Non-enzymatic compositions having p300 DNA binding domains for gene activation and associated methods

Abstract

Provided herein are non-enzymatic systems comprising peptides having a DNA binding domain (DBD) fused to a p300 recruiter domain of DUX4 (p300R domain) and associated methods for modifying gene and protein levels in a cell. The p300R-DBD peptides selectively bind to a locus that are altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands. The systems may further comprise one or more guide RNA and may be formulated in lipid nanoparticles.

Description

Attorney Docket No.: 184F-414450-WONON-ENZYMATIC COMPOSITIONS HAVING P300 DNA BINDING DOMAINS FOR GENE ACTIVATION AND ASSOCIATED METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 780,054, filed March 28, 2025, which is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains an ST.26 compliant Sequence Listing, which is submitted concurrently in xml format and hereby incorporated by reference in its entirety. The .xml copy, created on March 28, 2026, is titled “184F-414450-WO.xml” and is 213,923 bytes in size.FIELD OF INVENTION

[0003] The invention generally relates to methods and compositions for modulating gene expression.BACKGROUND

[0004] Gene therapy may offer a powerful approach to address genetic disorders by directly modifying the DNA sequence. However, the complexity of many diseases, involving intricate networks of gene interactions, necessitates a more flexible therapeutic strategy. Epigenetic therapeutics, which may modulate gene expression without altering the underlying DNA sequence, hold the promise of addressing a broader spectrum of indications. However, current epigenetic therapeutic approaches may result in dysregulated or over-extended gene activation, lacking the ability to modulate or discontinue treatment in response to evolving patient needs. This, coupled with safety concerns arising from off-target effects, underscores the need for nextgeneration epigenetic therapies that offer greater control and enhanced safety profiles.SUMMARY

[0005] In some embodiments, the present technology comprises a system for increasing expression of one or more proteins in a cell, relative to a control, the systemAttorney Docket No.: 184F-414450-WOcomprising: (a) a non-naturally occurring peptide comprising a DNA binding domain (DBD) fused to a p300 Recruiter domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more guide RNA (gRNA)s.

[0006] In some embodiments, the present technology comprises a system for increasing expression of a Laminin Subunit Alpha 1 (LAMA1), an Insulin Like Growth Factor 1 (IGF1), IL-10, Leptin, GSTK1 , SMCHD1 , Utrophin, CD16, AFP, LRIF1 or an Adiponectin (ADIPOQ) protein in a cell, relative to a control, the system comprising: (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a LAMA1 , an IGF1, IL-10, Leptin, GSTK1 , SMCHD1 , Utrophin, CD16, AFP, LRIF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

[0007] In some embodiments, the present technology comprises a lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

[0008] In some embodiments, the present technology comprises a lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs comprising or consisting of the nucleotide sequence GTCCGAAGGGCCTGCGCACC (SEQ ID NO.8).

[0009] In some embodiments, the present technology comprises a method of increasing expression of one or more proteins in a cell, relative to a control, the method comprising the steps of: (i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the cell for aAttorney Docket No.: 184F-414450-WOperiod of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0010] In some embodiments, the present technology comprises a method of increasing a LAMA1 protein level, an IGF1 protein level, or an ADIPOQ protein level in a cell, relative to a control, the method comprising the steps of: (i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1 , an IGF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0011] In some embodiments, the present technology comprises an engineered cell having increased expression of one or more proteins, relative to a control, the engineered cell generated by the steps of: (i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0012] In some embodiments, the present technology comprises an engineered cell having an increased LAMA1 , IGF1 , or an ADIPOQ protein level, relative to a control, the engineered cell generated by the steps of: (i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1, an IGF1, or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0013] In some embodiments, the engineered cell is an engineered mammalian cell.Attorney Docket No.: 184F-414450-WO

[0014] In some embodiments, the engineered mammalian cell is an engineered human cell or an engineered murine cell.

[0015] In some embodiments, the engineered human cell or the engineered murine cell is a myoblast.

[0016] In some embodiments, the engineered mammalian cell is an engineered kidney cell, an engineered liver cell, or an engineered muscle cell.

[0017] In some embodiments, the DBD comprises a clustered regularly interspaced short palindromic repeats associated protein (Cas) domain, a zinc finger domain, a helix-turn-helix (HTH) domain, a leucine zipper domain, and a basic helixloop-helix (bHLH) domain.

[0018] In some embodiments, the Cas domain comprises an inactive nuclease domain.

[0019] In some embodiments, the Cas domain comprising an inactive nuclease domain is a dead Cas (dCas) domain.

[0020] In some embodiments, the locus comprises a nucleotide sequence encoding a gene.

[0021] In some embodiments, the locus comprises a nucleotide sequence that is a cis regulatory element of a gene.

[0022] In some embodiments, the locus comprises a nucleotide sequence that is a trans regulatory element of a gene.

[0023] In some embodiments, the gene is LAMA1.

[0024] In some embodiments, the gene is selected from the group consisting of SMCHD1 , LRIF1 , MATR3, IGF1 , LAMA1 , ADIPOQ, IGF1 , PML, AFP, RPL30, CCND1 , MyoD1 , and LDLR.

[0025] In some embodiments, the locus comprises or consists of a promoter.

[0026] In some embodiments, the promoter is a LAMA1 promoter.

[0027] In some embodiments, the Cas domain or the dCas domain comprises a Cas1 domain, a Cas2 domain, a Cas3 domain, a Cas9 domain, a Casio domain, a Cas12a domain, or a Cas13 domain.Attorney Docket No.: 184F-414450-WO

[0028] In some embodiments, the Cas domain or the dCas domain comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179.

[0029] In some embodiments, the gRNA specifically directs the p300R-DBD peptide to the locus.

[0030] In some embodiments, the gRNA mediates selective binding of the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-dCas peptide to the locus.

[0031] In some embodiments, the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-dCas peptide is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

[0032] In some embodiments, the gRNA is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

[0033] In some embodiments, the one or more gRNAs comprise two or more gRNAs.

[0034] In some embodiments, the one or more gRNAs comprises a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to one or more of SEQ ID NOs: 2-136.

[0035] In some embodiments, the p300R domain comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 1 .

[0036] In some embodiments, the p300R domain is fused to the DBD domain, the Cas domain, or the dCas domain by a peptide linker.

[0037] In some embodiments, the peptide linker comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

[0038] In some embodiments, the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands has a reductionAttorney Docket No.: 184F-414450-WOin expression of the gene by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the control.

[0039] In some embodiments, the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands is repressed by the methylation.

[0040] In some embodiments, the one or more histones are selected from the group consisting of H2A, H2B, H3, and H4.

[0041] In some embodiments, the one or more histones are methylated at a lysine (K) residue or an arginine (R) residue.

[0042] In some embodiments, the lysine residue is a K4, K5, K8, K9, K14, K16, K18, K20, K23, K27, K36, K56, K119, or a K120 residue.

[0043] In some embodiments, the arginine residue is an R2, R3, R8, R17, R23, or an R26 residue.

[0044] In some embodiments, the locus altered by DNA methylation of one or more CpG islands is repressed by the methylation.

[0045] In some embodiments, the one or more CpG islands are located in a promoter region, a 5' untranslated region, or a cis regulatory element of a gene.

[0046] In some embodiments, the non-naturally occurring peptide or the gRNA is encoded by a nucleotide sequence comprised within a vector.

[0047] In some embodiments, the vector is a viral vector.

[0048] In some embodiments, the viral vector is a lentiviral vector.

[0049] In some embodiments, the vector comprises a detectable marker or a reporter gene.

[0050] In some embodiments, the vector comprises an antibiotic-inducible gene expression system.

[0051] In some embodiments, the antibiotic is doxycycline or tetracycline.

[0052] In some embodiments, the antibiotic-inducible gene expression system is a Tet-ON system.Attorney Docket No.: 184F-414450-WO

[0053] In some embodiments, the system, the non-naturally occurring peptide, the gRNA, or the vector is present in a delivery vehicle.

[0054] In some embodiments, the delivery vehicle comprises a nanoparticle.

[0055] In some embodiments, the nanoparticle is a lipid nanoparticle.

[0056] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject in need thereof.

[0057] In some embodiments, the subject is mammalian.

[0058] In some embodiments, the subject is human.

[0059] In some embodiments, the methylation level of the one or more histones and / or the DNA methylation level of the one or more CpG islands is altered by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0060] In some embodiments, the expression level of the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0061] In some embodiments, the expression level of a gene encoding the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 illustrates Laminin Subunit Alpha 1 (LAMA1) protein expression in murine myoblasts transfected with exemplary p300 recruiter (p300R) domain peptides comprising a DNA binding domain (p300R-DBD) and selected LAM A1 -targeting guide RNAs (gRNAs) of the present technology.

[0063] FIG. 2 illustrates quantified LAMA1 gene expression in myoblasts transfected with exemplary p300R domain peptides and selected LAMA1 -targeting gRNAs of the present technology.Attorney Docket No.: 184F-414450-WO

[0064] FIGS. 3A and 3B illustrate ADIPOQ (FIG. 3A) and LAMA1 (FIG. 3B) gene expression levels in AML12 murine hepatocytes and C2C12 murine myoblasts treated with 3R-ON mRNA and a CX037 (CybeRNAx) lipid nanoparticle-encapsulated ADIPOQ- or LAMA1 -targeting gRNA, relative to controls.

[0065] FIGS. 4A and 4B illustrate LAMA1 gene expression levels in C2C12 murine myoblasts treated with 3R-ON mRNA and LAMA1 -targeting gRNAs formulated in GenVoy lipid nanoparticles (FIG. 4A) or 0X319 lipid nanoparticles (FIG. 4B) over a five-day period.

[0066] FIGS. 5A and 5B illustrate LAMA1 gene expression levels in 02012 murine myoblasts treated with LAMA1 -targeting gRNA g9 formulated in GenVoy lipid nanoparticles (FIG. 5A) or CybeRNAx 0X319 lipid nanoparticles (FIG. 5B) across a dose range of 0 ng to 2,000 ng, with expression shown at Day 1 (d1) and Day 7 (d7) relative to a matched reference control.

[0067] FIGS. 6A-6C illustrate changes in Insulin-like growth factor 1 (IGF-1) and adiponectin (ADIPOQ) gene expression levels in murine hepatocytes transfected with exemplary p300R-DBD peptides and selected IGF-1 or ADIPOQ-targeting gRNAs of the present technology, compared to controls.

[0068] FIG. 7 illustrates human leptin (hLeptin) mRNA expression levels in HepG2 hepatocellular carcinoma cells transfected with 3R-ON mRNA and various gRNA combinations, shown as fold-change relative to a reference control lacking gRNA (-gRNA Ref). Data from three independent experimental runs are illustrated.

[0069] FIGS. 8A and 8B illustrate ADIPOQ mRNA expression levels in HepG2 human hepatocellular carcinoma cells subjected to an hADIPOQ gRNA screen. FIG. 8A shows expression levels following transfection with individual hADIPOQ-targeting gRNAs, and FIG. 8B shows expression levels following co-transfection of the same gRNAs with 3R-ON mRNA.

[0070] FIGS. 9A-9C illustrate hADIPOQ mRNA expression in HepG2 cells following Lipofectamine-mediated transfection with 3R-ON mRNA and hADIPOQ-targeting guide RNAs. FIG. 9A depicts the hADIPOQ gRNA screen across single-guide and double-guide conditions with negative controls. FIG. 9B depicts the same dataset with the screen conditions subsetted as shown in the figure. FIG. 90 depicts theAttorney Docket No.: 184F-414450-WOcorresponding raw RT-qPCR cycle threshold (Ct) values for ADIPOQ and GAPDH for the screen.

[0071] FIG. 10 illustrates glutathione kinase 1 (GSTK1) mRNA expression levels in HepG2 human hepatocellular carcinoma cells co-transfected with 3R-ON mRNA and a library of hGSTKI -targeting gRNAs — tested as single, dual, and triple combinations — using Lipofectamine.

[0072] FIGS. 11A-11C illustrate ADIPOQ gene activation achieved by 3R-ON mRNA in combination with the hADIPOQ-targeting gRNA g41 (3R-ON / g41) compared with VP-dCas9 in three cell types — HepG2 cells (FIG. 11 A), primary human myoblasts (FIG. 11B), and primary human hepatocytes (FIG. 11 C)— under single and double dosing regimens.

[0073] FIGS. 12A and 12B illustrate RT-qPCR comparisons of adiponectin (ADIPOQ) gene activation in AML12 murine hepatocytes and HepG2 human hepatocellular carcinoma cells transfected to assess activation potency of a 3R-ON system versus a VP64-dCas9 activator using lead ADIPOQ-targeting guide RNAs. FIG.12A depicts mADIPOQ expression in AML12 cells transfected with 3R-ON and the mADIPOQ-targeting dual-guide combination g3+g5, or with VP64-dCas9 and the same guides. FIG. 12B depicts hADIPOQ expression in HepG2 cells transfected with 3R-ON and the hADIPOQ-targeting guide g41 using LNP and non-LNP delivery conditions, or with VP64-dCas9. The figure further depicts negative control conditions including untreated (LIT), guide-only (-g), and scrambled (Scr) controls.

[0074] FIGS. 13A and 13B illustrate RT-qPCR measurement of human adiponectin (hADIPOQ) mRNA expression in primary human hepatocytes following Lipofectamine-mediated transfection with (i) 3R-ON mRNA + hADIPOQ-targeting guide RNA g41 (FIG. 13A) or (ii) VP64-dCas9 + hADIPOQ-targeting guide RNA g41 (FIG.13B), under three media conditions (media only, +FBS, and +ApoE). Expression is shown as fold change relative to an untreated reference at 24 hours and Day 7, and across the indicated RNA doses.

[0075] FIG. 14 illustrates changes in IGF-1 gene expression levels in murine hepatocytes transfected with a p300R-DBD of the present technology and one or more IGF-1 -targeting gRNAs, compared to controls.Attorney Docket No.: 184F-414450-WO

[0076] FIGS. 15A and 15B illustrate changes in IGF-1 gene expression levels across three replicates in murine hepatocytes (FIG. 15A) and murine myoblast cells (FIG. 15B) when transfected with a p300R-DBD peptide of the present technology and an IGF-1 -targeting gRNA, compared to controls.

[0077] FIGS. 16A and 16B illustrate changes in IGF-1 gene expression levels in murine hepatocytes (FIG. 16A) and murine myoblasts (FIG. 16B) when transfected with a p300R-DBD peptide of the present technology and one or two IGF-1 -targeting gRNAs, compared to controls.

[0078] FIG. 17 illustrates a screen of 3R-ON mRNA and guide RNA combinations in AML12 murine hepatocytes, showing fold-change in gene expression across the tested combinations and a side-by-side comparison of the 3R-ON platform and a VP64-dCas9 benchmark.

[0079] FIGS. 18A and 18B illustrate murine ADIPOQ (mADIPOQ) gene expression screen results in AML12 murine hepatocytes following Lipofectamine-mediated transfection relative to a Scr reference across single-guide and combinatorial guide conditions with controls. FIG. 18A illustrates combined results from AML123R-ON mADIPOQ Screens 1-3, and FIG. 18B illustrates combined results from AML12 VP64-dCas9 mADIPOQ Screens 1-3.

[0080] FIGS. 19A and 19B illustrate fold-change in mADIPOQ (FIG. 19A) and murine IGF-1 (mlGF-1) (FIG. 19B) mRNA expression in AML12 murine hepatocytes following Lipofectamine-mediated delivery of 3R-ON mRNA and the indicated targetspecific guide RNAs, including mADIPOQ-targeting g3 and mlGF-1 -targeting g6, g8, or a g6+g8 combination, with expression levels shown relative to controls.

[0081] FIG. 20 illustrates RT-qPCR measurement of human adiponectin (hADIPOQ) mRNA expression in HepG2 cells co-transfected via Lipofectamine with 3R-ON mRNA and hADIPOQ-targeting guide RNAs (single guides or dual-guide combinations), with cells harvested at 24 hours, 48 hours, and 72 hours posttransfection. Expression is presented on a Iog10 scale.

[0082] FIGS. 21A-21 E illustrate changes in gene expression levels in cells transfected with a p300R-DBD peptide and a LAMA1 or IGF-1 -targeting gRNA of the present technology, compared to controls. FIGS. 21 A and 21 B illustrate changes in IGF-Attorney Docket No.: 184F-414450-WO1 gene expression levels in murine hepatocytes (FIG. 21 A) and C2C12 cells (FIG. 21 B). FIGS. 210 and 21 D illustrate changes in LAMA1 gene expression levels in murine hepatocytes (FIG. 210) and murine myoblasts (FIG. 21 D). FIG. 21 E illustrates Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression levels with use of each guide in murine hepatocytes ("A") and murine myoblasts ("0").

[0083] FIGS. 22A and 22B illustrate day 1 gene expression levels of IGF-1 and LAMA1 in murine hepatocytes (FIG. 22A) and murine myoblasts (FIG. 22B) transfected with a p300R-DBD peptide and an IGF-1 or LAMA1 -targeting gRNA of the present technology, compared to controls.

[0084] FIGS. 23A and 23B illustrate gene expression levels of IGF-1 and LAMA1 in murine myoblasts (FIG. 23A) and murine hepatocytes (FIG. 23B) transfected with a p300R-DBD peptide and an IGF-1 or LAMA1 -targeting gRNA of the present technology, compared to controls.

[0085] FIG. 24 illustrates LAMA1 gene expression levels at day 1 (d1) and day 3 (d3) in murine myoblasts following transfection at different doses of a p300R-DBD peptide and a LAMA1 -targeting gRNA of the present technology, compared to controls.

[0086] FIGS. 25A-25D illustrate LAMA1 gene expression levels in murine myoblasts transfected with different lipid nanoparticle (LNP) formulations of the present technology, containing a p300R domain peptide and a LAMA1 -targeting gRNA. FIG.25A illustrates fold change in LAMA1 gene expression levels, relative to controls. FIGS.25B and 25C illustrate encapsulation efficiency of the p300R domain peptide and the gRNA and encapsulated RNA, respectively, with two different LNP formulations of the present technology. FIG. 25D illustrates day 4 LAMA1 gene expression levels in cells transfected with the LNP formulations of FIGS. 25B and 250.

[0087] FIGS. 26A-26D illustrate LAMA1 gene expression levels in murine myoblasts transfected with different LNP formulations of the present technology, containing a p300R-DBD peptide and a LAMA1 -targeting gRNA. FIGS. 26A and 26B illustrate day 1 and day 7 LAMA1 gene expression levels following transfection with LNP formulations containing different amounts of p300R-DBD peptides (3R-ON), respectively. FIGS. 26C and 26D illustrate day 1 (d1) compared to day 7 (d7) LAMA1 gene expression changes in cells transfected with Precision Nanosystems™ GenVoy™ or CybeRNAx™ (0X319) LNP formulations, respectively.Attorney Docket No.: 184F-414450-WO

[0088] FIGS. 27A-27C illustrate LAMA1 gene expression levels in murine myoblasts transfected with LNP formulations containing different amounts of p300R domain peptides and LAMA1 -targeting gRNAs of the present technology. FIG. 27A illustrates the LAMA1 gene expression levels when LNP formulations were assessed for stability. FIGS. 27B and 27C illustrate encapsulation efficiency of the p300R-DBD peptide and the gRNA and encapsulated RNA, respectively, with the two different LNP formulations of FIG. 27A.

[0089] FIGS. 28A-28D illustrate LAMA1 and Laminin Subunit Alpha 2 (LAMA2) levels in murine myoblasts transfected with a p300R domain peptide and a LAMA1-targeting gRNA of the present technology, compared to controls. FIGS. 28A and 28B illustrate LAMA1 and LAMA2 gene expression levels. FIG. 28C illustrates LAMA1 in vivo and in vitro protein levels. FIG. 28D illustrates LAMA1 gene expression levels in cells transfected with various amounts of a Precision Nanosystems™ GenVoy™ LNP formulation of the present technology, containing a p300R-DBD peptide and a gRNA.

[0090] FIGS. 29A and 29B illustrate LAMA1 gene expression changes, in murine myoblasts transfected with an LNP formulation of the present technology containing a p300R domain peptide and a LAMA1 targeting gRNA, compared to untreated cells (UT).

[0091] FIG. 30 illustrates gene expression levels at 24 hours and 72 hours in murine myoblasts transfected with an LNP formulation of the present technology containing a p300R-DBD peptide and a gRNA, compared to untreated cells.

[0092] FIG. 31 illustrates fold-change in human adiponectin (hADIPOQ) mRNA expression in primary human hepatocytes (PHH) and HepG2 cells following Lipofectamine-mediated transfection with 3R-ON mRNA and hADIPOQ-targeting gRNA g41 , as measured by RT-qPCR and normalized to controls.

[0093] FIG. 32 illustrates transcriptomic (RNA-seq) analysis of ADIPOQ expression in HepG2 cells 24 hours after transfection with 3R-ON mRNA and the hADIPOQ-targeting gRNA g41 , using either CybeRNAx lipid nanoparticles (0X037) or Lipofectamine delivery, and further illustrates a comparison condition using a VP64-dCas9 system under the same experimental timing.

[0094] FIG. 33 illustrates a three-step workflow for evaluating the functional bioactivity of 3R-ON mRNA in combination with mADIPOQ-targeting gRNAs g3+g5 (3R-Attorney Docket No.: 184F-414450-WOON / g3+5), including (Step 1) GenVoy lipid nanoparticle formulation and quality control, (Step 2) treatment of AML12 murine hepatocytes to generate adiponectin-containing conditioned media, and (Step 3) assessment of bioactivity by plate reader quantification of glucose uptake in recipient AML12 and C2C12 cells.

[0095] FIGS. 34A and 34B illustrate mADIPOQ production in AML12 murine hepatocytes following treatment with 3R-ON mRNA and mADIPOQ-targeting gRNAs g3+g5 (3R-ON / g3+5) formulated in CybeRNAx lipid nanoparticles (CX037). FIG.34A shows mADIPOQ mRNA expression across a dose range, and FIG. 34B shows time-course quantification of secreted adiponectin protein in extracellular media.

[0096] FIGS. 35A and 35B illustrate functional glucose uptake assays in AML12 murine hepatocytes (FIG. 35A) and C2C12 murine myoblasts (FIG. 35B) treated with conditioned media containing endogenously produced adiponectin generated by LNP-mediated delivery of 3R-ON, with comparisons to controls.

[0097] FIGS. 36A-36D illustrate titration of 3R-ON mRNA to gRNA mass ratios for target gene activation across multiple cell models, relative to controls. FIG. 36A illustrates AML12 murine hepatocytes transfected with 3R-ON mRNA and an mADIPOQ-targeting gRNA (g3). FIG. 36B depicts AML12 cells transfected with 3R-ON mRNA and mlGF-1 -targeting gRNAs (g6, g8, or g6+g8). FIG. 36C depicts C2C12 cells transfected with 3R-ON mRNA and a LAMA1 -targeting gRNA (g9). FIG. 36D depicts C2C12 murine myoblasts transfected with 3R-ON mRNA and mlGF-1 -targeting gRNAs (g6, g8, or g6+g8).

[0098] FIG. 37 illustrates epigenetic profiles at the human adiponectin (ADIPOQ) locus, showing genome browser tracks for repressive histone mark H3K27me3 and active histone marks H3K27ac, H3K4me3, and H3K4me1 in hepatocytes and beige adipocytes across the ADIPOQ promoter and gene body regions.

[0099] FIG. 38 illustrates ELISA quantification of mADIPOQ protein in AML12 murine hepatocytes transfected with 3R-ON mRNA and mADIPOQ-targeting guide RNAs g3+g5 (3R-ON / g3+5) at 125 ng and 625 ng. The figure illustrates absolute adiponectin concentrations (ng / mL) measured daily in cell lysates (intracellular) and conditioned media (extracellular secretion), normalized to untreated controls, and includes corresponding scrambled guide RNA control conditions.Attorney Docket No.: 184F-414450-WO

[0100] FIGS. 39A and 39B illustrate LNP-mediated delivery and activation of hADIPOQ in HepG2 cells across an LNP dose range of 0 ng to 2,000 ng using 0X037 LNPs formulated with 3R-ON mRNA and hADIPOQ-targeting gRNA g41. FIG.39A illustrates LNP transfection efficiency measured as the percentage of mCherry-positive cells by flow cytometry following delivery of 3R-ON mCherry mRNA. FIG.39B illustrates RT-qPCR measurement of hADIPOQ mRNA expression following LNP delivery.

