSVLP83 KSHV VIRF2 86 GLLAPPAGQSQTPFSPEGPVPSHVSGLDDCLPM (Q2HR71) VDHIEGCLLDLLSDVGQELPDLGDLGELLCETA E0.001 SPQGPMQSEGGEEGSTESVS84 KSHV VIRF2 98 IFPHETQGLLAPPAGQSQTPFSPEGPVPSHVSGL (Q2HR71) DDCLPMVDHIEGCLLDLLSDVGQELPDLGDLG E0.002 ELLCETASPQGPMQSEGGEEGSTESVSVLPAT 85 KSHV VIRF2 116 PTGGLPSPVIFPHETQGLLAPPAGQSQTPFSPEG(Q2HR71) E0.003 PVPSHVSGLDDCLPMVDHIEGCLLDLLSDVGQEAtorney Docket No. 232285-702601LPDLGDLGELLCETASPQGPMQSEGGEEGSTES VSVLPATHPLESSAPG86 KSHV VIRF2 134 VHTPTDLCLPTGGLPSPVIFPHETQGLLAPPAGQ (Q2HR71) SQTPFSPEGPVPSHVSGLDDCLPMVDHIEGCLL DLLSDVGQELPDLGDLGELLCETASPQGPMQSE GGEEGSTESVSVLPATHPLESSAPGASVMGSGQE0.004 E87 KSHV VIRF2 152 TSQVQGPLHVHTPTDLCLPTGGLPSPVIFPHETQ (Q2HR71) GLLAPPAGQSQTPFSPEGPVPSHVSGLDDCLPM VDHIEGCLLDLLSDVGQELPDLGDLGELLCETA SPQGPMQSEGGEEGSTESVSVLPATHPLESSAPE0.005 GASVMGSGQELPDLGDLSE88 KSHV VIRF2 225 PPAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHI (Q2HR71), EGCLLDLLSDVGQELPDLGDLGELLCETASPQG HHV6B IE1 PMQSEGGEEGSTESVSVLPGGGGSGGGGSMES (Q77PU6) AKDTTSTSMFILGKPSGNNMESNEERMQNYHP DPVVEESIKEILEESLKCDVSFESLLFPELEAFDL FIPESSNDIASKNVSYSSNVEEGASDEFKTLVAQE1.006 SVGNCIQSIGASVKAAMKQEQSNMED89 KSHV VIRF2 255 PPAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHI (Q2HR71), EGCLLDLLSDVGQELPDLGDLGELLCETASPQG KSHV VIRF3 PMQSEGGEEGSTESVSVLPGGGGSGGGGSTRH (F5HIC6) SASGVQPVDDANADSPGSGDEGPSTRHSDSQPP PADETTVHTDNVEDDLTLLDKESACALMYHV GQEMDMLMRAMCDEDLFDLLGIPEDVIATSQP GGDTDASGVVTEGSIAASAVGAGVEDVYLAGE1.007 ALEAQNVAGEYVLEISDEEVDDGAGLPPAS 90 KSHV VIRF2 225 PPAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHI (Q2HR71), EGCLLDLLSDVGQELPDLGDLGELLCETASPQG HSV2 VP16 PMQSEGGEEGSTESVSVLPGGGGSGGGGSAYS (P68336) RGRTRNNYGSTIEGLLDLPDDDDAPAEAGLVA PRMSFLSAGQRPRRLSTTAPITDVSLGDELRLD GEEVDMTPADALDDFDLEMLGDVESPSPGMTHE1.008 DPVSYGALDVDDFEFEQMFTDAMGIDDFGG 91 KSHV VIRF2 175 PPAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHI (Q2HR71), EGCLLDLLSDVGQELPDLGDLGELLCETASPQG SuHVl ICP4 PMQSEGGEEGSTESVSVLPGGGGSGGGGSMAD (P33479) DLFDFIETEGNFSQLLAAAAAAEEEGIASGPDG GSQGSRRRGSSGEDLLFGPGGLFSDDAAEAEAE1.009 AVLAAAAGATRP92 KSHV VIRF2 107 GGSLDGLDPLEQGVDSLLDLLCGEIHDVMPLC (Q2HR71) DDLSGGGLDGLDPLEQGVDSLLDLLCGEIHDV MPLCDDLSGGGLDGLDPLEQGVD SLLDLLCGEIE1.010 HDVMPLCDDL93 KSHV VIRF2 98 GGSDALDDFDLDMLGGSLDGLDPLEQGVDSLL (Q2HR71), DLLCGEIHDVMPLCDDLGGSDALDDFDLDMLG HSV1 VP16 GSLDGLDPLEQGVDSLLDLLCGEIHDVMPLCD (P06492) El. Oil DL94 CITED2 60 MPASVAHVPAAMLPPNVIDTDFIDEEVLMSLVI El.012 EMGLDRIKELPELWLGQNEFDFMTDFV 95 C3orf62 60 QDSPFKEEAWALLMDKSPQKATDADPGSLKQ El.013 AFDDHNIVETVLDLEEDYNVMTSFKYQIE 96 CITED 1 60 ESLSPSAGAQSPAIIDSDPVDEEVLMSLWELGL El.014 DRANELPELWLGQNEFDFTADFPS SC97 HHV6A IE2 110 MEPAKPSGNNMGSNDERMQDYRPDPMMEESI (Q77Z83) KEILEESLMCDTSFDDLIIPGLESFGLIIPESSNNI ESNNVEEGSDGELKTLAEQKCKQGNDNDVIQSEl.015 AMKLSGLYCDA98 KSHV VIRF4 120 NPEVSHLPTELERRECVCPGSGERPRVPLVSTY (Q2HR73) AGDRYAVGGYGPEQSLVPPPLGLPLTLSNLQGEDICTWEEGLGNILSELQEEPSSSTRQATDRRRPREl.016 SRSPHGRRTPVSHSGPEKPPAtorney Docket No. 232285-70260199 TP53 100 EEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPL PSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMP EAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQEl.017 K100 DUX4 110 AWEPQAGAAPPPQPAPPDASASARQGQMQGIP APSQALQEPAPWSALPCGLLLDELLASPEFLQQ AQPLLETEAPGELEASEEAASLEAPLSEEEYRALE1.018 LEEYRALLEEL101 MYBL1 120 GSFSSWSGSFLMDDNMSNTLNSLDEHTSEFYS MDENQPVSAQQNSPTKFLAVEANAVLSSLQTIP EFAETLELIESDPVAWSDVTSFDISDAAASPIKSEl.019 TPVKLMRIQHNEGAMECQFNV102 FOXO1 110 MNRLTQVKTPVQVPLPHPMQMSALGGYSSVSS CNGYGRMGLLHQEKLPSDLDGMFIERLDCDME SIIRNDLMDGDTLDFNFDNVLPNQSFPHSVKTTE1.020 THSKTTTHSWVSG103 NCOA2 150 SELEMNMGGPQYSQQQAPPNQTAPWPESILPID QASFASQNRQPFGSSPDDLLCPHPAAESPSDEG ALLDQLYLALRNFDGLEEIDRALGIPELVSQSQ AVDPEQFSSQDSNIMLEQKAPVFPQQYASQAQE1.021 MAQGSYSPMQDPNFHTMGQ104 MYBL1 140 RSSSKLQHKPCAAMDHMQTQNQFYIPVQIPGY QYVSPEGNCIEHVQPTSAFIQQPFIDEDPDKEKK IKELEMLLMSAENEVRRKRIPSQPGSFSSWSGSF LMDDNMSNTLNSLDEHTSEFYSMDENQPVSAQE1.022 QNSPTKFL105 ATF4 190 TEMSFLSSEVLVGDLMSPFDQSGLGAEESLGLL DDYLEVAKHFKPHGFSSDKAKAGSSEWLAVD GLVSPSNNSKEDAFSGTDWMLEKMDLKEFDLD ALLGIDDLETMPDDLLTTLDDTCDLFAPLVQET NKQPPQTVNPIGHLPESLTKPDQVAPFTFLQPLPE1.023 LSPGVLSSTPDHSFSLELGSEVDITEG106 MYB 130 LPATGQPTVNNDYSYYHISEAQNVSSHVPYPV ALHVNIVNVPQPAAAAIQRHYNDEDPEKEKRIK ELELLLMSTENELKGQQVLPTQNHTCSYPGWHE1.024 STTIADHTRPHGD S APVSCLGEHHSTPSLPADP 107 CEBPD 110 ARGAPWPAEPAPFYEPGRAGKPGRGAEPGALG EPGAAAPAMYDDESAIDFSAYIDSMAAVPTLEL CHDELFADLFNSNHKAGGAGPLELLPGGPARPE1.025 LGPGPAAPRLLKR108 HSV2 VP16 130 AYSRGRTRNNYGSTIEGLLDLPDDDDAPAEAG (P68336) LVAPRMSFLSAGQRPRRLSTTAPITDVSLGDEL RLDGEEVDMTPADALDDFDLEMLGDVESPSPG MTHDPVSYGALDVDDFEFEQMFTDAMGIDDFE1.026 GG109 KSHV VIRF2 150 GPLHVHTPTDLCLPTGGLPSPVIFPHETQGLLAP (Q2HR71) PAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHIE GCLLDLLSDVGQELPDLGDLGELLCETASPQGP MQSEGGEEGSTESVSVLPATHPLESSAPGASVME1.027 GSGQELPDLGDLSELLC110 KSHVRTA 161 ALTVPEADTTPSTTTPGTSLGSITTPQDVHATDV (F5HCV3) ATSEGPSEAQPPLLSLPPPLDVDQSLFALLDEAG PETWDVGSPLSPTDDALLSSILQGLYQLDTPPPL RSPSPASFGPESPADIPSPSGGEYTQLQPVRATSE1.028 ATPANEVQESGTLYQLHQWRNYFRD111 HHV6A IE2 110 MEPAKPSGNNMGSNDERMQDYRPDPMMEESI (Q8BB47) QQILEDSLMCDTSFDDLILPGLESFGLIIPESSNNI ESNNVEEGSNEDLKTLAEHKCKQGNDNDVIQSE1.029 AMKLSGLYCDA112 HPV5 E6 90 MAEGAEHQQKLTEKDKAELPLSIRDLAEALGIP (P06930) VIDCLIPCNFCGNFLNYLEACEFDYKRLSLIWKE1.030 DYCVFACCRVCCGATATYEFNQFYAtorney Docket No. 232285-702601113 HAdV7 E1A 130 MRHLRFLPQEIIS SETGIEILEF WNTLMGDDPEP (P03256) PVQPFDPPTLHDLYDLEVDGPEDPNEGAVNGFF TDSMLLAADEGLDINPPPETLVTPGVWESGRGE1.031 GKKLPDLGAAEMDLRCYEEGFPPSDDEDG 114 HHV6B IE1 130 MESAKDTTSTSMFILGKPSGNNMESNEERMQN (Q77PU6) YHPDPWEESIKEILEESLKCDVSFESLLFPELEA FDLFIPESSNDIASKNVSYSSNVEEGASDEFKTLE1.032 VAQSVGNCIQSIGASVKAAMKQEQSNMED 115 HHV6A IE1 110 MEPAKPSGNNMGSNDERMQDYRPDPMMEESI (Q69567) KEILEESLMCDTSFDDLIIPGLESFGLIIPESSNNI ESNNVEEGSDGELKTLAAQSAGNCIQSIGASVKE1.033 AAMKQEQSDM116 SFVBEL1 90 KHHKPRPKRSRKRSIDHESCASSGDTVANESGP (Q87042) LCTNTFWTPGPVLQGLLGESSNLPDLEVHMSG E1.034 GPFWKEVYGDSILGPPSGSGEHSVL117 HHV6B IE1 130 MESAKDTTSNSMFILGKPSGNNMESNEERMQN (Q6XA69) YHPDPWEESIKEILEESLKCDVSFESLLFPELEA FDLFIPESSNDIASKNVSYSSNVEEGASEEFKALE1.035 VAQSVGNCIQSIGASVKAAMKQEQSNMED 118 KSHV VIRF3 160 TRHSASGVQPVDDANADSPGSGDEGPSTRHSD (F5HIC6) SQPPPADETTVHTDNVEDDLTLLDKESACALM YHVGQEMDMLMRAMCDEDLFDLLGIPEDVIA TSQPGGDTDASGWTEGSIAASAVGAGVEDVY LAGALEAQNVAGEYVLEISDEEVDDGAGLPPAE1.036 S119 KSHV VIRF3 90 CALMYHVGQEMDMLMRAMCDEDLFDLLGIPE (Q9DUN1) DVIATSQPGGDTDASGWTEGSIAASAVGAGV E1.037 EDVYLAGALEAQNVAGEYVLEISDEEV 120 HAdV9 E1A 130 MRHLRLLPSTVPGELAVLMLEDFVDTVLEDEL (Q9YLA0) HPSPFELGPTLQDLYDLEVDAHDDDPNEEAVN LIFPESMILQADIANESTPLHTPTLSPIPELEEEDEE1.038 LDLRCYEEGFPPSDSEDERGPVSEDELSPS 121 KSHVRTA 150 ALTVPEADTTPSTTTPGTSLGSITTPQDVHATDV (F5HCV3) ATSEGPSEAQPPLLSLPPPLDVDQSLFALLDEAG PETWDVGSPLSPTDDALLSSILQGLYQLDTPPPL RSPSPASFGPESPADIPSPSGGEYTQLQPVRATSE1.039 ATPANEVQESGTLY122 SARS-CoV 90 SWLMWFIISIVQMAPVSAMVRMYIFFASFYYIW NSP3 KSYVHIMDGCTSSTCMMCYKRNRATRVECTTI (POC6X7|PRO VNGMKRSFYVYANGGRGFCKTHNWN 0000037311) E1.040123 HCoV-229E 90 NTCPQKKVWGIGEHCPGLGINEEKCGTQLNHS NSP12 SCSCSPDAFLGWSFDSCISNNRCNIFSNFIFNGIN (POC6X1 PRO SGTTCSNDLLYSNTEVSTGVCV 0000037303) E1.041124 MERS-CoV 110 PTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAP Spike EPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTG (K9N5Q8) IDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLT E1.042 YEMLSLQ125 HCoV-HKUl- 90 CNGTDIDYCVRAFDVYNKDASFIGKNLKSNCV N5 NSP4 RFKNVDKDDAFYIVKRCIKSVMDHEQSMYNLL (POC6X4|PRO KGCNAVAKHDFFTWHEGRTIYGNVSR 0000297791) E1.043126 EBVEBNA2 101 PVLGLHQGQGAGDSPTPGPSNAAPVCRNSHTA (Q3KSV2) TPNVSPIHEPESHNSPEAPILFPDDWYPPSIDPAD E1.044 LDESWDYIFETTESPSSDEDYVEGPSKRPRPSIQ 127 HSV1 VP16 80 STAPPTDVSLGDELHLDGEDVAMAHADALDDF (P06492) DLDMLGDGDSPGPGFTPHDSAPYGALDMADFE E1.045 FEQMFTDALGIDEYGG128 HSV2 VP16 80 YGSTIEGLLDLPDDDDAPAEAGLVAPRMSFLSA (P68336) GQRPRRLSTTAPITDVSLGDELRLDGEEVDMTPE1.046 ADALDDFDLEMLGDAtorney Docket No. 232285-702601129 HCMV IE2 80 KATTFLQTMLRKEVNSQLSLGDPLFPELAEESL (P19893) KTFEQVTEDCNENPEKDVLAELGDILAQAVNH E1.047 AGIDSSSTGPTLTTH130 KSHVRTA 80 SEAQPPLLSLPPPLDVDQSLFALLDEAGPETWD (F5HCV3) VGSPLSPTDDALLSSILQGLYQLDTPPPLRSPSP E1.048 ASFGPESPADIPS131 KSHV VIRF2 80 SHVSGLDDCLPMVDHIEGCLLDLLSDVGQELP (Q2HR71) DLGDLGELLCETASPQGPMQSEGGEEGSTESVS E1.049 VLP ATHPLES S APGA132 KSHV VIRF3 80 TRHSASGVQPVDDANADSPGSGDEGPSTRHSD (F5HIC6) SQPPPADETTVHTDNVEDDLTLLDKESACALM E1.050 YHVGQEMDMLMRAMCD 133 HHV6B IE1 80 MESAKDTTSTSMFILGKPSGNNMESNEERMQN (Q77PU6) YHPDPWEESIKEILEESLKCDVSFESLLFPELEA E1.051 FDLFIPESSNDIA134 KSHV VIRF4 80 LERRECVCPGSGERPRVPLVSTYAGDRYAVGG (Q2HR73) YGPEQSLVPPPLGLPLTLSNLQGEDICTWEEGL E1.052 GNILSELQEEPSSST135 SuHVl ICP4 80 MADDLFDFIETEGNFSQLLAAAAAAEEEGIASG (P33479) PDGGSQGSRRRGSSGEDLLFGPGGLFSDDAAEA E1.053 EAAVLAAAAGATRP136 KSHV VIRF2 80 ELWVGAQGFVGRDMHSLFFEEPEPSGFGSSGQ (Q2HR71) SSSLLAPDSPRPSTSQVQGPLHVHTPTDLCLPTG E1.054 GLPSPVIFPHETQG137 HCoV-HKUl- 80 WLSNLLYILFFISLICFILLWALLPTYSVYKSDI N5 NSP4 HLPAYASFKVIDNGWRDISVNDLCFANKFFQF (POC6U5PRO DQWYESTFGSVY0000338222) E1.055138 HHV6A U95 80 MSAHPDDLFWQQILNMDPTELLSDDVISSTCDE (Q89882) NTAAGTDIMQPLCVDMSVQPTTSGCHTGAMM E1.056 SDSYFSNDTPDQNCQS139 HHV6B IE1 300 MESAKDTTSTSMFILGKPSGNNMESNEERMQN (Q77PU6), YHPDPWEESIKEILEESLKCDVSFESLLFPELEA KSHV VIRF3 FDLFIPESSNDIASKNVSYSSNVEEGASDEFKTL (F5HIC6) VAQSVGNCIQSIGASVKAAMKQEQSNMEDGG GGSGGGGSTRHSASGVQPVDDANADSPGSGDE GPSTRHSDSQPPPADETTVHTDNVEDDLTLLDK ESACALMYHVGQEMDMLMRAMCDEDLFDLL GIPEDVIATSQPGGDTDASGWTEGSIAASAVG AGVEDVYLAGALEAQNVAGEYVLEISDEEVDDE1.057 GAGLPPAS140 KSHV VIRF2 170 ELWVGAQGFVGRDMHSLFFEEPEPSGFGSSGQ (Q2HR71), SSSLLAPDSPRPSTSQVQGPLHVHTPTDLCLPTG KSHV VIRF4 GLPSPVIFPHETQGGGGGSGGGGSLERRECVCP (Q2HR73) GSGERPRVPLVSTYAGDRYAVGGYGPEQSLVP PPLGLPLTLSNLQGEDICTWEEGLGNILSELQEEE1.058 PSSST141 KSHV VIRF2 170 SHVSGLDDCLPMVDHIEGCLLDLLSDVGQELP (Q2HR71), DLGDLGELLCETASPQGPMQSEGGEEGSTESVS KSHV VIRF4 VLPATHPLESSAPGAGGGGSGGGGSLERRECV (Q2HR73) CPGSGERPRVPLVSTYAGDRYAVGGYGPEQSL VPPPLGLPLTLSNLQGEDICTWEEGLGNILSELQE1.059 EEPSSST142 KSHV VIRF2 280 GPLHVHTPTDLCLPTGGLPSPVIFPHETQGLLAP (Q2HR71), PAGQSQTPFSPEGPVPSHVSGLDDCLPMVDHIE KSHV VIRF4 GCLLDLLSDVGQELPDLGDLGELLCETASPQGP (Q2HR73) MQSEGGEEGSTESVSVLPATHPLESSAPGASVM GSGQELPDLGDLSELLCGGGGSGGGGSNPEVS HLPTELERRECVCPGSGERPRVPLVSTYAGDRY AVGGYGPEQSLVPPPLGLPLTLSNLQGEDICTW EEGLGNILSELQEEPSSSTRQATDRRRPRSRSPHE1.060 GRRTPVSHSGPEKPPAtorney Docket No. 232285-702601143 KSHV VIRF4 280 NPEVSHLPTELERRECVCPGSGERPRVPLVSTY (Q2HR73), AGDRYAVGGYGPEQSLVPPPLGLPLTLSNLQGE KSHV VIRF2 DICTWEEGLGNILSELQEEPSSSTRQATDRRRPR (Q2HR71) SRSPHGRRTPVSHSGPEKPPGGGGSGGGGSGPL HVHTPTDLCLPTGGLPSPVIFPHETQGLLAPPAG QSQTPFSPEGPVPSHVSGLDDCLPMVDHIEGCL LDLLSDVGQELPDLGDLGELLCETASPQGPMQS EGGEEGSTESVSVLPATHPLESSAPGASVMGSGE1.061 QELPDLGDLSELLC144 KSHV VIRF4 170 LERRECVCPGSGERPRVPLVSTYAGDRYAVGG (Q2HR73), YGPEQSLVPPPLGLPLTLSNLQGEDICTWEEGL KSHV VIRF2 GNILSELQEEPSSSTGGGGSGGGGSSHVSGLDD (Q2HR71) CLPMVDHIEGCLLDLLSDVGQELPDLGDLGELL CETASPQGPMQSEGGEEGSTESVSVLPATHPLEE1.062 SSAPGA145 KSHV VIRF4 170 LERRECVCPGSGERPRVPLVSTYAGDRYAVGG (Q2HR73), YGPEQSLVPPPLGLPLTLSNLQGEDICTWEEGL KSHV VIRF2 GNILSELQEEPSSSTGGGGSGGGGSELWVGAQG (Q2HR71) FVGRDMHSLFFEEPEPSGFGSSGQSSSLLAPDSP RPSTSQVQGPLHVHTPTDLCLPTGGLPSPVIFPHE1.063 ETQG146 SuHVl ICP4 220 MADDLFDFIETEGNFSQLLAAAAAAEEEGIASG (P33479), PDGGSQGSRRRGSSGEDLLFGPGGLFSDDAAEA HSV2 VP16 EAAVLAAAAGATRPGGGGSGGGGSAYSRGRT (P68336) RNNYGSTIEGLLDLPDDDDAPAEAGLVAPRMS FLSAGQRPRRLSTTAPITDVSLGDELRLDGEEV DMTPADALDDFDLEMLGDVESPSPGMTHDPVSE1.064 YGALDVDDFEFEQMFTDAMGIDDFGG 147 KSHV VIRF3 300 TRHSASGVQPVDDANADSPGSGDEGPSTRHSD (F5HIC6), SQPPPADETTVHTDNVEDDLTLLDKESACALM HHV6B IE1 YHVGQEMDMLMRAMCDEDLFDLLGIPEDVIA (Q77PU6) TSQPGGDTDASGWTEGSIAASAVGAGVEDVY LAGALEAQNVAGEYVLEISDEEVDDGAGLPPA SGGGGSGGGGSMESAKDTTSTSMFILGKPSGN NMESNEERMQNYHPDPWEESIKEILEESLKCD VSFESLLFPELEAFDLFIPESSNDIASKNVSYSSN VEEGASDEFKTLVAQSVGNCIQSIGASVKAAME1.065 KQEQSNMED148 HSV2 VP16 220 AYSRGRTRNNYGSTIEGLLDLPDDDDAPAEAG (P68336), LVAPRMSFLSAGQRPRRLSTTAPITDVSLGDEL SuHVl ICP4 RLDGEEVDMTPADALDDFDLEMLGDVESPSPG (P33479) MTHDPVSYGALDVDDFEFEQMFTDAMGIDDF GGGGGGSGGGGSMADDLFDFIETEGNFSQLLA AAAAAEEEGIASGPDGGSQGSRRRGSSGEDLLFE1.066 GPGGLFSDDAAEAEAAVLAAAAGATRP 149 CTCF 262 DAVEAIVEESETFIKGKERKTYQRRREGGQEED ACHLPQNQTDGGEWQDVNSSVQMVMMEQL DPTLLQMKTEVMEGTVAPEAEAAVDDTQIITL QWNMEEQPINIGELQLVQVPVPVTVPVATTSV EELQGAYENEVSKEGLAESEPMICHTLPLPEGF QWKVGANGEVETLEQGELPPQEDPSWQKDPD YQPPAKKTKKTKKSKLRYTEEGKDVDVSVYDF EEEQQEGLLSEVNAEKVVGNMKPPKPTKIKKKE1.067 GVKKT150 CTCF 151 CAGPDGVEGENGGETKKSKRGRKRKMRSKKE DSSDSENAEPDLDDNEDEEEPAVEIEPEPEPQPV TPAPPPAKKRRGRPPGRTNQPKQNQPTAIIQVE DQNTGAIENIIVEVKKEPDAEPAEGEEEEAQPAE1.068 ATDAPNGDLTPEMILSMMDR151 CTCF 120 SKCGKTFTRRNTMARHADNCAGPDGVEGENGE1.069 GETI<I<SI<RGRI<RI<MRSI<I<EDSSDSENAEPDLDAtorney