Crispr sam biosensor cells and cell lines and methods of use thereof

Abstract

The disclosure provides cells or cell lines that express a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)-based synergistic activation mediator (SAM) complex (CRISPR SAM complex) that include a guide RNA (gRNA) that specifically targets a GJB2 regulatory element (e.g., a GJB2 promoter or enhancer), in which GJB2 is not normally expressed in these cells or cell lines. The disclosure also provides methods of measuring the ability of a vector to transfer a nucleic acid molecule into these cells or cell lines.

Description

PATENT ATTORNEY DOCKET NO.: 51471-019WO2 CRISPR SAM BIOSENSOR CELLS AND CELL LINES AND METHODS OF USE THEREOF Sequence Listing This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 1, 2025, is named “51471-019WO2_Sequence_Listing_10_1_25.xml” and is 228,839 bytes in size. Field of the Invention The present invention is related to cells and cell lines that express a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)-based synergistic activation mediator (SAM) complex (CRISPR SAM complex), which includes a guide RNA (gRNA) that specifically targets a promoter and / or enhancer of a gene, in which the gene is not normally expressed in these cells or cell lines and the complex is capable of inducing expression from cell-type specific regulatory elements (e.g., promoters and / or enhancers) packaged in vectors, particularly viral vectors such as adeno-associated virus (AAV), adenovirus or lentivirus vectors. The present invention also relates to methods of measuring the ability of a vector to transfer nucleic acid molecules into the cells and cell lines of the present invention. Background The use of cell type-specific regulatory elements (e.g., promoters and / or enhancers) in gene therapy provides a higher degree of specificity and results in a safer product. However, there is a lack of available immortalized cell lines that are both easily transduced by common gene therapy viruses such as AAVs and express cell-type specific regulatory elements. In the absence of such cell lines, all in vitro potency assays and validation of vector performance must be done in vivo. Accordingly, there is a need for cells and cell lines that can be used for potency assays and validation of vector performance for vectors containing cell type-specific regulatory elements. Summary of the Invention The invention provides compositions and methods for use in a cell-based potency assay for a gene therapy program, such as a gene therapy program using a GJB2 regulatory element (e.g., a GJB2 promoter or enhancer). The compositions and methods described herein relate to cells and cell lines that express a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)-based synergistic activation mediator (SAM) complex (CRISPR SAM complex), which includes a guide RNA (gRNA) that specifically targets a GJB2 regulatory element, such as a promoter and / or an enhancer. The present invention also relates to methods of measuring the ability of a vector to transfer a nucleic acid molecule (for example, a nucleic acid molecule that encodes GJB2) into the cells and cell lines of the present invention. In a first aspect, the invention provides a cell that expresses a CRISPR SAM complex that includes a chimeric Cas protein with reduced or eliminated nuclease activity, a gRNA that specificallyPATENT ATTORNEY DOCKET NO.: 51471-019WO2 targets a promoter and / or an enhancer of GJB2, wherein the gRNA includes a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102, and a chimeric adaptor protein. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is a HeLa cell. In another aspect, the invention provides a HeLa cell line that expresses a CRISPR SAM complex, in which the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 promoter or enhancer. The CRISPR SAM complex includes a chimeric Cas protein that has reduced or eliminated nuclease activity and a chimeric adaptor protein. The gRNA includes a nucleic acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments of either of the above aspects, the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 promoter. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 enhancer. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 promoter and enhancer. In some embodiments of either of the above aspects, the promoter is a human GJB2 (hGJB2) promoter. In some embodiments of either of the above aspects, the enhancer is an hGJB2 enhancer. In some embodiments of either of the above aspects, the gRNA includes a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO: 84. In some embodiments of either of the above aspects, the chimeric Cas protein with reduced or eliminated nuclease activity includes a dead Cas9 (dCas9) protein. In some embodiments, the chimeric dCas9 protein has a nuclease activity that is reduced or eliminated by at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to a wild-type Cas protein. In some embodiments, the chimeric dCas9 protein with reduced or eliminated