[0101] FIGS. 40A and 40B illustrate a 7-day time-course of hADIPOQ induction following treatment with LNP-encapsulated 3R-ON mRNA and hADIPOQ-targeting gRNA g41, with comparisons to an LNP-encapsulated scrambled gRNA control. FIG.40A depicts secreted adiponectin protein levels in conditioned media over time, and FIG. 40B depicts hADIPOQ mRNA expression measured by RT-qPCR.

[0102] FIGS. 41 A and 41 B illustrate functional glucose uptake in HepG2 cells following induction of hADIPOQ using LNP-encapsulated 3R-ON mRNA and hADIPOQ-targeting gRNA g41, with comparisons to an LNP-encapsulated scrambled gRNA control. FIG. 41 A illustrates relative intracellular glucose levels measured 3 days after LNP treatment (autocrine). FIG. 41 B illustrates relative intracellular glucose levels measured 2 hours after treatment with adiponectin-containing conditioned media (paracrine).

[0103] FIG. 42 illustrates dose-dependent mADIPOQ mRNA expression in AML12 murine hepatocytes following treatment with increasing concentrations of 3R-ON mRNA and mADIPOQ-targeting gRNAs g3+g5 (3R-ON / g3+5) formulated in CX037 LNPs, as measured by RT-qPCR at 24 hours post-transfection and presented as fold change relative to untreated controls.

[0104] FIG. 43 illustrates a dose-response comparison of ADIPOQ gene activation in cells treated with 3R-ON mRNA and an ADIPOQ-targeting guide RNA g3 (3R-ON / g3) delivered using either Lipofectamine or CX037 LNPs across a dose range of 3 ng to 3,000 ng.

[0105] FIG. 44 illustrates RT-qPCR-measured mADIPOQ gene expression in cells treated with 3R-ON mRNA and an mADIPOQ-targeting guide RNA (g3) delivered using GenVoy LNPs or CX037 (CybeRNAx) LNPs, across the indicated RNA doses (e.g., 30 ng and 3,000 ng) and time points (Day 1 and Day 3).Attorney Docket No.: 184F-414450-WO

[0106] FIGS. 45A-45D illustrate RT-qPCR analysis of dCas9 (3R-ON) mRNA and human adiponectin (hADIPOQ) mRNA expression at Day 1 following lipid nanoparticle (LNP)-mediated transfection of HepG2 cells and primary human hepatocytes (PHH) with 3R-ON (dCas9) mRNA and hADIPOQ-targeting guide RNA g41 using either 0X037 or GenVoy LNPs across a dose range of 125 ng to 1 ,000 ng. FIG. 45A depicts HepG2 dCas9 expression, FIG. 45B depicts HepG2 hADIPOQ expression, FIG. 45C depicts PHH dCas9 expression, and FIG. 45D depicts PHH hADIPOQ expression, each presented as fold change relative to untreated (UT) controls.

[0107] FIG. 46 illustrates RT-qPCR measurement of mADIPOQ mRNA expression in AML12 murine hepatocytes and C2C12 murine myoblasts following treatment with CX037 LNPs comprising 3R-ON mRNA and mADIPOQ-targeting guide RNAs g3, g5, or a multiplexed g3+g5 combination, with comparisons to controls.

[0108] FIGS. 47A-47D illustrate an experimental schema (FIG. 47A) and corresponding gene expression levels at 24 hours and 72 hours in mice treated with an LNP formulation of the present technology containing a p300R-DBD peptide and a LAMA1 -targeting gRNA, compared to untreated mice (FIGS. 47B-47D) (n=3).

[0109] FIGS. 48A and 48B illustrate 24-hour and 72-hour LAMA1 gene expression level differences in the mice of FIGS. 47A-47D.

[0110] FIGS. 49A-49C illustrate additional assessments of LAMA1 gene expression level changes in the mice of FIGS. 47A-47D.

[0111] FIGS. 50A and 50B illustrate gene expression levels with various p300R-DBD peptides (3R-ON) and gRNA amounts for the mice of FIGS. 47A-47D.

[0112] FIG. 51 illustrates ADIPOQ mRNA and dCas9 transcript levels in C57BL / 6 mice following a single tail vein injection of 3R-ON mRNA and an ADIPOQ-targeting guide RNA g3 (3R-ON / g3) formulated in GenVoy / Cytiva LNPs at low dose (0.1 mg / kg) or high dose (1 mg / kg).

[0113] FIG. 52 illustrates hepatic transcriptomic (RNA-seq) analysis of ADIPOQ expression in C57BL / 6 mice following a single tail vein injection of 3R-ON mRNA with ADIPOQ-targeting guide RNAs g3+g5 formulated in Aouitas LNPs at doses of 0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg. Liver lysates were collected at 24-48 hours postinjection, and ADIPOQ expression is presented in transcripts per million (TPM).Attorney Docket No.: 184F-414450-WO

[0114] FIG. 53 illustrates hepatic murine adiponectin (ADIPOQ) mRNA and dCas9 transcript levels in high-fat diet (HFD) C57BL / 6 male mice following tail vein injection of 3R-ON mRNA with ADIPOQ-targeting guide RNAs g3+g5 formulated in Acuitas lipid nanoparticles (LNPs) at doses of 0.1 mg / kg and 0.5 mg / kg. Liver tissue was harvested at 24 hours, 72 hours, and 11 days post-injection, lysed, and analyzed by RT-qPCR, with results presented as fold change.

[0115] FIGS. 54A and 54B illustrate LAMA1 gene expression levels at day 1 (d1) and day 3 (d3) in murine myoblasts transfected with a p300R-DBD peptide and LAMA1 -targeting gRNA or an alternate domain peptide (VP-ON) and a LAMA1 -targeting gRNA in cells treated with a p300 inhibitor (iP300w), normalized to GAPDH gene expression levels.

[0116] FIGS. 55A-55D illustrate additional LAMA1 gene expression measurements for the same cells of FIGS. 54A and 54B.

[0117] FIGS. 56A and 56B illustrate LAMA1 gene expression levels in AML12 and murine myoblasts transfected with a p300R-DBD peptide and a LAMA1 -targeting gRNA of the present technology, normalized to GAPDH gene expression levels.

[0118] FIGS. 57A-57D illustrate LAMA1 gene expression levels at day 1 (d1 ), day 3 (d3), and day 5 (d5) in murine myoblasts transfected with various amounts of LNP formulation containing a p300R-DBD peptide and a LAMA1 -targeting gRNA of the present technology, relative to untreated controls. FIGS. 57A-57C illustrate GAPDH gene expression levels at d1 , d3, and d5, respectively. FIG. 57D illustrates LAMA1 gene expression levels overdl , d3, and d5, normalized to the GAPDH gene expression levels of FIGS. 57A-57C.

[0119] FIGS. 58A-58E illustrate LAMA1 gene expression changes in the same cells of FIGS. 57A-57D treated with 0.1 pM, 1.0 pM, or 10 pM of a histone deacetylase inhibitor (HDACi).

[0120] FIGS. 59A and 59B illustrate LAMA1 gene expression changes in the same cells of FIGS. 57A-57D treated with 0.1 pM, 1.0 pM, or 10 pM of a p300 inhibitor (iP300w).

[0121] FIG. 60 illustrates TIP ChlP-seq analysis of dCas9 occupancy at the human adiponectin (ADIPOQ) promoter in the genomic region targeted by hADIPOQ guideAttorney Docket No.: 184F-414450-WORNA g41 , showing dCas9 enrichment and corresponding input tracks across an approximately 36 kb window on chromosome 3.

[0122] FIG. 61 illustrates RT-qPCR time-course analysis of dCas9 (3R-ON) mRNA and human adiponectin (hADIPOQ) mRNA expression in primary human hepatocytes following transfection with lipid nanoparticle (LNP)-encapsulated 3R-ON (dCas9) mRNA and hADIPOQ-targeting guide RNA g41 , monitored over a 5-day period, with dCas9 expression plotted on the left y-axis and hADIPOQ induction plotted on the right y-axis.

[0123] FIGS. 62A and 62B illustrate RT-qPCR specificity analysis of human adiponectin (hADIPOQ) induction in primary human hepatocytes (PHH) (FIG. 62A) and HepG2 cells (FIG. 62B) following transfection with CX037 lipid nanoparticles (LNPs) containing 3R-ON mRNA and hADIPOQ-targeting guide RNA g41 at doses ranging from 125 ng to 1 ,000 ng, further depicting expression of a panel of DUX4-related downstream genes (DUX4, LDLR, MBD3L2, PRAMEF12, and ZSCAN4).

[0124] FIGS. 63A-63C illustrate RT-qPCR time-course analysis of transcriptional specificity in primary human hepatocytes (PHH) and HepG2 cells transfected with CX037 lipid nanoparticles (LNPs) delivering 3R-ON mRNA and either the hADIPOQ-targeting guide RNA g41 or a scrambled (Scr) guide RNA control over a 7-day period, with expression normalized to untreated (UT) references. FIG. 63A depicts PHH results, FIG. 63B depicts HepG2 results, and FIG. 63C depicts HepG2 results for the scrambled-guide control condition, including measured expression of hADIPOQ, dCas9, and a panel of DUX4-related genes (DUX4, LDLR, MBD3L2, PRAMEF12, and ZSCAN4).

[0125] FIGS. 64A-64D illustrate ELISA-based measurements of adiponectin protein species in HepG2 conditioned media collected on Day 6 following 3R-ON mRNA delivery with the hADIPOQ-targeting guide RNA g41, including quantification of high molecular weight (HMW) adiponectin and additional adiponectin oligomer formats as shown across FIGS. 64A-64D.

[0126] FIG. 65 illustrates RT-qPCR measurement of human adiponectin (ADIPOQ) mRNA expression in HepG2 cells treated with 3R-ON mRNA and an hADIPOQ-targeting guide RNA in the presence or absence of a p300 inhibitor (p300i) across the indicated 3R-ON dose conditions, and further depicts a VP64-dCas9Attorney Docket No.: 184F-414450-WOcomparator condition under the same experimental settings, with expression presented as fold change relative to appropriate controls.

[0127] FIGS. 66A and 66B illustrate RT-qPCR time-course measurement of human adiponectin (hADIPOQ) mRNA expression in HepG2 cells following lipid nanoparticle (LNP)-mediated treatment across a range of input RNA doses, with expression presented as fold change relative to controls. FIG. 66A depicts cells treated with 3R-ON mRNA, and FIG. 66B depicts cells treated with VP64-dCas9, with hADIPOQ expression monitored over multiple days.

[0128] FIG. 67 illustrates a kinetic analysis of murine adiponectin (mADIPOQ) induction in AML12 murine hepatocytes following treatment with 3R-ON mRNA and mADIPOQ-targeting guide RNAs g3+g5 (3R-ON / g3+5), showing RT-qPCR-measured mADIPOQ mRNA fold change (left y-axis) and ELISA-measured adiponectin protein levels in conditioned media and cell lysates (right y-axis) over a multi-day time course, with corresponding scrambled (Scr) guide RNA control conditions.

[0129] FIGS. 68A-68E illustrate Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), IGF-1, and LRIF1 gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and various combinations of SMCHD1 -targeting gRNAs, compared to controls.

[0130] FIGS. 69A-69D illustrate Ligand Dependent Nuclear Receptor Interacting Factor 1 (LRIF1) and SMCHD1 gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and an LRIF1 -targeting gRNA combination or a SMCHD1-targeting gRNA combination, compared to controls.

[0131] FIGS. 70A-70F illustrate Methyl-CpG binding domain protein 3 like 2 (MBD3L2) (FIG. 70A), IGF-1 (FIG. 70B), SMCHD1 (FIG. 70C), Zinc Finger And SCAN Domain Containing 4 (ZSCAN4) (FIG. 70D), Leucine Twenty Homeobox (LEUTX) (FIG.70E), and solute carrier family 34 member 2 (SLC34A2) (FIG. 70F) gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and an IGF-1 -targeting gRNA or a SMCHD1 -targeting gRNA.

[0132] FIGS. 71 A and 71 B illustrate matrin 3 (MATR3) (FIG. 71 A) and IGF-1 (FIG.71 B) gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and an IGF-1 -targeting gRNA or an MATR3-targeting gRNA combination.Attorney Docket No.: 184F-414450-WO

[0133] FIGS. 72A and 72B illustrate LRIF1 gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and various LRIF1 -targeting gRNA combinations.

[0134] FIGS. 73A and 73B illustrate MBD3L2 gene expression levels in murine myoblasts 72 hours following transfection with a p300R-DBD peptide and a MBD3L2-targeting gRNA.

[0135] FIGS. 74A-74E illustrate IGF-1 (FIG. 74A), SMCHD1 (FIGS. 74B-74D), and LAMA1 (FIG. 74E) gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and a SMCHD1 , LAMA1 , or IGF-1 -targeting gRNA.

[0136] FIG. 75 illustrates MATR3 gene expression levels in murine myoblasts transfected with a p300R-DBD peptide and a MATR3-targeting gRNA of the present technology.

[0137] FIGS. 76A and 76B illustrate IGF-1 (FIG. 76A) and Trinucleotide Repeat Containing Adaptor 6B (TNRC6B) (FIG. 76B) gene expression levels in murine myocytes transfected with a peptide comprising a DBD fused to an embryonic ectoderm development polypeptide binder (EB) domain peptide and an IGF-1 or TNRC6B-targeting gRNA, relative to controls.

[0138] FIG. 77 illustrates (top) a genomic schematic of the murine structural maintenance of chromosomes flexible hinge domain containing 1 (mSMCHDI) locus depicting CpG island density, ReMap density peaks, and identified target regions R1-R7, including regions targeted with pools of three guide RNAs (gRNAs), and (bottom) a qPCR-based screen of SMCHD1 gene expression in C2C12 cells following Lipofectamine-mediated transfection with 3R-ON mRNA and combinations of gRNAs targeting regions R1-R7, with expression presented as fold change relative to controls.

[0139] FIG. 78 illustrates murine utrophin (Utrn) mRNA expression in C2C12 cells following transfection with identical concentrations of either 3R-ON or VP64-dCas9 effector systems and Utrn promoter-targeting guide RNAs (mUTRO g8, g12, or g8+g12). The figure further depicts control conditions including untreated (UT) cells, a Scramble gRNA reference, and a non-relevant LAMA1 -targeting guide RNA (g9), with Utrn expression quantified at 24 hours (Day 1) by RT-qPCR and presented as fold change relative to the Scramble control (1 .0).Attorney Docket No.: 184F-414450-WO

[0140] FIG. 79 illustrates LAMA1 gene expression levels in C2C12 murine myoblasts following co-transfection with EB-ON and 3R-ON and combinations of LAMA1 proximal promoter-targeting guide RNAs (g9— g12), with expression quantified at Day 1 , Day 3, and Day 5 by RT-qPCR and presented as LAMA1 fold change relative to an untreated (UT) reference.

[0141] FIGS. 80A-80C illustrate differentiation of THP-1 monocytes into macrophages and RT-qPCR screening of IL-10 activation following guide RNA (gRNA) transfection. FIG. 80A depicts a differentiation and transfection workflow in which THP-1 monocytes (Day 0) are treated with phorbol 12-myristate 13-acetate (PMA) to generate adherent macrophages by Day 3, followed by transfection with single or combinatorial gRNAs and RNA harvest 24 hours post-transfection (Day 4) for transcriptional analysis. FIG. 80B depicts IL-10 mRNA expression (fold change) in differentiated macrophages following Lipofectamine-mediated transfection of mRNA and IL-10-targeting gRNAs (single and combinations), shown relative to untreated (UT) and / or scrambled (Scr) controls. FIG. 80C depicts a secondary RT-qPCR screen of gRNA combinations in differentiated macrophages, with IL-10 fold change shown on the primary y-axis and, for selected samples including hADIPOQ g41 combinations, ADIPOQ fold change shown on the secondary y-axis.

[0142] FIG. 81 illustrates secreted interleukin-10 (IL-10) protein levels over a 5-day period following PMA-mediated differentiation of THP-1 monocytes and subsequent transfection with 3R-ON and selected IL-10-targeting guide RNA combinations (1 +2 and 1+6), with protein levels compared to VP64-dCas9 control conditions and quantified in pg / mL.

[0143] FIGS. 82A and 82B illustrate flow-cytometry analysis of NK92 cells transfected with varying amounts (1 pg, 5 pg, and 12.5 pg) of 3R-ON or EB-ON mRNA together with CD16-targeting guide RNAs. FIG. 82A depicts the CD16-targeting guide RNA conditions tested with each effector system across the dose range. FIG.82B depicts transfection / activation readouts presented as the percentage of FITC-positive cells (% FITC+), and further includes an eGFP positive control condition.

[0144] FIGS. 83A and 83B illustrate flow-cytometry analysis of NK92 cells transfected with effector mRNAs and a CD16-targeting guide RNA (g6). FIG.83A depicts delivery / transfection readouts, including an eGFP control and mCherryAttorney Docket No.: 184F-414450-WOexpression in cells transfected with VP-ON + g6 or 3R-ON + g6 at a 1 :1 ratio. FIG.83B depicts the percentage of CD16-positive cells measured at Day 4 in unsorted and sorted cell populations to assess persistence of CD16 protein expression across the indicated transfection conditions.

[0145] FIGS. 84A-84C illustrate flow-cytometry analysis of CD16 surface expression in cells at Day 4 (d4) following electroporation (EP) with 3R-ON or VP-ON effector mRNA and a CD16-targeting guide RNA (g6). FIG. 84A depicts the percentage of FITC-positive and mCherry-positive cells across the indicated EP and control conditions. FIG. 84B depicts the percentage of CD16-positive cells, including comparisons between unsorted and sorted cell populations and baseline “No EP -Ab” and “No EP” controls. FIG. 84C depicts mean fluorescence intensity (MFI) data for the indicated conditions.

[0146] FIGS. 85A-85C illustrate 3R-ON-mediated activation of a cell-surface protein in NK-92 cells. FIG. 85A depicts a schematic of a 3R-ON construct fused to mCherry via a 2A sequence, in which translation yields 3R-ON and mCherry at an approximately 1 :1 ratio following cleavage. FIG. 85B depicts flow-cytometry measurement of target protein surface expression in NK-92 cells following delivery of 3R-ON or VP64-dCas9. FIG. 85C depicts flow-cytometry detection of 3R-ON-mediated target protein surface expression in mCherry-negative cells.

[0147] FIG. 86 illustrates gene expression levels in cells transfected using Lipofectamine with 3R-ON mRNA and a panel of ten target-specific guide RNAs (g1-g10), with comparisons to untreated (UT), 3R-ON without guide RNA (3R -g), and 3R-ON with a scrambled guide RNA (3R Scr) control conditions, with expression presented as fold change relative to the indicated control.

[0148] FIGS. 87A-87E illustrate gene expression and morphology in FSHD1 (54-2) myotubes following transfection of FSHD1 myoblasts on Days 1 and 3 with 0.1 pg 3R-ON mRNA alone or in combination with single guide RNAs (sgRNAs), followed by induction of differentiation on Day 4 for 72 hours. Conditions include untransfected cells, 3R-ON mRNA alone, scrambled sgRNA + 3R-ON mRNA, and a target-gene sgRNA combination (g2+g5+g30) + 3R-ON mRNA. FIGS. 87A-87D depict RT-qPCR expression levels (fold change) of the indicated transcripts, and FIG. 87E depictsAttorney Docket No.: 184F-414450-WOrepresentative microscopy images of differentiated myotubes under the corresponding conditions.DETAILED DESCRIPTION

[0149] Gene therapy may provide a promising method for correcting genetic disorders by directly modifying or activating expression of genetic sequences. However, the complexity of many diseases, characterized by intricate networks of gene interactions, necessitates a more adaptable therapeutic strategy. Traditional gene editing technologies, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-nuclease-based technologies, may involve enzyme-mediated changes to DNA, which may lead to unintended off-target effects and long-term safety concerns including unwanted genetic edits. These methods often lack the ability to precisely control the duration and extent of gene activation, making it difficult to adapt treatment to the evolving needs of patients. In contrast, the present technology may include methods of controlling gene activation without altering an underlying DNA sequence by promoting euchromatin formation. These methods utilize epigenetic approaches to modulate gene expression, offering a safer and more adaptable alternative. By avoiding enzyme-mediated DNA modifications, the present technology may reduce the risk of off-target effects with greater control over gene activation, relative to conventional technologies. As such, the present technology may comprise a safer and / or more effective method for gene activation, relative to such conventional technologies.

[0150] While the present technology is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present technology is to be considered as an exemplification of the technology and is not intended to limit the present technology to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the present technology in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0151] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." It is to be understood, although not always explicitly stated, that allAttorney Docket No.: 184F-414450-WOnumerical designations are preceded by the term "about." It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0152] To the extent any materials incorporated by reference herein conflict with the present technology, the present technology controls.Definitions

[0153] The term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0154] "Amino acids" are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N-CHR-COOH, wherein R represents a side chain group. The various a-amino acids differ in the side-chain moiety that is attached to the a-carbon. The "amino acids" of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. As set forth above, the term "amino acid" also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids,Attorney Docket No.: 184F-414450-WOderivatized amino acids, constructs or structures designed to mimic amino acids, and the like.

[0155] Amino acids, including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs, or structures designed to mimic amino acids (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as "residues."

[0156] "Non-naturally occurring amino acids," as used herein, may refer to an amino acid that is a non-proteinogenic or unnatural amino acid. This may comprise synthetic or modified forms of amino acids that are not naturally present in biological systems and may be chemically synthesized or comprise a modified version of a naturally occurring amino acid. Nonlimiting examples of non-naturally occurring amino acids include Norleucine, Homocysteine, Beta-Alanine, Azetidine-2-carboxylic acid (Aze), Diaminopimelic acid (OAP), Alpha-Aminoisobutyric acid (Aib), 2-Aminoisobutyric acid (Aib), 4-Thiaproline.

[0157] As used herein, "conservative amino acid substitution" means a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g., antigen-binding activity and specificity of a native or reference polypeptide is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3)Attorney Docket No.: 184F-414450-WOacidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0158] Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0159] The term "expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. "Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral -based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.

[0160] The term "subject" refers to a mammalian subject, preferably a human. A "subject in need thereof" may refer to a subject who has been diagnosed with a disease, or is at an elevated risk of developing a disease. The phrases "subject" and "patient" are used interchangeably herein.Attorney Docket No.: 184F-414450-WO

[0161] A "therapeutically effective amount" as used herein is an amount that produces a desired effect in a subject for treating a disease. In some embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications), the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a composition, a vector, or a pharmaceutical composition containing the same, and adjusting the dosage accordingly. For additional guidance, see, e.g., Remington, The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, 2012, and Goodman & Gilman, The Pharmacological Basis of Therapeutics, 12th Edition, McGraw-Hill, New York, NY, 2011 , the entire disclosures of which are incorporated by reference herein.

[0162] A "clinically effective amount," "clinically effective concentration," or "clinically effective dose" refers to a concentration or dose of a composition containing the same that is shown to be effective in clinical trials or is predicted to be effective based on early phase or pre-clinical trials. In some embodiments, a "clinically effective amount" is the same as a "therapeutically effective amount." In some embodiments, a "clinically effective amount" is higher or lower than a "therapeutically effective amount." For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, the entire disclosure of which is incorporated by reference herein.Attorney Docket No.: 184F-414450-WO

[0163] The term "p300 recruiter" refers to a peptide that specifically binds to and / or recruits a p300 protein. This includes the C-terminal p300 binding domain of a Double Homebox 4 (DLIX4) protein.

[0164] The term "p300R-DBD" refers to a peptide comprising or consisting of a p300 recruiter domain of DUX4 (p300R domain) that is fused to a DNA binding domain (DBD), forming a fusion peptide. The p300R domain comprises or consists of a C-terminal p300 binding domain of a DUX4 protein, and specifically recruits and binds to p300. The p300R-DBD peptide selectively binds to target loci in DNA, which may be activated after the recruitment of endogenous p300 to the p300R. The p300R-DBD as used herein may also be referred to "engineered peptides," "non-naturally occurring engineered peptides," "p300R peptides," and "peptides."