Docket No. 232285-702601DNEDEEEPAVEIEPEPEPQPVTPAPPPAKKRRGR PPGRTNQPKQNQPTAIIQVEDQN152 CTCF 208 EKPYACSHCDKTFRQKQLLDMHFKRYHDPNFV PAAFVCSKCGKTFTRRNTMARHADNCAGPDG VEGENGGETKKSKRGRKRKMRSKKEDSSDSEN AEPDLDDNEDEEEPAVEIEPEPEPQPVTPAPPPA KKRRGRPPGRTNQPKQNQPTAIIQVEDQNTGAI ENIIVEVKKEPDAEPAEGEEEEAQPAATDAPNGE1.070 DLTPEMILSMMDR153 CTCF 139 HKCPDCDMAFVTSGELVRHRRYKHTHEKPFKC SMCDYASVEVSKLKRHIRSHTGERPFQCSLCSY ASRDTYKLKRHMRTHSGEKPYECYICHARFTQ SGTMKMHILQKHTENVAKFHCPHCDTVIARKSE1.071 DLGVHLRKQH154 CTCF 313 DAVEAIVEESETFIKGKERKTYQRRREGGQEED ACHLPQNQTDGGEWQDVNSSVQMVMMEQL DPTLLQMKTEVMEGTVAPEAEAAVDDTQIITL QWNMEEQPINIGELQLVQVPVPVTVPVATTSV EELQGAYENEVSKEGLAESEPMICHTLPLPEGF QWKVGANGEVETLEQGELPPQEDPSWQKDPD YQPPAKKTKKTKKSKLRYTEEGKDVDVSVYDF EEEQQEGLLSEVNAEKVVGNMKPPKPTKIKKK GVKKTFQCELCSYTCPRRSNLDRHMKSHTDERE1.072 PHKCHLCGRAFRTVTLLRNHLNTH155 KSHV VIRF2 357 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SDAVEAIVEESETFIKGKERKTYQRRREGGQEE CTCF DACHLPQNQTDGGEWQDVNSSVQMVMMEQ LDPTLLQMKTEVMEGTVAPEAEAAVDDTQIITL QWNMEEQPINIGELQLVQVPVPVTVPVATTSV EELQGAYENEVSKEGLAESEPMICHTLPLPEGF QWKVGANGEVETLEQGELPPQEDPSWQKDPD YQPPAKKTKKTKKSKLRYTEEGKDVDVSVYDF EEEQQEGLLSEVNAEKVVGNMKPPKPTKIKKKE1.073 GVKKT156 KSHV VIRF2 246 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SCAGPDGVEGENGGETKKSKRGRKRKMRSKK CTCF EDSSDSENAEPDLDDNEDEEEPAVEIEPEPEPQP VTPAPPPAKKRRGRPPGRTNQPKQNQPTAIIQV EDQNTGAIENIIVEVKKEPDAEPAEGEEEEAQPE1.074 AATDAPNGDLTPEMILSMMDR157 KSHV VIRF2 215 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SSKCGKTFTRRNTMARHADNCAGPDGVEGEN CTCF GGETKKSKRGRKRKMRSKKEDSSDSENAEPDL DDNEDEEEPAVEIEPEPEPQPVTPAPPPAKKRRGE1.075 RPPGRTNQPKQNQPTAIIQVEDQN158 KSHV VIRF2 303 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SEKPYACSHCDKTFRQKQLLDMHFKRYHDPNF CTCF VPAAFVCSKCGKTFTRRNTMARHADNCAGPD GVEGENGGETKKSKRGRKRKMRSKKEDSSDSE NAEPDLDDNEDEEEPAVEIEPEPEPQPVTPAPPP AKKRRGRPPGRTNQPKQNQPTAIIQVEDQNTG AIENIIVEVKKEPDAEPAEGEEEEAQPAATDAPNE1.076 GDLTPEMILSMMDRAtorney Docket No. 232285-702601159 KSHV VIRF2 234 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SHKCPDCDMAFVTSGELVRHRRYKHTHEKPFK CTCF CSMCDYASVEVSKLKRHIRSHTGERPFQCSLCS YASRDTYKLKRHMRTHSGEKPYECYICHARFT QSGTMI<MHILQI<HTENVAI<FHCPHCDTVIARI< E1.077 SDLGVHLRKQH160 KSHV VIRF2 408 LDDCLPMVDHIEGCLLDLLSDVGQELPDLGDL (Q2HR71), GGSDALDDFDLDMLGSDALDDFDLDMLGSDA HSV1 VP16 LDDFDLDMLGSDALDDFDLDMLGGGGSGGGG (P06492), SDAVEAIVEESETFIKGKERKTYQRRREGGQEE CTCF DACHLPQNQTDGGEWQDVNSSVQMVMMEQ LDPTLLQMKTEVMEGTVAPEAEAAVDDTQIITL QWNMEEQPINIGELQLVQVPVPVTVPVATTSV EELQGAYENEVSKEGLAESEPMICHTLPLPEGF QWKVGANGEVETLEQGELPPQEDPSWQKDPD YQPPAKKTKKTKKSKLRYTEEGKDVDVSVYDF EEEQQEGLLSEVNAEKVVGNMKPPKPTKIKKK GVKKTFQCELCSYTCPRRSNLDRHMKSHTDERE1.078 PHKCHLCGRAFRTVTLLRNHLNTH161 IAFNSNIDDAAAITNALSNKTTKQAITSTLKDVG Caijivirus DDILHGVRAEWTEGVEEAINYIASQDGLYNGK communis E1.079 85 KVFDKDIPQQTIKDYLQDP162 PSAITTANIRPVSPGISDANDHDLREPSGIKNPG GASASARVSSWDSQSYDIEQICLGASNPPWAST LEUTX E1.080 90 LFEIDEFVKIYDLPGEDDTSSLN163 MLSLREQQLQVWFKNRRAKLARERRLQQQPQ RVPGQRGRGARAAPLVPAASASAPQRGPSGILP TPRX1 E1.081 80 AAEPTICSLHQAWGGP164 ARAAPLVPAASASAPQRGPSGILPAAEPTICSLH QAWGGPGCRAQKGIPAALSPGPGPIPAPIPGPA TPRX1 E1.082 80 QIPGPLPGSIPGP165 GCRAQKGIPAALSPGPGPIPAPIPGPAQIPGPLPG SIPGPIPGPAQIPSPIPAPIPGPISGPVQIPGPFRGPI TPRX1 E1.083 80 PGPISGP166 IPGPAQIPSPIPAPIPGPISGPVQIPGPFRGPIPGPIS GPAPIPGPISGPFSGPNPGPIPGPNPGPIPGPISGPI TPRX1 E1.084 80 PGPIS167 APIPGPISGPFSGPNPGPIPGPNPGPIPGPISGPIPG PISVPIPGPIPGPISGPISGPNPGPIPGPIPGPISGPN TPRX1 E1.085 80 PGPIP168 VPIPGPIPGPISGPISGPNPGPIPGPIPGPISGPNPGP IPGPISGPNPGLIPGPIPGPISGPGPIIGPIPSPAQIP TPRX1 E1.086 80 GPGR169 GPISGPNPGLIPGPIPGPISGPGPIIGPIPSPAQIPGP GRLQGPGPILSPGRMRSPGSLPGLAPILGPGSGP TPRX1 E1.087 80 GSGSVPAP170 LQGPGPILSPGRMRSPGSLPGLAPILGPGSGPGS GSVPAPIPGPGSLPAPAPLWPQSPDASDFLPDTQ TPRX1 E1.088 80 LFPHFTELLLPL171 IPGPGSLPAPAPLWPQSPDASDFLPDTQLFPHFT ELLLPLDPLEGSSVSTMTSQYQEGDDSMGKKH TPRX1 E1.089 91 SGSQPQEEGGSVNENHSGPRLLLDL172 IYPHDDKHREKALRRSLRKKAQREAARKQAAA VATPTTSSAAEVSSRSQSEDTESSDSENELWVG AQGFVGRDMHSLFFEEPEPSGFGSSGQSSSLLA PDSPRPSTSQVQGPLHVHTPTDLCLPTGGLPSPV IFPHETQGLLAPPAGQSQTPFSPEGPVPSHVSGL DDCLPMVDHIEGCLLDLLSDVGQELPDLGDLG KSHV VIRF2 ELLCETASPQGPMQSEGGEEGSTESVSVLPATH(Q2HR71) E1.090 260 PLESSAPGASVMGSGQELPDLGDLSELLCAtorney Docket No. 232285-702601173 IYPHDDKHREKALRRSLRKKAQREAARKQAAA VATPTTSSAAEVSSRSQSEDTESSDSENELWVG AQGFVGRDMHSLFFEEPEPSGFGSSGQSSSLLA KSHV VIRF2 PDSPRPSTSQVQGPLHVHTPTDLCLPTGGLPSPV (Q2HR71) E1.091 140 IFPHETQG174 RKRSIDHESCASSGDTVANESGPLCTNTFWTPG SFVBEL1 PVLQGLLGESSNLPDLEVHMSGGPFWKEVYGD (Q87042) E1.092 80 SILGPPSGSGEHSVL175 KHHKPRPKRSRKRSIDHESCASSGDTVANESGP SFVBEL1 LCTNTFWTPGPVLQGLLGESSNLPDLEVHMSG (Q87042) E1.093 80 GPFWKEVYGDSILGP176 MKQGKKNPYGDKGDAALQYAYQVKRGCKAG SFVBET CLASPVLNFKALQFHRTLMADLTNPKIGQGYL(Q87043) E1.094 85 AHGYHAAMEAYGPQRGSSEERVW177 MKQGKKNPYGDKGDAALQYAYQVKRGCKAG CLASPVLNFKALQFHRTLMADLTNPKIGQGYL AHGYHAAMEAYGPQRGSSEERVWWNATRNQ GKKDEYYREGGEEPHYPNTPAPHKKTWEERH KVLKLSPFATPSDIQRWTTRALPYGWKVIDGN SFVBET GDDYTSRRNIRTLTEMTQEEIRKRWERGYCDPF(Q87043) E1.095 208 IDSGSDSDGPLSKPQTVKVW178 KHHKPRQKRPRRRSIDNESCASSSDTMANEPGS HFV BEL1 LCTNPLWNPGPLLSGLLEESSNLPNLEVHMSGG (P14353) E1.096 90 PFWEEVYGDSILGPPSGSGEHSVL179 NNYGSTIEGLLDLPDDDAPEEAGLAAPRLSFLP AGHTRRLSTAPPTDVSLGDELHLDGEDVAMAH HSV1 VP16 ADALDDFDLDMLGDGDSPGPGFTPHDSAPYGA (P06492) E1.097 120 LDMADFEFEQMFTDALGIDEYGG180 FLPAGHTRRLSTAPPTDVSLGDELHLDGEDVA HSV1 VP16 MAHADALDDFDLDMLGDGDSPGPGFTPHDSA (P06492) E1.098 80 PYGALDMADFEFEQMFT181 PPPLDVDQSLFALLDEAGPETWDVGSPLSPTDD KSHVRTA ALLSSILQGLYQLDTPPPLRSPSPASFGPESPADI(F5HCV3) E1.099 80 PSPSGGEYTQLQ182 EDFVDTVLEDELHPSPFELGPTLQDLYDLEVDA HAdV9 E1A HDDDPNEEAVNLIFPESMILQADIANESTPLHTP (Q9YLA0) El.100 80 TLSPIPELEEEDE183 DLTLLDKESACALMYHVGQEMDMLMRAMCD KSHV VIRF3 EDLFDLLGIPEDVIATSQPGGDTDASGVVTEGSI (Q9DUN1) El.101 80 AASAVGAGVEDVYLAG184 MDMLMRAMCDEDLFDLLGIPEDVIATSQPGGD KSHV VIRF3 TDASGWTEGSIAASAVGAGVEDVYLAGALEA (Q9DUN1) El.102 80 QNVAGEYVLEISDEEV185 QNYHPDPWEESIKEILEESLKCDVSFESLLFPEL HHV6B IE1 EAFDLFIPESSNDIASKNVSYSSNVEEGASEEFK (Q6XA69) El.103 80 ALVAQSVGNCI186 MGSNDERMQDYRPDPMMEESIKEILEESLMCD HHV6A IE1 TSFDDLIIPGLESFGLIIPESSNNIESNNVEEGSDG (Q69567) El.104 80 ELKTLAAQSAGN187 MRHLRFLPQEIIS SETGIEILEF WNTLMGDDPEP PVQPFDPPTLHDLYDLEVDGPEDPNEGAVNGFF TDSMLLAADEGLDINPPPETLVTPGVWESGRG GKKLPDLGAAEMDLRCYEEGFPPSDDEDGETE QSIHTAVNEGVKAASDVFKLDCPELPGHGCKS HAdV7 E1A CEFHRNNTGMKELLCSLCYMRMHCHFIYSPVS (P03256) El.105 210 DDESPSPDSTTSP188 IISSETGIEILEFVVNTLMGDDPEPPVQPFDPPTL HAdV7 E1A HDLYDLEVDGPEDPNEGAVNGFFTD SMLL AAD (P03256) El.106 80 EGLDINPPPETLV189 HAdV7 E1A AEMDLRCYEEGFPPSDDEDGETEQSIHTAVNEG(P03256) El.107 100 VKAASDVFKLDCPELPGHGCKSCEFHRNNTGMAtorney Docket No. 232285-702601KELLCSLCYMRMHCHFIYSPVSDDESPSPDSTT SP190 GFPPSDDEDGETEQSIHTAVNEGVKAASDVFKL HAdV7 E1A DCPELPGHGCKSCEFHRNNTGMKELLCSLCYM (P03256) El.108 80 RMHCHFIYSPVSDDE191 LTEKDKAELPLSIRDLAEALGIPVIDCLIPCNFCG HPV5 E6 NFLNYLEACEFDYKRLSLIWKDYCVFACCRVC (P06930) El.109 80 CGATATYEFNQFY192 MGSNDERMQDYRPDPMMEESIQQILEDSLMCD HHV6A IE2 TSFDDLILPGLESFGLIIPESSNNIESNNVEEGSNE (Q8BB47) El.110 80 DLKTLAEHKCKQ193 MRHLRDLPDEEIIIASGSEILELWNATMGDDHP EPPTPFGTPSLHDLYDLEVDVPEDDPNEKAVND LFSDAALLAAEEASSPSSDSDSSLHTPRHDRGE KEIPGLKWEKMDLRCYEECLPPSDDEDEQAIQN AASHGVQAVSESFALDCPPLPGHGCKSCEFHRI HAdV4 E1A NTGDKAVLCALCYMRAYNHCVYSPVSDADDE (P10407) El. Ill 220 TPTTESTLSPPEIGTSPSDNIVR194 MRHLRDLPDEEIIIASGSEILELWNATMGDDHP EPPTPFGTPSLHDLYDLEVDVPEDDPNEKAVND HAdV4 E1A LFSDAALLAAEEASSPSSDSDSSLHTPRHDRGE (P10407) El.112 130 KEIPGLKWEKMDLRCYEECLPPSDDEDEQA 195 MRHLRDLPDEEIIIASGSEILELWNATMGDDHP HAdV4 E1A EPPTPFGTPSLHDLYDLEVDVPEDDPNEKAVND (P10407) El.113 80 LFSDAALLAAEEA196 MDLRCYEECLPPSDDEDEQAIQNAASHGVQAV SESFALDCPPLPGHGCKSCEFHRINTGDKAVLC HAdV4 E1A ALCYMRAYNHCVYSPVSDADDETPTTESTLSPP (P10407) El.114 110 EIGTSPSDNIVR197 AVSESFALDCPPLPGHGCKSCEFHRINTGDKAV HAdV4 E1A LCALCYMRAYNHCVYSPVSDADDETPTTESTL (P10407) El.115 80 SPPEIGTSPSDNIVR198 MRMLPDFFTGNWDDMFQGLLETEYVFDFPEPS EASEEMSLHDLFDVEVDGFEEDANQEAVDGMF PERLLSEAESAAESGSGDSGVGEELLPVDLDLK HAdV40 E1A CYEDGLPPSDPETDEATEAEEEAAMPTYVNEN (P10541) El.116 130 E199 MRMLPDFFTGNWDDMFQGLLETEYVFDFPEPS HAdV40 E1A EASEEMSLHDLFDVEVDGFEEDANQEAVDGMF (P10541) El.117 80 PERLLSEAESAAESGS200 MESSAKRKMDPDNPDEGPSSKVPRPETPVTKA TTFLQTMLRKEVNSQLSLGDPLFPELAEESLKT HCMV IE2 FEQVTEDCNENPEKDVLAELGDILAQAVNHAG (P19893) El.118 120 IDSSSTGPTLTTHSCSVSSAPLN201 SMPELSPVLGLHQGQGAGDSPTPGPSNAAPVC RNSHTATPNVSPIHEPESHNSPEAPILFPDDWYP EBVEBNA2 PSIDPADLDESWDYIFETTESPSSDEDYVEGPSK (P12978) El.119 107 RPRPSIQ202 LHQGQGAGDSPTPGPSNAAPVCRNSHTATPNV EBVEBNA2 SPIHEPESHNSPEAPILFPDDWYPPSIDPADLDES (P12978) El.120 80 WDYIFETTESPSS203 PVDLDLKCYEDGLPPSDPETDEATEAEEEAAMP TYVNENENELVLDCPENPGRGCRACDFHRGTS HAdV40 E1A GNPEAMCALCYMRLTGHCIYSPISDAEGESESG (P10541) El.121 120 SPEDTDFPHPLTATPPHGIVRT204 AMPTYVNENENELVLDCPENPGRGCRACDFHR HAdV40 E1A GTSGNPEAMCALCYMRLTGHCIYSPISDAEGES (P10541) El.122 80 ESGSPEDTDFPHPLT205 MRTEMTPLVLSYQEADDILEHLVDNFFNEVPSD DDLYVPSLYELYDLDVESAGEDNNEQAVNEFF HAdV12 E1A PESLILAASEGLFLPEPPVLSPVCEPIGGECMPQL(P03259) El.123 170 HPEDMDLLCYEMGFPCSDSEDEQDENGMAHVAtorney Docket No. 232285-702601SASAAAAAADREREEFQLDHPELPGHNCKSCE HHRNSTG206 SYQEADDILEHLVDNFFNEVPSDDDLYVPSLYE HAdV12 E1A LYDLDVESAGEDNNEQAVNEFFPESLILAASEG (P03259) El.124 80 LFLPEPPVLSPVCE207 PVLGLHQGQGAGDSPTPGPSNAAPVCRNSHTA EBVEBNA2 TPNVSPIHEPESHNSPEAPILFPDDWYPPSIDPAD (Q3KSV2) El.125 80 LDESWDYIFETTE208 MECNLGTEHPSTDTWNRSKTEQAWDAFDESL VZVVP16 FGDVASDIGFETSLYSHAVKTAPSPPWVASPKIL (P09265) El.126 100 YQQLIRDLDFSEGPRLLSCLETWNEDLFSCFPIN 209 MECNLGTEHPSTDTWNRSKTEQAWDAFDESL VZVVP16 FGDVASDIGFETSLYSHAVKTAPSPPWVASPKIL (P09265) El.127 80 YQQLIRDLDFSEGP210 ERTNYGSSIEGLLELPDDADYDPDAPPPPPRLSF ATGSPRRRRSTVPRTDVSLGDELCLDASPSMTPCeHV2 VP16 SEALDDFDLTLLGDDDHAGTLDAAAYASLDM (Q5Y0Q4) El.128 118 ADFEFEQMFTDALGIDEFGG211 ERTNYGSSIEGLLELPDDADYDPDAPPPPPRLSF CeHV2 VP16 ATGSPRRRRSTVPRTDVSLGDELCLDASPSMTP (Q5Y0Q4) El.129 80 SEALDDFDLTLLG212 MESSAKRKMDPDNPDEGPSSKVPRPETPVTKA HCMV IE1 TTFLQTMLRKEVNSQLSLGDPLFPELAEESLKT (P03169) El.130 90 FERVTEDCNENPEKDVLAELVKQIK213 PDNPDEGPSSKVPRPETPVTKATTFLQTMLRKE HCMV IE1 VNSQLSLGDPLFPELAEESLKTFERVTEDCNEN (P03169) El.131 80 PEKDVLAELVKQIK214 MRHIICHGGVITEEMAASLLDQLIEEVLADNLPP PSHFEPPTLHELYDLDVTAPEDPNEEAVSQIFPD SVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPEQP EQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDE DEEGEEFVLDYVEHPGHGCRSCHYHRRNTGDP HAdV5 E1A DIMCSLCYMRTCGMFVYSPVSEPEPEPEPEPEP (P03255) El.132 220 ARPTRRPKMAPAILRRPTSP215 QPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSD DEDEEGEEFVLDYVEHPGHGCRSCHYHRRNTG HAdV5 E1A DPDIMCSLCYMRTCGMFVYSPVSEPEPEPEPEP (P03255) El.133 120 EP ARPTRRPKMAPAILRRPTSP216 EFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCS HAdV5 E1A LCYMRTCGMFVYSPVSEPEPEPEPEPEPARPTR (P03255) El.134 80 RPKMAPAILRRPTSP217 MRHIICHGGVITEEMAASLLDQLIEEVLADNLPP HAdV5 E1A PSHFEPPTLHELYDLDVTAPEDPNEEAVSQIFPD (P03255) El.135 100 SVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPE 218 ITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLH HAdV5 E1A ELYDLDVTAPEDPNEEAVSQIFPDSVMLAVQEG (P03255) El.136 80 IDLLTFPPAPGSP219 MGSNDERMQDYRPDPMMEESIKEILEESLMCD HHV6A IE2 TSFDDLIIPGLESFGLIIPESSNNIESNNVEEGSDG (Q77Z83) El.137 80 ELKTLAEQKCKQ220 RTNYGSSIEGLLELPDDTDYDPDAPPPPPRLSFA AAPPRRRSAAPRTDVSLGDELRFDGDVSMTPSECeHVl VP16 ALDDFDLTLLGDDDHAGGLDDAAAYASLDMA (Q805Y1) El.138 117 DFEFEQMFTDALGIDDIGG221 RTNYGSSIEGLLELPDDTDYDPDAPPPPPRLSFA CeHVl VP16 AAPPRRRSAAPRTDVSLGDELRFDGDVSMTPSE (Q805Y1) El.139 80 ALDDFDLTLLGDD222 EMADTVIPQKEEAAICGQMDLSHPPPRGHLDEL EBVRTA TTTLESMTEDLNLDSPLTPELNEILDTFLNDECL(P03209) El.140 85 LHAMHISTGLSIFDTSLFAtorney Docket No. 232285-702601223 VIPQKEEAAICGQMDLSHPPPRGHLDELTTTLES EBVRTA MTEDLNLD SPLTPELNEILDTFLNDECLLH AMH(P03209) El.141 80 ISTGLSIFDTSLF224 ASPKILYQQLIRDLDFSEGPRLLSCLETWNEDLF SCFPINEDLYSDMMVLSPDPDDVISTVSTKDHV VZVVP16 EMFNLTTRGSVRLPSPPKQPTGLPAYVQEVQDS (P09265) El.142 110 FTVELRAREE225 IRDLDFSEGPRLLSCLETWNEDLFSCFPINEDLY VZVVP16 SDMMVLSPDPDDVISTVSTKDHVEMFNLTTRG (P09265) El.143 80 SVRLPSPPKQPTGL226 MVNFVSAGLFRCLPVSCPEDLLVEELVDGLLSL EEELKDKEEEEAVLDGLLSLEEESRGRLRRGPP HTLV1 HBZ GEKAPPRGETHRDRQRRAEEKRKRKKEREKEE (P0C746) El.144 100 EK227 MVNFVSAGLFRCLPVSCPEDLLVEELVDGLLSL HTLV1 HBZ EEELKDKEEEEAVLDGLLSLEEESRGRLRRGPP (P0C746) El.145 80 GEKAPPRGETHRDR228 YGSTIEGLLDLPDDDDAPAEAGLVAPRMSFLSA GQRPRRLSTTAPITDVSLGDELRLDGEEVDMTP HSV2 VP16 ADALDDFDLEMLGDVESPSPGMTHDPVSYGAL (Q69113) El.146 120 DVDDFEFEQMFTDALGIDDFGG229 LSAGQRPRRLSTTAPITDVSLGDELRLDGEEVD HSV2 VP16 MTPADALDDFDLEMLGDVESPSPGMTHDPVSY (Q69113) El.147 80 GALDVDDFEFEQMFT230 MADDLFDFIETEGNFSQLLAAAAAAAEEEGIAS SuHVl ICP4 GPDGGSQGSRRRGSSGEDLLFGPGGLFSDDAAE(G3G929) El.148 80 AEAEAAVLAAAAGA