nuclease activity is fused to one or more transcriptional activation domains. In some embodiments, the one or more transcriptional activation domains in the CRISPR SAM complex are different from each other. In some embodiments of either of the above aspects, the gRNA is a single gRNA (sgRNA). In some embodiments of either of the above aspects, the chimeric dCas9 protein with reduced or eliminated nuclease activity is a dCas9-VP64 fusion protein, in which the dCas9 protein is fused to the transcriptional activator VP64. In some embodiments, the gRNA further includes an MS2 bacteriophage protein (MS2) aptamer. In some embodiments, the gRNA includes two MS2 RNA aptamers. In some embodiments, the chimeric adaptor protein includes an MS2 coat protein (MCP) fused to a NF-κB trans- activating subunit p65 and an activation domain of Heat Shock Factor 1 (MCP-p65-HSF1). In another aspect, the invention provides a gRNA including a nucleic acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleicPATENT ATTORNEY DOCKET NO.: 51471-019WO2 acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO: 84. In another aspect, the invention provides a method of measuring the ability of a vector to transfer a nucleic acid molecule into a cell including the steps of: a) introducing the nucleic acid molecule using the vector into the cell of the first aspect, in which the nucleic acid molecule encodes a gene, or a fragment thereof operably linked to the GJB2 promoter or enhancer that binds the gRNA expressed by the cell; and b) measuring the expression of the gene. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the vector is a lipid nanoparticle. In some embodiments, the gene is a reporter gene. In some embodiments, the reporter gene is selected from the group consisting of: a gene encoding beta-galactosidase, a bacterial chloramphenicol acetyltransferase gene, a firefly luciferase gene, a gene encoding beta-glucuronidase, and a gene encoding a fluorescent protein. In some embodiments, the reporter gene encodes an enhanced green fluorescent protein (eGFP). In some embodiments, the gene is GJB2. Definitions The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure. Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (--NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (--COOH). The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers including purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases. Nucleic acids are said to have “5' ends” and “3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circularPATENT ATTORNEY DOCKET NO.: 51471-019WO2 DNA molecule, discrete elements are referred to as being “upstream” or 5' of the “downstream” or 3' elements. As used herein, a “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term “vector” includes an autonomously replicating plasmid or a virus. “Vector” may also include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds liposomes, lipid nanoparticles, non-lipid nanoparticles, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. Preferably, the vector is an AAV vector or a lentiviral vector. The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted, and other elements added without sacrificing the necessary expression. The term “targeting vector” refers to a recombinant nucleic acid that can be introduced by homologous recombination, non-homologous-end-joining-mediated ligation, or any other means of recombination to a target position in the genome of a cell. The term “wild type” or “wild-type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles). The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal). “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions. The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein includes at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein includes two or more sub-sequencesPATENT ATTORNEY DOCKET NO.: 51471-019WO2 that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can include a heterologous label or a heterologous secretion or localization sequence. The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome of an organism. For example, a “GJB2 locus” may refer to the specific location of a GJB2 gene, GJB2 DNA sequence, GJB2- encoding sequence, or GJB2 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. A “GJB2 locus” may include a regulatory element of a GJB2 gene, including, for example, an enhancer, a promoter, 5' and / or 3' untranslated region (UTR), or a combination thereof. The term “gene” refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product) and includes the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the full-length mRNA (including the 5' and 3' untranslated sequences). The term “gene” also includes other non-coding sequences including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions. These sequences may be close to the coding region of the gene (e.g., within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene. As used herein, the term “regulatory element” refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Regulatory elements or transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990). As used herein, the term “enhancer” refers to a class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Many enhancer sequences are now known from mammalian genes. Enhancers are polynucleotides that can be operably linked to a promoter to regulate gene expression. The enhancer described herein can be operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product to increase the expression level of the expression product.