[0165] The term "gRNA" (guide RNA) refers to an RNA molecule designed to guide a DNA-binding enzyme to a specific genomic location.

[0166] The term "dCas" refers to a modified version of a Cas nuclease enzyme or at least a portion thereof that is catalytically inactive. The dCas may bind to a target DNA sequence guided by a gRNA, without introducing a double-strand break or another nuclease activity. The dCas may be fused with other peptide domains for modulating gene expression without altering the DNA sequence.

[0167] "Promoting" of euchromatin as used herein refers to the initiation or increase of euchromatin formation, relative to baseline or a control.

[0168] The term "control" refers to any cell or subject used as a basis for comparison. A control subject includes, but is not limited to, the subject of the present technology at baseline or a subject that has not been administered a composition of the present technology. The control may also comprise a system, composition, formulation, method, or lipid nanoparticle that lacks one or more features of the present technology.Compositions

[0169] The present technology comprises peptides having p300R domains fused to DNA binding domains (i.e., "p300R-DBD) that selectively recruit p300 proteins, including native p300 proteins (i.e., the endogenous p300 proteins expressed in an engineered cell, a host cell or a subject of the present technology), to the site at which the p300R domain is present. Recruitment of p300 promotes acetylation of proteinsAttorney Docket No.: 184F-414450-WO(e.g., histone or non-histone proteins) at or proximal to the p300R domain site. This acetylation promotes euchromatin formation, thereby activating and / or increasing expression of genes whose expression may be altered by post translational modifications of the proteins. The euchromatin formation and / or activation or increase in gene expression may occur in less than about 1 second, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 1 day.

[0170] Rather than expressing an exogenous p300 construct to acetylate histones, the p300R peptides of the present technology recruit native p300 to the p300R domain. Without intending to be bound by any particular theory, the p300R peptides of the present technology may reduce the risk of off-target epigenetic or gene expression changes that are otherwise observed with exogeneous p300 expression thereby circumventing use of an overexpression system to deliver exogenous p300. In this way, the p300R peptides of the present technology may reduce toxicity and off-target p300 acetylation activity due to the increase in p300 expression, relative to baseline.

[0171] In some embodiments, the p300R peptides are fused to another peptide comprising a DNA binding domain (DBD), referred to herein as "p300R-DBD peptides". The DBD selectively binds specific regions of the genome, guiding the p300R domains to these regions. This further reduces off-target acetylation events that are observed with the expression of exogenous p300 constructs, such as the acetylation of nonhistone proteins.

[0172] Nonlimiting examples of p300R domains and DBD domains of p300R-DBD peptides are further detailed under the "p300R Domains" and "DNA Binding Domains" headers.

[0173] The present technology further includes compositions and pharmaceutical compositions comprising a p300R-DBD peptide, a vector encoding a p300R-DBD peptide and, optionally, a gRNA, a host cell comprising the vector, the composition, and / or the pharmaceutical composition, and associated methods of making and using the same.

[0174] The peptides of the present technology (e.g., p300R-DBD peptides) may comprise one or more features of the peptides disclosed in W02020185850A1, suchAttorney Docket No.: 184F-414450-WOas the DBD peptides disclosed therein, which is incorporated by reference in its entirety herein.P300R Domains

[0175] The p300R domains of the present technology specifically bind to and / or recruit p300 proteins. The p300R domains may be comprised of an amino acid sequence of a p300 binding domain. Nonlimiting examples of proteins having p300 binding domains useful with the present technology include DUX4 (NCBI Accession(s): AUA60624.1 , AUA60623.1 , AUA60622.1, AUA60621.1, AUA60620.1 , AUA60367.1), NUT (NCBI Accession(s): NP_001271221.2, NP_786883.2, NP_001271222.2), p53 (NCBI Accession: AAC12971.1), SP1 (NCBI Accession(s): P08047.3, CBM42955.1, NP 612482.2, NP_003100.1 , NP_001238754.1 ), MEF2 (NCBI Accession(s): 002078.1 , AAB25838.1 , CAA48517.1 ), CITED2 (NCBI Accession(s): NP_006070.2, NP 001161860.1, NP 001161861.2, 099967.2), RelA (NCBI Accession(s): NP_068810.3, NP 001138610.1 , NP_001230913.1 , NP_001230914.1, NP 001391586.1 ), and HIF1a (NCBI Accession(s): 016665.1, NP_001230013.1, NP 851397.1, NP 001521.1 ).

[0176] In some embodiments, the p300R domains comprise or consist of at least a portion of the C-terminal domain of the DUX4 p300 binding domain. In some embodiments, the p300R domains comprise or consist of at least a portion of the p300 binding or p300 recruitment domains of a protein selected from the group consisting of NUT, p53, SP1, MEF2, CITED2, RelA, and HIF1a.

[0177] In some embodiments, the p300R domains of the present technology may comprise or consist of the amino acid sequence MSARQGQMQGIPAPSQALQEPAPWSALPCGLLLDELLASPEFLQQAQPLLETEAPG ELEASEEAASLEAPLSEEEYRALLEEL (SEQ ID NO: 1).

[0178] In some embodiments, the p300R domain comprises or consists of an amino acid sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

[0179] In some embodiments, the p300R domain comprises or consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,Attorney Docket No.: 184F-414450-WO92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

[0180] In some embodiments, the p300R domain comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

[0181] Any p300R domain amino acid sequence of the present technology may comprise one or more substitution variants. Substitution variants involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Such substitutions may be conservative amino acid substitutions.

[0182] Any p300R domain amino acid sequence of the present technology may be modified to comprise one or more non-naturally occurring amino acids. In some embodiments, the p300R domains comprise or consist of an amino acid sequence having one or more non-naturally occurring amino acids. In some embodiments, the p300R domains comprise or consist of an amino acid sequence having two or more non-naturally occurring amino acids. In some embodiments, the modification of the amino acid sequences to comprise non-naturally occurring amino acids is a conservative substitution.

[0183] The p300R domains may be shorter than the p300R domain amino acid sequences of the present technology that described above. For example, the p300R domain may comprise an amino acid sequence about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% shorter or reduced by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids relative to the p300R domain amino acid sequences described above.DNA Binding DomainsAttorney Docket No.: 184F-414450-WO

[0184] The p300R domains of the present technology may be fused to a DBD, forming the p300R-DBD peptide (i.e., a fusion peptide). The DBD may be positioned N-terminally relative to the p300R domain, C-terminally relative to the p300R domain, at or near the N-terminal end of the fusion peptide relative to the p300R domain, at or near the C-terminal end of the fusion peptide relative to the p300R domain. The DBDs bind specific genomic regions, thereby guiding the p300R domain to a precise site. DBDs may comprise a clustered regularly interspaced short palindromic repeats associated protein (Cas) domain. Non-limiting examples of Cas domains include a Cast domain, a Cas2 domain, a Cas3 domain, Cas9 domain, a Casio domain, a Cas12a domain (e.g., ErCasI 2a domain), or a Cas13 domain. Other DBDs may comprise a zinc finger domain, a helix-turn-helix (HTH) domain, a leucine zipper domain, and a basic helixloop-helix (bHLH) domain. In some embodiments, the Cas domain comprises a responsive portion which interacts with a gRNA that selectively binds to the locus.

[0185] The Cas domain may comprise an inactive nuclease domain. In some embodiments, the inactive nuclease domain is a dead Cas (dCas) domain.

[0186] In some embodiments, the Cas domain comprises or consists of an amino acid sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179 (Table 1).

[0187] In some embodiments, the Cas domain comprises or consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179.

[0188] In some embodiments, the Cas domain comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179.Table 1 : Cas domain amino acid sequencesName Amino Acid Sequence SEQ ID NOAttorney Docket No.: 184F-414450-WOdCas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 157RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR ICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLI NGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE D IQKAQVSGQG DSLHEHIAN LAGSP Al KKG I LQTVKVVD ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD MYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY GG FDSPTVAYS VLVVAKVE KG KSKKLKS VKE LLG IT I M E RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDAttorney Docket No.: 184F-414450-WOKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDStreptococcus MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNT 162 thermophiles SKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRN Cas 9 RILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKY PIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYL ALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAIF ESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSG IFSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDL ETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEA PLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVFKDDT KNGYAGYIDGKTNQEDFYVYLKKLLAEFEGADYFLEKID REDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFL AKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKI TPWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHS LLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRL YFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLST YHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQR LSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEK SGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQKAQIIG DEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGG RKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKEL GSKILKENIPAKLSKIONNALQNDRLYLYYLQNGKDMYT GDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNR GKSDDVPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTK AERGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFN NKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREIND FHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNS FRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVN EETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQNH GLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKK YGGYAGISNSFTVLVKGTIEKGAKKKITNVLEFQGISILO RINYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRAttorney Docket No.: 184F-414450-WOMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTI NENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKL LNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRG SAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYET RIDLAKLGEGA0A1L8R GR8 MKQNKELVNIGFDIGIASVGWSVVSKQSGKILETGVSIF 163 Enterococcus PSGTASKNEERRSFRQARRLLRRRKNRISDLKILLEENG FRIAKLNQLVTPYELRVRGLNEQLSKEELSVALLHLVKRcanisRGISYSLEDSEGEGDNQTSYKQSVSINQKLLKEKTPGEI QLERLEKYGKIRGQVKDLQEENAAVLMNVFPNTAYVRE AELILLKQKEYYSEITDNFIKEATALISRKREYFVGPGSEK SRTDYGIYRTDGTKLDNLFEILIGKDKIFPNEFRAAGNSY TAQLYNLLNDLNNLKIKTLEDGKLTKDQKLSIIEELKTTTK KVNMMQLIKKIAKAEESDISGYRIDRNDKPEIHSMAIFYK VRKKFLEQEIDINDWPIDFLDILGRVLTLNTENGEIRRSLT ELKKDYIFLDETLIELIINSKDSFKLTSNQKWHRFSLKTM QLLIPELLNSSKEQMTILTELGLLHENKQDYSNKTKIDVK NLTENIYNPVVRKSVKQAMDIFNSLFKKYPNIAYLVVEM PRDEAEDEVEQKKQAQKFQKENEAEKEKSLKEFQELA GVSDSQLENQIYKRRKLRMKIRLWYQQLGKCPYSGKTI AAEDLFWTDHLFEIDHVIPLSISYDDGQNNKVLCYSEMN QEKGQKTPYGFMQSGKGQGFSALQAMLKSNSRMSGA KKRNLLFTEDINDIEVRKRFIARNLVDTRYASRIVLNELQ QFTRSKQLDTKVTVIRGKFTSKLRETWRLNKSRETHHH HAVDATIIAVSPMLKLWERNAEIIPMKVNENVVDIKTGEIL TDKVYQEEMYQLPYASLLEDIAVMENKIKFHHQVDKKM NRKVSDATIYATRSAKVGKDKEPQNYVLGKIKDIYDTKE YENFKKIYDKDKSKFLMQQLDPMTFEKLEKVLKEYPDF EEVQQDNGRVKRIPISPFELYRREKGPITKFAKRNNGPA IKSVKYYDSKMGSAIDITPQTAKNKKVVLQSLKPWRTDV YFNQETKEYEIMGIKYSDMQYLNGNYGITNERYKEIQRE EGVADNSEFMMSLYRGDRIKVIDTNSDESVELLFGSRTIAttorney Docket No.: 184F-414450-WOPTKKGYVELKPIEKTKFDSKEIVSFYGQVTPNGQFVKKF TRNGYRLLKVNTNILGNPYYISKEGINPRNILDTGFKGJ7RUA5 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN 164VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTStaphylococcusDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRaureus Cas9GVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQ LERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQ LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYE MLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITR DENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDI KGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGT HNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQ QKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKE NAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEV DHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSS SDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINR FSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVK VKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANA DFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQE YKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYS TRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLM YHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTK YSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVK LSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVN NDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQS IKKYSTDILGNLYEVKSKKHPQIIKKGG3ECR1 MLFNKCIIISINLDFSNKEKCMTKPYSIGLDIGTNSVGWA 165 Streptococcus VITDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFAttorney Docket No.: 184F-414450-WOthermophilus QRLDDSFLVPDDKRDSKYPIFGNLVEEKVYHDEFPTIYH Cas9 LRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNS KNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKI SKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCF NLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKK LYDAILNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKNLL AEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEM RAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGN SDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRMTSF DLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDY QFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGI ELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEII HTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRHYTGW GKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHD DALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGIL QSIKIVDELVKVMGGRKPESIVVEMARENQYTNQGKSN SQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDR LYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLKDN SIDNKVLVSSASNRGKSDDFPSLEVVKKRKTFWYQLLK SKLISQRKFDNLTKAERGGLLPEDKAGFIQRQLVETRQI TKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQF RKDFELYKVREINDFHHAHDAYLNAVIASALLKKYPKLE PEFVYGDYPKYNSFRERKSATEKVYFYSNIMNIFKKSIS LADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYP QVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSN ENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKK KITNVLEFQGISILDRINYRKDKLNFLLEKGYKDIELIIELP KYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLSQK FVKLLYHAKRISNTINENHRKYVENHKKEFEELFYYILEF NENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTG SERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKD ATLIHQSVTGLYETRIDLAKLGEGAttorney Docket No.: 184F-414450-WOQ99ZW2 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD 166 Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR pyogenes Cas 9 ICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLI NGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE D IQKAQVSGQG DSLHEHIAN LAGSP Al KKG I LQTVKVVD ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY GG FDSPTVAYS VLVVAKVE KG KSKKLKS VKE LLG IT I M E RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDAttorney Docket No.: 184F-414450-WOKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDC9X1G5 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDL 167 Neisseria GVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHR meningitidis LLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAA Cas9 ALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKEL GALLKGVAGNAHALQTGDFRTPAELALNKFEKESGHIR NQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGL KEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAK NTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDE PYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEAST LMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTA FSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLK ALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLP PIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIET AREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPN FVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKG YVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPY EYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFD EDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVAC STVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKT HFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRT LLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHME TVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPK LYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVK AVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDK YYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFN FKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIH DLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRL KKRPPVRAttorney Docket No.: 184F-414450-WOA0Q5Y3 MNFKILPIAIDLGVKNTGVFSAFYQKGTSLERLDNKNGK 168 Francisella VYELSKDSYTLLMNNRTARRHQRRGIDRKQLVKRLFKLI novicida Cas9 WTEQLNLEWDKDTQQAISFLFNRRGFSFITDGYSPEYL NIVPEQVKAILMDIFDDYNGEDDLDSYLKLATEQESKISE IYNKLMQKILEFKLMKLCTDIKDDKVSTKTLKEITSYEFEL LADYLANYSESLKTQKFSYTDKQGNLKELSYYHHDKYNI QEFLKRHATINDRILDTLLTDDLDIWNFNFEKFDFDKNEE KLQNQEDKDHIQAHLHHFVFAVNKIKSEMASGGRHRSQ YFQEITNVLDENNHQEGYLKNFCENLHNKKYSNLSVKN LVNLIGNLSNLELKPLRKYFNDKIHAKADHWDEQKFTET YCHWILGEWRVGVKDQDKKDGAKYSYKDLCNELKQKV TKAGLVDFLLELDPCRTIPPYLONNNRKPPKCQSLILNP KFLDNQYPNWQQYLQELKKLQSIQNYLDSFETDLKVLK SSKDQPYFVEYKSSNQQIASGQRDYKDLDARILQFIFDR VKASDELLLNEIYFQAKKLKQKASSELEKLESSKKLDEVI ANSQLSQILKSQHTNGIFEQGTFLHLVCKYYKQRQRAR DSRLYIMPEYRYDKKLHKYNNTGRFDDDNQLLTYCNHK PRQKRYQLLNDLAGVLQVSPNFLKDKIGSDDDLFISKWL VEHIRGFKKACEDSLKIQKDNRGLLNHKINIARNTKGKC EKEIFNLICKIEGSEDKKGNYKHGLAYELGVLLFGEPNE ASKPEFDRKIKKFNSIYSFAQIQQIAFAERKGNANTCAV CSADNAHRMQQIKITEPVEDNKDKIILSAKAQRLPAIPTR IVDGAVKKMATILAKNIVDDNWQNIKQVLSAKHQLHIPIIT ESNAFEFEPALADVKGKSLKDRRKKALERISPENIFKDK NNRIKEFAKGISAYSGANLTDGDFDGAKEELDHIIPRSH KKYGTLNDEANLICVTRGDNKNKGNRIFCLRDLADNYKL KQFETTDDLEIEKKIADTIWDANKKDFKFGNYRSFINLTP QEQKAFRHALFLADENPIKQAVIRAINNRNRTFVNGTQR YFAEVLANNIYLRAKKENLNTDKISFDYFGIPTIGNGRGI AEIRQLYEKVDSDIQAYAKGDKPQASYSHLIDAMLAFCI AADEHRNDGSIGLEIDKNYSLYPLDKNTGEVFTKDIFSQI KITDNEFSDKKLVRKKAIEGFNTHRQMTRDGIYAENYLPI LIHKELNEVRKGYTWKNSEEIKIFKGKKYDIQQLNNLVYAttorney Docket No.: 184F-414450-WOCLKFVDKPISIDIQISTLEELRNILTTNNIAATAEYYYINLKT QKLHEYYIENYNTALGYKKYSKEMEFLRSLAYRSERVKI KSIDDVKQVLDKDSNFIIGKITLPFKKEWQRLYREWQNT TIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEGKF LVKRKTWDNNFIYQILNDSDSRADGTKPFIPAFDISKNEI VEAIIDSFTSKNIFWLPKNIELQKVDNKNIFAIDTSKWFEV ETPSDLRDIGIATIQYKIDNNSRPKVRVKLDYVIDDDSKIN YFMNHSLLKSRYPDKVLEILKQSTIIEFESSGFNKTIKEM LGMKLAGIYNETSNNQ73QW6 MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDL 169 Treponema WGMRCFETAETAEVRRLHRGARRRIERRKKRIKLLQEL denticola Cas9 FSQEIAKTDEGFFQRMKESPFYAEDKTILQENTLFNDKD FADKTYHKAYPTINHLIKAWIENKVKPDPRLLYLACHNIIK KRGHFLFEGDFDSENQFDTSIQALFEYLREDMEVDIDA DSQKVKEILKDSSLKNSEKQSRLNKILGLKPSDKQKKAIT NUSGNKINFADLYONPDLKDAEKNSISFSKDDFDALSDD LASILGDSFELLLKAKAVYNCSVLSKVIGDEQYLSFAKVK IYEKHKTDLTKLKNVIKKHFPKDYKKVFGYNKNEKNNNN YSGYVGVCKTKSKKLIINNSVNQEDFYKFLKTILSAKSEI KEVNDILTEIETGTFLPKQISKSNAEIPYQLRKMELEKILS NAEKHFSFLKQKDEKGLSHSEKIIMLLTFKIPYYIGPINDN HKKFFPDRCWVVKKEKSPSGKTTPWNFFDHIDKEKTAE AFITSRTNFCTYLVGESVLPKSSLLYSEYTVLNEINNLQIII DGKNICDIKLKQKIYEDLFKKYKKITQKQISTFIKHEGICN KTDEVIILGIDKECTSSLKSYIELKNIFGKQVDEISTKNML EEIIRWATIYDEGEGKTILKTKIKAEYGKYCSDEQIKKILN LKFSGWGRLSRKFLETVTSEMPGFSEPVNIITAMRETQ NNLMELLSSEFTFTENIKKINSGFEDAEKQFSYDGLVKP LFLSPSVKKMLWQTLKLVKEISHITQAPPKKIFIEMAKGA ELEPARTKTRLKILQDLYNNCKNDADAFSSEIKOLSGKIE NEDNLRLRSDKLYLYYTQLGKCMYCGKPIEIGHVFDTS NYDIDHIYPQSKIKDDSISNRVLVCSSCNKNKEDKYPLK SEIQSKQRGFWNFLQRNNFISLEKLNRLTRATPISDDETAttorney Docket No.: 184F-414450-WOAKFIARQLVETRQATKVAAKVLEKMFPETKIVYSKAETV SMFRNKFDIVKCREINDFHHAHDAYLNIVVGNVYNTKFT NNPWNFIKEKRDNPKIADTYNYYKVFDYDVKRNNITAW EKGKTIITVKDMLKRNTPIYTRQAACKKGELFNQTIMKK GLGQHPLKKEGPFSNISKYGGYNKVSAAYYTLIEYEEK GNKIRSLETIPLYLVKDIQKDQDVLKSYLTDLLGKKEFKIL VPKIKINSLLKINGFPCHITGKTNDSFLLRPAVQFCCSNN EVLYFKKIIRFSEIRSQREKIGKTISPYEDLSFRSYIKENL WKKTKNDEIGEKEFYDLLQKKNLEIYDMLLTKHKDTIYK KRPNSATIDILVKGKEKFKSLIIENQFEVILEILKLFSATRN VSDLQHIGGSKYSGVAKIGNKISSLDNCILIYQSITGIFEK RIDLLKVU2UMQ6 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEE 170 Acidaminococcu DKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSA s AsCas12a AIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLT (Cpf1) DAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIE EVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLN EVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFIL EEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSE ILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLL DWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILF VKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYY DYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALC KWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAE LNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYAttorney Docket No.: 184F-414450-WOVNHRLSHDLSDEARALLPNVITKEVSHEIIKORRFTSDKF FFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDR GERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREK ERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQA VVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLV LKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYV PAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFL HYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEK NETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANE LIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSV LQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWP MA0A182D WE3 AASKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVE 171 Lachnospiracea DEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISe Cas12a LFRKKTRTEKENKELENLEINLRKEIAKAFKGAAGYKSLF (Cpf1) KKDIIETILPEAADDKDEIALVNSFNGFTTAFTGFFDNRE NMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDK HEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYN AIIGGFVTESGEKIKGLNEYINLYNAKTKQALPKFKPLYK QVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSS IKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNLI RDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFS LEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLF DADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFG EGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQK PYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGS KYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPN KMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFN LNOCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIA GFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIY NKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAE LFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYD VYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDAttorney Docket No.: 184F-414450-WODNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNF NGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAG YISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQ VYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNK FESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYT SIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADY IKKWKLYSYGNRIRIFAAAKKNNVFAWEEVCLTSAYKEL FNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQ MRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAIL PKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAIS NKEWLEYAQTSVKC7NBY4 MKVTKVGGISHKKYTSEGRLVKSESEENRTDERLSALL 172 Leptotrichia NMRLDMYIKNPSSTETKENQKRIGKLKKFFSNKMVYLK DNTLSLKNGKKENIDREYSETDILESDVRDKKNFAVLKKIbuccalisGas 13a (C2c2) YLNENVNSEELEVFRNDIKKKLNKINSLKYSFEKNKANY QKINENNIEKVEGKSKRNIIYDYYRESAKRDAYVSNVKE AFDKLYKEEDIAKLVLEIENLTKLEKYKIREFYHEIIGRKN DKENFAKIIYEEIQNVNNMKELIEKVPDMSELKKSQVFYK YYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNI SNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYL QDGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILE TENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENK KNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIR HGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKL KLKIFRQLNSANVFRYLEKYKILNYLKRTRFEFVNKNIPF VPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEIIDAQ IYLLKNIYYGEFLNYFMSNNGNFFEISKEIIELNKNDKRNL KTGFYKLQKFEDIQEKIPKEYLANIQSLYMINAGNQDEE EKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTS LAEKKQEFDKFLKKYEQNNNIKIPYEINEFLREIKLGNILK YTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAF SDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKV KDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEAttorney Docket No.: 184F-414450-WOKIADKAGYKISIEELKKYSNKKNEIEKNHKMQENLHRKY ARPRKDEKFTDEDYESYKQAIENIEEYTHLKNKVEFNEL NLLQGLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIE EIFNFENKKNVKYKGGQIVEKYIKFYKELHQNDEVKINKY SSANIKVLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENL RKLLSYDRKLKNAVMKSVVDILKEYGFVATFKIGADKKI GIQTLESEKIVHLKNLKKKKLMTDRNSEELCKLVKIMFEY KMEEKKSEN PODOC6 MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYIL 173 Leptotrichia NINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAG shahii Cas13a NILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLK (C2c2) ALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDE YTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIE KIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIR EKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNIN VOLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRR NRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSI KEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYK VNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKV RLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLR HNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELN KIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRD LDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQG TQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKI NDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFD TIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQEL KKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKK VIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKT YERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFF ATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNL EEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEF KDDINGCDVLEKKLEKMFDDETKFEIDKKSNILQDEQR KLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKAttorney Docket No.: 184F-414450-WOKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKN LFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNK ISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNI QNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDI NWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISR AYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGID LSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRV SNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKL IGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIEN TNDTL QP 897 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT 174 Campylobacter GESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLN jejuni Cas9 YEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDF ARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLAN YQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIA QSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRA LKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLL NNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLG LSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLN EIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHL NISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKD FLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGK VHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELE CEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQD EKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQT PFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDK EQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENT KLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNN HLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKI SELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKP SGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVN GKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLP NKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDAttorney Docket No.: 184F-414450-WOMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILF KNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQR EDFKKP 14727 MDPIRSRTPSPARELLPGPQPDGVQPTADRGVSPPAG 175 Xanthomonas GPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQ euvesicataria FDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRA ADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPA AQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHG FTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIA KRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDG GKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPQQVVAIASNSGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLC QAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQV VAIASHOGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG GKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNSGGKQALETV QRLLPVLCQAHGLTPEQVVAIASHOGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHOGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNGGGRPALETVQRLLPVLCQAHGLTPE QVVAIASHOGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACL GGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADH AQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQL FRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKP SPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTR ASSRKRSRSDRAVTGPSAQQSFEVRVPEQRDALHLPLAttorney Docket No.: 184F-414450-WOSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQD EDPFAGAADDFPAFNEEELAWLMELLPQB2SU53 MDPIRSRTPSPARELLPGPQPDRVQPTADRGGAPPAG 176 Xanthomonas GPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQ oryzae pv. FDPSLLDTSLLDSMPAVGTPHTAAAPAECDEVQSGLRA Oryzae ADDPPPTVRVAVTAARPPRAKPAPRRRAAQPSDAS PAAQVDLRTLGYSQQQQEKIKPKVGSTVAQHHEALVG HGFTHAHIVALSRHPAALGTVAVKYQDMIAALPEATHED IVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLVK IAKRGGVTAVEAVHASRNALTGAPLNLTPAQVVAIASNN GGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQ ALETMQRLLPVLCQAHGLPPDQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPDQVVAIASHGGGKQALETVQRLL PVLCQAHGLTPDQVVAIASHOGGKQALETVQRLLPVLC QAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAH GLTPDQVVAIASNGGKQALETVQRLLPVLCQAHGLTPD QVVAIASHDGGKQALETVQRLLPVLCQTHGLTPAQVVAI ASHDGGKQALETVQQLLPVLCQAHGLTPDQVVAIASNI GGKQALATVQRLLPVLCQAHGLTPDQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTQVQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPAQVVAIASHOGGKQALETVQRLLPVL CQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAH GLTQEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP DQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVV AIASNIGGKQALETVQRLLPVLCQDHGLTLAQVVAIASNI GGKQALETVQRLLPVLCQAHGLTQDQVVAIASNIGGKQ ALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETV QRLLPVLCQDHGLTLDQVVAIASNGGKQALETVQRLLP VLCQDHGLTPDQVVAIASNSGGKQALETVQRLLPVLCQ DHGLTPNQVVAIASNGGKQALESIVAQLSRPDPALAALT NDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIG ERTSHRVADYAQVVRVLEFFQCHSHPAYAFDEAMTQFAttorney Docket No.: 184F-414450-WOGMSRNGLVQLFRRVGVTELEARGGTLPPASQRWDRIL QASGMKRAKPSPTSAQTPDQASLHAFADSLERDLDAP SPMHEGDQTGASSRKRSRSDRAVTGPSAQHSFEVRV PEQRDALHLPLSWRVKRPRTRIGGGLPDPGTPIAADLA ASSTVMWEQDAAPFAGAADDFPAFNEEELAWLMELLP QSGSVGGTIA0A158R FF2 INPWFLTGFIDGEGCFRISVTKINRAIDWRVQLFFQINLH 177 Gremmeniella EKDRALLESIKDYLKVGKIHISGKNLVQYRIQTFDELTILIK abietina HLKEYPLVSKKRADFELFNTAHKLIKNNEHLNKEGINKL VSLKASLNLGLSESLKLAFPNVISATRLTDFTVNIPDPHW LSGFASAEGCFMVGIAKSSASSTGYQVYLTFILTQHVRD ENLMKCLVDYFNWGRLARKRNVYEYQVSKFSDVEKLL SFFDKYPILGEKAKDLQDFCSVSDLMKSKTHLTEEGVA KIRKIKEGMNRGQ94AD9 MRTPMSDTQHVQSSLVSIRSSDKIEDAFRKMKVNETGV 178 Arabidopsis EELNPYPDRPGERDCQFYLRTGLCGYGSSCRYNHPTH thaliana LPQDVAYYKEELPERIGQPDCEYFLKTGACKYGPTCKY HHPKDRNGAQPVMFNVIGLPMRLGEKPCPYYLRTGTC RFGVACKFHHPQPDNGHSTAYGMSSFPAADLRYASGL TMMSTYGTLPRPQVPQSYVPILVSPSQGFLPPQGWAP YMAASNSMYNVKNQPYYSGSSASMAMAVALNRGLSE SSDQPECRFFMNTGTCKYGDDCKYSHPGVRISQPPPS LINPFVLPARPGQPACGNFRSYGFCKFGPNCKFDHPML PYPGLTMATSLPTPFASPVTTHQRISPTPNRSDSKSLSN GKPDVKKESSETEKPDNGEVQDLSEDASSPdAsCas12a MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEE 179 (D908A) DKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSA AIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLT DAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIE EVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNAttorney Docket No.: 184F-414450-WOEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFIL EEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALGDHWDTLRNALYERRISELTGKI TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSE ILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLL DWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILF VKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYY DYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALC KWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAE LNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDY VNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKF FFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIAR GERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREK ERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQA VVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLV LKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYV PAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFL HYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEK NETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANE LIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSV LQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWP MDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQD WLAYIQELRN