[0085] In some embodiments, the polypeptide provided herein further comprises a linker. A linker is a molecular entity that can directly or indirectly connect at two parts of a composition, e.g., from the first protein construct to the second protein construct and the second protein construct to a third protein construct, and so on. Linkers can be configured according to a specific need, e.g., stability or length between two amino acid sequences. In some embodiments, linkers can be configured to allow multimerization of the nuclease with a transcriptional activation domain provided herein (e.g., to from a di-, tri-, tetra-, penta-, or higher multimeric complex) while retaining biological activity (e.g., cleavage of a target nucleic acid or set of target nucleic acids).

[0086] In some embodiments, linkers are configured to facilitate expression and purification of the polypeptides provided herein. In some embodiments, a polypeptide provided herein comprises a cleavable linker or a non-cleavable linker between the DNA-binding domain and the transcriptional activation domain. For example, a linker can be a polypeptide linker, such as a linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids long. Generally, the number of additional elements in the engineered protein will depend on a number of factors, including, e.g., size or molecule weight of the protein for delivery to a cell, or translocation of the protein desired in a cell type of interest.Attorney Docket No. 232285-702601

[0087] In some embodiments, the Cas protein and the transcriptional activation domain can be fused in a single polypeptide sequence (“a fusion protein”). In some embodiments, the Cas protein and the transcriptional activation domain can be fused directly to one another. Alternatively, the Cas protein and the transcriptional activation domain can be fused via a peptide linker (or an amino acid linker) that is heterologous to the Cas protein and the transcriptional activation domain. The peptide linker can be derived from a natural polypeptide sequence. Alternatively, the peptide linker can be a synthetic sequence. The peptide linker can have a length of at least or up to about 1 amino acid residue, at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 90 amino acid residues, or at least or up to about 100 amino acid residues. In some embodiments, the peptide linker can be a GS linker. The term “GS linker” or “GS linker sequence,” as used interchangeably herein, generally refers to a peptide linker that mainly comprises glycine and serine residues. Particularly, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95% or substantially about 100% of the amino acid residues in the GS linker sequence can be selected from glycine and serine residues. The GS linker sequence according to the present invention can, for example, comprise from about 1 to about 50 amino acid residues, from about 1 to about 45 amino acid residues, from about 1 to about 40 amino acid residues, from about 1 to about 35 amino acid residues, or from about 1 to about 30 amino acid residues, in total. In some cases, the GS linker sequence may not comprise about 10, about 5, about 4, about 3, about 2 or about 1 amino acid residue (s) other than glycine or serine. In some embodiments, the Cas protein and the transcriptional activation domain are fused via a linker comprising an amino acid sequence of any one of SEQ ID NOS: 231-249.

[0088] In some embodiments, a polypeptide described herein comprises a purification tag. For example, the purification tag comprises, or may include, at least one tag selected from the group consisting of a His tag, a FLAG tag, an AU1 epitope tag, an AU5 epitope tag, a bacteriophage T7 tag, a bacteriophage V5 epitope tag, a Bluetongue virus tag (B-tag), a Glu-Glu tag (EE-tag), an HSV epitope tag, a KT3 epitope tag, a Myc epitope tag, a PDZ ligand tag, a polyarginine tag, a polyaspartate tag, a poly cysteine tag, a polyphenylalanine tag, a protein C tag, an SI -tag, an S-tag,Atorney Docket No. 232285-702601a Step-tag, and a VSV-G tag. In some embodiments, a polypeptide described herein comprises a FLAG epitope tag. IN some embodiments, the FLAG epitope tag comprises an amino acid sequence of SEQ ID NO: 250.

[0089] In some embodiments, a polypeptide described herein further comprises a nuclear localization signal (NLS). In some embodiments, aNLS described herein comprises an amino acid sequence of SEQ ID NO: 251 or SEQ ID NO: 498

[0090] In Some embodiments, the NLS, the purification tag, and / or the linker are located N-terminal to the N terminus of the DNA binding domain. In some embodiments, the NLS, the purification tag, and / or the linker are fused or linked to the C terminus of the DNA binding domain. In some embodiments, the NLS, the purification tag, and / or the linker are fused or linked to the N-terminus and the C-terminus of the DNA binding domain. In some embodiments, the NLS / purification tag and the DNA binding domain are linked via a linker described herein.Table 4. Linkers and TagsSEQ ID NO: Functions Sequences231 Linker GGGS232 Linker GGGGS233 Linker GSGSGGSGGS234 Linker KESGSVSSEQLAQFRSLD235 Linker EGKSSGSGSESKST236 Linker GSAGSAAGSGEF237 Linker EAAAK238 Linker EAAAR239 Linker PAPAP240 Linker AEAAAKEAAAKA241 Linker MSRPDPA242 Linker MKIIEQLPSA243 Linker VRHKLKRVGS244 Linker SIVAQLSRPDPA245 Linker GHGTGSTGSGSS246 Linker GSAGSAAGSGEF247 Linker VPFLLEPDNINGKTC248 Linker SGSETPGTSESATPES249 Linker SGGSSGGSSGSETPGTSESATPESSGGSSGGSS250 Epitope Tag DYKDHDGDYKDHDIDYKDDDDK251 Bipartite nuclear KRTADGSEFESPKKKRKVlocalizationsignal498 Nuclear KRPAATKKAGQAKKKKlocalizationsignalAttorney Docket No. 232285-702601

[0091] Further provided herein are nucleic acids comprising a polynucleotide sequence encoding for a polypeptide described herein. In some embodiments, the polynucleotide encoding for the polypeptide comprises DNA. In some embodiments, the polynucleotide encoding for the polypeptide comprises RNA. In some embodiments, the RNA is mRNA.b. Guide nucleic acids

[0092] Provided herein are guide nucleic acids comprising: a spacer region that has complementarity to a target sequence of a gene encoding for an exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and a nuclease binding region, wherein the nuclease binding region comprises a secondary structure that binds to the nuclease. In some embodiments, the nuclease of the polypeptide described herein binds to a guide nucleic acid provided herein. In some embodiments, the nuclease of the polypeptide binds to a target sequence within a gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine as described herein to form a complex. In some embodiments, the complex further comprises a portion of the guide nucleic acid sequence. In some embodiments, the complex further comprises a portion of the guide nucleic acid sequence. For example, the nuclease binding region and the targeting region of the guide nucleic acid form a complex between the nuclease region of a polypeptide provided herein and the regulatory region of the target polynucleotide via the targeting sequence.

[0093] In some embodiments, the guide nucleic acid described herein is a single guide sequence. In some embodiments, the guide nucleic acid comprises ribonucleosides. In some embodiments, the guide nucleic acid comprises ribonucleotides. In some embodiments, the guide nucleic acid comprises a combination of ribonucleosides and deoxyribonucleosides. In some embodiments, the guide nucleic acid comprises a combination of ribonucleotides and deoxyribonucleotides. In some embodiments, the guide polynucleotide comprises nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). The nucleobase of a nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In an embodiment, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base. Guide polynucleotides provided herein can comprise non-naturally occurring sequences or engineered sequences.

[0094] In some embodiments, a guide nucleic acid used in compositions and methods of the disclosure can comprise a spacer sequence that can bind to an endogenous target polynucleotide described herein. The spacer sequence can be, for example, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at leastAttorney Docket No. 232285-70260128, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 nucleotides.