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 A “promoter” is a regulatory region of DNA usually including a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally include other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell- specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes. Promoters used in AAV vectors include, for example, an AAV p5 promoter. Promoters include, but are not limited to, GJB2, CAG, SYN1, CMV, NSE, CBA, PDGF, SV40, RSV, LTR, SV40, dihydrofolate reductase promoter, beta-actin promoter, PGK, EF1alpha, GRK, MT, MMTV, TY, RU486, RHO, RHOK, CBA, chimeric CMV-CBA, MLP, RSV, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, functional fragments thereof, etc. In AAV packaged with heterologous DNA, a promoter normally associated with heterologous nucleic acid can be used, or a promoter normally associated with the AAV vector, or a promoter not normally associated with either, can be used. A constitutive promoter is one that is active in all tissues or particular tissues at all developing stages. Examples of constitutive promoters include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, functional fragments thereof, or combinations thereof. Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet- Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example, temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter). Tissue-specific promoters can be, for example, neuron-specific promoters, glia-specific promoters, muscle cell-specific promoters, heart cell-specific promoters, kidney cell-specific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell- specific promoters (e.g., a B cell promoter or a T cell promoter). Developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 As used herein, the term “GJB2-expressing inner ear cell” refers to a cell within the inner ear that endogenously expresses GJB2. GJB2-expressing cells within the ear are found in both the cochlea and the vestibule. Cochlear GJB2-expressing cells include inner phalangeal cells, inner border cells, inner pillar cells, outer pillar cells, Deiter cells, Hensen’s cells, Claudius cells, interdental cells, inner sulcus cells, outer sulcus cells, cells of the spiral limbus, spiral prominence cells, root cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, fibrocytes of the spiral limbus and spiral ligament, and mesenchymal cells lining the scala vestibuli. Vestibular GJB2-expressing cells include supporting cells, dark cells, fibrocytes, and mesenchymal cells. “Operable linkage” or being “operably linked” or “under transcriptional control” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence). “Complementarity” of nucleic acids means that a nucleotide sequence in one strand of nucleic acid, due to orientation of its nucleobase groups, forms hydrogen bonds with another sequence on an opposing nucleic acid strand. The complementary bases in DNA are typically A with T and C with G. In RNA, they are typically C with G and U with A. Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. “Substantial” or “sufficient” complementary means that a sequence in one strand is not completely and / or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm (melting temperature) of hybridized strands, or by empirical determination of Tm by using routine methods. Tm includes the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured (i.e., a population of double-stranded nucleic acid molecules becomes half dissociated into single strands). At a temperature below the Tm, formation of a hybridization complex is favored, whereas at a temperature above the Tm, melting or separation of the strands in the hybridization complex is favored. Tm may be estimated for a nucleic acid having a known G+C content in an aqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41(% G+C), although other known Tm computations consider nucleic acid structural characteristics. Hybridization condition includes the cumulative environment in which one nucleic acid strand bonds to a second nucleic acid strand by complementary strand interactions and hydrogen bonding to produce a hybridization complex. Such conditions include the chemical components and their concentrations (e.g., salts, chelating agents, formamide) of an aqueous or organic solution containing the nucleic acids, and the temperature of the mixture. Other factors, such as the length of incubation time orPATENT ATTORNEY DOCKET NO.: 51471-019WO2 reaction chamber dimensions may contribute to the environment. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2.sup.nd ed., pp.1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), herein incorporated by reference in its entirety for all purposes. Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables which are well known. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides) the position of mismatches becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Illustrative minimum lengths for a hybridizable nucleic acid include at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. Furthermore, the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. The sequence of a polynucleotide disclosed herein need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). A polynucleotide (e.g., gRNA) can include at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, a gRNA in which 18 of 20 nucleotides are complementary to a target region, and would therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al. (1990) J. Mol. Biol.215(3):403-410; Zhang and Madden (1997) Genome Res.7(6):649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (1981) Adv. Appl. Math.2(4):482-489. The methods and compositions provided herein employ a variety of different components. Some components throughout the present disclosure can have active variants and fragments. Such components include, for example, Cas proteins, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNAs, tracrRNAs, and guide RNAs (gRNAs). Biological activity for each of these components is described herein. The term “functional” refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. Such biological activitiesPATENT ATTORNEY DOCKET NO.: 51471-019WO2 or functions can include, for example, the ability of a Cas protein to bind to a gRNA and to a target DNA sequence. The biological functions of functional fragments or variants may be the same or may be changed (e.g., with respect to their specificity or selectivity or efficacy) in comparison to the original molecule, but with retention of the molecule's basic biological function. The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid). The term “fragment,” when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment,” when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, when referring to a protein fragment, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminal and C-terminal ends of the protein). A fragment can be, for example, when referring to a nucleic acid fragment, a 5' fragment (i.e., removal of a portion of the 3' end of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' end of the nucleic acid), or an internal fragment (i.e., removal of a portion each of the 5' and 3' ends of the nucleic acid). “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif). “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including anyPATENT ATTORNEY DOCKET NO.: 51471-019WO2 algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. As one of skill in the art would understand, as used herein, for the purpose of determining “percent sequence identity”, a uridine nucleoside in an RNA molecule is considered equivalent to a thymidine nucleoside in a DNA molecule. Therefore, an RNA equivalent may be considered to have 100% sequence identity to a DNA polynucleotide if the RNA equivalent and DNA polynucleotide differ from one another only by the substitution of uridine nucleosides in the RNA equivalent with thymidine nucleosides in the DNA polynucleotide. A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution. The term “in vitro” includes artificial environments and processes or reactions that occur within an artificial environment (e.g., a test tube or in isolated cell or cell line). The term “in vivo” includes natural environments (e.g., a cell or organism or body) and processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells. The term “reporter gene” refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantifiably assayed when a construct containing the reporter gene sequence operably linked to an endogenous or heterologous promoter and / or enhancer element is introduced into cells containing (or which can be made to contain) the factors necessary for the activationPATENT ATTORNEY DOCKET NO.: 51471-019WO2 of the promoter and / or enhancer elements. Examples of reporter genes include, but are not limited, to genes encoding green fluorescent protein (GFP)¸ enhanced green fluorescent protein (eGFP), beta- galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) genes, firefly luciferase genes, genes encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. A “reporter protein” refers to a protein encoded by a reporter gene. The term “fluorescent reporter protein” as used herein means a reporter protein that is detectable based on fluorescence in which the fluorescence may be either from the reporter protein directly, activity of the reporter protein on a fluorogenic substrate, or a protein with affinity for binding to a fluorescent tagged compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, eGFP, GFP-2, tagGFP, turboGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, and ZsGreen1), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellow1), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, and T- sapphire), cyan fluorescent proteins (e.g., CFP, eCFP, Cerulean, CyPet, AmCyan1, and Midoriishi-Cyan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed- Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, and tdTomato), and any other suitable fluorescent protein whose presence in cells can be detected by flow cytometry methods. Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of'' means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of'' when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.” “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not. Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value. The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The term “or” refers to any one member of a particular list and also includes any combination of members of that list.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof. Unless otherwise indicated, statistically significant means p<0.05. As used herein, “treatment” refers to any delivery, administration, or application of a therapeutic for a disease or condition. Treatment may include curing the disease, inhibiting the disease, slowing or stopping the development of the disease, ameliorating one or more symptoms of the disease, or reducing the likelihood of or preventing the recurrence of one or more symptoms of the disease. As used herein, “AAV” refers to an adeno-associated virus. AAV is a non-enveloped virus that is icosahedral, is about 20 to 24 nm long with a density of about 1.40-1.41 g / cc and contains a single stranded linear genomic DNA molecule approximately 4.7 kb in length. The single stranded AAV genomic DNA can be either a plus strand, or a minus strand. In certain embodiments, the term “AAV” or “AAV vector” refers to an AAV that has been modified so that a therapeutic, such as for example, a CRISPR complex, replaces the Rep and Cap open reading frames between the inverted terminal repeats (ITRs) of the AAV genome. As used herein, “AAV serotype” means a sub-division of AAV that is identifiable by serologic or DNA sequencing methods and can be distinguished by its antigenic character. As used herein, “RNA” refers to a molecule including one or more ribonucleotide residues. A “ribonucleotide” is a nucleotide with a hydroxyl group at the 2' position of the beta-D-ribofuranose moiety. The term “RNA” includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g. partially purified RNA), essentially pure RNA, synthetic RNA, and recombinantly produced RNA. The term “RNA” also refers to modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. As used herein, a “stable expression” of a transfected or transduced gene in a host cell means the integration of the gene in the genome of the host cell and as a result, is able to express the transfected genetic material. As used herein, “gene editing” and “nucleic acid editing” refers to modification or modulation of the nucleic acid sequence of a target gene. Gene editing or nucleic acid editing may be modulation of DNA or RNA expression or translation. As used herein, “nucleic acid editing system” and “gene editing system” refers to a method that can be used for performing gene editing or nucleic acid editing. Nucleic acid editing systems and gene editing systems include CRISPR systems and interfering RNAs. As used herein, “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated (Cas)-based synergistic activation mediator (SAM) complex (CRISPR SAM complex)” refers to a complex including a chimeric Cas protein with reduced or eliminated nuclease activity, a gRNA that specifically targets a promoter and / or an enhancer of a gene (e.g., promoter and / or an enhancer of GJB2), and a chimeric adaptor protein. In some embodiments, the chimeric Cas protein with reduced or eliminated nuclease activity includes a dead Cas9 (dCas9) protein. In some embodiments, the chimericPATENT ATTORNEY DOCKET NO.: 51471-019WO2 dCas9 protein with reduced or eliminated nuclease activity is a dCas9-VP64 fusion protein, in which the dCas9 protein is fused to the transcriptional activator VP64. In some embodiments, the gRNA further includes an MS2 bacteriophage protein (MS2) aptamer. In some embodiments, the chimeric adaptor protein includes an MS2 coat protein (MCP) fused to a NF-κB trans-activating subunit p65 and an activation domain of Heat Shock Factor 1 (MCP-p65-HSF1). Brief Description of the Drawings FIG.1 is a bar graph showing relative fold change of eGFP expression for several gRNAs as compared to an un-transfected control reporter cell line in which GFP is under the control of a GJB2 regulatory element (SEQ ID NO: 144). The relative fold change was used to identify active gRNAs. The arrows above the bars indicate guides selected for further study. n=4 replicate wells. FIG.2 is a bar graph showing expression of human GJB2 under the control of the GJB2 regulatory element of SEQ ID NO: 144 in an AAV1 vector in gRNA