[0189] Any DBD amino acid sequence of the present technology may be modified to comprise one or more non-naturally occurring amino acids. In some embodiments, the DBDs comprise or consist of an amino acid sequence having one or more non-naturally occurring amino acids. In some embodiments, the DBDs comprise or consist of an amino acid sequence having two or more non-naturally occurring amino acids. InAttorney Docket No.: 184F-414450-WOsome embodiments, the modification of the amino acid sequences to comprise non-naturally occurring amino acids is a conservative substitution.

[0190] In some embodiments, the p300R-DBD peptide comprises or consists of the amino acid sequence MSARQGQMQGIPAPSQALQEPAPWSALPCGLLLDELLASPEFLQQAQPLLETEAPG ELEASEEAASLEAPLSEEEYRALLEELSGGGGSRGGGSGGGGSGGGGSGGGGSG GGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPG EKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNEL TKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY LQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST KEVLDATLIHQSITGLYETRIDLSQLGGDAYPYDVPDYASLGSGSPKKKRKVEDPKKK RKVDGIGSGSNGSSGSATNFSLLKQAGDVEENPGPMVSKGEEDNMAIIKEFMRFKV HMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAY VKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFAttorney Docket No.: 184F-414450-WOPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK KPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO: 137).

[0191] In some embodiments, the p300R-DBD peptide comprises or consists of an amino acid sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137.

[0192] In some embodiments, the p300R-DBD peptide comprises or consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137.

[0193] In some embodiments, the p300R-DBD peptide comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137.

[0194] Any fusion peptide amino acid sequence of the present technology may be modified to comprise one or more non-naturally occurring amino acids. In some embodiments, the fusion peptides comprise or consist of an amino acid sequence having one or more non-naturally occurring amino acids. In some embodiments, the fusion peptides comprise or consist of an amino acid sequence having two or more non-naturally occurring amino acids. In some embodiments, the modification of the amino acid sequences to comprise non-naturally occurring amino acids is a conservative substitution.Additional Peptide Features

[0195] Localization domains: In some embodiments, the peptides of the present technology, such as the p300R-DBD peptides, further comprise a localization domain. Any suitable localization domain can be used, including but not limited to any nuclear localization domain. The localization domain may comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of YPYDVPDYASLGSGSP KKKR KVE DPKKKRKVDGIGSGSNGSSGSATNFSLLKQAGDAttorney Docket No.: 184F-414450-WOVEENPGP (SEQ ID NO: 158), wherein the underlined residues are optional. In some embodiments, the optional residues are present. In some embodiments, the optional residues are absent, in whole or in part.

[0196] Detectable domains: In some embodiments, the p300R-DBD peptides of the present technology further comprise a detectable domain. The detectable domain may comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 159, 160, or 161 (Table 2).Table 2: Detectable Domain Amino Acid Sequences Name Amino Acid Sequence SEQ ID NODetectable MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGR 159 Domain A PYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPA DIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEF IYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDI TSHNEDYTIVEQYERAEGRHSTGGMDELYKDetectable SGGGGSRGGGSGGGGSGGGGSGGGGSGGGGMDKKYSIG 160 Domain B LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVAttorney Docket No.: 184F-414450-WOYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIK KGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKOLilKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Detectable SGGGGSRGGGSGGGGSGGGGSGGGGSGGGGMDKKYSIG 161 Domain C LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRW TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYAttorney Docket No.: 184F-414450-WONELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQ LKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFL DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQWIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYWFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDYPYDVPDYASLGSGSPKKKR KVEDPKKKRKVDGIGSGSNGSSGSATNFSLLKQAGDVEENP GPMVSKGEEDNMAIIKEFMRFKVHMEGSWGHEFEIEGEGE GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKH PADIPDYLKLSFPEGFKWERVMNFEDGGWTVTQDSSLQDG EFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNWIKL DITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0197] In some embodiments, the residues of SEQ ID NO: 160 or SEQ ID NO: 161 are not modified.Attorney Docket No.: 184F-414450-WO

[0198] The DBD may be fused to p300R domain by a peptide linker. The peptide linkers of the present technology may vary from 2 to 31 amino acids of any primary sequence in length and do not impose any constraints on the conformation or interactions of the linked partners. In some embodiments, the linkers vary from about 2-30, 2-29, 2-28, 2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21 , 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2- 14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-31 , 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21 , 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-31 , 4-30, 4-29, 4-28, 4-27, 4-26, 4- 25, 4-24, 4-23, 4-22, 4-21, 4- 20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-31, 5- 30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21 , 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5- 14, 5-13, 5-12,5-11 , 5-10, 5-9, 5-8, 5-7, 5-6, 6-31 , 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21 , 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, 6-7, 7-31 , 7-30, 7-29, 7-28, 7-27, 7- 26, 7-25, 7-24, 7-23, 7-22, 7-21 , 7-20, 7-19, 7-18, 7-17, 7- 16, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, 7-8, 8-31 , 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21 , 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11 , 8-10, 8-9, 9-31 , 9-30, 9-29, 9- 28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-22, 9-21 , 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9- 13, 9-12, 9-11 , 9-10, 10-31 , 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10- 22, 10-21 , 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 , 11- 31 , 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21 , 11-20, 11- 19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-31 , 12-30, 12-29, 12-28, 12- 27, 12-26, 12-25,12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-31 , 13-30, 13-29, 13-28, 13-27, 13-26, 13-25, 13-24, 13-23, 13-22, 13-21 , 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-31 , 14-30, 14-29, 14-28, 14- 27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14- 15, 15-31 , 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21 , 15- 20, 15-19, 15-18, 15-17, 15-16, 16-31 , 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16- 24, 16-23, 16-22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-31 , 17-30, 17-29, 17-28, 17- 27, 17-26, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-31 , 18-30, 18- 29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 18-19, 19-31 , 19- 30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21 , 19-20, 20-31 , 20- 30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21 , 21-31 , 21-30, 21- 29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-31 , 22-30, 22-29, 22-28, 22-Attorney Docket No.: 184F-414450-WO27, 22-26, 22-25, 22-24, 22-23, 23-31 , 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23- 24, 24-31 , 24-30, 24-29, 24-28, 24-27, 24-26, 24-25, 25-31 , 25-30, 25-29, 25-28, 25- 27, 25-26, 26-31 , 26-30, 26-29, 26-28, 26-27, 27-31 , 27-30, 27-29, 27-28, 28-31 , 28- 30, 28-29, 29-31 , 29-30, or 30-31 amino acids of any primary sequence in length. The peptide linkers may be designed as appropriate for an intended use.

[0199] The peptide linkers may comprise one or more of a Gly-rich linkers (e.g., a flexible linker connecting various domains in a single protein without interfering with the function of each domain; a linker forming stable covalently linked dimers; a linker to connect two independent domains that create a ligand-binding site or recognition sequence), a Serine linker (e.g., a coiled structure linker); a coiled structure linker comprising a Gin, Arg, Glu, Ser, and / or Pro amino acids; a rigid space linker comprising one or more of a Pro, Arg, Phe, Thr, Glu, and / or Gin residues; a linker comprising a flexible Gly-rich regions that may can generate loops connecting domains; or a linker comprising a Thr, Ser, Gly, and / or Ala residue.

[0200] In some embodiments, the peptide linker comprises an amino acid sequence listed in Table 3.

[0201] In some embodiments, the peptide linker comprises an amino acid sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

[0202] In some embodiments, the peptide linker comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

[0203] In some embodiments, the peptide linker comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

[0204] Any peptide linker amino acid sequence of the present technology may be modified to comprise one or more non-naturally occurring amino acids. In some embodiments, the peptide linkers comprise or consist of an amino acid sequence havingAttorney Docket No.: 184F-414450-WOone or more non-naturally occurring amino acids. In some embodiments, the peptide linkers comprise or consist of an amino acid sequence having two or more non-naturally occurring amino acids.Table 3: Peptide Linker Amino Acid Sequences Name Amino Acid SEQ ID NO Linker A (GGS)I-5138 Linker B GGGGSLVPRGSGGGGS 139 Linker C GSGSGS 140 Linker D GS8141 Linker E GGSGGHMGSGG 142 Linker F GGSGGSGGSGG 143 Linker G GGSGG 144 Linker H GGSGGGGG 145 Linker I GSGSGSGS 146 Linker J GSGSGSGSGSGSGSGSGSGSGSGSGSGSGSG 147 Linker K GGGSEGGGSEGGGSEGGG 148 Linker L AAGAATAA 149 Linker M GGGGG 150 Linker N GGSSG 151 Linker 0 GSGGGTGGGSG 152 Linker P GT - Linker Q GSGSGSGSGGSG 154 Linker R GSGGSGSGGSGGSG 155 Linker S SGGGGSRGGGSGGGGSGGGGSGGGGSGGGG 156**One or more of the underlined residues in SEQ ID NO: 156 are optional.Attorney Docket No.: 184F-414450-WO

[0205] In another aspect the disclosure provides nucleic acids encoding the polypeptide of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure. Systems

[0206] The present technology comprises non-enzymatic systems for increasing or otherwise modulating expression of one or more genes or proteins in a cell, relative to a control. In some embodiments, the systems modify or alter methylation of one or more histones and / or DNA methylation of one or more CpG islands in the cell.

[0207] The systems may comprise a composition of the present technology or at least a p300R domain peptide or a p300R-DBD thereof and one or more gRNAs. In some embodiments, the systems comprise two or more gRNAs.gRNAs

[0208] The gRNAs of the present technology may comprise a guanine nucleotide at the 5' end of its polynucleotide sequence. The guanine nucleotide in 5' may be present when the gRNA is expressed under the control of the 116 promoter. The gRNAs may be of varying lengths. The gRNA may comprise a gRNA guide sequence of at least 10 nucleotides (nts), at least 11 nts, at least a 12 nts, at least a 13 nts, at least a 14 nts, at least a 15 nts, at least a 16 nts, at least a 17 nts, at least a 18 nts, at least a 19 nts, at least a 20 nts, at least a 21 nts, at least a 22 nts, at least a 23 nts, at least a 24 nts, at least a 25 nts, at least a 30 nts, or at least a 35 nts of a target sequence in the gene target. In embodiments, the "gRNA guide sequence" or "gRNA target sequence" may be least 10 nucleotides long; in some embodiments 10-40 nts long (e.g., 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,Attorney Docket No.: 184F-414450-WO37, 38, 39 or 40 nts long). In other embodiments, gRNA guide sequence is between 17-30, 17-22, 10-40, 10-30, 12-30, 15-30, 18-30, or 10-22 nucleotides long.

[0209] The number of gRNAs administered to or expressed in a cell of the present technology may be at least 1 gRNA, at least 2 gRNAs, at least 3 gRNAs at least 4 gRNAs, at least 5 gRNAs, at least 6 gRNAs, at least 7 gRNAs, at least 8 gRNAs, at least 9 gRNAs, at least 10 gRNAs, at least 11 gRNAs, at least 12 gRNAs, at least 13 gRNAs, at least 14 gRNAs, at least 15 gRNAs, at least 16 gRNAs, at least 17 gRNAs, or at least 18 gRNAs. In some embodiments, use of 2 or more gRNAs increases a gene expression or protein level in the cell, relative to use of 1 gRNA.

[0210] In some embodiments, when at least a complete match (e.g., a 100% complementary match) between the gRNA guide sequence and the DNA sequence on the target locus does not occur, a mismatch between a gRNA guide sequence and target sequence on the gene sequence of interest is also permitted in the event it still allows hybridization of the gRNA with the complementary strand of the gRNA target polynucleotide sequence on the targeted gene.

[0211] Any gRNA guide sequence may be selected in the target gene, as long as it allows introducing at the proper location, the desired modification(s). Accordingly, the gRNA guide sequence or target sequence of the present invention may be in coding or non-coding regions of the target gene.

[0212] In some embodiments, the gRNA directs the peptides of present technology to and / or binds a loci. The loci may include one or more features that deem the loci candidates for gRNA binding. For example, the loci may comprise euchromatin or heterochromatin for gRNA and p300R-DBD peptide accessibility. In some embodiments, the loci contain epigenetic marks such as DNA methylation or histone modifications that influence gene expression patterns and accessibility or binding efficiency of the gRNA or the p300R-DBD of the present technology, including, for example, silencing marks such as H3K9me3 and / or H3K27me3. In some embodiments, loci comprise or are associated with one or more regulatory elements, such as promoters, enhancers, or silencers, that regulate expression of a target gene within the loci.

[0213] In some embodiments, the loci is selected from the group consisting of a Laminin Subunit Alpha 1 (LAMA1) loci, a Structural Maintenance of ChromosomesAttorney Docket No.: 184F-414450-WOflexible Hinge Domain Containing 1 (SMCHD1) loci, a LRIF1 loci, a (Matrin 3) MATR3 loci, an Insulin-like Growth Factor 1 (IGF1 ) loci, a promyelocytic leukemia protein (PML) loci, an adiponectin (ADIPOQ) loci, an Alpha-fetoprotein (AFP) loci, a Methyl-CpG binding domain protein 3 like 2 (MBD3L2) loci, a Zinc Finger And SCAN Domain Containing 4 (ZSCAN4) loci, a Leucine Twenty Homeobox (LEUTX) loci, a solute carrier family 34 member 2 (SLC34A2) loci, and / or a Trinucleotide Repeat Containing Adaptor 6B (TNRC6B) loci.

[0214] The loci may comprise a nucleotide sequence encoding a gene, a nucleotide sequence that is a cis regulatory element of a gene, a nucleotide sequence that is a trans regulatory element of a gene, and / or a promoter region. Nonlimiting examples of genes include LAMA1 (NCBI GENE ID: 284217; NCBI Protein Accession(s): P25391.2, AAH62318.1, AAH39051.1), SMCHD1 (NCBI GENE ID: 23347; NCBI Protein Accession: NP 056110.2), LRIF1 (NCBI GENE ID: 55791 ; NCBI Protein Accession(s): NP_001006946.1 , NP_060842.3, Q5T3J3.1 , XP_054193607.1, XP 054193607.1 ), MATR3 (NCBI GENE ID: 9782; NCBI Protein Accession: P43243.2), IGF1 (NCBI GENE ID: 3479; NCBI Protein Accession: CAG46659.1 ), PML (NCBI GENE ID: 5371 ; NCBI Protein Accession: AAB19601.2), ADIPOQ (NCBI GENE ID: 9370; NCBI Protein Accession: AAH54496.1), AFP (NCBI GENE ID: 174; NCBI Protein Accession: P02771.1), MBD3L2 (NCBI GENE ID: 125997; NCBI Accession(s): NP_653215.2. AAM28154.2), ZSCAN4 (NCBI GENE ID: ZNF494; NCBI Accession(s): NP_001093656.1 ), LEUTX (NCBI GENE ID: 342900; NCBI Accession(s): A8MZ59.4, NP 001137304.1 ), SLC34A2 (NCBI GENE ID: 10568; NCBI Accession: AAI46667.1 ), and TNRC6B (NCBI GENE ID: 23112; NCBI Accession(s) AAH28626.1 ; Q9UPQ9.4).

[0215] The gRNAs of the present technology may be used to target one or more genes. Genes may be targeted to increase a gene expression level thereof, relative to a control. In some embodiments, the gRNA or at least a nucleotide thereof, binds at or near a transcription start site (TSS) of a gene within and / or associated with the loci. In some embodiments, the gRNA or at least the nucleotide thereof binds at least within 10 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 30, nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, 213 nucleotides, 267 nucleotides, 300 nucleotides, 353 nucleotides, 392Attorney Docket No.: 184F-414450-WOnucleotides, 400 nucleotides, 442 nucleotides, 445 nucleotides, 500 nucleotides, 570 nucleotides, 571 nucleotides, 588 nucleotides, 600 nucleotides, 608 nucleotides, 626 nucleotides, 649 nucleotides, 681 nucleotides, 700 nucleotides, 702 nucleotides, 705 nucleotides, 710 nucleotides, 750 nucleotides, 783 nucleotides, 790 nucleotides, 799 nucleotides, 800 nucleotides, 846 nucleotides, 900 nucleotides, 904 nucleotides, 930 nucleotides, 967 nucleotides, 1000 nucleotides, 1100 nucleotides, 1118 nucleotides, 1158 nucleotides, 1180 nucleotides, 1188 nucleotides, 1200 nucleotides, 1250 nucleotides, 1300 nucleotides, 1331 nucleotides, 1400 nucleotides, 1434 nucleotides, 1500 nucleotides, 1535 nucleotides, 1573 nucleotides, 1600 nucleotides, 1670 nucleotides, 1700 nucleotides, 1735 nucleotides, 1800 nucleotides, 1900 nucleotides, 2000 nucleotides, 2025 nucleotides, 2181 nucleotides, 2500 nucleotides, 3000 nucleotides, 4000 nucleotides, or 5000 nucleotides of the TSS of the gene within and / or associated with the loci.

[0216] In some embodiments, the gRNA sequence comprises a nucleotide sequence listed in Table 4.

[0217] In some embodiments, the gRNA sequence comprises a nucleotide sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to any one of SEQ ID NOs: 2-136.

[0218] In some embodiments, the gRNA sequence comprises a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to any one of SEQ ID NOs: 2-136.