[0095] In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure is at most at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, or at most 40 nucleotides.

[0096] In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure is between about 8 and about 40 nucleotides, between about 10 and about 40 nucleotides, between about 11 and about 40 nucleotides, between about 12 and about 40 nucleotides, between about 13 and about 40 nucleotides, between about 14 and about 40 nucleotides, between about 15 and about 40 nucleotides, between about 16 and about 40 nucleotides, between about 17 and about 40 nucleotides, between about 18 and about 40 nucleotides, between about 19 and about 40 nucleotides, between about 20 and about 40 nucleotides, between about 22 and about 40 nucleotides, between about 24 and about 40 nucleotides, between about 26 and about 40 nucleotides, between about 28 and about 40 nucleotides, between about 30 and about 40 nucleotides, between about 8 and about 30 nucleotides, between about 10 and about 30 nucleotides, between about 11 and about 30 nucleotides, between about 12 and about 30 nucleotides, between about 13 and about 30 nucleotides, between about 14 and about 30 nucleotides, between about 15 and about 30 nucleotides, between about 16 and about 30 nucleotides, between about 17 and about 30 nucleotides, between about 18 and about 30 nucleotides, between about 19 and about 30 nucleotides, between about 20 and about 30 nucleotides, between about 22 and about 30 nucleotides, between about 24 and about 30 nucleotides, between about 26 and about 30 nucleotides, between about 28 and about 30 nucleotides, between about 8 and about 25 nucleotides, between about 10 and about 25 nucleotides, between about 11 and about 25 nucleotides, between about 12 and about 25 nucleotides, between about 13 and about 25 nucleotides, between about 14 and about 25 nucleotides, between about 15 and about 25 nucleotides, between about 16 and about 25 nucleotides, between about 17 and about 25 nucleotides, between about 18 and about 25 nucleotides, between about 19 and about 25 nucleotides, between about 20 and about 25 nucleotides, between about 22 and about 25 nucleotides, between about 24 and about 25 nucleotides, between about 8 and about 20 nucleotides, between about 10 and about 20 nucleotides, between about 11 and about 20Attorney Docket No. 232285-702601nucleotides, between about 12 and about 20 nucleotides, between about 13 and about 20 nucleotides, between about 14 and about 20 nucleotides, between about 15 and about 20 nucleotides, between about 16 and about 20 nucleotides, between about 17 and about 20 nucleotides, between about 18 and about 20 nucleotides, between about 19 and about 20 nucleotides, between about 8 and about 18 nucleotides, between about 10 and about 18 nucleotides, between about 11 and about 18 nucleotides, between about 12 and about 18 nucleotides, between about 13 and about 18 nucleotides, between about 14 and about 18 nucleotides, between about 15 and about 18 nucleotides, between about 16 and about 18 nucleotides, between about 8 and about 16 nucleotides, between about 10 and about 16 nucleotides, between about 11 and about 16 nucleotides, between about 12 and about 16 nucleotides, between about 13 and about 16 nucleotides, between about 14 and about 16 nucleotides, or between about 15 and about 16 nucleotides.

[0097] In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure is from 18 to 25 nucleotides. In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure is 20 nucleotides. In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure is 23 nucleotides. In some embodiments, a guide nucleic acid can be a guide RNA (gRNA) or a part thereof. In some embodiments, a gRNA comprises a CRISPRRNA (crRNA). In some embodiments, a gRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) that binds to the crRNA to form a complex with a Cas protein. In some embodiments, a crRNA described herein comprises a space sequence that specifically binds to a target polynucleotide described herein.

[0098] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 252-313 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 252-313. In someAtorney Docket No. 232285-702601embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 252-313.Table 5. FST Spacer Sequences.SEQ ID NO Sequence252 AAAGCTCCCCACCCCCAAGGAGC253 GGAGGTGCCCGAAATCAAAACGG254 TTTTCTCCCCTTTCACTTCG255 GAGCGGAGGAGAGGCAGCACGGA256 TGGGGCTGTGGGAGGGCGGCCGC257 CCACCTTCTGGAAACCCTTCCCA258 GGGCTGTGGGAGGGCGGCCGCGA259 GGGCACCTCCGAAAGATAATTGG260 CAAAAGTCTCTTTGGAGCGG261 TTTGCTTCCGACTAGTCCAGACG262 AAGCGCGGCTGCGCCCGGCT263 CGCGGCTCCTTGGGGGTGGGGAG264 CAGAAGGTGGGAAATGTCACCTG265 ACTTCGCGGCGTCTGGAGTCGGA266 CAGTGTGTGGGGCTGTGGGAGGG267 AAAAGTTCAGTGTGAATCAG268 CGTAGGAGCGAGAGGGAGGGGGA269 AAGCGCGGCTGCGCCCGGCTTCT270 CGTAGGAGCGAGAGGGAGGG271 TTGGGGGTGGGGAGCTTTCAAAA272 GGCAGCCCGGGGCTCTTCGG273 TGTCTGGGTCTGTGAGTGCCTGA274 CAAACGGCCGAAGAGCCCCG275 CAAGGAGCCGCGCACACCCTCGC276 ACTTCGCGGCGTCTGGAGTC277 GAGCGGAGGAGAGGCAGCAC278 ACACCTCAGGCCCGGTCTCC279 CCACCCCCAAGGAGCCGCGCACA280 CAGCCACCCCCAGCCCAGAGGTG281 TCTCCGTGCTGCCTCTCCTC282 ACCCCCAAGGAGCCGCGCACACC283 GGAGGTGCCCGAAATCAAAA284 TTTTCTCCCCTTTCACTTCGCGG285 TTTGCTTCCGACTAGTCCAG286 CAGCCACCCCCAGCCCAGAG287 ATTTCGGGCACCTCCGAAAGATA288 AATCTAGACGGGGGTCTCCG289 AAAGCTCCCCACCCCCAAGG290 GCGCACACCCTCGCTCGCGGCCG291CTTCCGACTAGTCCAGACGAAGGAtorney Docket No. 232285-702601292 TGTCTGGGTCTGTGAGTGCC293 TCCCCTTTCACTTCGCGGCG294 CCTCCTGCTGCTGCTGCTCTGCC295 ATTTCGGGCACCTCCGAAAG296 AATCTAGACGGGGGTCTCCGCGG297 CTCGCTCGCGGCCGCCCTCCCAC298 GGGCACCTCCGAAAGATAAT299 GAAAGCTCCCCACCCCCAAGGAG300 CCACCTTCTGGAAACCCTTC301 ACACCTCAGGCCCGGTCTCCCCG302 CGGTGGGCGGTCTCCCAGTGTGT303 CTTCCGACTAGTCCAGACGA304 AAAAGTTCAGTGTGAATCAGGTG305 TCTCCGTGCTGCCTCTCCTCCGC306 TCCCCTTTCACTTCGCGGCGTCT307 CAGAAGGTGGGAAATGTCAC308 CGGTGGGCGGTCTCCCAGTG309 CAAACGGCCGAAGAGCCCCGGGC310 GGCAGCCCGGGGCTCTTCGGCCG311 CAAAAGTCTCTTTGGAGCGGAGG312 AAGGAGCCGCGCACACCCTCGCT313GGAGGGCGGCCGCGAGCGAGGGT

[0099] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 314-343 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 314-343. In some embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 314-343.Table 6. FGF21 Guide SequencesSEQ ID NO Guide Name FGF21 spacer SequencesAtorney Docket No. 232285-702601314 FGF21 20-01 AGCCACCCCTCCCCCCAGGT315 FGF21 20-02 ACCAGAGAAGCCATCCCAGG316 FGF21 20-03 AGGAGCACCTGCCTGGGCCT317 FGF21 20-04 TTTCACCAGAGAAGCCATCC318 FGF21 20-05 CTGGAGCCCCTAAGGGTGCA319 FGF21 20-06 GGGGGAGGGCGTGATCTGAC320 FGF21 20-07 CAGGAGCCCAGCACCCCTCC321 FGF21 20-08 TGCGCCCCATCTGAATTTCT322 FGF21 20-09 TCTTGTTCTGCGTGATCTGC323 FGF21 20 10 ATACAGCAATTGCCGCCTTG324 FGF21 20 11 TAAGTTTGGGGGGAGGGCGT325 FGF21 23-01 AGCCACCCCTCCCCCCAGGTTAT326 FGF21 23-02 GGGGGAGGGCGTGATCTGACACT327 FGF21 23-03 TACCCAGACAGGCCCGCCCAATC328 FGF21 23-04 CAGGAGCCCAGCACCCCTCCTCC329 FGF21 23-05 ACCCATTTGGCAGGAGCTACAGA330 FGF21 23-06 GGATAGATGCAGAAGCTCCAGAA331 FGF21 23-07 ACCAGAGAAGCCATCCCAGGCCC332 FGF21 23-08 TGCGCCCCATCTGAATTTCTAAG333 FGF21 23-09 GCAGGAGCTACAGAATTCCATCG334 FGF21 23-10 GAATTGGTGCCAGATTCCAGTTG335 FGF21 23-11 GGCTGGATCCTCAGCTGACAGAA336 FGF21 23-12 ACCATCATCTCCCCAGGCTCGGG337 FGF21 23-13 CTGGAGCCCCTAAGGGTGCAAGG338 FGF21 23-14 AGGAGCACCTGCCTGGGCCTGGG339 FGF21 23-15 ACCTGGGTCCCAGCTAACCCGAT340 FGF21 23-16 ATACAGCAATTGCCGCCTTGCTG341 FGF21 23-17 AATGGAGGGCCAGACTCCTGGGT342 FGF21 23-18 TTTCACCAGAGAAGCCATCCCAG343 FGF21 23-19 GTCTGGACTCCTGGGTCTGAGGG

[0100] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 344-403 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identityAtorney Docket No. 232285-702601to a polynucleotide sequence selected from any one of SEQ ID NOS: 344-403. In some embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 344-403.Table 7. Apelin Guide SequencesSEQ ID NO Guide Name Apelin Spacer Sequences344 APLN 20-01 CCCAACTCCACACAGTCAAG345 APLN_20-02 TTGTTCCCTGGAGCTGTCCT346 APLN_20-03 CTGACAGCTGTTAAGGAAGC347 APLN 20-04 CAGAGACCTGAAGGATTTCA348 APLN_20-05 GAGAGATCCAGAAGAGTGAG349 APLN 20-06 ATATGTGTTGAGTGCCACCT350 APLN_20-07 CCTTTCCAGACTCGATTGTT351 APLN 20-08 CTTGAAGTGTTAAGTCCAGA352 APLN 20-09 GAAGCGGGTCCTGAAGTTGT353 APLN 20-10 GTCAAGACAACCAAACACTG354 APLN 20-11 TGTAAATCTGGACTTAACAC355 APLN_20-12 GTTGTCTTGACCAAACCCTT356 APLN 20-13 CGTGTACTTGCGATTCCGCT357 APLN 20-14 AGGTGCTGCTGCAAAGAATG358 APLN 20-15 TGGCACACACGCACCCTGTC359 APLN 20-16 CACAAAAGATACATTGGACA360 APLN_20-17 AAATCCTTCAGGTCTCTGCA361 APLN 20-18 AGCAAGCCAGGCAGTTTGCT362 APLN 20-19 GGGCGGTGGAGGGGCACACT363 APLN_20-20 CAGCAGCACCTCAAATCCCA364 APLN_20-21 CAGACTCGATTGTTAGCTGC365 APLN_20-22 AATTGGGGGTGACTGCCTAG366 APLN_20-23 TCACCTTGCTTCCTTAACAG367 APLN_20-24 AAACACAGGAAGCATTTGGG368 APLN_20-25 AACTGGCTTTGAATTGGGGG369 APLN_20-26 GATTCAGTCTTTTAATATGT370 APLN_20-27 TTGGCCTCCGGGCGCCCGAC371 APLN_20-28 TTGTGGGGCCACCTCGGGGT372 APLN_20-29 TAGCTTGTGGCTTGGCCGCG373 APLN_20-30 TTAGCGGCTGGCTGCTCGGC374 APLN 23-01 GAGAGATCCAGAAGAGTGAGAAG375 APLN_23-02 CCCAACTCCACACAGTCAAGGCT376 APLN_23-03 CAGAGACCTGAAGGATTTCAAAC377 APLN 23-04 CAGCAGCACCTCAAATCCCATCG378 APLN_23-05 GTCAAGACAACCAAACACTGGCC379 APLN 23-06 GGGCGGTGGAGGGGCACACTTAA380 APLN_23-07 TGGCACACACGCACCCTGTCCAA381 APLN_23-08 AAATCCTTCAGGTCTCTGCAAAC382APLN 23-09 CACAAAAGATACATTGGACAGGGAttorney Docket No. 232285-702601383 APLN 23-10 AGGTGCTGCTGCAAAGAATGAAT384 APLN 23-11 AAATGTTGCAAAAATCCTATATA385 APLN_23-12 GAAGCGGGTCCTGAAGTTGTTTG386 APLN_23-13 CAGACTCGATTGTTAGCTGCTTC387 APLN 23-14 AGCAAGCCAGGCAGTTTGCTTGA388 APLN_23-15 TGTAAATCTGGACTTAACACTTC389 APLN 23-16 AATATGCCATGGTCTCTCATGTC390 APLN_23-17 CTGACAGCTGTTAAGGAAGCAAG391 APLN 23-18 GTTGTCTTGACCAAACCCTTGAA392 APLN 23-19 TCACCTTGCTTCCTTAACAGCTG393 APLN_23-20 AAACACAGGAAGCATTTGGGGCG394 APLN_23-21 CTGCCTAAATTCAGGTAGCAGGA395 APLN_23-22 ATATGTGTTGAGTGCCACCTATG396 APLN_23-23 AATTGGGGGTGACTGCCTAGGCT397 APLN_23-24 TAGCTTGTGGCTTGGCCGCGAGC398 APLN_23-25 CGTGTACTTGCGATTCCGCTTTA399 APLN_23-26 TTGTGGGGCCACCTCGGGGTTCC400 APLN_23-27 TTGTTCCCTGGAGCTGTCCTCAT401 APLN_23-28 CCTTTCCAGACTCGATTGTTAGC402 APLN_23-29 TTGGCCTCCGGGCGCCCGACCTC403APLN_23-30 CTTGAAGTGTTAAGTCCAGATTT

[0101] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 404-430 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 404-430. In some embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 404-430.Table 8. FNDC5 Guide SequencesSEQ ID NO Guide Name FNDC5 Spacer Sequences404 FNDC5 20-01 TGGCTGGGGAGGAGCTGGCC405 FNDC5 20-02 ATCCCCATAGTGAGGGCAGT406FNDC5 20-03 CAGGGTTGCCCTGAGAATTAAttorney Docket No. 232285-702601407 FNDC5 20-04 CCAGGGTAGAGAGAGGACAA408 FNDC5 20-05 AACACAGGCTGATCTGACTC409 FNDC5 20-06 GGAGTCAGATCAGCCTGTGT410 FNDC5 20-07 CAGTGGGGTTGGGAAGAGGC411 FNDC5 20-08 TGCCCCCACCCCTAAGCGCG412 FNDC5 20-09 TGCCCTTTCCCAGGGTAGAG413 FNDC5 20-10 TCTGGCTGAGATGAGGGAAG414 FNDC5 20-11 CACAGGAGGAGCCTGCAGCT415 FNDC5 20-12 CATTATGTGGCTTAATTCTC416 FNDC5 20-13 CTTCATTTTCATCCCCATAG417 FNDC5 20-14 TTTGCATTATGTGGCTTAAT418 FNDC5 23-01 GGAGTCAGATCAGCCTGTGTTCA419 FNDC5 23-02 TGCCCCCACCCCTAAGCGCGCCT420 FNDC5 23-03 CCAGGGTAGAGAGAGGACAAGTG421 FNDC5 23-04 CACAGGAGGAGCCTGCAGCTTGG422 FNDC5 23-05 AACACAGGCTGATCTGACTCCCA423 FNDC5 23-06 CAGGGTTGCCCTGAGAATTAAGC424 FNDC5 23-07 TGCCCTTTCCCAGGGTAGAGAGA425 FNDC5 23-08 ATCCCCATAGTGAGGGCAGTAAT426 FNDC5 23-09 TGGCTGGGGAGGAGCTGGCCCCG427 FNDC5 23-10 CAGTGGGGTTGGGAAGAGGCTGC428 FNDC5 23-11 TCTGGCTGAGATGAGGGAAGGGG429 FNDC5 23-12 CATTATGTGGCTTAATTCTCAGG430FNDC5 23-13 CTTCATTTTCATCCCCATAGTGA

[0102] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 431-457 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 431-457. In some embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 431-457.Table 9. HSPA1A Guide SequencesAtorney Docket No. 232285-702601SEQ ID NO Guide Name HSPA1A Spacer Sequences431 HSPA1A 20 01 GAGAGTGACTCCCGTTGTCC432 HSPA1A 20 02 GCCTCCCGTCCTGCCCCCCA433 HSPA1A 20 03 CCTTCTGAGCCAATCACCGA434 HSPA1A 20 04 GCCATGCCGGTTCCCTGCTC435 HSPA1A 20 05 CAGCCCCCAATCTCAGAGCG436 HSPA1A 20 06 TGTTGACACCGCCCGGCCAC437 HSPA1A 20 07 CGGCGTCCGGAAGGACCGAG438 HSPA1A 20 08 TAAGTCGTCACGGAGACCCG439 HSPA1A 20 09 ATTGTGGGGGCTTGCTGGGC440 HSPA1A 23 01 AGACGGCTCCAACTCAGTAATCT441 HSPA1A 23 02 GGAAAGTACTGGAAAGTTCCCTA442 HSPA1A 23 03 CAGCCCCCAATCTCAGAGCGGAG443 HSPA1A 23 04 CCAAACAAGGACCTACTGAGCCT444 HSPA1A 23 05 GAAACGTCCAGATTACGCAGCCC445 HSPA1A 23 06 GCCATGCCGGTTCCCTGCTCTCT446 HSPA1A 23 07 CGGCGTCCGGAAGGACCGAGCTC447 HSPA1A 23 08 ATATTGAGGGAGCCCCCACCTAC448 HSPA1A 23 09 CAGGTGATCAACGACGGAGACAA449 HSPA1A 23 10 AGGTTCACAATCAATCAGATCCC450 HSPA1A 23 11 GAGAGTGACTCCCGTTGTCCCAA451 HSPA1A 23 12 CAAACTGGCCCATGAGGTCAGAG452 HSPA1A 23 13 GCCTCCCGTCCTGCCCCCCAGCC453 HSPA1A 23 14 CAAACTGAAGCGAAGGCGTCGGG454 HSPA1A 23 15 GAAAAAGATTACTGAGTTGGAGC455 HSPA1A 23 16 CAGTACTTTCCCAAACAAGGACC456 HSPA1A 23 17 TGTTGACACCGCCCGGCCACGTT457 HSPA1A 23 18 CCTTCTGAGCCAATCACCGAGCT