clones. The relative fold change of hGJB2 expression in HeLa SAM gRNA clones as compared to HeLa SAM D73 infected cells is shown in the graph. Ordinary one-way ANOVA, p<0.0001 with Dunnett's multiple comparisons test was used to statically analyze the data in this experiment. n=3 replicate wells. FIG.3 includes a set of graphs showing sensitivity of the expression of human GJB2 under the control of the GJB2 regulatory element of SEQ ID NO: 144 in an AAV1 vector in gRNA clones. The relative fold change of hGJB2 expression in HeLa SAM gRNA_1 and gRNA_23 clones at multiple multiplicities of infection (MOIs); 2x106, 1x106, 5x105, 2.5x105, 1.25x105, 6.25x104, 3.13x104, and 0 vg / cell is shown in the graphs. The graph on the left shows the relative fold change of hGJB2 expression in HeLa SAM gRNA_1 clones. The graph on the right shows the relative fold change of hGJB2 expression in HeLa SAM gRNA_23 clones. n=4 replicate wells. FIG.4 is a map of the full pLenti gRNA plasmid including gRNA23. FIG.5 is a graph showing sensitivity of the mRNA expression of human GJB2 under the control of the GJB2 regulatory element of SEQ ID NO: 144 in an AAV1 vector in gRNA_23 clone G31. The relative fold change of GJB2 mRNA expression is shown at multiple MOIs over the range of 1.5 × 10⁴ to 1.5 × 10⁶ MOI. Error bars represent the standard deviation of n=3. FIG.6 is a graph showing sensitivity of the protein expression of human GJB2 under the control of the GJB2 regulatory element of SEQ ID NO: 144 in an AAV1 vector in gRNA_23 clone G31. Mean fluorescence intensity (MFI) is shown at multiple MOIs over the range of 4 × 10⁴ to 1 × 10⁷ MOI. Open circles and closed circles represent the presence or absence of 250 nM etoposide, respectively. Error bars represent the standard deviation of n=3. Detailed Description Described herein are compositions and methods for use in a cell-based potency assay for a gene therapy program, such as a gene therapy program using a GJB2 regulatory element. The present disclosure features a cell-based potency assay developed for a GJB2 gene therapy program that uses an engineered cell type-specific regulatory element containing a GJB2 promoter and enhancer. ThePATENT ATTORNEY DOCKET NO.: 51471-019WO2 compositions and methods described herein relate to cells and cell lines that express a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)-based synergistic activation mediator (SAM) complex (CRISPR SAM complex), which includes a guide RNA (gRNA) that specifically targets GJB2 regulatory elements such as a promoter and / or an enhancer. The GJB2 gene is not normally expressed in these cells or cell lines expressing the CRISPR SAM complex, and the complex is capable of inducing expression from cell-type specific regulatory elements (e.g., promoters and / or enhancers) packaged in vectors, particularly viral vectors, such as adeno-associated virus (AAV), adenovirus, or lentivirus vectors. The present disclosure also relates to methods of measuring the ability of a vector to transfer a nucleic acid molecule (for example, a nucleic acid molecule that encodes GJB2) into the cells and cell lines of the present disclosure. CRISPR SAM Complex The cells or cell lines disclosed herein use transfected CRISPR SAM complexes for use in in vitro testing of vector performance, in which the vector transfers a nucleic acid molecule that codes for a gene activated by a promoter and / or enhancer that binds the gRNA expressed by the cell or cell line. The CRISPR SAM complex described herein includes, for example, chimeric Cas proteins with reduced or eliminated nuclease activity, chimeric adaptor proteins, and gRNAs as described herein to activate transcription of target genes. Chimeric Cas proteins (e.g., chimeric Cas proteins, such as chimeric Cas9 proteins, such as a chimeric Streptococcus pyogenes Cas9 protein, a chimeric Campylobacter jejuni Cas9 protein, or a chimeric Staphylococcus aureus Cas9 protein (e.g., a chimeric Cas9 protein derived from a Streptococcus pyogenes Cas9 protein, a Campylobacter jejuni Cas9 protein, or a Staphylococcus aureus Cas9 protein)) and chimeric adaptor proteins (e.g., including an adaptor protein that specifically binds to an adaptor-binding element within a gRNA and one or more heterologous transcriptional activation domains) are described in further detail herein. In one example for the preparation of the cell lines of the present disclosure, the chimeric Cas protein and the chimeric adaptor protein are delivered in a single multicistronic or bicistronic nucleic acid (e.g., DNA or mRNA) (referred to as SAM cassette or SAM mRNA). For example, the sequence encoding the chimeric Cas protein and the sequence encoding the chimeric adaptor protein can be linked by a sequence encoding a 2A protein as described in more detail herein. In a specific example, the chimeric Cas protein (e.g., NLS-Cas9-NLS-VP64 in which, for example, the 5' NLS is monopartite and the 3' NLS is bipartite) can be provided as a multicistronic or bicistronic mRNA (e.g., in vitro transcribed