[0219] In some embodiments, the gRNA sequence comprises a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to any one of SEQ ID NOs: 2-136.Table 4: gRNA sequencesName Nucleotide Sequence SEQ ID NOgRNA 1 GCGTTATGAATACGTTTGCG 2 gRNA2 CCCTCTTACATTGAGTCGTA 3 gRNA3 GATATAGGGTCCCGCAAGAA 4Attorney Docket No.: 184F-414450-WOgRNA4 GCCCTTTAAACAGGTACGGG 5 gRNA5 GCGAAATTATCACACACACG 6 gRNA6 GAAAATTTCACAACAGCACA 7 LAMA1 gRNA9 GTCCGAAGGGCCTGCGCACC 8 LAMA1 gRNAIO GGGCGCGGCCAGGCAGGCGG 9 LAMA1 gRNA11 CCCTGAATGCTCACCCCAAC 10 LAMA1 gRNA12 GCGCCCAGGTCTGTCCTCCA 11 ADIPOQgRNA 1 TAAGATGTGAGTCCGCCGAG 12 ADIPOQgRNA 2 ATGGGATCCGGTCTAGCAAG 13 ADIPOQgRNA 3 GACCCAAGCTGGATTAAACC 14 ADIPOQgRNA 4 GGAGCAAGGGGCCCACTCAT 15 ADIPOQgRNA 5 CCTTTCCCATAAGTTTGTCC 16 IGF1 gRNA1 CCAGGGTAAACTAGATCGAA 17 IGF1 gRNA2 ATCCATGGGGCAGCGTAAAG 18 IGF1 gRNA3 TCAGATACTCAACAGACGTC 19 IGF1 gRNA4 AAAGCACTAACTTACACGAA 20 IGF1 gRNA5 GTATTCCAAATAATACCAGC 21 IGF1 gRNA6 GCTCCAGTTTTTAAGAGCGA 22 IGF1 gRNA7 GTGTGCTAATACACCTCACC 23 IGF1 gRNA8 TTAGCAGACATTAAATACTG 24 hl_RIF1_588_g1 TAGTTTCGATGAACGTAGGC 25 hLRIF1_392_g2 GAGAGAAAAACGTACGCTAG 26 hLRIF1_790_g3 GTCCCTCATCTTGACGCCAT 27 hl_RIF1_1573_g4 TACTGTAGAGCAAGCTTACT 28 hLRIF1_213_g5 TCTTCCGGGGTCGTCACGTC 29Attorney Docket No.: 184F-414450-WOhl_RIF1_1158_g6 GTGTGTTTAGCCAGACAACC 30 hl_RIF1_967_g7 GGTGGTAAAATAATGGGTGG 31 hl_RIF1_1331_g8 AGAGAAGCAAGTCATAAGTG 32 hLRIF1_2025_g9 TCCCCATTGCTCTTATGATA 33 hLRIF1_705_g10 ATAAAGAGCATCAACTGCAA 34 hMATR3_904_g1 ACGCTGTGACAGCCGGATGT 35 hMATR3_2181_g2 ACTAGCTACCCAATTAGAGT 36 hMATR3_1434_g3 GTCAAAAAGGTTGACTATGA 37 hMATR3_118_g4 TCCCCTCGTGATCCCGAGCT 38 hMATR3_353_g5 GAGAGGGTTCATTGTTCGCT 39 hMATR3_702_g6 GCAAGAGACCCTAGGCTGTA 40 hMATR3_1670_g7 GAGGTCAGCTTTAAGTTATG 41 hMATR3_1250_g8 ACCAATAATCTGGTGATGAC 42 hMATR3_267_g9 GCTAGACTTGCCCTGCACCA 43 hMATR3_445_g10 TTGGGGGGCGGGTGTCGGAA 44 LRIF1 gRNA1 GGAGTCTGGAGAGTTGTCGT 45 LRIF1 gRNA2 TGGGGCGAGAACCAGAGCGA 46 LRIF1 gRNA3 GTTGCCCACAGCAACTGTGA 47 LRIF1 gRNA4 CTGAAGGTCCAGCGCCACCA 48 LRIF1 gRNA5 GACAACTCTCCAGACTCCGC 49 LRIF1 gRNA6 GCGACAGCGGGCTCTCGAGA 50 LRIF1 gRNA7 GTTTCAGGAAGACCCTCCGT 51 LRIF1 gRNA8 GCACTGACCAGGCTCTGTTG 52 LRIF1 gRNA9 GCGGACCGAGTAGATCTCCA 53 LRIF1 gRNAW GCCCTACGCTGAGAGAGAAT 54Attorney Docket No.: 184F-414450-WOLRIF1 gRNA11 AGAAACCGGAAGGCTCCTGG 55 LRIF1 gRNA12 GAGGGGTTCGACCTTAACGG 56 hSMCHD1_283_g5 TCGGCCCTCGCGAAGTTGGA 57 hSMCHD1_217_g13 GCGAATCCAGCAGGCCGCGC 58 586(196) GCCUCUGAGGACUACCCGCA 59 592(190) GAGGACUACCCGCAGGGCGC 60 hSMCHD1_534_g3 TTCAGGCCGGCGGTACTCGC 61 hSMCHD1_462_g6 TGAGCTTGTAGGTGCCGCGC 62 344 (424) GUCCACCAAAGCGAGCACGU 63 285 (497) AGUACUACUAAGAAAUGAGG 64 hSMCHD1_772_g2 CGACGGCATTCGGGGTACGG 65 hSMCHD1_1737_g9 AGAGGTGTGCTTGTATAATC 66 hSMCHD1_ 1664_g10 CTCAATTTGCTGAAGTAGAG 67 hlL10_73_g1 CAATCAAGGTTTCCCGGCAC 68 hlL10_566_g2 TGTGACCCCGCCTGTACTGT 69 hlL10_809_g3 GCACTGGTGTACCCTTGTAC 70 hlL10_27_g4 TTTATATTGTAAGCTCAGGG 71 hlL10_158_g5 GCATCGTAAGCAAAAATGAT 72 hlL10 332 g6 GTTCCTAGGTCACAGTGACG 73 275_hLeptin_g1 TCCGGCGCGACTATGGCGCA 74 67_hLeptin_g2 CTGGCGCTAGAAATGCGCCG 75 809_hLeptin_g3 CGGGGAACTACAAACGATAC 76 433_hLeptin_g4 GAATCGGGACTCGGCCCCGA 77 20_hLeptin_g5 TGATCGGGCCGCTATAAGAG 78 509_hLeptin_g6 GCTCCCCGACCCAGATTGCG 79Attorney Docket No.: 184F-414450-WO1313_ADIPOQ_g1 GGTCCCCCGGCTTAAATAAG 80 355_ADIPOQ_g2 TATTAGGGCAAATCTATGGG 81 1038_ADIPOQ1_g3 TTAAATTACGACCCCAGCAA 82 906_ADIPOQ1_g4 CTTTCTAGGAACACCTAAGT 83 1493_ADIPOQ1_g5 TAGAACATAAACTTCAGGTC 84 1476_ADIPOQ1_g6 CAAAATGTTATCCGAAATTT 85 475_ADIPOQ1_g7 TGTCAGAGGGGTCTGCAATC 86 2809_ADIPOQ1_g8 GACCTGTAGCTACGGTACCC 87 2641_ADIPOQ1_g9 CCACCACAAAGGCCGTCATG 88 3727_ADIPOQ1_g10 GCAAGACATGCAGTAAGCGC 89 44_hADIPOQ_g17 CCCTCACTGAGTTGGCCAAT 90 128_hADIPOQ_g41 AGCCCAAGCTGGGTTGTACC 91 203_hADIPOQ_g51 CTGTTGTAGGAGGCAAAATA 92 mSMCHD1_213_g1 TTCCGCGACCCGCGAACACA 93 mSMCHDI _468_g2 CGCGAAGGCCCCTAAGGCGC 94 mSMCHDI _702_g3 GAACCCACCCACGACGGCGA 95 mSMCHD1_4138_g4 AATCTGAGGTACTGAGCTAC 96 mSMCHDI _4245_g5 CCGGCAGCTGGCTAGAGTTA 97 mSMCHDI 4316 g6 CCAGTGATTGCAGGGTAAAA 98 mSMCHDI _5273_g7 CTTTTGTCTAGCTGGACTAG 99 mSMCHDI _5465_g8 GCGTTTAGAGACTGGCCATA 100 mSMCHDI _25561_g9 GACAAACATTCAAACGTAGC 101 mSMCHDI _8075_g 10 TGGAGCATAAGGCCAGTTGC 102 mSMCHDI _8353_g 11 AGAATATGAGGAGTCAACAG 103 mSMCHDI _8658_g 12 GACAGAAGGTCAAGCGAAAC 104Attorney Docket No.: 184F-414450-WOmSMCHD1_10240_g13 GGTTAATGCTTCTCAGAACT 105 mSMCHD1_10844_g14 CAAGGAGACAGTCAGTCGAG 106 mSMCHD1_11535_g15 CATGCCCCCAAACAGCACCC 107 mSMCHD1_14411_g16 TCAACCTCTGAAACTCAGCC 108 mSMCHD1_14520_g17 GACAGTCTACTTCTGCTTTG 109 mSMCHD1_14720_g18 AAACAAATCCTTTATTACTG 110 mSMCHD1_15518_g19 GGCCTCTTTGGCTCCTGAAC 111 mSMCHD1_15889_g20 CTTTAGCTCTCACGTCGCCA 112 mSMCHD1_16301_g21 TAGCCAAGATCTGAAGTTTG 113 Utro_1087_g8 CTGGCTCATAAATTAACCCTTGA 114 Utro_848_g12 CTTGGAAAAATAGAAAATAAATA 115 Utro_215_g1 TTCTCGCACCGCGCACCGCA 116 Utro_70_g2 AGGACCGAACCCGGAGCCGA 117 Utro_148_g3 GATTGACCGGGCGCGCGGTC 118 Utro_410_g4 AAT GGGGCGAATTGGCCAGA 119 CD16 1201_g1 TAGCACTTCGGATGCTTATC 120 CD16_567_g2 GTCTGTCAGCCTAGGAGCCG 121 CD16_335_g3 TAAAAAGTCAGTAGCTCCTG 122 CD16 1566 g4 TGAGTGTTGTTGGATTTCTA 123 CD16_225_g5 TAGTGGTCTGAAGATTTGAA 124 CD16_777_g6 CATCTGGGACAGGTCTCACC 125 AFP_795_g1 TGTACAAGGGTTCCCGATCT 126 AFP_1618_g2 GGAATCCAAACGGATAGACA 127 AFP_2086_g3 GATGATCAGATTGGATTACT 128 AFP 410_g4 GCAAGATGAAGAGTCTGAAT 129Attorney Docket No.: 184F-414450-WOAFP_688_g5 GTGTCTTTATAAAGTGGGTC 130 AFP 1004_g6 TATGGTACATTCATAAAGTA 131 hAFP_101_g7 GCCAATAATTAACAGCATAG 132 hAFP_30_g8 ACAAAAGGTTACTAGTTAAC 133 hAFP_71_g9 GTATTTCCTTAGGTTGAAAA 134 hAFP_141_g10 TCAAGGACACAGAGCTCTTT 135 hLRIF1_g30 GCAACCGGACGUGACGACCC 136MethodsLipofectamine Transfection

[0220] Cells were transfected using Lipofectamine™ MessengerMAX™ Reagent according to manufacturer’s instructions. Briefly, 0.5uL of Lipofectamine™ MessengerMAX™ Reagent was diluted in OptiMEM media and incubated at room temperature for 10 minutes. This diluted Lipofectamine™ MessengerMAX™ Reagent was added to an equal volume of OptiMEM containing 500ng mRNA and 500ng gRNA. RNA-Lipid complexes were allowed to form for 5 minutes at room temperature then added to 2e5 cells in suspension or pre-plated into assay-specific tissue culture plates. Cells were then incubated overnight at 37°C, 5% CO2.LNP Transfection

[0221] Cells were dosed with the desired payload of LNPs by diluting concentrated LNP preparation solutions in the appropriate media and adding to the cells either in suspension or pre-plated into assay-specific tissue culture plates. LNP preparation solution concentrations were determined using standard Quant-iT™ RiboGreen® RNA Reagent protocols prior to dilution. Cells were then incubated overnight at 37°C, 5% CO2.Electroporation

[0222] Cells were electroporated using the MaxCyte ATx electroporator according to the manufacturer’s instructions. Briefly, 5e6 cells resuspended in EP buffer containing RNase inhibitor were loaded per Process Assembly chamber with 5ug mRNA and 5ug gRNA payloads. The Process Assemblies were then loaded into the MaxCyte ATxAttorney Docket No.: 184F-414450-WOelectroporator and EP protocol NK-4 was run. Electroporated cells were then transferred to a tissue culture plate containing the appropriate media and incubated overnight at 37°C, 5% CO2.RNA Analysis

[0223] Total RNA was harvested from cell samples via lysis and subsequently purified using QIAGEN RNeasy extraction protocol involving gDNA elimination, ethanol binding, sequential buffer washes, and aqueous elution. The isolated RNA was measured, normalized and combined with TaqMan™ Fast Virus 1 -Step Master Mix and specific Thermo Scientific Gene Expression Assay target probes to generate a 10 pltotal reaction volume. One-step RT-qPCR amplification was then performed using a thermal cycling profile consisting of reverse transcription at 50°C for 5 minutes and activation at 95°C for 20 seconds, followed by cycling steps of denaturation at 95°C and annealing / extension at 60°C.ELISA

[0224] Protein lysates were first quantified via BCA assay and standardized to a uniform concentration to ensure consistent total protein loading across all samples for the ELISA. These normalized lysates were then processed through successive incubation, enzyme-conjugation, and substrate development steps on a pre-coated microplate. The reaction was finalized by adding stop solution and measuring the optical density to determine specific protein levels within each sample.Glucose Uptake

[0225] Samples were first deproteinized to remove interfering enzymes and then adjusted to a neutral pH to ensure reaction stability. Prepared samples and standards were then incubated with a specific reaction mix at 37°C for 30 minutes, allowing for the development of a measurable signal. Finally, the concentration was determined by measuring optical density or fluorescence and interpolating the results against a standard curve.Vectors

[0226] In some embodiments, the present technology comprises vectors comprising the nucleotide sequence of one or more gRNAs, one or more peptides, or one or more protein domains of the present technology (e.g., a p300R-DBD peptide, aAttorney Docket No.: 184F-414450-WOp300R domain, a DBD). In some embodiments, the present technology comprises vectors comprising a gRNA sequence of the present technology.

[0227] In some embodiments, the gRNA nucleotide sequence is operatively linked to a suitable control sequence. In one embodiment, the sequence encoding the gRNA is within 50-100 base pairs of a TATA box. In one such embodiment, the TATA box is 5' to the gRNA encoding sequence; in another embodiment, the TATA box is 3' to the gRNA encoding sequence. As described in the examples that follow, no sequence or structural gRNA requirements were found with regard to TATA box proximity, and no functional PAM specificity was found with regard to TATA box region.

[0228] The vector may be a viral vector (e.g., a lentiviral vector).

[0229] The vectors of the present technology may comprise a detectable marker and / or a reporter gene.

[0230] In some embodiments, the vector comprises an antibiotic-inducible gene expression system. Nonlimiting examples of antibiotics for use in the antibiotic-inducible gene expression system include doxycycline and tetracycline. In some embodiments, the antibiotic-inducible gene expression system is a Tet-ON system.Host Cells and Engineered Cells

[0231] The compositions or the vectors of the present technology may be present in a host cell. In some embodiments, the host cell is a mammalian host cell (e.g., a human host cell or a murine host cell). The host cell may be a myoblast. In some embodiments, the host cell is a kidney cell, a muscle cell, a liver cell, or a macrophage.

[0232] In some embodiments, the present technology comprises engineered cells having increased expression of one or more genes or proteins, relative to a control. Nonlimiting examples of such genes or proteins include Laminin Subunit Alpha 1 (LAMA1), an Insulin Like Growth Factor 1 (IGF1), or an Adiponectin (ADIPOQ), SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1, MyoD1, or LDLR locus. The engineered cells may have at least about 5%, 10%, 20%, 30%, 50%, 75%, 90%, 100%, 150%, 200%, 250%, 500%, or 1000% increased expression of the one or more genes or proteins, relative to a control.Attorney Docket No.: 184F-414450-WO

[0233] In some embodiments, the one or more genes or proteins are increased in the engineered cell for at least about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 72 hours, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more.

[0234] The engineered cells may be generated by the steps of: (i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD peptide (e.g., a p300R-Cas peptide, a p300R-dCas peptide), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control. In some embodiments, the period of time is about 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 10 hours, 12 hours, 18 hours, or 24 hours.

[0235] In some embodiments, the engineered cell is a mammalian host cell (e.g., a human engineered cell or a murine engineered cell). The engineered cell may be a myoblast. In some embodiments, the engineered cell is a kidney cell, a muscle cell, a liver cell, or a macrophage.Delivery Vehicles

[0236] The compositions (e.g., peptides and / or gRNAs) vectors, or host cells may be present in a delivery vehicle. In some embodiments, the delivery vehicle comprises a nanoparticle. Nonlimiting examples of nanoparticles include lipid nanoparticles and gold nanoparticles.

[0237] In some embodiments, the present technology comprises delivery vehicles (e.g., lipid nanoparticles) comprising a composition of the present technology or a p300R domain peptide or a p300R-DBD peptide (e.g., a p300R-Cas peptide or a p300R-dCas peptide) thereof and / or one or more gRNAs. In some embodiments, the nanoparticles comprise one or more nucleic acid sequences encoding a p300R domain peptide or a p300R-DBD peptide and / or one or more gRNAs. The nucleic acid sequence may be comprised within a vector. The p300R domain peptide or the p300R-DBD peptide may be encoded by a nucleic acid sequence derived from the same or distinct vector relative to the vector expressing the one or more gRNAs. In some embodiments, the one or more gRNAs comprise two or more gRNAs, which may beAttorney Docket No.: 184F-414450-WOeach encoded by a nucleic acid sequence on the same vector or may be expressed from separate vectors.

[0238] The delivery vehicles may be formulated for administration to a subject in need thereof. In some embodiments, the nanoparticles contain or deliver at least about 5ng, 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, 27 pg, 30 pg, 50 pg, 100 pg, or 500 pg of a peptide, vector, composition, or gRNA of the present technology.

[0239] In some embodiments, formulation of the p300R domain peptide, the p300R-DBD peptide (e.g., a p300R-Cas peptide or a p300R-dCas peptide), or the one more gRNAs in a delivery vehicle increases efficacy, efficiency, durability of effects, delivery, or stability of the peptides or gRNAs, relative to a control lacking a delivery vehicle.Scaffolds

[0240] In some embodiments, the composition, the gRNA, or the p300R-DBD peptide is carried by or otherwise bound to a scaffold, including but not limited to the nanoparticles of the present technology, a virus-like particle (VLP), or other polypeptide scaffold. In embodiments where the composition is a p300R-DBD peptide (i.e. , a fusion protein), the fusion protein may be further covalently linked to be expressed as part of a polypeptide scaffold. Alternatively, the composition or fusion protein may be linked to the scaffold via any suitable means, as will be apparent to those of skill in the art based on the teachings herein. Any suitable nanoparticle, VLP, or other polypeptide scaffold may be used as deemed appropriate for an intended use.Pharmaceutical Compositions, Formulations, and Administration

[0241] In some embodiments, the compositions, systems, vectors, and / or delivery vehicles of the present technology are present in a pharmaceutical composition. The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers and / or excipients such as, for example, buffers, binders, excipients, stabilizers, lubricants, oils, adjuvants, and antioxidants. For example, when the pharmaceutical composition is formulated for subcutaneous administration, the one or more pharmaceutically acceptable carriers and / or excipients may comprise water. The pharmaceutical composition may comprise a therapeutically effective amount and / or a clinically effective amount of the systems, compositions, vectors, engineered cells,Attorney Docket No.: 184F-414450-WOand / or delivery vehicles of the present technology. In some embodiments, two or more dosage units of the pharmaceutical composition comprise a therapeutically effective amount and / or a clinically effective amount of the compositions and / or vectors of the present technology.

[0242] In some embodiments, the pharmaceutical compositions of the present technology are formulated for delivery to a subject in need thereof. In some embodiments, the pharmaceutical compositions are formulated for intraperitoneal, intravenous, parenteral, subcutaneous, intramuscular, intracerebroventricular, or oral administration to the subject.

[0243] The compositions, systems, delivery vehicles, vectors, engineered cells, host cells, and pharmaceutical compositions of the present technology may be used to increase a gene or protein expression in a subject in need thereof. This may be accomplished by (1) promoting euchromatin formation at or near a loci which comprises the gene or a gene encoding the protein of interest, or (2) promoting euchromatin formation at or near a loci that comprises a gene encoding an expressional activator (e.g., a transcription factor) of the gene or protein of interest. In some embodiments, the subject has muscular dystrophy (e.g., Merosin-deficient congenital muscular dystrophy (MDC1 A) or facioscapulohumeral muscular dystrophy (FSHD)). In some embodiments, the subject has cancer.

[0244] In some embodiments, the present technology comprises method of increasing expression of one or more genes or proteins in a cell, relative to a control. Nonlimiting examples of genes and proteins include Laminin Subunit Alpha 1 (LAMA1 ), an Insulin Like Growth Factor 1 (IGF1), an Adiponectin (ADIPOQ), SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , and LDLR. The methods may alter a methylation level of one or more histones and / or a DNA methylation level of one or more CpG islands in the cell. The methylation of the one or more histones and / or the DNA methylation of the one or more CpG islands may impact gene expression of a locus, including a LAMA 1 , IGF1 , ADIPOQ, SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , or LDLR.

[0245] In some embodiments, the present technology comprises a method of increasing a level of LAMA1 gene or protein expression in a subject in need thereof relative to a control, the method comprising administering to the subject a composition,Attorney Docket No.: 184F-414450-WOa vector, a pharmaceutical composition, or a host cell of the present technology. In some embodiments, the subject has a LAMA2 mutation that reduces LAMA2 activity and / or gene expression or protein expression relative to a control. The methods may comprise (i) altering a locus altered by the methylation of one or more histones of the present technology, thereby increasing a level of LAMA1 , SMCHD1 , LRIF1, MATR3, IGF1 , or PML or (ii) increasing the level of LAMA1 gene or protein expression and / or activity, where (i) or (ii) may occur without the use of an enzyme or a nucleotide sequence encoding an enzyme in the composition, the vector, the system, the delivery vehicle, the engineered cell the pharmaceutical composition, or the host cell.

[0246] In some embodiments, the composition, the vector, the pharmaceutical composition, the delivery vehicle, the system, or the host cell is administered to the subject at a dose of about 0.5 ng to about 200 pg.

[0247] In some embodiments, the present technology comprises method of increasing a level of LAMA1, IGF1 , ADIPOQ, SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , or LDLR gene or protein expression in a subject in need thereof relative to a control, the method comprising administering to the subject a system, a composition, a delivery vehicle, a vector, an engineered cell or a pharmaceutical composition of the present technology.

[0248] In some embodiments, the level of gene or protein expression is increased by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a control. The gene or protein may be selected from the group consisting of LAMA1, IGF1 , ADIPOQ, SMCHD1, LRIF1 , MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , and LDLR.

[0249] In some embodiments, the methods target a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands of the present technology, thereby increasing a level of LAMA1, IGF1 , ADIPOQ, SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1 , MyoD1 or LDLR, gene or protein expression and / or activity. In some embodiments, the alteration of the locus and / or reduction in the level of LAMA1 , IGF1 , ADIPOQ, SMCHD1 , LRIF1 , MATR3, PML, AFP, RPL30, CCND1 , MyoD1, or LDLR gene or protein expression or activity occurs in a mammalian cell (e.g., a human cell or a murine cell). The mammalianAttorney Docket No.: 184F-414450-WOcell may be a myoblast. In some embodiments, the mammalian cell is a kidney cell, a muscle cell, a liver cell or a macrophage.

[0250] When the methods of the present technology comprise administering a vector having a reporter gene and / or a detectable marker, the methods may target a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands of the present technology, thereby increasing a level of LAMA1 , IGF1 , ADIPOQ, SMCHD1 , LRIF1, MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , or LDLR gene or protein expression and / or activity, when the reporter gene or the detectable marker is not expressed at a detectable level.

[0251] In some embodiments, the alteration of the locus is maintained for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months. The locus may have an increased maintenance in effects, relative to controls lacking one or more steps of the methods of the present technology.