[0103] In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence (e.g., a consecutive polynucleotide sequence) having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 458-487 In some embodiments, a spacer sequence of gRNA as described herein comprises a polynucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a polynucleotide sequence selected from any one of SEQ ID NOS: 458-487. In someAtorney Docket No. 232285-702601embodiments, a spacer sequence of the crRNA as described herein comprises a polynucleotide sequence of any one of SEQ ID NOS: 458-487.Table 10. NRTN Guide SequencesSEQ ID NO Guide Name HSPA1A Spacer Sequences458 NRTN 01 CCCGGCCCTCCCCGCCCTCC459 NRTN_02 GGCTCCCCTGCCCGTGAGCC460 NRTN_03 GGTCACCCCGCGGGGCGGGG461 NRTN 04 GGAGGTCTGGCAGAAAGGTC462 NRTN_05 CCATCCTCACTCCGGGGACC463 NRTN 06 TGCCAGACCTCCCAAACCTG464 NRTN_07 GGCAAGAGGAGCCGTCAGGG465 NRTN 08 GTGAAGGGACTTCTTGGCGC466 NRTN 09 TCCCCCGGCCCCGGGGACTC467 NRTN 10 CGTCTCAGTTCCCTGGAAGA468 NRTN 11 AGGGGGGCGCCGCTCTGGGG469 NRTN_12 TTGGGAGGGGGCTTCTTCAG470 NRTN 13 TCGCTCCTAGCTCCCGCCGG471 NRTN 14 GACGGCTTTCTCCCCCGGCC472 NRTN_23-01 GGAGGTCTGGCAGAAAGGTCAGA473 NRTN_23-02 TGCCAGACCTCCCAAACCTGTCC474 NRTN_23-03 GGCTCCCCTGCCCGTGAGCCTTG475 NRTN_23-04 GGCAAGAGGAGCCGTCAGGGGTC476 NRTN_23-05 TGGGGACGTGCATATGTTGGGGT477 NRTN_23-06 CCCGGCCCTCCCCGCCCTCCCCG478 NRTN_23-07 TCGCTCCCGCCGCTGAGGTTCTT479 NRTN_23-08 CCATCCTCACTCCGGGGACCCCT480 NRTN_23-09 TCCCCCGGCCCCGGGGACTCCGA481 NRTN_23-10 AGGGGGGCGCCGCTCTGGGGGCT482 NRTN 23-11 TTGGGAGGGGGCTTCTTCAGCTT483 NRTN_23-12 GTGAAGGGACTTCTTGGCGCCTT484 NRTN_23-13 TCGCTCCTAGCTCCCGCCGGCGC485 NRTN 23-14 TGATTCCAGCAGCCTGGGGGTTC486 NRTN_23-15 CAGGCGCTTTGTGGGGACGTGCA487 NRTN 23-16 GACGGCTTTCTCCCCCGGCCCCG

[0104] The guide nucleic acids provided herein can be produced, for example, by phosphoramidite chemical synthesis, in-vitro transcription (IVT) techniques, M13 bacteriophage methods, DNA isolation, RNA isolation, ligation, tagmentation, and combinations thereof. Furthermore, guide nucleic acids can be generated and expressed by transducing or transfecting cells with a plasmid DNA or a vector that comprises a polynucleotide encoding for an expression cassette.Attorney Docket No. 232285-702601

[0105] In some embodiments, the guide nucleic acids comprise a 5' cap. Free 5' hydroxyl groups can be capped by acetylation. In some embodiments, the guide nucleic acids comprise a 3' polyadenylated tail. In some embodiments, the guide nucleic acids comprise a polynucleotide linker or spacer regions.

[0106] In some embodiments, the guide nucleic acid comprises a modified nucleobase, a modified nucleotide, or a modified nucleoside. The modified nucleosides and modified nucleotides described herein, which can be incorporated into a target nucleic acid, can include a modified nucleobase. In some embodiments, the nucleobase or the nucleotide provided herein is chemically modified. Nucleobases and nucleotides provided herein can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into a target nucleic acid.

[0107] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m C), N4-acetyl-cytidine (act), 5- formylcytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5 -methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine (k C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0- methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f 5Cm), N4,N4,2'-0-trimethyl-cytidine (m4 2Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0108] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g, 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyl- adenosine (i A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosineAtorney Docket No. 232285-702601(ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m6Am), N6-Methyl-2'-deoxyadenosine, N6,N6,2'-0-trimethyl-adenosine (m62Am), l,2'-0-dimethyl-adenosine (i Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8- azidoadenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl) - adeno sine.

[0109] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1-methyl-inosine (iVl), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (o2yW), hydroxy wybuto sine (OHyW), undermodified hydroxy wybuto sine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQO, archaeosine (G+), 7-deaza-8 -aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1 -methyl-guanosine (m'G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2 ,7G), N2, N2,7-dimethyl-guanosine (m 2 ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0-methyl-guanosine (m 2"Gm), N2,N2-dimethyl-2'-0-methyl- guano sine (m 22Gm), l-methyl-2'-0-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl-guanosine (m",7Gm), 2'-0-methyl- inosine (Im), l,2'-0-dimethyl-inosine (m'lm), 06-phenyl-2'-deoxyinosine, 2'-0-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methyl- guanosine, 06-Methyl-2'-deoxy guanosine, Z -F-ara-guanosine, and 2'-F -guanosine.

[0110] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (y), pyridin- 4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (emo 5 U), uridineAttorney Docket No. 232285-7026015-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio- uridine (mcm5s2U), 5-aminomethyl-2 -thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno- uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (xcm5U), 1-taurinom ethyl -pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (rp'\| / ), 5-methyl-2- thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m xi / ), 4-thio- 1-methyl-pseudouridine, 3- methyl-pseudouridine (m y), 2-thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio- 1 -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy -pseudouridine, 4-methoxy-2-thio-pseudouridine, N 1-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp 3 U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 y), 5- (isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine(inm5s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-dimethyl-uridine (m5Um), 2'-0- methyl-pseudouridine (yrp), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mem 5Um), 5-carbamoylmethyl-2'-0-methyl-uridine (ncm 5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm 5 Um), 3,2'-0-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'- F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E- propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.[OHl] In some embodiments, the modified nucleobase is a modified thymine. In some embodiments, the modified nucleoside is a modified thymidine. Non-limiting examples of modified thymine and thymidine include: 6-(azo)thymine, 3 '-azido-3 '-deoxythymidine, 2', 3'-didehydro-2',3'-dideoxythymidine; l-(2,3-dideoxy-beta-D-glyceropent-2-enofuranosyl)thymine, 3-(2-chloroethyl)thymidine, 3 '-fluoro-3 '-deoxythymidine, P-L-2'-deoxythymidine, thieno[3,4-d]-pyrimidine T-mimic deoxynucleoside, l-(2-Deoxy-P-D-threo-pentofuranosyl)thymine, 5-ethynyl-2 '-deoxyuridine, bromodeoxyuridine, tritiated thymidine, 5-chlorodeoxyuridine (CldU), 5-iododeoxyuridine (IdU), 2-thiothymidine triphosphate, 5-(a-tert-butylortho-bromobenzyloxy) methyl-2'-deoxyuridine, 5-(a-methylbenzyloxy)methyluracil, and 5-ethyldeoxyuridine.Atorney Docket No. 232285-702601

[0112] Nucleic acids can be modified using various chemistries and modifications. In some embodiments, regular internucleosidic linkages between nucleotides can be altered by mono- or di-thioation of the phosphodiester bonds to yield phosphorothioate esters or phosphorodithioate esters, respectively. Other modifications of the internucleosidic linkages can include amidation or peptide linkers. A ribose sugar can be modified by substitution of the 2'-0 domain with a lower alkyl (Cl -4, such as 2'-0-Me), alkenyl (C2-4), alkynyl (C2-4), methoxy ethyl (2'-M0E), or other substituent. In some cases, substituents of the 2' OH group can comprise a methyl, methoxyethyl or 3,3 '-dimethylallyl group. In some cases, locked nucleic acid sequences (LNAs), comprising a 2'-4' intramolecular bridge (such as a methylene bridge between the 2' oxygen and 4' carbon) linkage inside the ribose ring, can be applied. Purine nucleobases and / or pyrimidine nucleobases can be modified to alter their properties, for example by amination or deamination of the heterocyclic rings. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the guide nucleic acids can contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. Several suitable methods can be used to produce nucleic acid molecules and nucleic acids containing modified nucleobases. For example, a guide nucleic acid that contains modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) a DNA that encodes for a guide RNA using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and the like, or mutants of these polymerases which allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction can contain nucleotides and modified nucleotides, and other components that support the activity of the selected polymerase, such as a suitable buffer, and suitable salts. The incorporation of nucleotide analogs into a guide nucleic acid may be engineered, for example, to alter the stability of such RNA / DNA molecules or to increase resistance against RNases.

[0113] In some embodiments, the guide nucleic acid comprises a secondary structure or a tertiary structure. In some embodiments, the secondary structure comprises a bulge, a stem, a stem loop, a loop, a tetraloop, a hairpin, a wobble base pair, a pseudoknot, a nexus, or a combination thereof. In some embodiments, the bulge, the stem loop, or the hairpin comprise an unpaired region of nucleotides within a nucleic acid duplex.c. Target polynucleotide

[0114] In some embodiments, the systems and methods provided herein can target at least one target polynucleotide within an exerkine gene or a regulatory region of an exerkine gene. In some embodiments, the systems and methods provided herein target the transcriptional start site (TSS),Attorney Docket No. 232285-702601CpG island, or other regulatory element site of an exerkine gene. In some embodiments, the systems and methods provided herein target one of the exons of an exerkine gene. In some embodiments, the systems and methods provided herein target Exon 1 of an exerkine gene. In some embodiments, the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1A (HSPA1A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins. In some embodiments, the exerkine is FST. In some embodiments, the target polynucleotide is within a DNA comprising a sequence or complementary to the following sequence:TGTATAAGGACTATACCATTTCTCTGAATAAAGAAGAAATAAATTCAGCTTTGGAACTCTGCCA TTGAGTGGCGGCTCCTGCAAAGGGGACAGTTTGCTATTAATTGTTAATGTCGTCTGCCATTAGA GGGCACTAAACAACTCTACATTAATGAGTTGGAGGGAAGAGAGAAAAGGAGACTGGAAAAGAAA GAGAGGGAGAAAAGGGAGGGAGAGTACGGGTAGAGTCTGGGTAGAGGGAGGGAAGGAGAGAAGA GGGGAAGGAGTAGGGGAAAGAGAGCAGAGGGAATGAGTTGGAGGACTGGAGGGAAAGGGAGAAG GGGAGAGAGACAGCGAGACAGAAACAGACAGAATGATTCTGGAAAGATTAAGAAGGATGTGGAG CCCCCACAGCCGTCTGAAGTGAGCAAAATCCAGTCTCCCCCGGGCCGCCCGAGCCCCTCCCCTC CCGTCCTCATGACGGTAATTATAAAGCGAAAAGCGGCGGCGAGGTCACCCGCTTTTACCAGCCT GCTGCACCGAATGCGCGCGGTGCAGGTGCGGCGGCCGCGGGGCTTTGTGATTCATGCCCACTTT CAAAGGGGGGCTTGGGCCCGTCTGAGAGGCACCCAAACAACTGTCTTCTAATATTAGCACGGCT GTATTTCGGGATCTATTATAGTCTGTCCAGACAGATCCCATTGTAATGGGATCTTTTATTAAAA GATTAGCACTATCTCCTGCAGTTGGTGGAAAAAAATCTGTTATCACTGAGTTATTTGCAGTTGT GGAGGGGGGAAGGTTGACGGAAGATGGGGGGTGGGAGGGCTAGAGGAGGACTGGGGGAGGGTCT GGCGGGGGGAGGGCAGTTGCAGGGGGGGAAGAAGGGCAGATTTGAAATTCGTAGAACAAATCCC AGTGACCTGCGAATGTCCAAGAATTTTGTCTGCATACTTCGCTATCTGTTGTCCTTCGGGCTTC AGATGCGAAAAGAACAAATTTAAAAGGCAGCTTTGGAATAAACAATATAGCATTTTCCGTGAAG TAATCGTTTTATATAAAAGGAAATATCGCCTCCTCAGTTTCATTCAGCGGTGCCAAAAATGTTA TAAATGAGAGACCCGCCAACTTTCAGCGAAGAATGCCAGGCACTGTACTTAATAAACTGAGGCT ACAGAAGTTCATTATGGATGTTGCAATCTTTTTTATTCCGCGAAAAGAGAGAAGACGAGACAGA AAGAGAGAGAGATTGGAGCCGAGGAGACTGAGAGACAGACAGAGGCACACAGGACAGAAACTGG GGAGTCTCCAGGCGGGAGAGGAAGGGGGGGCCAGACCGCCTACGTCGGCGCCCCCGCTCCGGGC TCCGACTCCAGACGCCGCGAAGTGAAAGGGGAGAAAAGAAAGGGAGAGGGCGAGGCTGTGCCGCAttorney Docket No. 232285-702601 GGGGAGACCGGGCCTGAGGTGTTAAACATTTTTGTTTGCTTCCGACTAGTCCAGACGAAGGGCC GCGTCTCGGTAGCGCTCTGCCAGGGTGGAAGGTGCCGGGGCCGGGGTTCCTAGCAACACCTCTG GGCTGGGGGTGGCTGCAAAGTCAGGCACTCACAGACCCAGACACAAAACCTCGCGGGTCCCGCG CCCAGGCTGCGGGTGCCCGGAACCGCCGCGAGGCCGGCGCGCTCCGACCCGACCCGGGGCGGGA TATTTGGGCAGCCCGGGGCTCTTCGGCCGTTTGCAAAAGTCTCTTTGGAGCGGAGGAGAGGCAG CACGGAGACAAACTCCCGGGTTCCCCCCGCCACCGCCTCCAGCGCCCCCACCGCGCCCTCCCTC TCACACTCGCGCGCGCGCGCACACACACTCACACACACACTCACACACACACCCGCCACCCCGG GCGCGCCGGCGCTGCCGGCGAGCGGCGGCGAGCAGGACTTGAAGTGGGTGTTCTTCCCCACTCC CCACCCCCGACGCGTAGCCCCCAACCCCCGCCCCGGTCGCCTTCCCCCTCCCTCTCGCTCCTAC GCAAATAAGAACTCGACGATTCCCTTCCAGCCGTTTTGATTTCGGGCACCTCCGAAAGATAATT GGGAAGGGTTTCCAGAAGGTGGGAAATGTCACCTGATTCACACTGAACTTTTGAAAGCTCCCCA CCCCCAAGGAGCCGCGCACACCCTCGCTCGCGGCCGCCCTCCCACAGCCCCACACACTGGGAGA CCGCCCACCGCAAACCGCGGAGACCCCCGTCTAGATTTAAAGCGCGGCTGCGCCCGGCTTCTGA CGTCCATTGAATCGCGCGGGCGGCCGGCGGCGAGCGCGGGGCTGCGCCGGGATCGCTGCGCCCT CCGCCGCTGGCCTCTGCGACGCGCGCCGCTCGCCCGAGCCACCCGCCGCCGCGCCGGCTCCCCG CGCCGCTGCGCTCCTCGCCCCGCGCCTGCCCCCAGGATGGTCCGCGCGAGGCACCAGCCGGGTG GGCTTTGCCTCCTGCTGCTGCTGCTCTGCCAGTTCATGGAGGACCGCAGTGCCCAGGGTAAGCG AGTGGGGATGCGCTGGGGAGGCTTGGCTGAGGACAGGGGGGTCGACTTTTCTGTGGTCTCCACT TGGTCTGTTCTGAGCAACGCTGCTCTCGGAAGATGTTGAACCAACTTGGGGACTCCGGGATGGA GAGGAGTGGGAA (SEQ ID NO: 488).

[0115] In some embodiments, the systems and methods provided herein can target at least one target polynucleotide within an exerkine gene or a regulatory region thereof. In some embodiments, the regulatory region is endogenous to a cell, for example, present in the cell’s genome, or endogenous to a subject, for example, present in the subject’s genome. In some embodiments, the at least one target nucleic acid comprises at least or up to about 1, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 6, at least or up to about 7, at least or up to about 8, at least or up to about 9, at least or up to about 10, at least or up to about 15, or at least or up to about 20 target polynucleotide sequence(s) within the specific regulatory region. In some embodiments, the at least one target polynucleotide can have a length of at least or up to about 6 nucleobases, at least or up to about 8 nucleobases, at least or up to about 10 nucleobases, at least or up to about 12 nucleobases, at least or up to about 16 nucleobases, at least or up to about 18 nucleobases, at least or up to about 20 nucleobases, at least or up to about 22 nucleobases, at least or up to about 24 nucleobases, at least or up to about 26 nucleobases, at least or up to about 28 nucleobases, at least or up to about 30 nucleobases, at least or up to about 32 nucleobases, at least or up to about 34 nucleobases, at least or up to aboutAtorney Docket No. 232285-70260136 nucleobases, at least or up to about 38 nucleobases, at least or up to about 40 nucleobases, at least or up to about 45 nucleobases, or at least or up to about 50 nucleobases.

[0116] In some embodiments, at least a portion of a positive-sense strand (+) of the target polynucleotide can be targeted. In some embodiments, at least a portion of a negative- sense strand (-) of the target polynucleotide can be targeted.

[0117] In some embodiments, binding of the nuclease or the fragments thereof is mediated by full or partial complementarity of the targeting region of the guide nucleic acid provided herein. In some embodiments, the polypeptide binds to a protospacer-adjacent motif (PAM) sequence. Enzymes and proteins from different bacteria can recognize different sequence motifs or PAMs. In some embodiments, a polypeptide described herein binds to the guide nucleic acid that binds to the target polynucleotide within 5 nucleobases of a PAM sequence, 10 nucleobases of a PAM sequence, 15 nucleobases of a PAM sequence, or 20 nucleobases of a PAM sequence. In some embodiment, the polypeptide described herein binds to the guide nucleic acid that binds to the target polynucleotide within 4 nucleobases of a PAM sequence.

[0118] In some embodiments, the target polynucleotide is part of a dsDNA. In some embodiments, dsDNA-targeting specificity is determined, at least in part, by two parameters: the gRNA spacer targeting a protospacer in the target dsDNA (the sequence in the target dsDNA corresponding to the gRNA spacer on the non-complementary DNA strand) and a short sequence, the PAM, located immediately 5' (upstream) of the protospacer on the non-complementary DNA strand. In some embodiments, the PAM comprises a sequence of 5'-TTTV-3', where V is G, A, C. In some embodiments, the PAM comprises a sequence selected from the sequences in listed Table 11. In some embodiments, the PAM is 5'-TTTG-3', 5'-TTTC-3', 5'-TTTA-3', or 5'-TTTT-3'.Table 11. PAM Sequences.PAM SequencesTTTG _TTTC _TTTA _TTTT _CACC _CTCC _CCCC _TGTG _AGCCDelivery vehicles and vectorsAtorney Docket No. 232285-702601

[0119] Provided herein are compositions comprising a system, a polypeptide, a polynucleotide, or a guide nucleic acid described herein and a delivery vehicle. The compositions and cells provided herein can be delivered to a target cell, tissue, organ, or subject by any suitable means.

[0120] In some embodiments the guide nucleic acids, the polypeptides or any polynucleotide encoding for a polypeptide provided herein can be admixed with a delivery vehicle that permits delivery of the system to the target nucleic acid sequence within a cell, tissue, or subject. In some embodiments, a polypeptide described herein and a guide nucleic acid described herein form a ribonucleoprotein (RNP) complex.

[0121] In some embodiments, the delivery vehicle comprises a vector, a lipid, a nanoparticle, a plasmid, a virus, a liposome, an extracellular vesicle, or a combination thereof. Additional nonlimiting examples of delivery vehicles include an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, a nanoparticle, a polymer, microcapsules, recombinant cells, cell culture medium, blood, or serum. Specific types of delivery vehicles that can be used in a composition provided herein are further described below.

[0122] In some embodiments, the delivery vehicle is a liposome. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core. Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles can also be used as a delivery vehicle.

[0123] In some embodiments, the delivery vehicle is a phospholipid. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposomes form. Physical characteristics of liposomes dependAtorney Docket No. 232285-702601on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a tightly packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.

[0124] In some embodiments, the delivery vehicle is a nanoparticle. Nanoparticle carriers that specifically target a tissue provided herein may also be used as a pharmaceutically acceptable carrier. In some embodiments, the nanoparticle is a gold nanoparticle, a platinum nanoparticle, an iron-oxide nanoparticle, a lipid nanoparticle, a selenium nanoparticle, a tumor-targeting glycol chitosan nanoparticle (CNP), a cathepsin B sensitive nanoparticle, a hyaluronic acid nanoparticle, a paramagnetic nanoparticle, or a polymeric nanoparticle. In some embodiments, the delivery vehicle is a lipid nanoparticle.

[0125] Provided herein are compositions comprising a lipid nanoparticle comprising: a system provided herein, a set of polynucleotides encoding the system provided herein, a vector provided herein, an engineered protein provided herein, a guide nucleic acid or a portion thereof, or any composition provided herein. In some embodiments, the lipid nanoparticle is a solid lipid nanoparticle (SLN) or a nanostructured lipid carrier (NLC).

[0126] The lipid nanoparticles provided herein can comprise cationic lipids, ionizable lipids, a mixture of lipids, zwitterionic lipids, and / or phospholipids. In some embodiments, the lipid nanoparticle comprises a cationic lipid selected from the group consisting of: 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1 ,2-dioleoyl-ST?-glycero-3- phosphoethanol amine (DOPE), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Dimethyldioctadecylammonium bromide (DDAB), and Ethylphosphatidylcholine (ePC). In some embodiments, the lipid nanoparticle comprises an ionizable lipid selected from the group consisting of: 2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide (DOGS; Transfectam), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), DC-Cholesterol and N4-cholesteryl-spermine (GL67), 9Z,12Z -octadecadienoic acid, 3 -[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), [(4-Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), bis(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)nonadecanedioate (Lipid A9), 5-(dimethylamino)-pentanoic acid, (6Z)-l,2-di-(4Z)-4-decen-l-yl-6-dodecen-l-yl ester (LipidAtorney Docket No. 232285-702601CL1), or 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5). In some embodiments, the lipid nanoparticle comprises a phosphatidylcholine. In some embodiments, the lipid nanoparticle comprises cholesterol or a cholesterol analog. In some embodiments, the lipid nanoparticle comprises a cholesterol analog selected from the group consisting of: P-sitosterol, Vitamin D3, Vitamin D2, calcipotriol, stigmasterol, betulin, lupeol, ursolic acid, oleanolic acid, stigmastanol, campesterol, fucosterol, brassicasterol, and ergosterol. In some embodiments, the lipid nanoparticle comprises polyethylene glycol (PEG). In some embodiments, the PEG is l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (PEG2000-DMG) or l,2-distearoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (PEG2000-DSG). In some embodiments, the lipid nanoparticle comprises N-acetylgalactosamine (GalNAc). In some embodiments, the GalNAc is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[tris-GalNAc-GABA-(polyethylene glycol)-2000] (Tri-GalNAc-PEG2000-DSPE).

[0127] In some embodiments, the composition comprising the lipid nanoparticle is in the form of a emulsion. In some embodiments, the composition comprising the lipid nanoparticle is in the form of a liquid. In some embodiments, the composition comprising the lipid nanoparticle is in the form of a gel. In some embodiments, the composition comprising the lipid nanoparticle is in the form of a solid. In some embodiments, the system provided herein, the set of polynucleotides encoding the system provided herein, the vector provided herein, the engineered protein provided herein, or the guide nucleic acid or a portion thereof are encapsulated by the lipid nanoparticle. In some embodiments, the system provided herein, the set of polynucleotides encoding the system provided herein, the vector provided herein, the engineered protein provided herein, or the guide nucleic acid or a portion thereof are in complex with the lipid nanoparticle.

[0128] The compositions provided herein can be delivered to a cell system using vectors, for example containing polynucleotide sequences encoding a system, a guide nucleic acid, an engineered protein, or a composition provided herein. In some embodiments, a system as described herein can be delivered absent a viral vector. Any vector systems can be used including, but not limited to, plasmid vectors, viral vectors, and oncolytic viral vectors. Furthermore, any of these vectors can comprise one or more transcription factor, transgene, or molecular tag.

[0129] In some embodiments, the vectors provided herein are viral vectors. Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno-associated viral vectors (AAV), adenoviral vectors, herpes simplex viral vectors, alphaviral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviral vectors, picomaviral vectors, and oncolytic viral vectors.Atorney Docket No. 232285-702601

[0130] In some embodiments, the viral vector comprises an AAV. AAVs can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. A recombinant AAV vector (rAAV) comprises an infectious, replication-defective virus composed of an AAV protein shell encapsulating a heterologous nucleotide sequence of interest that is flanked on both sides by AAV ITRs. An rAAV vector is produced in a suitable host cell comprising an AAV vector, AAV helper functions, and accessory functions. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery. In some embodiments, the AAV or the rAAV provided herein comprises a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, or any combination thereof. In some embodiments, the delivery vehicle comprises a hybrid AAV-lipid nanoparticle delivery system.

[0131] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of: a human immunodeficiency virus 1 (HIV-1); a human immunodeficiency virus 2 (HIV-2), a visna-maedi virus (VMV) virus; a caprine arthritis-encephalitis virus (CAEV); an equine infectious anemia virus (EIAV); a feline immunodeficiency virus (FIV); a bovine immune deficiency virus (BIV); and a simian immunodeficiency virus (SIV), fragments, derivatives, or variants thereof.

[0132] Conventional viral and non-viral based gene transfer methods can be used to introduce polynucleotides encoding for a composition, system, guide nucleic acid, or an engineered protein provided herein to cells (e.g., mammalian cells) and target tissues. Exemplary non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acids complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can also include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0133] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acidAtorney Docket No. 232285-702601delivery systems include those provided by AMAXA® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., TRANSFECTAM® and LIPOFECTIN®).

[0134] Delivery of the compositions and systems provided herein can be to cells (ex vivo administration) or target tissues (in vivo administration). Additional methods of delivery include the use of packaging the polynucleotides to be delivered into EnGenelC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis.

[0135] Vectors including viral and non-viral vectors containing nucleic acids encoding a nucleic editing system provided herein can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. More than one route can be used to administer a particular composition.

[0136] In some embodiments, a composition, protein, guide nucleic acid, or a system provided herein can be shuttled to a cellular nucleus. For example, a vector can contain a nuclear localization sequence (NLS). A vector or any composition provided herein can also be shuttled by a protein or protein complex. In some embodiments, a composition or a system provided herein can be introduced to a cell or a target tissue by a minicircle vector.

[0137] In some embodiments, a vector or a polynucleotide provided herein can be pre-complexed with an engineered protein provided herein prior to electroporation into a cell. An engineered protein that can be used for shuttling can be a nickase or a catalytically dead Cas protein. A nuclease that can be used for shuttling can be a nuclease-competent protein. In some embodiments, an engineered protein herein can be pre-mixed with a guide nucleic acid provided herein an any additional elements (e.g., transgenes or other engineered proteins).

[0138] A cell can be transfected with a mutant or chimeric adeno-associated viral vector encoding a system or a composition provided herein. For example, an AAV vector concentration can be from 0.5 nanograms to 50 micrograms.

[0139] A system or a composition provided herein can also be introduced to a cell via electroporation techniques. The amount of polynucleotides that can be introduced into the cell byAttorney Docket No. 232285-702601electroporation can be varied to optimize transfection efficiency and / or cell viability. In some embodiments, less than about 100 picograms of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). In some embodiments, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms, of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some embodiments, the amount of nucleic acid (e.g., dsDNA) required for optimal transfection efficiency and / or cell viability can be specific to the cell type. In some embodiments, the amount of nucleic acid (e.g, dsDNA) used for each sample can directly correspond to the transfection efficiency and / or cell viability. The transfection efficiency of cells with any of the nucleic acid delivery platforms described herein, for example, nucleofection or electroporation, can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%.

[0140] Viral particles, such as AAV, can be used to deliver a viral vector comprising a gene of interest or a transgene into a cell ex vivo or in vivo. In some embodiments, a mutated or chimeric adeno-associated viral vector as disclosed herein can be measured as pfu (plaque forming units). In some embodiments, the pfu of recombinant virus or mutated or chimeric adeno-associated viral vector of the compositions and methods of the disclosure can be about 108to about 5* 1010pfu. In some embodiments, recombinant viruses of this disclosure are at least about 1 x 108, 2* 108, 3 * 108, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, and 5xl010pfu. In some embodiments, recombinant viruses of this disclosure are at most about 1 x 108, 2x 108, 3 x 108, 4x 108, 5x 108, 6x 108, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10,Atorney Docket No. 232285-7026012*1O10, 3*1010, 4*1O10, and 5*1010pfu. In some aspects, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured as vector genomes. In some embodiments, recombinant viruses of this disclosure are IxlO10to 3*1012vector genomes, or IxlO9to 3*1013vector genomes, or lx 108to 3 *1014vector genomes, or at least about lx 101, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, 1 x 1017, and IxlO18vector genomes, or are 1 x 108to 3 x 1014vector genomes, or are at most about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, IxlO17, and IxlO18vector genomes.

[0141] In some embodiments, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured using multiplicity of infection (MOI). In some embodiments, MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered. In some embodiments, MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered. In some embodiments, the MOI can be IxlO6GC / mL. In some embodiments, the MOI can be IxlO5GC / mL to IxlO7GC / mL. In some embodiments, the MOI can be IxlO4GC / mL to IxlO8GC / mL. In some embodiments, recombinant viruses of the disclosure are at least about IxlO1GC / mL, IxlO2GC / mL, IxlO3GC / mL, IxlO4GC / mL, IxlO5GC / mL, IxlO6GC / mL, IxlO7GC / mL, IxlO8GC / mL, IxlO9GC / mL, IxlO10GC / mL, IxlO11GC / mL, IxlO12GC / mL, IxlO13GC / mL, IxlO14GC / mL, IxlO15GC / mL, IxlO16GC / mL, IxlO17GC / mL, and IxlO18GC / mL MOI. In some embodiments, a mutated or chimeric adeno-associated viruses of this disclosure are from about IxlO8GC / mL to about 3xl014GC / mL MOI, or are at most about IxlO1GC / mL, IxlO2GC / mL, IxlO3GC / mL, IxlO4GC / mL, IxlO5GC / mL, IxlO6GC / mL, IxlO7GC / mL, IxlO8GC / mL, IxlO9GC / mL, IxlO10GC / mL, IxlO11GC / mL, IxlO12GC / mL, IxlO13GC / mL, IxlO14GC / mL, IxlO15GC / mL, IxlO16GC / mL, IxlO17GC / mL, and IxlO18GC / mL MOI.

[0142] In some aspects, a non-viral vector or nucleic acid can be delivered without the use of a mutated or chimeric adeno-associated viral vector and can be measured according to the quantity of nucleic acid. Generally, any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure. In some embodiments, nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. In some embodiments, nucleic acid can be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300Attorney Docket No. 232285-702601pig, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.

[0143] Polypeptides, vectors, plasmids, compositions, systems, engineered proteins, and guide nucleic acids provided herein can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to construct any embodiment of the compositions provided herein include genetic engineering, recombinant engineering, and synthetic techniques. In some embodiments, polypeptides, vectors, plasmids, compositions, systems, engineered proteins, and guide nucleic acids provided herein can be delivered to liver. In some embodiments, polypeptides, vectors, plasmids, compositions, systems, engineered proteins, and guide nucleic acids provided herein can be delivered to muscle.Cells

[0144] Provided herein are cells comprising a system or a composition provided herein. In some embodiments, a polynucleotide encoding for an engineered protein provided herein and a polynucleotide encoding for a guide nucleic acid provided herein are administered to a cell or a population of cells.

[0145] The compositions, polynucleotides, engineered proteins, guide nucleic acids and systems provided herein can be delivered to any suitable cell. In some embodiments, compositions, polynucleotides, engineered proteins, guide nucleic acids and systems provided herein modulate a gene, a protein, and / or a functional phenotype of a cell.

[0146] Suitable cells can include but are not limited to eukaryotic and prokaryotic cells and / or cell lines. A suitable cell can be, for example, a human primary cell. A primary cell can be taken directly from living tissue (i.e., biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. A primary cell can be acquired from a variety of sources such as an organ, vasculature, buffy coat, whole blood, apheresis, plasma, bone marrow, tumor, cell-bank, cryopreservation bank, or a blood sample. A primary cell can be a stem cell.

[0147] Suitable cells that can contacted with a composition, a polynucleotide, an engineered protein, a guide nucleic acid, or a system provided herein include but are not limited to: epithelialAtorney Docket No. 232285-702601cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, leukocytes, lymphocytes (B, NK, and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, Leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, dopaminergic cells, embryonic stem cells, fibroblasts and fetal fibroblasts. Further, the one or more cells can be, for example, pancreatic islet cells and / or cell clusters or the like, including, but not limited to pancreatic a cells, pancreatic P cells, pancreatic 5 cells, pancreatic F cells (e.g., PP cells), or pancreatic a cells.

[0148] Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. Suitable cells can comprise any number of primary cells, such as human cells, nonhuman cells, and / or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from the subject to be treated (e.g., a subject with a disease, a subject in need of treatment, or a subject that is immunocompromised). Suitable cells can be derived from a human donor.

[0149] In some embodiments, the cell is genetically modified by the methods, systems, and compositions provided herein. Cells provided herein can be administered to a subject in need thereof, e.g., a subject with a disease or a condition in need of treatment.

[0150] A method of attaining suitable cells, such as human primary cells, can comprise selecting cells. In some embodiments, a cell can comprise a marker that can be selected for the cell. For example, such marker can comprise GFP, a resistance gene (for example, a gene conferring antibiotic resistance), a cell surface marker, an endogenous tag. Cells can be selected using anyAtorney Docket No. 232285-702601endogenous marker. Suitable cells can be selected using any technology. Such technology can comprise flow cytometry and / or magnetic columns. The selected cells can also be expanded to large numbers.

[0151] Delivery vehicles for in vivo and ex vivo use can include pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., a polynucleotide, a protein, a vector, or a cell), as well as by the particular method used to administer the composition.Pharmaceutical compositions, dosing, and administration

[0152] Provided herein are pharmaceutical compositions comprising: a composition, at least one guide nucleic acid, a polypeptide, or a polynucleotide provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. Provided herein is a pharmaceutical composition comprising a vector provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient.

[0153] In some embodiments, compositions provided herein (e.g., a nanoparticle comprising a system or a composition provided herein) are combined with pharmaceutically acceptable salts, excipients, and / or carriers to form a pharmaceutical composition. Pharmaceutical salts, excipients, and carriers may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug. A pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.

[0154] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol). Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agentsAtorney Docket No. 232285-702601in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0155] Compositions and systems provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage. A unit dosage form is a physically discrete unit of a composition provided herein appropriate for a subject to be treated. For any composition provided herein the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., EDso (the dose is therapeutically effective in 50% of the population) and LDso (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use.

[0156] Provided herein are pharmaceutical compositions for administering a composition or a system to a subject in need thereof (e.g., as a treatment of a disease). In some embodiments, pharmaceutical compositions provided herein are in a form that allows for compositions provided herein to be administered to a subject. In some embodiments, the pharmaceutical composition is formulated for intratumoral delivery. In some embodiments, administration of a pharmaceutical composition provided herein is local administration or systemic administration. In some embodiments, a pharmaceutical composition provided herein is formulated for administration / for use in administration via an intratumoral, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, or intracranial route. In some embodiments, the administering is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administering is at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours (1 day), at least about 48 hours (2 days), at least about 72 hours (3 days), at least about 96 hours (4 days), at least about 120 hours (5 days), at least about 144 hours (6 days), at least about 168 hours (7 days), at least about 336 hours (14 days), at least about 504 hours (21 days), at least about 672 hours (28 days), up to 744 hours (31 days). In some embodiments, the administering is every 744 hours (e.g., once per month) or once per year (365 days).Atorney Docket No. 232285-702601

[0157] Vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual subject (e.g., lymphocytes, T cells, bone marrow aspirates, or tissue biopsy), followed by reimplantation of the cells into a subject, usually after selection for cells which have incorporated the vector. Prior to or after selection, the cells can be expanded.

[0158] Provided herein are cells expressing a system or a composition provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a nucleic acid encoding for a composition or a system provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a vector encoding for an engineered protein provided herein, a system provided herein, or a composition provided herein.Kits

[0159] Provided herein are kits, wherein the kits comprise: a system, a composition, a polynucleotide, a vector, a guide nucleic acid, an engineered protein, or an engineered RNA encoding the engineered protein provided herein; and packaging and materials thereof. In some embodiments, the kit further comprises a scaffold provided herein. In some embodiments, the kit further comprises a cell-free system. In some embodiments, the kit further comprises a population of cells. In some embodiments, the cells are stored in a cryopreservation medium. In some embodiments, the cryopreservation medium comprises: dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises a buffer, an isotonic agent or an apoptosis inhibitor. Non-limiting examples of buffer elements include: citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and tris. Non-limiting examples of isotonic agents include, for example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and tris. Additional isotonic agents include sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trehalose, and glucose. The apoptosis inhibitor can include, for example, a Rho associated kinase (ROCK) inhibitor, catalase, and zVAD-fmk. In some embodiments, the kits comprise reagents. In some embodiments, the reagents comprise saccharides and saccharide derivatives (e.g., sodium carboxymethyl cellulose and cellulose acetate), detergents, glycols, polyols, esters, buffering agents, alginic acid, and organic solvents.Attorney Docket No. 232285-702601

[0160] In some embodiments, a formulation of a composition described herein is prepared in a single container for administration to a cell or a subject. In some embodiments, a formulation of a composition provided herein is prepared two containers for administration, separating the guide nucleic acid or polynucleotide encoding the guide nucleic acid and / or the polynucleotide encoding the engineered protein provided herein. As used herein, “container” includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi -well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and / or transported, and accessed to remove the contents. Examples of such containers include glass and / or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe. In some embodiments, the containers are RNase free.