[0252] In some embodiments, the increase in LAMA1 , IGF1 , ADIPOQ, SMCHD1 , LRIF1 , MATR3, PML, AFP, RPL30, CCND1 , MyoD1 , or LDLR gene expression, protein expression, and / or protein activity is maintained for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0253] In some embodiments, the methods comprise (i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD (e.g., a p300R-Cas peptide or a p300R-dCas peptide), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control. In some embodiments, the one or more gRNAs include two or more gRNAs. The two or more gRNAs may promote increased methylation changes or increased gene or protein expression changes, relative to use of one gRNA.Attorney Docket No.: 184F-414450-WO

[0254] In some embodiments, the period of time is about 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 10 hours, 12 hours, 18 hours, or 24 hours.

[0255] The methods of the present technology may lack or have reduced off-target effects in one or more tissues of a subject in need thereof, relative to a control. In some embodiments, the methods lack or have reduced off-target effects in the liver of a mammalian subject, such as a human or murine subject. In some embodiments, the off-target effects are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.Examples

[0256] The following examples are intended to illustrate various embodiments of the present technology. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the present technology. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of present technology, and it is understood that such equivalent embodiments, are to be included herein. Further, all references cited herein are hereby incorporated by reference in their entirety, as if fully set forth herein.Example 1 : Increasing LAMA1 Expression

[0257] This example assessed the efficacy of p300R domain peptides cotransfected with gRNAs targeting the LAMA1 promoter.

[0258] The C terminal p300 recruiter domain of DLIX4 (p300R) was fused to a DNA binding domain (p300R-DBD), specifically, a dCas9 domain. Domains were fused using a 30 amino acid residue 6x5 (SGGGG) linker. The catalytically inactive cassette of dCas9 had an NLS-2A-mCherry for peptide cleavage. The p300RdCas9-mCherry open reading frame (ORF) was 5343 base pairs (bp) with a molecular weight of 206kDa.

[0259] The expression vectors comprising nucleotide sequences encoding p300R-dCas9 were referred to as p300R-linker-dCas9-NLS-2A-mCherry (p300RdCas9). Vectors comprised a gRNA designed to guide the p300R-dCas9 peptides to the LAMA1 promoter or a p300 nucleotide sequence as a control. All vectors were transfected into C2C12 myoblasts and cells that were not transfected served as an additional control.Attorney Docket No.: 184F-414450-WO

[0260] The gRNAs assessed included gRNA 1 (SEQ ID NO:2), gRNA 2 (SEQ ID NO: 3), gRNA 3 (SEQ ID NO: 4), gRNA 4 (SEQ ID NO: 5), gRNA 5 (SEQ ID NO: 6), and gRNA 6 (SEQ ID NO: 7). Distances of each gRNA binding from the LAMA1 transcription start site are shown in Table 5.Table 5: Distance from LAMA1 Transcription Start Site gRNA Distance from TSSgRNA 1 681gRNA 2 1535 gRNA 3 442gRNA 4 930gRNA 5 1118 gRNA 6 1735

[0261] After transfections, cells were incubated for about 24 hours. To assess effects on LAMA1 expression, LAMA1 protein levels were assessed by immunofluorescence staining (FIG. 1), and LAMA1 gene expression levels were quantified by quantitative PCR (FIG. 2). Negative controls included no transfection and transfection using a p300 peptide only.

[0262] Use of gRNAs 1-6 in combination with the p300R-dCas9 peptides increased protein levels of LAMA1 (FIG. 1), and use of gRNAs 5 and 6 resulted in the highest LAMA1 gene expression levels.

[0263] AML12 (mouse hepatocytes) and C2C12 (mouse myoblasts) were treated with 3R-ON mRNA and target-specific guide RNAs (g3 for ADIPOQ; g9 for LAMA1) encapsulated in CX037 CybeRNAx Lipid Nanoparticles (LNPs). Gene expression was measured via RT-qPCR and expressed as fold change relative to a scramble control. ADIPOQ expression fold change across treatment groups is shown in FIG. 3A, and LAMA1 expression fold change across treatment groups is shown in FIG. 3B. Scramble and untreated groups served as negative controls. 0X037 LNP delivery of 3R-ON / g3 increased mADIPOQ expression in both cell lines, yielding a 10,632-fold increase in C2C12 myoblasts and a 1 ,032-fold increase in AML12 hepatocytes, indicating deliveryAttorney Docket No.: 184F-414450-WOefficiency across different cell lineages when targeting the ADIPOQ locus. LAMA1 activation was cell-type dependent; 3R-ON / g9 LNPs produced a 1 ,267-fold increase in LAMA1 expression in C2C12 cells but did not increase LAMA1 expression in AML12 cells (1-fold), suggesting that the LAMA1 promoter in AML12 hepatocytes may be subject to epigenetic barriers not overcome by the 3R-ON system under these conditions.

[0264] C2C12 cells were treated with 3R-ON mRNAand LAMA1 gRNAs (g9) using either GenVoy or CX319 LNP formulations over a five-day period. Various dosing regimens were assessed, including single dosing (dO), double dosing (dO+d1 ord0+d2), consecutive triple dosing (dO+d1 +d2), staggered triple dosing (d0+d2+d4), and five daily doses (d1— d4). LAMA1 gene expression was measured via RT-qPCR and expressed as fold change relative to controls. Single and double doses resulted in expression declines by day 5, and consecutive triple dosing showed reduced expression after day 3. Staggered triple dosing (d0+d2+d4) maintained LAMA1 expression over a longer duration relative to the other multi-dose regimens. Five daily doses (d1 — d4) produced an increase in LAMA1 expression over the treatment period, reaching over 500-fold change with GenVoy and over 1000-fold change with CX319 by day 5 (FIGS. 4A and 4B).

[0265] C2C12 cells were treated with 3R-ON mRNA and LAMA1 g9 formulated in either GenVoy or CybeRNAx (CX319) LNPs across a concentration gradient from 0 ng to 2000 ng. LAMA1 gene expression was measured via RT-qPCR and expressed as fold change relative to controls over a seven-day period. Both formulations produced dose-dependent increases in LAMA1 expression. GenVoy achieved a peak fold change of over 1250-fold at the 1000 ng dose by day 7 (FIG. 5A). CybeRNAx produced a peak fold change of approximately 350-fold at 2000 ng on day 1 , with expression levels tapering or stabilizing by day 7 across most doses tested (FIG. 5B).Example 2: Assessing Gene Expression in Hepatocytes

[0266] To assess the systems and methods of the present technology in hepatocytes, alpha mouse liver 12 (AML12) cells were transfected with a p300R-DBD peptide of the present technology and a gRNA selected from an ADIPOQ-targeting gRNA: TAAGATGTGAGTCCGCCGAG (ADIPOQ gRNA 1), ATGGGATCCGGTCTAGCAAG (ADIPOQ gRNA 2), GACCCAAGCTGGATTAAACCAttorney Docket No.: 184F-414450-WO(ADIPOQ gRNA 3); GGAGCAAGGGGCCCACTCAT (ADIPOQ gRNA 4); CCTTTCCCATAAGTTTGTCC (ADIPOQ gRNA 5); and an IGF-1 -targeting gRNA: TTAGCAGACATTAAATACTG (IGF1 gRNA 8). This demonstrated that the platforms in accordance with the present technology successfully increased ADIPOQ and IGF1 gene expression levels, relative to untreated cells (UT), cells administered peptide without a gRNA (-g), and scrambled gRNA controls (scramble) (FIGS. 6A-6C).

[0267] HepG2 cells were transfected with 3R-ON mRNA and various hLeptin gRNA combinations using Lipofectamine and harvested at 48 hours for RT-qPCR analysis. Fold change of hLeptin mRNA expression was normalized to a reference control (-gRNA Ref). Data represents three independent experimental runs. Among the combinations tested, gRNA combinations 3+2, 6+2, 4+2, and 2+1 produced increased hLeptin expression, with the 3+2 combination yielding the highest induction across all runs, reaching approximately 800-fold in Run 1 (FIG. 7).

[0268] HepG2 cells were transfected with 3R-ON mRNA and hADIPOQ gRNAs using Lipofectamine and harvested at 24 hours and 48 hours for adiponectin mRNA expression analysis via RT-qPCR. Among the gRNAs tested, g7 and g2 produced upregulation at 24 hours and 48 hours (FIG 8A). In an extended time-course assessment, fold change was monitored on day 1 (d1), day 3 (d3), and day 5 (d5). Peak induction for guides g5, g7, and g9 was observed at d1 (FIG. 8B).

[0269] HepG2 cells were co-transfected with 3R-ON mRNA and hADIPOQ gRNAs via Lipofectamine and harvested at 24 hours, 48 hours, and 72 hours for RT-qPCR analysis. Among the gRNAs tested, g41 alone or in combination with other guides produced activation of hADIPOQ gene expression, reaching up to approximately 8,000-fold induction over the 72-hour period. FIG. 9A depicts fold change across all gRNA combinations tested; FIG. 9B depicts g41 data alone; and FIG. 9C shows the corresponding raw Ct values for the g41 screen.

[0270] HepG2 cells were co-transfected with 3R-ON mRNA and a library of hGSTKI gRNAs (single, dual, and triple combinations) via Lipofectamine. Cells were harvested at 24h, 48h, and 96h for RT-qPCR analysis. Among the gRNAs tested, guide 4 produced the highest activation of GSTK1 expression, reaching up to approximately 140-fold induction over the 96-hour period (FIG. 10).Attorney Docket No.: 184F-414450-WO

[0271] HepG2 cells, primary human myoblasts, and primary human hepatocytes were transfected with 3R-ON mRNA and hADIPOQ g41 via Lipofectamine under singledose and double-dose regimens. RT-qPCR analysis indicated that 3R-ON / g41 produced approximately 100-fold higher hADIPOQ induction compared to VP-dCas9 across all cell types tested. No significant difference in activation was observed between single-dose and double-dose transfections in any of the three cell models (FIGS. HAUG).

[0272] AML12 (mouse) and HepG2 (human) cells were transfected with 3R-ON mRNA and lead gRNA candidates to compare activation of ADIPOQ against a VP64-dCas9 control. In AML12 cells, the dual-gRNA combination mAg3+5 delivered with 3R-ON produced approximately 1,800-fold induction of mADIPOQ, whereas the VP64 system showed negligible activation with the same guide RNAs (FIG. 12A). In HepG2 cells, hAg41 delivered via 3R-ON LNPs induced hADIPOQ expression by approximately 15,000-fold, compared to approximately 6,500-fold with 3R-ON non-LNP delivery; the VP64 system did not produce detectable induction (FIG. 12B). Untreated (UT), guide-only (-g), and Scrambled (Scr) controls showed no significant ADIPOQ expression in either cell line.

[0273] Primary human hepatocytes were transfected with 3R-ON mRNA and hADIPOQ g41 via Lipofectamine across three media conditions: Media Only, +FBS, and +ApoE. RT-qPCR analysis at 24 hours and Day 7 indicated that hADIPOQ mRNA induction was achieved in all groups, with supplementation with ApoE producing the highest peak activation (up to approximately 550-fold). Across all conditions, expression levels attenuated by Day 7 (FIG. 13A and 13B).Example 3: Assessing Impact of Multiple gRNAs

[0274] To assess impact of multiple gRNAs in the methods of the present technology, AML12 and C2C12 cells were each transfected with a p300R-DBD peptide and one of the following IGF1 -targeting gRNA combinations: GCTCCAGTTTTTAAGAGCGA (IGF1 gRNA 6; "g6") alone; TTAGCAGACATTAAATACTG (IGF1 gRNA 8; "g8") alone; ATCCATGGGGCAGCGTAAAG (IGF1 gRNA 2; "g2") in combination with TCAGATACTCAACAGACGTC (IGF1 gRNA 3; "g3"); g1 in combination with g6; g1 in combination with g8; g2 in combination with g6; g2 in combination with g8; g6 inAttorney Docket No.: 184F-414450-WOcombination with g8; g1 in combination with g2 and g6; g1 in combination with g2 and g8; g1 in combination with g6 and g8; g2 in combination with g6 and g8; and g1 in combination with g2, g6, and g8. While each of these successfully increased IGF1 gene expression in both AML 12 cells and C2C12 cells, some gRNA combinations increased IGF1 gene expression more than individual gRNA use (FIG. 14).

[0275] IGF1 gene expression changes were next assessed in both AML12 cells and C2C12 cells using a p300R-DBD peptide transfected with a gRNA selected from CCAGGGTAAACTAGATCGAA (IGF1 guide 1 ; "g1"), ATCCATGGGGCAGCGTAAAG (IGF1 guide 2; "g2"), TCAGATACTCAACAGACGTC (IGF1 guide 3; "g3"), AAAGCACTAACTTACACGAA (IGF1 guide 4; "g4"), GTATTCCAAATAATACCAGC (IGF1 guide 5; "g5"), GCTCCAGTTTTTAAGAGCGA (IGF1 guide 6; "g6"), GTGTGCTAATACACCTCACC (IGF1 guide 7; "g7"), and TTAGCAGACATTAAATACTG (IGF1 guide 8; "g8") (n=3 replicates) (FIGS. 15A and 15B). This demonstrated that the system of the present technology successfully increases IGF1 gene expression levels in murine hepatocyte and myocytes, relative to controls. This was repeated with twice the gRNA amount, demonstrating that doubled gRNA content increases IGF1 gene expression relative to lower amounts with some gRNAs of the present technology (FIGS. 16A and 16B).

[0276] AML12 (murine hepatocyte) cells were transfected with 3R-ON mRNA or VP64-dCas9 together with various guide RNA combinations targeting ADIPOQ via Lipofectamine. Gene expression was measured via RT-qPCR and expressed as fold change relative to controls. Among the combinations tested, g3+5 produced the highest activation in the 3R-ON condition, with peak induction exceeding 15,000-fold, whereas activation in the VP64-dCas9 condition peaked below 150-fold (FIG. 17).

[0277] AML12 (murine hepatocyte) cells were transfected with 3R-ON mRNA (FIG. 18A) or VP64-dCas9 (FIG. 18B) together with various guide RNA combinations targeting ADIPOQ via Lipofectamine. Gene expression was measured via RT-qPCR and expressed as fold change relative to controls. Among the combinations tested, g3+5 produced the highest activation in the 3R-ON guide screen, with peak induction exceeding 15,000-fold (FIG. 18A). In the corresponding VP64-dCas9 condition, ADIPOQ activation was substantially lower, peaking below ~150-fold under otherwise comparable conditions (FIG. 18B).Attorney Docket No.: 184F-414450-WO

[0278] AML12 cells were transfected with 3R-ON mRNA and target-specific gRNAs via Lipofectamine. Gene expression was measured via RT-qPCR and expressed as fold change relative to Scramble controls. Treatment with g3 produced an approximately 900-fold increase in mADIPOQ expression (FIG. 19A). For mlGF-1, individual guides g6 and g8 yielded approximately 28-fold and 18-fold increases, respectively, while the g6+g8 combination produced an approximately 85-fold increase (FIG. 19B). Non-target groups maintained baseline expression levels (near 1-fold).

[0279] HepG2 cells were co-transfected via Lipofectamine with 3R-ON mRNA and specific hADIPOQ gRNAs (single or dual combinations). Cells were harvested at 24h, 48h, and 72h post-transfection for RT-qPCR analysis. Among the gRNAs tested, guide 41 produced the highest activation, reaching up to approximately 10A6-fold induction of hADIPOQ over the 72-hour period. Data were plotted on a log 10 scale to accommodate the dynamic range of transcriptional activation observed (FIG. 20).Example 4: Assessing Gene Expression Between Cell Types

[0280] To compare results of the platforms of the present technology between cells, AML12 cells (FIGS. 21A and 21C) and c2c12 cells (murine myocytes) (FIGS.21 B and 21 D) were each transfected with a p300R-DBD peptide of the present technology in combination with an IGF1 gRNA g1 , g2, g3, g4, g5, g6, g7, or g8 of Example 3 or a LAMA1 gRNA (GTCCGAAGGGCCTGCGCACC; LAMA1 gRNA9). All gene expression levels were assessed relative to GAPDH (FIG. 21 E) and were assessed using two different analysis machines (Replicate 1 : Applied Biosystems™ 7300 Real Time PCR System and Replicate 2: Bio-Rad™ CFX384 Touch Real-Time PGR Detection System). This demonstrated that IGF1 increased in both cell types and LAMA1 was most increased in C2C12s. Trends were replicated with different machine types.

[0281] This analysis was repeated with an additional screen, which assessed LAMA1 and IGF1 gene expression in both AML12 and C2C12 cells after day 1 and day 3 of transfection. Similarly, IGF1 was increased in both cell types and LAMA1 was increased in C2C12 cells (FIGS. 22A and 22B; FIGS. 23A and 23B; FIG. 24).Example 5: Assessing Lipid Nanoparticle Formulations In VitroAttorney Docket No.: 184F-414450-WO

[0282] The platforms of the present technology were next assessed with lipid nanoparticle formations. LAMA1 gene expression levels were assessed in in C2C12 cells transected with different lipid nanoparticle (LNP) formulations of the present technology, containing a p300R-DBD peptide and a gRNA (GTCCGAAGGGCCTGCGCACC; LAMA1 gRNA9). LNP formulations were generated using Invitrogen™ Lipofectamine™ MessengerMAX™ (lipofection transfection reagent) ("Lipofectamine™"), Precision Nanosystems™ GenVoy™ (LNP transfection reagent) ("GenVoy™"), or CybeRNAx™ (0X319). Transfections were assessed with 0 ng, 1 ng, 10 ng, 100 ng, 1000 ng, or 2000 ng of LNP. Cells were harvested for RNA isolation and subsequent RT-qPCR analysis to assess LAMA1 gene expression levels at 24 hours post-transfection. Exemplary LNP formulation encapsulations and transfections are shown in Table 6 and Table 7, respectively.Table 6: LNP EncapsulationCondition of Buffer (uL) mRNA (uL) gRNA (uL) TE Buffer Total Formulation (uL) Volume (uL) GenVoy™ 695 75 75 0 845 3R-ON +LAMA1 g9CX3193R- 695 75 75 0 845 ON +LAMA1 g9Table 7: LNP TransfectionsNg LNP GenVoy™ LNP CX319 LNP Concentration (ng / uL) 202 1821111 1111 6.1043956044

[0283] LAMA1 gene expression increased with all LNP formulations with at least 100 ng and 1000 ng formulations (FIG. 25A). Encapsulation efficiency of the p300R-DBD peptide and the gRNA was comparable between LNP formulations assessed (FIG.25B) and encapsulated RNA levels were greatest with GenVoy™ formulations (FIG.25C). Day 4 LAMA1 fold changes were also assessed up to 2000 ng (FIG. 25D).Attorney Docket No.: 184F-414450-WOAssessments were repeated with day 1 and day 8 measurements (FIG. 26A-26D and 27A-27C).

[0284] LAMA1 and LAMA2 gene (FIGS. 28A and 28B) and protein (FIGS. 28C) levels were next assessed in C2C12 cells transfected with LNP formulations of the present technology, containing different amounts of a p300R-DBD peptide and a gRNA (GTCCGAAGGGCCTGCGCACC; LAMA1 gRNA9) (10 ng, 100 ng, or 1000 ng) (FIG.28D). This demonstrated that increasing LNP formulation component amounts correlated with increased LAMA1 expression.

[0285] Effects of LNP formulations were also assessed up to 72 hours. Specifically, LAMA1 and Alpha-fetoprotein (AFP) gene expression changes were measured in C2C12 cells transfected with an LNP formulation of the present technology containing a p300R-DBD peptide and a LAMA1 -targeting gRNA, compared to untreated cells (UT) at both 24 and 72 hours (FIGS. 29A and 29B; FIG. 30). This demonstrated that gene expression changes were detectable as soon as 24 hours for LAMA1 and at 72 hours for both genes.

[0286] Primary Human Hepatocytes (PHH) and HepG2 cells were transfected with 3R-ON mRNA and hADIPOQ g41 using Lipofectamine to assess gene activation across different cell models. Induction was measured via RT-qPCR and normalized to untreated controls. Negative controls included untreated cells (UT), 3R-ON without guide RNA (-g), and 3R-ON with scrambled gRNA. Treatment with 3R-ON and hADIPOQ g41 yielded an 18,829-fold increase in hADIPOQ expression in HepG2 cells. Primary Human Hepatocytes demonstrated activation with a 288-fold induction. All negative control conditions (UT, -g, and Scramble) maintained baseline or negligible expression levels (<2-fold change) in both cell types. Data are presented as fold change on a Iog10 scale (FIG. 31).

[0287] HepG2 cells were transfected with 3R-ON mRNA and hADIPOQ g41 using either CybeRNAx LNP (CX037) or Lipofectamine delivery platforms, and transcriptomic analysis was performed via RNA-seq at 24 hours post-transfection. RNA-seq data indicated that 3R-ON / g41 produced ADIPOQ upregulation in HepG2 cells with both delivery platforms. Under identical conditions, the VP64-dCas9 system did not produce detectable induction of the ADIPOQ transcript (FIG. 32).Attorney Docket No.: 184F-414450-WO

[0288] A three-step workflow was employed to evaluate the functional bioactivity of 3R-ON / g3+5. 3R-ON / g3+5 was formulated in GenVoy LNPs and subjected to quality control (Step 1). Murine AML12 cells were then treated with the formulated LNPs to generate adiponectin-containing conditioned media (Step 2). Bioactivity was assessed by measuring glucose uptake in recipient AML12 and C2C12 cells via plate reader quantification (Step 3) (FIG. 33).

[0289] Murine AML12 cells were treated with 3R-ON / g3+5 formulated in CybeRNAx LNP (CX037). RT-qPCR indicated dose-dependent mRNA upregulation, with the 2000 ng dose producing approximately 4,000-fold adiponectin upregulation (FIG. 34A). Time-course ELISA analysis of extracellular media indicated that Day 3 corresponded to peak secreted protein levels, measured at 1 ng / mL (FIG. 34B). Conditioned media harvested at the 2000 ng dose on Day 4 were used as the substrate for subsequent functional glucose uptake assays.

[0290] Bioactivity of LNP-delivered, 3R-ON-induced adiponectin was assessed via functional glucose uptake assays in AML12 (FIG. 35A) and C2C12 (FIG. 35B) cell lines. Conditioned media (CM) containing endogenously synthesized adiponectin (1 .2-2.4 ng / mL) produced increased intracellular glucose levels compared to recombinant monomeric adiponectin (rAd) and scramble controls.

[0291] AML12 and C2C12 cells were transfected with 3R-ON mRNA and gRNAs targeting ADIPOQ, IGF-1 , and LAMA1 at varying mRNA:gRNA mass ratios (1 :0.5 to 1 :4). In AML12 cells, mADIPOQ g3 at a 1 :1 ratio produced peak activation of approximately 500-fold. In C2C12 cells, mLAMAI g9 at a 1 :2 ratio produced approximately 1 ,000-fold activation, and the mlGF-1 combination (g6+g8) produced the highest activation at the 1 :0.5 ratio (FIGS. 36A-D).

[0292] Epigenetic characterization of the ADIPOQ locus in hepatocytes and adipocytes was performed using chromatin immunoprecipitation sequencing (ChlP-seq) for histone modifications. In hepatocytes, the ADIPOQ locus exhibited high enrichment of H3K27me3 across the gene body and promoter, with minimal signal for H3K27ac, H3K4me3, or H3K4me1 , indicating epigenetic repression. In beige adipocytes, H3K27me3 levels were near-absent, and the locus instead exhibited enrichment of H3K27ac, H3K4me3, and H3K4me1 at the TSS and upstream regulatoryAttorney Docket No.: 184F-414450-WOregions, consistent with an active transcriptional state. These data indicate differential epigenetic regulation of ADIPOQ between hepatocytes and adipocytes (FIG. 37).

[0293] Murine AML12 hepatocytes were transfected with 3R-ON / g3+5 to activate mADIPOQ at dosages of 125 ng and 625 ng. Intracellular and secreted adiponectin protein levels were quantified daily over five days using ELISA, with intracellular protein measured from cell lysates and secreted protein measured from conditioned media. Data represent absolute protein concentrations (ng / mL) normalized to untreated controls. Intracellular adiponectin levels exhibited a peak at Day 2, followed by a decline over the remaining time course. Secreted protein levels in the media increased over the study period, reaching a concentration of 0.08 ng / mL by Day 5. The 125 ng dosage produced higher protein levels in both lysate and media fractions compared to the 625 ng dose (FIG. 38).