[0161] Provided herein are kits comprising: a first container comprising: a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a spacer region that has complementarity to a target sequence within a gene encoding for an exerkine or a regulatory region of the exerkine a in a cell; and (ii) a nuclease binding region, wherein the nuclease binding region comprises a secondary structure that binds to the nuclease, and a second container comprising: an engineered protein or a polynucleotide encoding for the engineered protein, wherein the engineered protein comprises: a nuclease operably linked to a transcriptional activation domain. In some embodiments, a kit provided herein further comprises reagents for nucleic acid amplification, transcription, translation, or nucleic acid isolation. In some embodiments, a kit provided herein further comprises a reporter molecule provided herein.Methods for detecting epigenetic editing efficiency

[0162] The epigenetic editing activity or efficiency of a system or a composition provided herein may be assayed by various methods. In some embodiments, the activity of a system or a composition provided herein may be assayed using a cell expressing a reporter protein or containing a reporter gene. For example, a reporter gene may be engineered to contain an obstruction, such as a stop codon, a frameshift mutation, a spacer, a linker, or a transcriptional terminator; the system may then be used to remove the obstruction and the resultant functional reporter protein may be detected. Similarly, a reporter gene can be introduced into the target nucleic acid for detection of the target. In some embodiments, the reporter gene may be designed such that a specific sequence modification is required to restore functionality of the reporter protein. In other embodiments, the reporter gene may be designed such that any insertion or deletion which results in a frame shift of one or two bases in the target nucleic acid may beAttorney Docket No. 232285-702601sufficient to restore functionality of the reporter protein. Examples of reporter proteins encoded by a reporter gene include colorimetric enzymes, metabolic enzymes, fluorescent proteins, enzymes and transporters associated with antibiotic resistance, and luminescent enzymes. Examples of such reporter proteins include P-galactosidase, Chloramphenicol acetyltransferase, Green fluorescent protein, Red fluorescent protein, and Firefly and Renilla luciferase. Different detection methods may be used for different reporter proteins. For example, the reporter protein may affect cell viability, cell growth, fluorescence, luminescence, or expression of a detectable product. In some embodiments, the reporter protein may be detected using a colorimetric assay. In some embodiments, the reporter protein may be a fluorescent protein, and epigenetic editing may be assayed by measuring the degree of fluorescence in treated cells, or the number of treated cells with at least a threshold level of fluorescence. In some embodiments, transcript levels of a reporter gene may be assessed. In other embodiments, a reporter gene may be assessed by sequencing.

[0163] In some embodiments, to determine exerkine activation efficiency of a system or a composition provided herein, after administration of a system or a composition provided herein to a subject, a biopsy is taken for counting cells with exerkine expression ex vivo, which in turn provides tissue level and cellular level readouts for increased expression by analyzing cell populations within a tissue sample. In some embodiments, the assays for determining exerkine activation efficiency of a system or composition provided herein can include but is not limited to: RT-PCR, nucleic acid sequencing, T7 endonuclease 1 (T7E1) mismatch detection assays, tracking of indels by decomposition (TIDE) assays, and indel detection by amplicon analysis (IDAA) assays. The indel pattern that is induced at the target site of a programmable nuclease or nickase may also be determined by PCR-amplifying the respective region and subsequent next generation sequencing. To obtain a quantitative single-cell view of gene editing efficiency, cell surface markers can be targeted and the loss of signal that occurs as a consequence of indel formation by flow cytometry can be quantified. Single-cell sequencing can also be employed to directly assess the gene-editing efficiency.Use of the Systems and Compositions

[0164] Provided herein are methods of increasing expression of exerkines (e.g., FST) in a cell, where in the methods comprise: administering to a cell, a tissue, or a subject a composition described herein. In some embodiments, upon administration, the spacer region binds to the nuclease to form a complex with the polypeptide, thereby bringing in the DNA binding domain and the transcriptional activation domain together to bind a target sequence within a gene encoding for an exerkine or a regulatory region of the gene encoding for the exerkine a in the cell, initiatingAtorney Docket No. 232285-702601transcription of the target gene, and consequently increasing an expression level of the exerkine in the cell as compared to the expression level of FST in the cell prior to the introduction of the system. In some embodiments, a transcriptional activation domain described herein interacts with other proteins (e.g., coactivators) to stimulate the process of transcription by facilitating the assembly of the RNA polymerase complex at the promoter region of an exerkine gene, thereby effectively turning on gene expression of the exerkine. Further provided herein are methods of increasing expression of at least two exerkines in a cell, where in the methods comprise: administering to a cell, a tissue, or a subject a composition described herein. In some embodiments, without bound by any theory, targeting multiple exerkines is achieved using a dCasl2a system, wherein multiplexed guide arrays of crRNA spacers targeting two or more loci. In some embodiments, the multiplexed guide arrays of crRNA spacers are separated by a direct repeat (DR) region.

[0165] In some embodiments, an expression level is an RNA expression level that can be measured by, for example, RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, an expression level is a protein expression level that can be measured by, for example, Western Blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, etc.

[0166] In some embodiments, a method provided herein increases the expression of the target gene encoding for an exerkine (e.g., FST) by at least about 1.01 fold to about 5,000 fold (e.g., as compared to a control cell lacking the polypeptide and / or the guide nucleic acid). In some embodiments, the method increases the expression of FST by at least about 1.01 fold to about 1.05 fold, about 1.01 fold to about 1.1 fold, about 1.01 fold to about 1.5 fold, about 1.01 fold to about 2 fold, about 1.01 fold to about 5 fold, about 1.01 fold to about 10 fold, about 1.01 fold to about 50 fold, about 1.01 fold to about 100 fold, about 1.01 fold to about 500 fold, about 1.01 fold to about 1,000 fold, about 1.01 fold to about 5,000 fold, about 1.05 fold to about 1.1 fold, about 1.05 fold to about 1.5 fold, about 1.05 fold to about 2 fold, about 1.05 fold to about 5 fold, about 1.05 fold to about 10 fold, about 1.05 fold to about 50 fold, about 1.05 fold to about 100 fold, about 1.05 fold to about 500 fold, about 1.05 fold to about 1,000 fold, about 1.05 fold to about 5,000 fold, about 1.1 fold to about 1.5 fold, about 1.1 fold to about 2 fold, about 1.1 fold to about 5 fold, about 1.1 fold to about 10 fold, about 1.1 fold to about 50 fold, about 1.1 fold to about 100 fold, about 1.1 fold to about 500 fold, about 1.1 fold to about 1,000 fold, about 1.1 fold to about 5,000 fold, about 1.5 fold to about 1 fold, about 1.5 fold to about 5 fold, about 1.5 fold to about 10 fold, about 1.5 fold to about 50 fold, about 1.5 fold to about 100 fold, about 1.5 fold to about 500 fold, about 1.5 fold to about 1,000 fold, about 1.5 fold to about 5,000 fold, about 1 fold to about 5 fold,Attorney Docket No. 232285-702601about 1 fold to about 10 fold, about 1 fold to about 50 fold, about 1 fold to about 100 fold, about 1 fold to about 500 fold, about 1 fold to about 1,000 fold, about 1 fold to about 5,000 fold, about 5 fold to about 10 fold, about 5 fold to about 50 fold, about 5 fold to about 100 fold, about 5 fold to about 500 fold, about 5 fold to about 1,000 fold, about 5 fold to about 5,000 fold, about 10 fold to about 50 fold, about 10 fold to about 100 fold, about 10 fold to about 500 fold, about 10 fold to about 1,000 fold, about 10 fold to about 5,000 fold, about 50 fold to about 100 fold, about 50 fold to about 500 fold, about 50 fold to about 1,000 fold, about 50 fold to about 5,000 fold, about 100 fold to about 500 fold, about 100 fold to about 1,000 fold, about 100 fold to about 5,000 fold, about 500 fold to about 1,000 fold, about 500 fold to about 5,000 fold, or about 1,000 fold to about 5,000 fold (e.g., as compared to a control cell lacking the heterologous polypeptide and / or the guide nucleic acid). In some embodiments, the method increases the expression of an exerkine by at least about 1.01 fold, about 1.05 fold, about 1.1 fold, about 1.5 fold, about 1 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold (e.g., as compared to a control cell lacking the polypeptide and / or the guide nucleic acid). In some embodiment, the target sequence is within an endogenous target gene (e.g., FST or regulatory region of FST). In some embodiments, the method increases the expression of the endogenous target gene encoding the target protein by at least at least about 1.01 fold, about 1.05 fold, about 1.1 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, or about 1,000 fold (e.g., as compared to a control cell lacking the heterologous polypeptide and / or the guide nucleic acid). In some embodiments, the method increases the expression of an exerkine by at least at most about 1.05 fold, about 1.1 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold (e.g., as compared to a control cell lacking the polypeptide and / or the guide nucleic acid).

[0167] In some embodiments, the exerkines is FST. In some embodiments, the increase in the expression level of FST in a subject after administration of a composition as provided herein results in enhanced (or increased) muscle mass, as compared to the subject prior to the administration. The increase in the muscle mass (e.g., average muscle mass) can be at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%, as compared to such control subject. The increase in the muscle mass (e.g., average muscle mass) can be at leastAtorney Docket No. 232285-702601or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.5- fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3 -fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 150-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold, as compared to that in the subject prior to administration.

[0168] In some embodiments, the increase in the expression level of the F ST in a subject after administration of a composition as provided herein results in enhanced (or increased) muscle function, as compared to the subj ect prior to the administration. The increase in the muscle function can be at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%, as compared to such control subject. The increase in the muscle mass (e.g., average muscle mass) can be at least or up to about 1.1 -fold, at least or up to about 1.2-fold, at least or up to about 1.5- fold, at least or up to about 1.8-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 150-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold, as compared to that in the subject prior to administration.

[0169] In some embodiments, the methods described herein can activate expression of an exerkine for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least aboutAtorney Docket No. 232285-70260118 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, or at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, or more.

[0170] In some embodiments, the methods described herein can activate expression of an exerkine to above a certain threshold for at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 14 hours, at most about 18 hours, at most about 20 hours, at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 14 days, at most about 21 days, at most about 28 days, at most about 5 weeks, at most about 6 weeks, at most about 7 weeks, at most about 8 weeks, at most about 9 weeks, at most about 10 weeks, at most about 12 weeks, at most about 14 weeks, at most about 18 weeks, at most about 20 weeks, at most about 26 weeks, or at most about 5 months, at most about 6 months, at most about 9 months, at most about 12 months, or more.

[0171] In some embodiments, the systems and methods described herein can activate expression of an exerkine to above a certain threshold for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.

[0172] Also provided herein are methods of increasing muscle mass or improving muscle function comprising administering to a cell, a tissue, or a subject a composition described herein. In some embodiments, the method comprises administering to a subject having a disorder or disease or at risk of developing a disease or disorder. In some embodiments, the disease or disorder is associated with low FST expression. In some embodiments, the disease or disorder is associated with anAttorney Docket No. 232285-702601abnormal amount, development, or metabolic activity of muscle in a subject. In some embodiments, the disease or disorder is a neuromuscular or muscular degenerative disorder. In some embodiments, the disease or disorder is Lou Gehrig's disease (ALS), cancer anorexiacachexia syndrome, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease (and muscle wasting associated with COPD), muscle wasting syndrome, sarcopenia, or cachexia. Muscular dystrophy refers to a group of degenerative muscle diseases characterized by gradual weakening and deterioration of skeletal muscles and sometimes the heart and respiratory muscles. Exemplary muscular dystrophies that can be treated with a composition described herein include Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also known as Landouzy -Dejerine), Myotonic Dystrophy (MMD) (also known as Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD). In some embodiments, the disease or disorder is a metabolic disorder. In some embodiment, a metabolic disorder described herein is obesity and type II diabetes. In some embodiments, the disease or disorder is cancer or tumor. In some embodiments, the cancer or tumor is breast cancer (e.g., triple-negative breast cancer (“TNBC”)), squamous cell carcinoma, lung cancer, ovarian cancer, head and neck cancer, gastric cancer, colorectal cancer, or thymic epithelial tumors.

[0173] In some embodiments, a subject being treated with a composition described herein are patients with undesirably low red blood cell or hemoglobin levels caused by a muscular dystrophy, such as patients having an anemia, and those that are at risk for developing undesirably low red blood cell or hemoglobin levels, such as those patients that are about to undergo major surgery or other procedures that may result in substantial blood loss, such as having blood drawn and stored for a later transfusion. Patients and candidate patients may also include those patients in need of an increase in red blood cells and / or hemoglobin levels that do not respond well to Epo. When observing hemoglobin levels in humans, a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account. Potential causes of anemia include blood-loss, nutritional deficits, medication reaction, various problems with the bone marrow and many diseases. More particularly, anemia has been associated with a variety of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, bone marrow transplantation. Anemia may also be associated with the following conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e.g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkins lymphomas); tumors of the hematopoietic system (e.g., leukemia, myelodysplastic syndrome, multiple myeloma); radiation therapy; chemotherapy (e.g., platinum containingAtorney Docket No. 232285-702601regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); acute or chronic renal disease or failure including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; situations where transfusion of red blood cells is not possible due to patient allo- or auto-antibodies and / or for religious reasons (e.g., some Jehovah's Witnesses); infections (e.g., malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemias; drug use or abuse, e.g., alcohol misuse; pediatric patients with anemia from any cause to avoid transfusion; and elderly patients or patients with underlying cardiopulmonary disease with anemia who cannot receive transfusions due to concerns about circulatory overload.EXAMPLESExample 1. Screening System.

[0174] The epigenetic editing system for FST upregulation was assayed in mouse and human liver cells. HepG2 cells were chosen as the screening system to perform a screening of guides for Casl2a and Cas9 that can access genomic FST region with known potent viral activators like VP128, mVPR and VIRF2 (FIG. 1A). Targeting, non-targeting, or safe-targeting gRNA-encoding DNA fragments were chemically synthesized and inserted by Golden Gate assembly into either the same all-in-one destination vector downstream of the hU6 promoter, or into a gRNA-only plasmid with the identical expression cassette structure. crRNA guides were designed to bind within the upstream region of FST gene (FIG. 3), encompassing transcriptional start site (TSS), CpG island, and regulatory element sites. Plasmids containing the guides and activators were transiently delivered into HepG2 cells. Supernatant was collected for FST ELISA measurement at days 3, 6, 9, 12, 15, and 18. As shown in FIG. 3, Guides are labeled such that “ l_fst-5” denotes the highest activity, “3_fst-49” denotes third highest, etc., and guides designed using the alternative PAM range are denoted as “alt-”.

[0175] crRNA guide screening results on day 3 are shown in FIGS. 4A-4C, FIG.5, and FIG.6A-6B. The guides are ranked by the fold of activation in Table 12 below. FIGS. 4A-4C illustrate the observed relative on-target activity rankings of each Casl2a crRNA guide, when co-delivered effector was held constant, as measured by ELISA detection of human follistatin protein secreted by HepG2 cells at 6 days post-delivery. FIG. 5 illustrates the ranked performance of 62 individual spacers, as tested in FIGS. 4A-4B, with targeting guides (dots) and non-targeting (NT) controlsAtorney Docket No. 232285-702601(boxes). To test FST activation fold changes at mRNA and protein levels, RT-qPCR and ELISA were performed (FIGS. 6A-6B). Guides were tested in single-plex (FST) or using multiplex arrayed guides (FST and a second gene co-targeted). Non-targeting (NT) guides and safe-targeting guides (AAVS-1 safe harbor locus) served as baseline controls for calculating fold on-target activities. Replicate RT-qPCR experiments were conducted for accuracy, using two distinct reference genes (TBP and GUSB), and fold mRNA was calculated using the delta-delta Ct method.

[0176] The guides were ranked by the fold change in activation as shown in Table 12 below. FST 5, FST 2, FST 49, and FST 3 were among the top-performing Casl2a guides for FST. Out of 62 guides screened, a total of 9 significant hits for Casl2a were identified, including 7 guides with TTTN PAM sequence (N=A, T, G, or C), 2 guides with TGTG PAM sequence. In addition, it was observed that 23 bp was a preferable spacer length to 20 bp.Table 12. Casl2a guides for FST ActivationGuide ID PAM Spacer Sequence Length Strand Activation (SEQ ID NO:) (fold) FST 5 TTTG 95 23 (+) 3.572 FST _2 TTTC 96 23 (-) 3.569 FST 49 TTTC 97 20 (-) 3.207 FST 3 TTTG 98 23 (+) 3.184 FST_alt_5 TGTG 99 23 (-) 2.937 FST 6 TTTC 100 23 (-) 2.884 FST_alt_8 TTTG 101 23 (-) 2.88 FST 4 TTTC 102 23 (+) 2.775 FST_47 TTTG 103 20 (+) 2.485 FST 36 TTTG 104 23 (+) 2.448 FST 35 TTTA 105 20 (+) 2.413 FST_alt_7 TGTG 106 23 (-) 2.316 FST 8 TTTC 107 23 (+) 2.274 FST 42 TTTC 108 23 (-) 2.256 FST_alt_6 CTCC 109 23 (-) 2.066 FST 34 TTTC 110 20 (-) 1.971Atorney Docl <ct No. 232285-702601FST_25 TTTG 111 23 (-) 1.95 FST 33 TTTA 112 23 (+) 1.939 FST 28 TTTG 113 20 (-) 1.902 FST alt l CTCC 114 23 (-) 1.892 FST 46 TTTG 115 20 (+) 1.882 FST 14 TTTG 116 23 (-) 1.851 FST 45 TTTG 117 20 (-) 1.788 FST_alt_2 CCCC 118 23 (+) 1.752 FST_32 TTTC 119 20 (-) 1.67 FST 43 TTTG 120 20 (+) 1.665 FST_22 TTTA 121 20 (-) 1.642 FST_alt_4 CTCC 122 23 (+) 1.633 FST 1 TTTG 123 23 (-) 1.622 FST 41 TTTG 124 20 (-) 1.609 FST alt l l CCCC 125 23 (+) 1.594 FST_7 TTTC 126 20 (-) 1.585 FST 48 TTTC 127 23 (-) 1.578 FST 44 TTTG 128 20 (+) 1.571 FST 40 TTTG 129 20 (-) 1.568 FST 15 TTTG 130 23 (+) 1.542 FST_27 TTTA 131 20 (-) 1.531 FST l l TTTG 132 20 (+) 1.51 FST_alt_9 AGCC 133 23 (+) 1.47 FST 31 TTTG 134 23 (+) 1.458 FST 39 TTTG 135 20 (-) 1.446 FST_37 TTTC 136 20 (-) 1.368 FST_17 TTTG 137 23 (+) 1.355Atorney Docket No. 232285-702601FST 24 TTTG 138 20 (+) 1.331 FST_23 TTTA 139 23 (-) 1.324 FST alt lO CACC 140 23 (+) 1.314 FST 10 TTTC 141 20 (+) 1.287 FST_alt_3 TTTT 142 23 (+) 1.284 FST 12 TTTC 143 20 (-) 1.281 FST 13 TTTA 144 23 (-) 1.253 FST 19 TTTG 145 23 (-) 1.138 FST 38 TTTG 146 20 (+) 1.102 FST 30 TTTC 147 23 (-) 1.079 FST 9 TTTG 148 23 (-) 1.078 FST 29 TTTC 149 23 (-) 1.037 FST 16 TTTC 150 20 (+) 1.027 FST 26 TTTG 151 20 (-) 1.023 FST 21 TTTG 152 23 (-) 0.865 FST 18 TTTG 153 23 (+) 0.675 FST_20 TTTG 154 23 (+) 0.605 FST_alt_12 CCCC 155 23 (+) 0.391 FST_alt_13 TGTG 156 23 (-) 0.386EXAMPLE 2. Screening of Transcriptional Activators for FST Upregulation

[0177] Screening was performed with the top-performing guides from Example 1 to identify the top-performing transcriptional activators for FST upregulation with regards to both potency and durability, based on the FST expression fold changes on day 6 and day 12, respectively. Target activities were measured while the plasmid was detectable from day 3 to day 6 (3-6 d) are defined as potency, while target activities from day 9 to day 18 (9-18 d) are defined as durability. Fold changes in FST protein concentration were calculated with respect to non-targeting (NT) guide controls. Benchmark effector domains as described in prior work, for comparison, included the miniaturized fusion VP64-p65-Rta (VPR), the extended 150-amino acid domain from the viralAtorney Docket No. 232285-702601protein Q2HR71, the fusion NCOA3 [1045-1092], FOXO3 FOXO-TAD [604-644], and ZNF473 KRAB [5-48] (NFZ), and the VP16-derived VP128 transactivation domain. The results suggest, in general, domains of viral origin outperform domains of human origin for target gene activation, both in potency (3-6 d) and durability (9-18 d)FST expression fold changes in the presence of transcriptional activators or control (NT) are shown in FIG. 7, among the four known activators mVPR, VIRF2 (“Q2HR71”), NFZ, and VP128, VP128 is the top performer on day 6, which increased FST expression by about 7-folds. On day 12, however, VIRF2 performed slightly better than VP 128 and NFZ, which showed increased FST expression by about 3.8-fold. Among the engineered activators designed in house, L1EO_1.32|L1_CTCF_4 showed the best potency on day 6 (over 8-fold), while L1T31 showed the best durability on day 12 (about 4.7-fold).EXAMPLE 3. Activation of additional exerkines

[0178] To assay the activation of fibroblast growth factor 21 (FGF21), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), Neurturin (NRTN), heat shock protein family A (Hsp70) Member 1 A (HSPA1 A), targeting, non-targeting, or safe-targeting gRNA-encoding DNA fragments were chemically synthesized and inserted by Golden Gate assembly into either the same all-in-one destination vector downstream of the hU6 promoter or into a gRNA-only plasmid with the identical expression cassette structure. Similar to the steps in EXAMPLE 1, Guide and editor plasmids were transiently delivered into HEK293T cells. RT-qPCR measurements were performed 5-6 days after transfection. Fold activation of FGF21, apelin, FNDC5, NRTN, and HSPA1A at mRNA level with different gRNAs are shown in FIGS. 8-12.

[0179] The activation level fine-tuning capabilities conferred by adjustment of the crRNA spacer length was also observed following crRNA screens against six human gene targets (FST, NRTN, FGF21, HSPA1 A, FNDC5, and APLN). In these experiments, the dCas-effector was kept constant across experiments to allow separate evaluation of crRNA spacer efficacies in arrayed screens in HEK293T and HepG2 cells. In the majority of cases, greater activation is achieved by the 23-nt version relative to the 20-nt version (FIG. 14A-14G).EXAMPLE 4. Co-upregulation of FST with another exerkine

[0180] To assay the co-upregulation of FST with other exerkines, multiplexed Casl2a guide arrays containing targeting, non-targeting, or safe-targeting gRNA-encoding DNA fragments were chemically synthesized and inserted by Golden Gate assembly into either the same all-in-one destination vector downstream of the hU6 promoter or into a gRNA-only plasmid with the identical expression cassette structure. Guides and editor plasmids were transiently delivered into HEK293T cells. RT-qPCR measurements were performed 5-6 days after transfection. Delivery ofAtorney Docket No. 232285-702601FST guide and apelin guide together results in 4.8-fold activation of FST and 8-fold activation of apelin (FIG. 13A). Delivery of FST guide and irisin guide together results in 2.4-fold activation of FST and 1.9-fold activation of FNDC5 (FIG. 13B), respectively.EXAMPLE 5. Safety of epigenetic effectors sourced from human and viral proteins

[0181] To compare the safety of epigenetic effectors sourced from human proteins with those from viral proteins, cell death and cell counts were measured following delivery of compositions comprised of epigenetic effector domains sourced from human and viral proteins. Safety measures indicate that among tested effectors, human and viral fragments are similarly tolerated by contacted cells (FIG. 15C). Compared to human effectors, viral effectors caused less cell death and greater cell survival (FIG. 15A and FIG. 15B).EXAMPLE 6. Potency and durability of the engineered transcriptional activators

[0182] Validation of the engineered transcriptional activators was performed on fluorescent reporter under a weak promoter, TRE3G. FIG. 10 shows absolute activation fold changes of TRE3G-GFP in HEK293T cells at Potency time points (3-6 d) for benchmark effectors and selected engineered effector compositions. A few engineered effectors outperformed the benchmark effectors, including E1.028, El.078, El.075, E1.039, E1.048, and E1.073. FIG. 11A shows absolute activation folds at Durability time points (9-18d) for benchmark effectors and selected engineered effector compositions. Notably, most of the engineered effectors outperformed the benchmark effectors. FIG. 11B shows the comparison of potency versus durability scores for all effectors in terms of TRE3G-GFP activation. Absolute activation folds at Durability time points (3-6d) (FIG. 16A) (9-18d) (FIG. 16B) for benchmark effectors and selected engineered effector compositions also suggested most selected engineered effectors showed better activation.EXAMPLE 7. Engineered transcriptional effectors with fused CTCF protein

[0183] To assay the functional effects on activation dynamics, various fusion proteins were created by fusing functionally discrete sub-domains of the endogenous human CTCF protein to C-terminus of a benchmark activator, vBi. Independently synthesized fragments encoding the N-terminal domain (NTD), C-terminal domain (CTD), intrinsically disordered region (IDR), RNA-binding domain (RBD), Zinc Fingers 1-2, and the Zinc Fingers 3-7 DNA-binding domain were fused C-terminally to a potent bipartite benchmark activator (vBi) in order to determine their functional impact on activation dynamics of this benchmark activator (FIG. 17A). FIG. 17B illustrates absolute fold activation relative to baseline over a full experimental time series (0-18d) at TRE3G-GFP in HEK293T cells. The Y axis represents fold change of the fluorescent reporterAtorney Docket No. 232285-702601(GFP), whereas the X axis represents time (days). Dark line indicates the benchmark effector vBi without fusion, and faint lines indicate individual vBi-CTCF domain fusion effectors. The Potency (3-6d) and Durability (9-18d) scores for vBi and each vBi-CTCF fusion effector are shown in FIG.17C. Dashed lines indicate the activation values achieved by the reference vBi effector alone. FIG.17D illustrates the effector size of each vBi-CTCF domain fusion relative to the levels achieved by vBi effector alone, over a full experimental time series (0-18d). The plotted relative folds were calculated with respect to the absolute activation by vBi without fusion. Dark lines indicate fusions that significantly augment activation potency and / or durability at the indicated time points.EXAMPLE 8. Effector Tiles versus Domains

[0184] To investigate engineered functional enhancements to activator potency, activation at a synthetic TRE3G-GFP promoter in HEK293T cells were tested with 39 examples of hit individual tiles (grey) and their respective domains (black). Full domains outperformed the respective screen hit tiles by an average 3.4-fold higher activation potency, some domains over 20-fold higher than individual tiles, with an overall -82% success rate of the design strategy across n=39 such tests (FIG. 18A). Merging of two adjacent full domains further enhances potency in a super-additive manner, on average >18-fold compared to either respective full domain, and exceeding the sum of both domains across 8 such cases tested (FIG. 18B). Design strategy for domains, merged, and bipartite domains base on HT screen hit tiles (FIG. 18C).EXAMPLE 9. Effects of activation effectors on mice

[0185] To confirm the effects of FST activation effectors on mice, C57B1 / 6J mice (males, 8-10 weeks) were injected intravenously at 2 mg / kg with LNP formulations containing mRNA-encoded dCas9-activators and FST-targeting sgRNAs. Blood draws were collected at day 1 post-dose and processed to serum prior to FST detection by ELISA according to manufacturer protocols. Fold activation was calculated relative to baseline (saline-injected mice). For comparison, the benchmark effector VPH-SS18 was also included. Fold activation was calculated relative to baseline (saline-injected mice). On day 1 post-dose, effectors El.039, El.057, and El.034 showed about 3-fold activation, 8-fold activation, and about 12-fold activation, respectively (FIG. 19A).At day 4 post-dose, effector N06-C02 showed higher fold activation as compared to the benchmark effector VPH-SS18 (FIG. 19B). Comparison of amino acid length among effectors tested in vivo suggests that while the benchmark effector VPH-SS18 contains more amino acid residues, N06-C02 with shorter length still outperformed it (FIG. 19C). N06-C02 is a construct encoding dCas9 and two identical effector domains, each having the sequence of SEQ ID NO: 172.Attorney Docket No. 232285-702601

[0186] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 232285-702601CLAIMS WHAT IS CLAIMED IS:

1. A system for upregulating expression of an exerkine in a cell, the system comprising:(a) a polypeptide comprising:(i) a DNA binding domain that comprises a nuclease that substantially lacks DNA cleavage activity,(ii) a transcriptional activation domain, and(b) a nucleic acid comprising:(i) a spacer region that is about 18 to 25 bases and has complementarity to a target sequence within a gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and (ii) a nuclease binding region that binds to the nuclease,wherein upon introduction of the system to the cell, the spacer region binds to the nuclease to form a complex with the polypeptide, thereby bringing the DNA binding domain and the transcriptional activation domain together to bind the target sequence or the regulatory region in the cell, initiating transcription of the gene, and consequently increasing an expression level of the exerkine in the cell as compared to the expression level of the exerkine in the cell prior to the introduction of the system to the cell.

2. The system of claim 1, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids.

3. The system of claim 2, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids.

4. The system of claim 2, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids.

5. The system of claim 1, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230.

6. The system of claim 1, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230.Atorney Docket No. 232285-7026017. The system of claim 1, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP128.

8. The system of claim 1, wherein the nuclease is dCas 9 or dCas 12.

9. The system of claim 1, wherein the nucleic acid is a guide RNA (gRNA).

10. The system of claim 1, wherein the length of the spacer region is about 20 bases.

11. The system of claim 1, wherein the length of the spacer region is about 23 bases.

12. The system of claim 1, wherein the spacer comprises a nucleic acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 252-313.

13. The system of claim 1, wherein the spacer comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313.

14. The system of claim 1, wherein the expression level of the exerkine in the cell is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold on day 6 following introduction of the system to the cell.

15. The system of claim 1, wherein the expression level of the exerkine in the cell is increased by about 8-fold on day 6 following introduction of the system to the cell.

16. The system of claim 1, wherein the expression level of the exerkine in the cell is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, or about 7-fold on day 12 following introduction of the system to the cell.

17. The system of claim 1, wherein the expression level of the exerkine in the cell is increased by about 6-fold on day 12 following introduction of the system to the cell.

18. The system of claim 1, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1 A (HSPA1A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins.

19. The system of claim 18, wherein the exerkine is FST.

20. The system of claim 19, wherein the cell is a muscle cell or liver a cell.Atorney Docket No. 232285-70260121. The system of claim 20, wherein the liver cell is a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal endothelial cell.

22. The system of any one of claims 1 to 21, wherein the system is in the form of a ribonucleoprotein complex (RNP-complex).

23. An engineered protein, comprising:a DNA binding domain; anda transcriptional activation domain,wherein the engineered protein binds a target gene encoding for an exerkine or a regulatory region of the target gene encoding for the exerkine.

24. The engineered protein of claim 23, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1 A (HSPA1 A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins.

25. The engineered protein of claim 23, wherein the exerkine is FST.

26. The engineered protein of claim 23, wherein the DNA binding domain is a zinc finger protein (ZFP).

27. The engineered protein of claim 23, wherein the DNA binding domain is dCas 9 or dCas 12.

28. The engineered protein of claim 23, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP128.

29. The engineered protein of claim 23, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids.

30. The engineered protein of claim 23, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids.

31. The engineered protein of claim 23, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids.Atorney Docket No. 232285-70260132. The engineered protein of claim 23, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230.

33. The engineered protein of claim 23, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230.

34. A nucleic acid encoding for the engineered protein of any one of claims 23 to 33.

35. A composition comprising the nucleic acid of claim 34, wherein the nucleic acid is incorporated into a delivery vehicle.

36. The composition of claim 35, wherein the delivery vehicle is a viral vector.

37. The composition of claim 36, wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.

38. The composition of claim 37, wherein the viral vector is an adeno-associated virus (AAV) vector.

39. The composition of claim 35, wherein the delivery vehicle is a nanoparticle.

40. The composition of claim 39, wherein the nanoparticle is a lipid nanoparticle.

41. A composition, comprising:(a) the engineered protein of any one of claims 23 to 33; and (c) a nucleic acid, wherein the nucleic acid nucleic acid is about 18 to 25 bases in length and comprises:(i) a spacer region that has complementarity to an endogenous target sequence within a gene encoding for the exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and(ii) a nuclease binding region, wherein the nuclease binding region comprises a secondary structure that binds to a nuclease.

42. The composition of claim 41, wherein the nucleic acid is a guide RNA.

43. The composition of claim 41, wherein the length of the spacer region is about 20 bases.

44. The composition of claim 41, wherein the length of the spacer region is about 23 bases.

45. The composition of claim 41, wherein the spacer region comprises a nucleic acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to any one of SEQ ID NOS: 252-313.Attorney Docket No. 232285-70260146. The composition of claim 41, wherein the spacer region comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313.

47. The composition of claim 41, wherein the engineered protein is complexed with the nucleic acid to form a ribonucleoprotein complex (RNP-complex).

48. A composition, comprising:(a) a first nucleic acid encoding for a polypeptide comprising:(i) a DNA binding domain, wherein the DNA binding domain comprises a nuclease that substantially lacks DNA cleavage activity; and(ii) a transcriptional activation domain; and(b) a second nucleic acid, wherein the nucleic acid nucleic acid is about 18 to 25 bases in length and comprises:(i) a spacer region that has complementarity to an endogenous target sequence within a gene encoding for an exerkine or a regulatory region of the gene encoding for the exerkine in a cell; and(ii) a nuclease binding region, wherein the nuclease binding region comprises a secondary structure that binds to the nuclease.

49. The composition of claim 48, wherein the exerkine is follistatin (FST), fibroblast growth factor 21 (FGF21), vascular endothelial growth factor (VEGF), apelin, Fibronectin Type III Domain-Containing Protein 5 (FNDC5), heat shock protein family A (Hsp70) Member 1 A (HSPA1A), cathepsin B, clusterin (CLU), humanin, glycosylphosphatidylinositol-specific phospholipase DI (Gpldl), neurturin, brain-derived neurotrophic factor (BDNF), musclin, carboxylesterase 2A (CES2A), heat shock proteins (HSP60 and HSP72), adropin, osteopontin (OPN), platelet factor 4 (PF4), angiopoietin-like 4 (ANGPTL4), meteorin-like protein (METRNL), myonectin, adipsin, irisin, mesencephalic astrocyte-derived neurotrophic factor (MANF), interleukin 6 (IL-6), or sestrins.

50. The composition of claim 48, wherein the exerkine is FST.

51. The composition of claim 48, wherein the first nucleic acid is an mRNA.

52. The composition of claim 48, wherein the second nucleic acid is a guide RNA.

53. The composition of claim 48, wherein the first nucleic acid is an mRNA and the second nucleic acid is a guide RNA.

54. The composition of claim 48, wherein the DNA binding domain is a zinc finger protein.

55. The composition of claim 48, wherein the DNA binding domain is dCas 9 or dCas 12.Atorney Docket No. 232285-70260156. The composition of claim 48, wherein the transcriptional activation domain comprises VP64-p65-Rta (VPR) or VP128.

57. The composition of claim 48, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 500 amino acids.

58. The composition of claim 48, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 300 amino acids.

59. The composition of claim 48, wherein the transcriptional activation domain comprises one or more transcriptional activator fragments each comprising an amino acid sequence having a length that is equal to or less than 200 amino acids.

60. The composition of claim 48, wherein the transcriptional activation domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 74-230.

61. The composition of claim 48, wherein the transcriptional activation domain comprises an amino acid sequence of any one of SEQ ID NOS: 74-230.

62. The composition of claim 48, wherein the length of the spacer region is about 20 bases.

63. The composition of claim 48, wherein the length of the spacer region is about 23 bases.

64. The composition of claim 48, wherein the spacer region comprises a nucleic acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to any one of SEQ ID NOS: 252-313.

65. The composition of claim 48, wherein the spacer region comprises a nucleic acid sequence of any one of SEQ ID NOS: 252-313.

66. The composition of any one of claims 48 to 65, wherein the first nucleic acid and the second nucleic acid are incorporated into a delivery vehicle.

67. The composition of any one of claims 48 to 65, wherein the first nucleic acid and the second nucleic acid are incorporated into two separate delivery vehicles.

68. The composition of claim 66 or 67, wherein each delivery vehicle is a viral vector.Atorney Docket No. 232285-70260169. The composition of claim 68, wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.

70. The composition of claim 69, wherein the viral vector is an adeno-associated virus (AAV) vector.

71. The composition of claim 66 or 67, wherein each delivery vehicle is a nanoparticle.

72. The composition of claim 71, wherein the nanoparticle is a lipid nanoparticle.

73. Use of the composition of any one of claims 35 to 72 in the manufacture of a medicament for treating or preventing a disease in a subject.

74. The use of claim 73, wherein the disease is muscular dystrophy.

75. A method of activating expression of FST in a cell, comprising administering to a cell, a tissue, or a subject the composition of any one of claims 35 to 72,wherein the second nucleic acid forms a complex with the DNA binding domain, wherein the complex binds to the endogenous target gene encoding for follistatin (FST) or the regulatory region of the gene encoding for FST, thereby activating expression of FST.

76. A method of improving muscle function, comprising administering to a cell, a tissue, or a subject the composition of any one of claims 35 to 72, wherein after administration, the level of FST in the cell, the tissue, or the subject is increased as compared to that before administration.

77. A method of treating muscular dystrophy, comprising administering to a cell, a tissue, or a subject the composition of any one of claims 35 to 72, wherein after administration, the level of FST in the cell, the tissue, or the subject is increased as compared to that before administration.

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