[0294] HepG2 cells were transfected with LNPs formulated with 3R-ON mRNA and hADIPOQ gRNA (g41) or a Scrambled (Scr) control across a dose range of 0 ng to 2000 ng. Transfection efficiency was quantified by flow cytometry measuring mCherry expression from the 3R-ON mCherry mRNA delivered via LNPs. CX037 hADIPOQ g41 LNPs produced higher percentages of mCherry-positive cells compared to the Scr control, reaching approximately 50% mCherry-positive cells at the 500 ng dose. Transfection efficiency increased from 125 ng to 500 ng, followed by a plateau and slight decline at 1000-2000 ng (FIG 39A). RT-qPCR analysis of hADIPOQ mRNA levels, normalized to the Scr reference, indicated that the 500 ng LNP dose produced the highest transcriptional activation, yielding an approximately 28,000-fold increase in hADIPOQ expression. The 125 ng and 250 ng doses produced approximately 15,000-fold induction, while the 2000 ng dose produced lower induction than the 500 ng dose (FIG. 39B).

[0295] LNP-encapsulated 3R-ON mRNA and gRNA41 were used to induce adiponectin expression over a 7-day time course. Secreted protein levels in the conditioned media were measured and compared against a scrambled gRNA control. A dose-dependent effect was observed between the 12.5 ng and 125 ng RNA treatments, with the 125 ng dose yielding adiponectin concentrations of 10 ng / mL (FIGS. 40A and 40B).Attorney Docket No.: 184F-414450-WO

[0296] LNP-encapsulated 3R-ON mRNA and gRNA41 were used to induce adiponectin expression. Conditioned media containing secreted protein were harvested and applied to HepG2 target cells to assess functional glucose uptake compared to a scrambled control (FIG. 41 A and 41 B).

[0297] Murine AML12 hepatocytes were treated with increasing concentrations of 3R-ON / g3+5 formulated in CX037 LNPs. RT-qPCR analysis at 24 hours posttransfection indicated a dose-dependent correlation between LNP dosage and mADIPOQ mRNA levels, with the 2 pg dose producing a peak induction of approximately 3,200-fold relative to untreated controls (FIG. 42).

[0298] AML12 cells were treated with 3R-ON / g3 at concentrations ranging from 3 ng to 3000 ng using either Lipofectamine or CX037 LNP delivery systems. Gene expression was measured via RT-qPCR and expressed as fold change relative to untreated (UT) controls. Lipofectamine produced peak activation of approximately 780-fold at the 30 ng dose, with fold change values plateauing or declining at concentrations from 300 ng to 3000 ng. CX037 LNPs produced lower activation at 3 ng and 30 ng doses relative to Lipofectamine, but exhibited a dose-dependent increase in expression, reaching approximately 220-fold at 300 ng and approximately 500-fold at 3000 ng (FIG.43).

[0299] AML12 cells were treated with 3R-ON / g3 encapsulated in either CX037 or GenVoy LNPs. RT-qPCR analysis indicated that CX037 produced approximately 1 ,035-fold activation of mADIPOQ at Day 1 , compared to approximately 325-fold with GenVoy. By Day 3, CX037-treated cells maintained approximately 270-fold activation, whereas GenVoy-treated cells returned to baseline levels. In a dose-response comparison against Lipofectamine, Lipofectamine produced peak activation of approximately 780-fold at 30 ng, with declining fold change at higher doses, while CX037 produced a dosedependent increase reaching approximately 500-fold at 3,000 ng (FIG. 44).

[0300] HepG2 cells and Primary Human Hepatocytes (PHH) were transfected with CX037 or GenVoy LNPs to deliver 3R-ON (dCas9) mRNA and hADIPOQ g41 . Gene expression was analyzed at day 1 post-transfection via RT-qPCR across a dose range of 125 ng to 1000 ng. In HepG2 cells, both platforms produced dose-dependent increases in dCas9 mRNA levels, with GenVoy at 1000 ng producing the highest expression, exceeding a 550-fold increase relative to untreated controls (FIG. 45A). ForAttorney Docket No.: 184F-414450-WOhADIPOQ induction, peak activation occurred at lower doses, with CX037 yielding approximately 4,000-fold induction at 250 ng; the 1000 ng dose resulted in a slight reduction in fold change to approximately 3,600-fold (FIG. 45B). In PHH, dCas9 transcript levels were higher than in HepG2 cells, with GenVoy reaching approximately 100,000-fold at the 1000 ng dose (FIG. 45C). hADIPOQ activation in PHH was more modest, with CX037 at 250 ng reaching approximately 120-fold induction (FIG. 45D). In both cell types, the 1000 ng dose for both platforms produced lower hADIPOQ activation compared to mid-range doses.

[0301] AML12 and C2C12 cells were transfected with 3R-ON / g3+5 formulated in 0X037 LNPs or delivered via Lipofectamine. RT-qPCR analysis at 24 hours indicated that 0X037 LNP delivery produced approximately 145,000-fold induction of mADIPOQ mRNA in 02012 myoblasts. Both delivery platforms produced mADIPOQ induction in AML12 hepatocytes (FIG. 46).Example 6: Assessing Lipid Nanoparticle Formulations In Vivo

[0302] To assess the LNP formulations of the present technology in vivo, mice were treated with LNP formulations comprising a p300R-DBD peptide and a gRNA (GTCCGAAGGGCCTGCGCACC; LAMA1 gRNA9) (27pg) by right tibialis anterior injection (n=3) (control comprised PBS injection to the left tibialis anterior) (FIG. 47A). Tissues were assessed at both 24 and 72 hours. This revealed that murine tibialis anterior tissues on average had increased LAMA 1 gene expression at both 24 and 72 hours, compared to controls (FIGS. 47B-47D; FIGS. 48A and 48B; FIGS. 49A-49C; FIGS. 50A and 50B), demonstrating successful in vivo delivery. LAMA1 was not detected in the control group tissues and unrelated, non-LAMA1 gene expression changes were also not detected.

[0303] C57BL / 6 mice received a single tail vein injection of 3R-ON / g3 formulated in GenVoy / Cytiva LNPs at either a low dose (0.1 mg / kg) or high dose (1 mg / kg). Liver tissues were harvested at 24 hours and 72 hours post-injection, followed by lysis and RT-qPCR analysis to quantify hepatic ADIPOQ mRNA and dCas9 transcript fold change (FIG. 51).

[0304] C57BL / 6 mice received a single tail vein injection of 3R-ON with g3+g5 guide RNAs using Acuitas LNPs at doses of 0.1 mg / kg (low), 0.5 mg / kg (med), and 1 mg / kg (high). RNA-seq transcriptomic analysis of liver lysates at 24 hours to 48 hoursAttorney Docket No.: 184F-414450-WOpost-injection indicated ADIPOQ upregulation, with the high-dose group producing peak mRNA induction at 10 transcripts per million (TPM) in mouse liver (FIG. 52).

[0305] High-fat diet (HFD) C57BL / 6 male mice (12 weeks) were administered 3R-ON with g3+g5 guide RNAs via tail vein injection using Acuitas LNPs at doses of 0.1 mg / kg (low) and 0.5 mg / kg (high). Liver tissues were harvested at 24 hours, 72 hours, and 11 days post-injection, followed by lysis and RT-qPCR analysis to quantify hepatic ADIPOQ mRNA and dCas9 transcript fold change (FIG. 53).Example 7: Assessing Platform Mechanisms

[0306] To further assess mechanisms of the platforms of the present technology, LAMA1 gene expression levels at day 1 (d1) and day 3 (d3) were measured in C2C12 cells transfected with a p300R-DBD peptide (3R-ON) and LAMA1 -targeting gRNA, GTCCGAAGGGCCTGCGCACC (LAMA1 gRNA9; "g9"), or an alternate domain peptide (VP-ON) comprising a domain encoded by the nucleotide sequence GACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGAT TTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGC TCGGCAGTGACGCCCTTGATGATTTTGACCTGGACATGCTG, and the same LAMA1 -targeting gRNA in cells treated with a p300 inhibitor (iP300w), normalized to GAPDH gene expression levels (FIGS. 54A and 54B and FIGS. 55A-55D). These results showed that the p300R-DBD peptides may be distinct from other potential gene activators due to the iP300w response.

[0307] Next, AML12 cells were transfected with p300R-DBD peptide and a gRNA. gRNAs used included GTCCGAAGGGCCTGCGCACC (LAMA1 gRNA9; "g9"); GGGCGCGGCCAGGCAGGCGG (LAMA1 gRNA10; "g10"); CCCTGAATGCTCACCCCAAC (LAMA1 gRNA11 ; "g11"); and GCGCCCAGGTCTGTCCTCCA (LAMA1 gRNA12; "g12"). (FIGS. 56A and 56B). Cells were not treated with an inhibitor and LAMA1 levels were assessed, demonstrating no off-target effects were observed in AML12 cells when LAMA1 was activated with a platform of the present technology.

[0308] LAMA1 levels were next measured in C2C12 cells transfected with p300R peptide and g9, with or without use of LNP formulations, over 5 days (FIGS. 57A-57D). LAMA1 gene expression changes were assessed also assessed the same C2C12 protocol of FIGS. 57A-57D, but cells were treated with 0.1 pM, 1.0 pM, or 10 pM of aAttorney Docket No.: 184F-414450-WOhistone deacetylase inhibitor (HDACi) (FIGS. 58A-58E) or with 0.1 pM, 1.0 |_iM, or 10 pM of a p300 inhibitor (iP300w) (FIGS. 59A and 59B). This demonstrated that LAMA1 gene expression levels trended similarly regardless of whether an inhibitor was used.

[0309] TIP-ChIP was performed to assess binding and spatial aggregation of dCas9 at the human ADIPOQ promoter site, targeting the region identified for gRNA g41. TIP ChlP-seq tracks indicated a localized peak of dCas9 enrichment at the ADIPOQ promoter regulatory region. Signal accumulation was observed at the g41 target site, indicating recruitment of the 3R-ON complex to the locus. Input tracks exhibited minimal background signal. The analysis covered a 36 kb window on Chr3, and dCas9 occupancy was localized to the intended target site without significant proximal off-target binding (FIG. 60).

[0310] Primary Human Hepatocytes were transfected with LNP-encapsulated 3R-ON (dCas9) mRNA and hADIPOQ g41. Transcriptional kinetics for both the delivered mRNA and the target gene were monitored over a 5-day time course via RT-qPCR. dCas9 mRNA levels reached a peak of greater than 6xlOA6-fold at Day 2, followed by a decline by Day 3 and a return toward baseline by Day 5. In contrast, hADIPOQ mRNA levels increased from Day 1 through Day 5, reaching approximately 450-fold change at the final timepoint. hADIPOQ expression continued to increase as dCas9 mRNA levels declined (FIG. 61).

[0311] Primary Human Hepatocytes (PHH) and HepG2 cells were transfected with CX037 LNPs containing 3R-ON mRNA and hADIPOQ g41 at doses ranging from 125 ng to 1000 ng. To assess specificity of the activation complex, hADIPOQ expression was compared against a panel of DUX4-related downstream genes (DUX4, LDLR, MBD3L2, PRAMEF12, and Zscan4) via RT-qPCR. In both cell models, hADIPOQ exhibited dose-dependent induction, with peak activation occurring at the 250 ng dose in PHH (approximately 120-fold) and HepG2 cells (greater than 4,000-fold). Expression levels of downstream DUX4 targets remained at or near baseline (<2-fold change) across most doses and conditions. A transient increase in MBD3L2 (approximately 13-fold) was observed in PHH at 500 ng; however, this did not correspond to a general activation of the DUX4 pathway. In HepG2 cells, expression of DUX4, LDLR, PRAMEF12, and Zscan4 showed negligible changes compared to the untreated (UT) reference. Ultimately, these results demonstrate that the 3R-ON / g41 complexAttorney Docket No.: 184F-414450-WOselectively activates the silenced ADIPOQ locus without triggering the broader DUX4 transcriptional program, confirming high target specificity in hepatic models (FIGS. 62A and 62B).

[0312] PHH and HepG2 cells were transfected with CX037 LNPs delivering 3R-ON mRNA and either hADIPOQ g41 or a Scrambled (Scr) control to evaluate transcriptional specificity over a 7-day time course. Gene expression was quantified via RT-qPCR and normalized to Untreated (UT) references. In both PHH and HepG2 cell models, transfection with 3R-ON and hADIPOQ g41 produced sustained activation of ADIPOQ. In HepG2 cells, ADIPOQ levels maintained a plateau of approximately 15 log2(Fold Change) from Day 1 through Day 7. dCas9 expression peaked within the first 48 hours (reaching greater than 2 Iog2 fold change) before exhibiting a gradual decline across the 7-day period in all experimental groups. No significant activation of DUX4-related downstream genes, including DUX4, LDLR, MBD3L2, PRAMEF12, and ZSCAN4, was observed. These off-target markers remained near baseline levels (typically less than 2 Iog2 fold change) throughout the 7-day duration. In the 3R-ON with Scrambled control group in HepG2 cells, dCas9 expression was detected without corresponding induction of ADIPOQ or any DUX4-related targets, indicating that gene activation was dependent on the presence of the target-specific g41 guide (FIGS. 63A-C).

[0313] HepG2 conditioned media were collected at Day 6 and analyzed by ELISA to assess adiponectin protein oligomerization. High molecular weight (HMW) forms constituted approximately 2.73% of total secreted adiponectin (FIGS. 64A-D).

[0314] HepG2 cells were transfected with 3R-ON and hADIPOQ g41 and treated with a p300 inhibitor (p300i) at varying concentrations. RT-qPCR analysis indicated dose-dependent reduction of ADIPOQ induction following p300i treatment. At 3R-ON dosages up to 1000 ng, p300 inhibition attenuated transcriptional activation, indicating that ADIPOQ induction by the 3R-ON complex is dependent on p300 acetyltransferase activity. Under identical conditions, VP64-dCas9 did not produce detectable ADIPOQ activation (FIG. 65).

[0315] HepG2 cells were transfected with 3R-ON and hADIPOQ g41 via LNPs across a range of concentrations. RT-qPCR analysis indicated dose-dependent hADIPOQ mRNA upregulation, with peak expression occurring between Day 1 and DayAttorney Docket No.: 184F-414450-WO2 across all concentrations tested. Expression levels declined following the initial peak but remained detectable through Day 7. Lower dosages (e.g., 64 ng to 16 ng) maintained higher relative induction at later time points compared to the 1000 ng dose (FIG 66A). VP64-dCas9 did not produce comparable activation under the same conditions (FIG. 66B).

[0316] AML12 cells were transfected with 3R-ON / g3+5, and kinetic analysis of mADIPOQ mRNA and secreted adiponectin protein was performed over a 7-day time course. RT-qPCR indicated approximately 2,000-fold induction of hepatic mADIPOQ mRNA expression at 24 hours, followed by a decline in intracellular mRNA levels over subsequent days. In contrast, secreted adiponectin protein levels in the conditioned media increased over the same period, reaching a concentration of approximately 1 .2 ng / mL by Day 3 and remaining at that level through Day 7 (FIG. 67).Example 8: Assessing Additional gRNA Combinations in Human Myoblasts

[0317] To assess alternate gRNA combinations and gene targets with the platforms of the present technology, Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), IGF-1, and LRIF1 gene expression levels in FSHD1 (54-2) human myoblasts transfected with a p300R-DBD peptide and various combinations of SMCHD1 -targeting gRNAs, were assessed (FIGS. 68A-68D). gRNAs included those having close proximity to the target gene's corresponding transcription start site (TSS), where the selected gRNAs were designed to bind a target various nucleotide distances from the target gene TSS (FIG. 68A) and those having the TSS binding distances shown on the X-axis of FIGS. 68B, 68D, and 68E, compared to controls (FIG. 68C). SMCHD1 -targeting gRNA combinations used in Example 8 are shown in Table 8.Table 8: SMCHD1 -targeting gRNA combinations Combination gRNAs Nucleotide Sequence Combo g5, g13, 586* or hSMCHD1_283*_g5 TCGGCCCTCGCGAAGTT 1 592 GGA (SEQ ID NO: 57) hSMCHD1_217*_g13 GCGAATCCAGCAGGCCG CGC (SEQ ID NO: 58) 586* (196) GCCUCUGAGGACUACCC GCA (SEQ ID NO: 59) 592* (190) GAGGACUACCCGCAGGGCGC (SEQ ID NO: 60)Attorney Docket No.: 184F-414450-WOCombo g6, g3,344* hSMCHD1_534*_g3 TTCAGGCCGGCGGTACT 2 CGC (SEQ ID NO: 61) hSMCHD1_462*_g6 TGAGCTTGTAGGTGCCG CGC (SEQ ID NO: 62) 344* (424) GUCCACCAAAGCGAGCA CGU (SEQ ID NO: 63) Combo g3, 344*, 285*, hSMCHD1_534*_g3 TTCAGGCCGGCGGTACT 3 g6 CGC (SEQ ID NO: 61) hSMCHD1_ 462*_g6 TGAGCTTGTAGGTGCCG CGC (SEQ ID NO: 62) 344* (424) GUCCACCAAAGCGAGCA CGU (SEQ ID NO: 63) 285* (497) AGUACUACUAAGAAAUG AGG (SEQ ID NO: 64) Combo 285*, g6, g2 hSMCHD1_772*_g2 CGACGGCATTCGGGGTA 4 CGG (SEQ ID NO: 65) hSMCHD1_ 462*_g6 TGAGCTTGTAGGTGCCG CGC (SEQ ID NO: 62) 285 (497) AGUACUACUAAGAAAUG AGG (SEQ ID NO: 64) Combo g9*, g10 hSMCHD1_1737*_g9 AGAGGTGTGCTTGTATAA 5 TC (SEQ ID NO: 66) hSMCHD1_1664*_g10 CTCAATTTGCTGAAGTAGAG (SEQ ID NO: 67) *denotes TSS binding distance for target gene

[0318] Combination assessments were repeated for Ligand Dependent Nuclear Receptor Interacting Factor 1 (LRIF1) and SMCHD1 gene expression levels in FSHD1 (54-2) human myoblasts transfected with a p300R-DBD peptide and an LRIF1 -targeting gRNA combination or a SMCHD1 -targeting gRNA combination, compared to controls (FIGS. 69A-69D). This demonstrated that certain TSS proximity binding by gRNAs increased target gene expression levels relative to those binding at alternate TSS distances. LRIF-1 targeting gRNA binding from the LRIF-1 TSS are shown in the X-axis of FIGS. 69A-69D. LRIF-1 -targeting gRNAs used in Example 8 are shown in Table 9.Table 9: LRIF1 -targeting gRNAsgRNA LRIF 1 Nucleotide Sequence LRIF1 TSS Guide Binding Distance NumberhLRIF1_588_g1 1 TAGTTTCGATGAACGTAGGC 588(SEQ ID NO: 25)Attorney Docket No.: 184F-414450-WOhLRIF1_392_g2 2 GAGAGAAAAACGTACGCTAG 392(SEQ ID NO: 26)hLRIF1_790_g3 3 GTCCCTCATCTTGACGCCAT 790(SEQ ID NO: 27)hLRIF1_1573_g4 4 TACTGTAGAGCAAGCTTACT 1573(SEQ ID NO: 28)hLRIF1_213_g5 5 TCTTCCGGGGTCGTCACGTC 213(SEQ ID NO: 29)hLRIF1_1158_g6 6 GTGTGTTTAGCCAGACAACC 1158(SEQ ID NO: 30)hLRIF1_967_g7 7 GGTGGTAAAATAATGGGTGG 967(SEQ ID NO: 31)hLRIF1_1331_g8 8 AGAGAAGCAAGTCATAAGTG 1331(SEQ ID NO: 32)hLRIF1_2025_g9 9 TCCCCATTGCTCTTATGATA 2025(SEQ ID NO: 33)hLRIF1_705_g10 10 ATAAAGAGCATCAACTGCAA 705(SEQ ID NO: 34)

[0319] Methyl-CpG binding domain protein 3 like 2 (MBD3L2) (FIG. 70A), IGF-1 (FIG. 70B), SMCHD1 (FIG. 70C), Zinc Finger And SCAN Domain Containing 4 (ZSCAN4) (FIG. 70D), Leucine Twenty Homeobox (LELITX) (FIG. 70E), and solute carrier family 34 member 2 (SLC34A2) (FIG. 70F) gene expression levels were next assessed in FSHD1 (54-2) human myoblasts transfected with a p300R-DBD peptide and an IGF-1 -targeting gRNA (IGF-1 guide 7: GTGTGCTAATACACCTCACC) or a SMCHD1 -targeting gRNA combination of FIG. 68A (FIGS. 70A-70F) (Table 8). IGF-1 gene expression increased in most targets other than SMCHD1 in cells transfected with IGF-1 -targeting gRNAs relative to those transfected with the SMCHD1 -targeting gRNA combinations.Attorney Docket No.: 184F-414450-WO

[0320] IGF-1 -targeting gRNAs were next assessed against matrin 3 (MATR3)-targeting gRNA combinations (FIGS. 71 A and 71 B). MATR3-targeting gRNA combinations used in Example 8 are shown in Table 10. FSHD1 (54-2) human myoblasts were transfected with a p300R-DBD peptide and an IGF-1 -targeting gRNA (GTGTGCTAATACACCTCACC), an MATR3-targeting gRNA combination, or an SMCHD1 -targeting combination (Table 8). MATR3 gene expression levels were increased in all MATR3 and SMCHD1 -targeting gRNA transfected groups, relative to controls. Similarly, LRIF1 gene expression levels were assessed in FSHD1 (54-2) human myoblasts transfected with a p300R-DBD peptide and various combinations of LRIF1 -targeting gRNA combinations of FIG. 68E, wherein LRIF1 -targeting gRNA combinations increased LRIF1 gene expression levels, relative to controls (FIGS. 72A and 72B) (Table 9).Table 10: MATR3-targeting gRNAsCombination gRNAs Nucleotide Sequence Binding Distance from TSS hMATR3_2181_g2 ACTAGCTACCCAATTAGAGT 2181 (SEQ ID NO: 36)Combo 1hMATR3_904_g1 ACGCTGTGACAGCCGGATGT 904 (SEQ ID NO: 35)hMATR3353 q5 GAGAGGGTTCATTGTTCGCT 353 (SEQ ID NO: 39)Combo 2 hMATR3_118_g4 TCCCCTCGTGATCCCGAGCT 118 (SEQ ID NO: 38)hMATR3353 _g5 GAGAGGGTTCATTGTTCGCT 353 (SEQ ID NO: 39)Combo 3 hMATR3_1434_g3 GTCAAAAAGGTTGACTATGA 1434 (SEQ ID NO: 37)hMATR3 1250 g8 ACCAATAATCTGGTGATGAC 1250 (SEQ ID NO: 42)Attorney Docket No.: 184F-414450-WOhMATR3_1434_g3 GTCAAAAAGGTTGACTATGA 1434 (SEQ ID NO: 37)Combo 4hMATR3_1250_g8 ACCAATAATCTGGTGATGAC 1250 (SEQ ID NO: 42)hMATR3904 _g1 ACGCTGTGACAGCCGGATGT 904 (SEQ ID NO: 35) hMATR3_904_g1 ACGCTGTGACAGCCGGATGT 904 (SEQ ID NO: 35)Combo 5hMATR3_702_g6 GCAAGAGACCCTAGGCTGTA 702 (SEQ ID NO: 40) hMATR3_267_g9 GCTAGACTTGCCCTGCACCA 267 (SEQ ID NO: 43)hMATR3445 _g10 TTGGGGGGCGGGTGTCGGAA 445 (SEQ ID NO: 44)

[0321] MBD3L2 gene expression levels were also assessed in FSHD (54-2) human myoblasts following 72 hours after transfection with a p300R-DBD peptide and a MBD3L2-targeting gRNA (FIGS. 73A and 73B). This demonstrated that the MBD3L2-targeting platform successfully increased target gene expression, relative to controls. Similarly, IGF-1 (FIG. 74A), SMCHD1 (FIGS. 74B-74D), LAMA1 (FIG. 74E), and MATR3 (FIG. 75) gene expression levels in human myoblasts transfected with a p300R-DBD peptide and a SMCHD1, LAMA1 (GTCCGAAGGGCCTGCGCACC), IGF-1 (GTGTGCTAATACACCTCACC), or MATR3- targeting gRNA, demonstrating successful and / or upward-trending upregulation of the target gene, relative to controls. As an additional control, IGF-1 (FIG. 76A) and Trinucleotide Repeat Containing Adaptor 6B (TNRC6B) (FIG. 76B) gene expression levels in FSHD1 (54-2) human myoblasts transfected with a peptide comprising a DBD fused to an embryonic ectoderm development polypeptide binder (EB) domain peptide and an IGF-1 or TNRC6B-targeting gRNA, relative to controls.Attorney Docket No.: 184F-414450-WO

[0322] A schematic representation of the mSMCHDI locus was generated showing CpG island density, ReMap density peaks, and identified target regions R1 through R7. Each region (R1 , R2, and R4) was targeted using a pool of three specific guide RNAs (gRNAs). C2C12 cells were transfected via Lipofectamine with 3R-ON mRNA and various combinations of gRNAs targeting regions R1-R7. SMCHD1 fold change relative to controls was measured by qPCR. Controls included untreated (UT), scrambled (Scr), and mRNA-only (-gRNA) conditions. Certain combinations, including R1+R4 and R1+R2+R4, produced increases in SMCHD1 expression of up to approximately 4-fold change relative to controls (FIG. 77).

[0323] C2C12 cells were transfected with identical concentrations of either the 3R-ON or VP64-dCas9 effector systems targeting the mouse Utrophin (Utrn) promoter via specific guide RNAs (mUTRO g8, g12, or g8+g12). Experimental controls included untreated (UT) cells, a Scramble gRNA reference, and a non-relevant LAMA1 (g9) guide to establish baseline expression and assess off-target effects. Following a 24-hour incubation, Utrn expression was quantified via RT-qPCR, with results expressed as fold change relative to the Scramble control (FIG. 78).

[0324] C2C12 cells were transfected with combinations of guide RNAs (g9-g12) targeting the proximal promoter of LAMA1 , utilizing co-transfection of EB-ON and 3R-ON. Co-transfection with EB-ON and 3R-ON resulted in increased LAMA1 expression at the Day 1 time point for the majority of guides tested relative to 3R-ON alone. Approximately 10-fold activation was maintained at Day 5 in select gRNA treatment conditions (FIG. 79).Example 9: Assessing Gene Expression in Macrophages.

[0325] THP-1 monocytes were treated with Phorbol 12-myristate 13-acetate (PMA) to induce differentiation into adherent macrophages by Day 3 (D3). Cells were then transfected with specific single guide RNA (gRNA) or combinations thereof. Total RNA was harvested 24 hours post-transfection (D4) for transcriptional analysis via RT-qPCR. Various gRNA combinations were screened to assess their effect on IL-10 mRNA expression levels. mRNA and gRNA(s) were delivered to macrophages using Lipofectamine. Guide combinations (1+2), (1+3), and (1+6) produced IL-10 induction averaging approximately 400-fold relative to untreated controls. Data are presented as IL-10 fold change relative to Untreated (UT) or Scrambled (Scr) controls. A secondaryAttorney Docket No.: 184F-414450-WOscreen of gRNA combinations in differentiated macrophages assessed both IL-10 and ADIPOQ expression. Combinations (1+2) and (1+6) multiplexed with Adiponectin g41 produced concurrent induction of both IL-10 and ADIPOQ (FIGS. 80A-C).

[0326] Following PMA-mediated differentiation of THP-1 monocytes, transfection with 3R-ON and selected guide combinations (1+2 and 1+6) resulted in increased secreted IL-10 protein levels compared to VP64 controls. Secreted IL-10 protein levels exceeded 10,000 pg / mL by Day 5 over the 5-day study period (FIG. 81).Example 10: Assessing Gene Expression in Natural Killer (NK) Cells

[0327] NK92 cells were transfected with varying concentrations (1 pg, 5 pg, and 12.5 pg) of 3R-ON or EB-ON mRNA together with specific guide RNAs targeting CD16. Results were assessed as the percentage of FITC-positive cells (% FITC+), with an eGFP positive control indicating transfection efficiency of 96% at 12.5 pg. The 3R-ON + CD16 #2 combination yielded 63% FITC+ cells at the 5 pg dose (FIGS. 82A and 82B).

[0328] NK92 cells were electroporated with 3R-ON or VP-ON mRNA at defined ratios together with CD16-targeting guide RNA g6. On Day 1 , delivery efficiency was confirmed via a 1 pg eGFP control (92% FITC+), while cells transfected with 1 :1 VP-ON + g6 and 1 :1 3R-ON + g6 exhibited mCherry expression at 68% and 61%, respectively. By Day 4, the percentage of CD16-positive cells was assessed in both unsorted and sorted populations. The 1 :1 3R-ON + g6 condition yielded 67% CD16 positivity in the sorted population, compared to the 1 :33R-ON condition and the 1 :1 VP-ON + g6 condition, the latter of which declined to 10% or less by the final time point (FIGS. 83A and 83B).

[0329] Flow cytometric analysis at Day 4 post-electroporation assessed CD16 surface expression persistence. The 1 :1 3R-ON + g6 condition exhibited 52% CD16 positivity in the unsorted population and 67% following sorting. By comparison, the 1 :3 3R-ON + g6 condition yielded 21% in the unsorted population, and the 1 :1 VP-ON + g6 condition yielded 7% unsorted and 10% sorted. Baseline background levels were established via "No EP -Ab" and "No EP" controls, which registered at 3% and 2%, respectively (FIGS. 84A-C).

[0330] CD16 protein expression was further assessed in NK-92 cells using a construct in which 3R-ON was fused to mCherry via a 2A self-cleaving peptideAttorney Docket No.: 184F-414450-WOsequence, such that mCherry was cleaved from 3R-ON at a 1 :1 ratio following translation. Cells transfected with 3R-ON, but not VP64-dCas9, exhibited detectable CD16 surface protein expression. CD16 surface expression was also detected in mCherry-negative cells, indicating that 3R-ON-mediated gene activation occurred at expression levels below the threshold of mCherry detection (FIGS. 85A-C).Example 11: Assessing Gene Expression in Human Embryonic Kidney (HEK) Cells

[0331] HEK cells were transfected using Lipofectamine with 3R-ON mRNA and a panel of ten target-specific guide RNAs (g1— g10) targeting Alpha-Fetoprotein (AFP). Comparisons were made against untreated cells (LIT), cells transfected with 3R-ON mRNA without guide RNA (3R -g), and cells transfected with 3R-ON mRNA and a scrambled guide RNA (3R Scr). As demonstrated in FIG. 86, multiple gRNAs increased AFP expression relative to controls, with g3 and g6 showing the highest induction, achieving approximately 14-fold and 10-fold increases in AFP expression, respectively.Example 12: Assessing Gene Expression in FSHD Cell Lines

[0332] FSHD1 (54-2) myoblasts were transfected on days 1 and 3 with 0.1 pg of 3R-ON mRNA alone or in combination with guide RNAs (gRNAs). On day 4, cells were induced to differentiate for 72 hours to myotubes. Conditions included a non-transfected control (Untransfected), 3R-ON mRNA transfected alone (0.1 3RON), a scramble control gRNA transfected with 3R-ON mRNA (Scramble + 3RON), and a target gene guide combination transfected with 3R-ON mRNA (g2+5+30 + 3RON). 3R-ON-mediated upregulation of LRIF1 expression resulted in decreased expression of DUX4 target genes in FSHD1 myotubes, while preserving differentiation capacity (FIGS. 87A-87E).Additional Embodiments

[0333] Various embodiments of the present technology are set forth below in paragraphs

[0334] to

[0390] :

[0334] 1 . A system for increasing expression of one or more proteins in a cell, relative to a control, the system comprising:(a) a non-naturally occurring peptide comprising a DNA binding domain (DBD) fused to a p300 Recruiter domain (p300R-DBD), the p300R-DBD peptideAttorney Docket No.: 184F-414450-WOselectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more guide RNA (gRNAs).

[0335] 2. A system for increasing expression of a Laminin Subunit Alpha 1 (LAMA1), an Insulin Like Growth Factor 1 (IGF1 ), or an Adiponectin (ADIPOQ) protein in a cell, relative to a control, the system comprising:(a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a LAMA1 , an IGF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

[0336] 3. A lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

[0337] 4. A lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs comprising or consisting of the nucleotide sequence GTCCGAAGGGCCTGCGCACC (SEQ ID NO. 8).

[0338] 5. A method of increasing expression of one or more proteins in a cell, relative to a control, the method comprising the steps of:(i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and(ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.Attorney Docket No.: 184F-414450-WO

[0339] 6. A method of increasing a LAMA1 protein level, an IGF1 protein level, or an ADIPOQ protein level in a cell, relative to a control, the method comprising the steps of:(i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1, an IGF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0340] 7. An engineered cell having increased expression of one or more proteins, relative to a control, the engineered cell generated by the steps of:(I) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and(ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0341] 8. An engineered cell having an increased LAMA1, IGF1, or an ADIPOQ protein level, relative to a control, the engineered cell generated by the steps of:(i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1 , an IGF1 , a or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

[0342] 9. The engineered cell of embodiment 7 or 8, wherein the engineered cell is an engineered mammalian cell.Attorney Docket No.: 184F-414450-WO

[0343] 10. The engineered cell of embodiment 9, wherein the engineered mammalian cell is an engineered human cell or an engineered murine cell.

[0344] 11. The engineered cell of embodiment 10, wherein the engineered human cell or the engineered murine cell is a myoblast.

[0345] 12. The engineered cell of embodiment 10, wherein the engineered mammalian cell is an engineered kidney cell, an engineered liver cell, or an engineered muscle cell.

[0346] 13. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1 -7, wherein the DBD comprises a clustered regularly interspaced short palindromic repeats associated protein (Cas) domain, a zinc finger domain, a helix-turn-helix (HTH) domain, a leucine zipper domain, and a basic helix-loop-helix (bHLH) domain.

[0347] 14. The system, lipid nanoparticle, method, or engineered cell of embodiment 13, wherein the Cas domain comprises an inactive nuclease domain.

[0348] 15. The system, lipid nanoparticle, method, or engineered cell of embodiment 14, wherein the Cas domain comprising an inactive nuclease domain is a dead Cas (dCas) domain.

[0349] 16. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-15, wherein the locus comprises a nucleotide sequence encoding a gene.

[0350] 17. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-15, wherein the locus comprises a nucleotide sequence that is a cis regulatory element of a gene.

[0351] 18. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-15, wherein the locus comprises a nucleotide sequence that is a trans regulatory element of a gene.

[0352] 19. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 16-18, wherein the gene is LAMA1 .

[0353] 20. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 16-18, wherein the gene is selected from the group consisting ofAttorney Docket No.: 184F-414450-WOSMCHD1 , LRIF1 , MATR3, IGF1 , LAMA1 , ADIPOQ, IGF1 , PML, AFP, RPL30, CCND1 , MyoD1 , and LDLR.

[0354] 21 . The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1 -17, wherein the locus comprises or consists of a promoter.

[0355] 22. The system, lipid nanoparticle, method, or engineered cell of embodiment 21 , wherein the promoter is a LAMA1 promoter.

[0356] 23. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 13-22, wherein the Cas domain or the dCas domain comprises a Cas1 domain, a Cas2 domain, a Cas3 domain, a Cas9 domain, a Casio domain, a Cas12a domain, or a Cas13 domain.

[0357] 24. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 13-23, wherein the Cas domain or the dCas domain comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179.

[0358] 25. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-24, wherein the gRNA specifically directs the p300R-DBD peptide to the locus.

[0359] 26. The system, lipid nanoparticle, method, or engineered cell of embodiment 25, wherein the gRNA mediates selective binding of the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-dCas peptide to the locus.

[0360] 27. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1 -26, wherein the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-dCas peptide is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

[0361] 28. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-27, wherein the gRNA is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

[0362] 29. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-26, wherein the one or more gRNAs comprise two or more gRNAs.Attorney Docket No.: 184F-414450-WO

[0363] 30. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-29, wherein the one or more gRNAs comprises a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to one or more of SEQ ID NOs: 2-136.

[0364] 31 . The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-30, wherein the p300R domain comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 1 .

[0365] 32. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-31 , wherein the p300R domain is fused to the DBD domain, the Cas domain, or the dCas domain by a peptide linker.

[0366] 33. The system, lipid nanoparticle, method, or engineered cell of embodiment 32, wherein the peptide linker comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

[0367] 34. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-20, wherein the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands has a reduction in expression of the gene by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the control.

[0368] 35. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-34, wherein the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands is repressed by the methylation.

[0369] 36. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-35, wherein the one or more histones are selected from the group consisting of H2A, H2B, H3, and H4.Attorney Docket No.: 184F-414450-WO

[0370] 37. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-36, wherein the one or more histones are methylated at a lysine (K) residue or an arginine (R) residue.

[0371] 38. The system, lipid nanoparticle, method, or engineered cell of embodiment 37, wherein the lysine residue is a K4, K5, K8, K9, K14, K16, K18, K20, K23, K27, K36, K56, K119, or a K120 residue.

[0372] 39. The system, lipid nanoparticle, method, or engineered cell of embodiment 37, wherein the arginine residue is an R2, R3, R8, R17, R23, or an R26 residue.

[0373] 40. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-39, wherein the one or more CpG islands are located in a promoter region, a 5' untranslated region, or a cis regulatory element of a gene.

[0374] 41 . The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-40, wherein the non-naturally occurring peptide or the gRNA is encoded by a nucleotide sequence comprised within a vector.

[0375] 42. The system, lipid nanoparticle, method, or engineered cell of embodiment 41 , wherein the vector is a viral vector.

[0376] 43. The system, lipid nanoparticle, method, or engineered cell of embodiment 42, wherein the viral vector is a lentiviral vector.

[0377] 44. The system, lipid nanoparticle, method, or engineered cell of embodiment 42 or 43, wherein the vector comprises a detectable marker or a reporter gene.

[0378] 45. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 41-44, wherein the vector comprises an antibiotic-inducible gene expression system.

[0379] 46. The system, lipid nanoparticle, method, or engineered cell of embodiment 45, wherein the antibiotic is doxycycline or tetracycline.

[0380] 47. The system, lipid nanoparticle, method, or engineered cell of embodiment 45 or 46, wherein the antibiotic-inducible gene expression system is a Tet-ON system.Attorney Docket No.: 184F-414450-WO

[0381] 48. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1 -39, wherein the system, the non-naturally occurring peptide, the gRNA, or the vector is present in a delivery vehicle.

[0382] 49. The system, lipid nanoparticle, method, or engineered cell of embodiment 48, wherein the delivery vehicle comprises a nanoparticle.

[0383] 50. The system, lipid nanoparticle, method, or engineered cell of embodiment 49, wherein the nanoparticle is a lipid nanoparticle.

[0384] 51 . A pharmaceutical composition comprising the system or the vector of any one of embodiments 1 or 13-50.

[0385] 52. The pharmaceutical composition of embodiment 51 , wherein the pharmaceutical composition is formulated for delivery to a subject in need thereof.

[0386] 53. The pharmaceutical composition of embodiment 52, wherein the subject is mammalian.

[0387] 54. The pharmaceutical composition of embodiment 53, wherein the subject is human.

[0388] 55. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-50, wherein the methylation level of the one or more histones and / or the DNA methylation level of the one or more CpG islands is altered by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0389] 56. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-50 or 55, wherein the expression level of the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0390] 57. The system, lipid nanoparticle, method, or engineered cell of any one of embodiments 1-50, 55, or 56, wherein an expression level of a gene encoding the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.Attorney Docket No.: 184F-414450-WO

[0391] From the foregoing, it will be appreciated that specific embodiments of the present technology have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the present technology. Accordingly, the present technology is not limited except as by the appended claims.

Claims

Attorney Docket No.: 184F-414450-WOCLAIMS1 . A system for increasing expression of one or more proteins in a cell, relative to a control, the system comprising:(a) a non-naturally occurring peptide comprising a DNA binding domain (DBD) fused to a p300 Recruiter domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more guide RNA (gRNAs).

2. A system for increasing expression of a Laminin Subunit Alpha 1 (LAMA1), an Insulin Like Growth Factor 1 (IGF1), or an Adiponectin (ADIPOQ) protein in a cell, relative to a control, the system comprising:(a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a LAMA1 , an IGF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

3. A lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs.

4. A lipid nanoparticle comprising (a) a non-naturally occurring peptide comprising a DBD fused to a p300R domain (p300R-DBD), the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs comprising or consisting of the nucleotide sequence GTCCGAAGGGCCTGCGCACC (SEQ ID NO. 8).

5. A method of increasing expression of one or more proteins in a cell, relative to a control, the method comprising the steps of:Attorney Docket No.: 184F-414450-WO(i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and(ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

6. A method of increasing a LAMA1 protein level, an IGF1 protein level, or an ADIPOQ protein level in a cell, relative to a control, the method comprising the steps of:(i) introducing, to the cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1, an IGF1 , or an ADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (ii) culturing the cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

7. An engineered cell having increased expression of one or more proteins, relative to a control, the engineered cell generated by the steps of:(i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a p300R-DBD, the p300R-DBD peptide selectively binding to a locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and(ii) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

8. An engineered cell having an increased LAMA1 , IGF1, or an ADIPOQ protein level, relative to a control, the engineered cell generated by the steps of:(i) introducing, to a cell, (a) a non-naturally occurring peptide comprising a P300R-DBD, the p300R-DBD peptide selectively binding to a LAMA1, an IGF1 , or anAttorney Docket No.: 184F-414450-WOADIPOQ locus that is altered by methylation or acetylation of one or more histones and / or DNA methylation of one or more CpG islands and (b) one or more gRNAs; and (II) culturing the engineered cell for a period of time sufficient to alter a methylation level of the one or more histones and / or a DNA methylation level of the one or more CpG islands, relative to the control.

9. The engineered cell of claim 7 or 8, wherein the engineered cell is an engineered mammalian cell.

10. The engineered cell of claim 9, wherein the engineered mammalian cell is an engineered human cell or an engineered murine cell.

11. The engineered cell of claim 10, wherein the engineered human cell or the engineered murine cell is a myoblast.

12. The engineered cell of claim 10, wherein the engineered mammalian cell is an engineered kidney cell, an engineered liver cell, or an engineered muscle cell.

13. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-7, wherein the DBD comprises a clustered regularly interspaced short palindromic repeats associated protein (Gas) domain, a zinc finger domain, a helix-turn-helix (HTH) domain, a leucine zipper domain, and a basic helix-loop-helix (bHLH) domain.

14. The system, lipid nanoparticle, method, or engineered cell of claim 13, wherein the Gas domain comprises an inactive nuclease domain.

15. The system, lipid nanoparticle, method, or engineered cell of claim 14, wherein the Gas domain comprising an inactive nuclease domain is a dead Gas (dCas) domain.

16. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-15, wherein the locus comprises a nucleotide sequence encoding a gene.

17. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-15, wherein the locus comprises a nucleotide sequence that is a cis regulatory element of a gene.Attorney Docket No.: 184F-414450-WO18. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-15, wherein the locus comprises a nucleotide sequence that is a trans regulatory element of a gene.

19. The system, lipid nanoparticle, method, or engineered cell of any one of claims 16-18, wherein the gene is LAMA1.

20. The system, lipid nanoparticle, method, or engineered cell of any one of claims 16-18, wherein the gene is selected from the group consisting of SMCHD1, LRIF1 , MATR3, IGF1 , LAMA1 , ADIPOQ, IGF1 , PML, AFP, RPL30, CCND1 , MyoD1, and LDLR.21 . The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-17, wherein the locus comprises or consists of a promoter.

22. The system, lipid nanoparticle, method, or engineered cell of claim 21, wherein the promoter is a LAMA1 promoter.

23. The system, lipid nanoparticle, method, or engineered cell of any one of claims 13-22, wherein the Gas domain or the dCas domain comprises a Cas1 domain, a Cas2 domain, a Cas3 domain, a Cas9 domain, a Casio domain, a Cas12a domain, or a Cas13 domain.

24. The system, lipid nanoparticle, method, or engineered cell of any one of claims 13-23, wherein the Gas domain or the dCas domain comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 157 or 162-179.

25. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -24, wherein the gRNA specifically directs the p300R-DBD peptide to the locus.

26. The system, lipid nanoparticle, method, or engineered cell of claim 25, wherein the gRNA mediates selective binding of the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-dCas peptide to the locus.

27. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-26, wherein the p300R-DBD peptide, the p300R-Cas peptide, or the p300R-Attorney Docket No.: 184F-414450-WOdCas peptide is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

28. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -27, wherein the gRNA is present in an amount of about 10 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, 25 pg, or 27 pg.

29. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-26, wherein the one or more gRNAs comprise two or more gRNAs.

30. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -29, wherein the one or more gRNAs comprises a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to one or more of SEQ ID NOs: 2-136.31 . The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-30, wherein the p300R domain comprises or consists of an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 1 .

32. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-31 , wherein the p300R domain is fused to the DBD domain, the Gas domain, or the dCas domain by a peptide linker.

33. The system, lipid nanoparticle, method, or engineered cell of claim 32, wherein the peptide linker comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 138-152, 154-156, and GT.

34. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-20, wherein the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands has a reduction in expression of the gene by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the control.Attorney Docket No.: 184F-414450-WO35. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-34, wherein the locus altered by the methylation of the one or more histones and / or by DNA methylation of one or more CpG islands is repressed by the methylation.

36. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-35, wherein the one or more histones are selected from the group consisting of H2A, H2B, H3, and H4.

37. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -36, wherein the one or more histones are methylated at a lysine (K) residue or an arginine (R) residue.

38. The system, lipid nanoparticle, method, or engineered cell of claim 37, wherein the lysine residue is a K4, K5, K8, K9, K14, K16, K18, K20, K23, K27, K36, K56, K119, or a K120 residue.

39. The system, lipid nanoparticle, method, or engineered cell of claim 37, wherein the arginine residue is an R2, R3, R8, R17, R23, or an R26 residue.

40. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-39, wherein the one or more CpG islands are located in a promoter region, a 5' untranslated region, or a cis regulatory element of a gene.41 . The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-40, wherein the non-naturally occurring peptide or the gRNA is encoded by a nucleotide sequence comprised within a vector.

42. The system, lipid nanoparticle, method, or engineered cell of claim 41, wherein the vector is a viral vector.

43. The system, lipid nanoparticle, method, or engineered cell of claim 42, wherein the viral vector is a lentiviral vector.

44. The system, lipid nanoparticle, method, or engineered cell of claim 42 or 43, wherein the vector comprises a detectable marker or a reporter gene.

45. The system, lipid nanoparticle, method, or engineered cell of any one of claims 41-44, wherein the vector comprises an antibiotic-inducible gene expression system.Attorney Docket No.: 184F-414450-WO46. The system, lipid nanoparticle, method, or engineered cell of claim 45, wherein the antibiotic is doxycycline or tetracycline.

47. The system, lipid nanoparticle, method, or engineered cell of claim 45 or 46, wherein the antibiotic-inducible gene expression system is a Tet-ON system.

48. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-40, wherein the system, the non-naturally occurring peptide, the gRNA, or the vector is present in a delivery vehicle.

49. The system, lipid nanoparticle, method, or engineered cell of claim 48, wherein the delivery vehicle comprises a nanoparticle.

50. The system, lipid nanoparticle, method, or engineered cell of claim 49, wherein the nanoparticle is a lipid nanoparticle.51 . A pharmaceutical composition comprising the system or the vector of any one of claims 1 or 13-50.

52. The pharmaceutical composition of claim 51 , wherein the pharmaceutical composition is formulated for delivery to a subject in need thereof.

53. The pharmaceutical composition of claim 52, wherein the subject is mammalian.

54. The pharmaceutical composition of claim 53, wherein the subject is human.

55. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1-50, wherein the methylation level of the one or more histones and / or the DNA methylation level of the one or more CpG islands is altered by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

56. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -50 or 55, wherein the expression level of the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.Attorney Docket No.: 184F-414450-WO57. The system, lipid nanoparticle, method, or engineered cell of any one of claims 1 -50, 55, or 56, wherein an expression level of a gene encoding the one or more proteins is increased by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

Images