mRNA) that also encodes a chimeric adaptor protein (e.g., MS2(MCP)-NLS-p65-HSF1). The nucleic acids encoding the chimeric Cas protein and the chimeric adaptor protein can be linked by a nucleic acid encoding a 2A protein. As one example, the mRNA can include from 5' to 3': NLS-Cas9-NLS-VP64-2A-MS2(MCP)-NLS- p65-HSF1. The mRNA can be capped at the 5' end (e.g., a cap 1 structure in which the +1 ribonucleotide is methylated at the 2'O position of the ribose), can be polyadenylated (poly(A) tail), and can optionally also be modified to be fully substituted with pseudouridine. CRISPR SAM complexes include transcripts and other elements involved in the expression of, or directing the activity of, Cas genes. A CRISPR SAM complex can be, for example, a type I, a type II, aPATENT ATTORNEY DOCKET NO.: 51471-019WO2 type III system, or a type V system (e.g., subtype V-A or subtype V-B). CRISPR SAM complexes used in the cell lines of the present disclosure can be non-naturally occurring. A “non-naturally occurring” system includes anything indicating the involvement of the hand of man, such as one or more components of the system being altered or mutated from their naturally occurring state, being at least substantially free from at least one other component with which they are naturally associated in nature or being associated with at least one other component with which they are not naturally associated. For example, some CRISPR SAM complexes employ a gRNA and a Cas protein that do not naturally occur together, employ a Cas protein that does not occur naturally, or employ a gRNA that does not occur naturally. In one embodiment, the methods and compositions disclosed herein employ the CRISPR SAM complexes that are expressed in the cell lines of the present disclosure by using or testing the ability of CRISPR SAM complexes (including a gRNA complexed with a chimeric Cas protein and a chimeric adaptor protein) to induce transcriptional activation of a target gene transduced using a viral vector such as an AAV, adenovirus, or lentivirus. Chimeric Cas Proteins Provided herein are chimeric Cas proteins with reduced or eliminated nuclease activity that can bind to the gRNAs disclosed herein to activate transcription of target genes. Such chimeric Cas proteins can include: (a) a DNA-binding domain that is a CRISPR-associated (Cas) protein or a functional fragment or variant thereof that is capable of forming a complex with a gRNA and binding to a target sequence; and (b) one or more transcriptional activation domains or functional fragments or variants thereof. For example, such fusion proteins can include 1, 2, 3, 4, 5, or more transcriptional activation domains (e.g., two or more heterologous transcriptional activation domains or three or more heterologous transcriptional activation domains). In one example, the chimeric Cas protein can include a catalytically inactive Cas protein (e.g., dCas9) and a VP64 transcriptional activation domain or a functional fragment or variant thereof. For example, such a chimeric Cas protein can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dCas9-VP64 chimeric Cas protein sequence set forth in SEQ ID NO: 1. However, chimeric Cas proteins in which the transcriptional activation domains include other transcriptional activation domains or functional fragments or variants thereof and / or in which the Cas protein includes other Cas proteins (e.g., catalytically inactive Cas proteins) are also provided. Examples of other suitable transcriptional activation domains are provided herein. SEQ ID NO: 1 is shown below: MKRPAATKKAGQAKKKKDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKPATENT ATTORNEY DOCKET NO.: 51471-019WO2 IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSGRADALDDFDLDMLGS DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINCT (SEQ ID NO: 1). The transcriptional activation domain(s) can be located at the N-terminus, the C-terminus, or anywhere within the Cas protein. For example, the transcriptional activation domain(s) can be attached to the Rec1 domain, the Rec2 domain, the HNH domain, or the PI domain of a Streptococcus pyogenes Cas9 protein or any corresponding region of an orthologous Cas9 protein or homologous or orthologous Cas protein when optimally aligned with the S. pyogenes Cas9 protein. For example, the transcriptional activation domain can be attached to the Rec1 domain at position 553, the Rec1 domain at position 575, the Rec2 domain at any position within positions 175-306 or replacing part of or the entire region within positions 175-306, the HNH domain at any position within positions 715-901 or replacing part of or the entire region within positions 715-901, or the PI domain at position 1153 of the S. pyogenes Cas9 protein. See, e.g., WO 2016 / 049258, herein incorporated by reference in its entirety for all purposes. The transcriptional activation domain may be flanked by one or more linkers on one or both sides as described herein. Chimeric Cas proteins can also be operably linked or fused to additional heterologous polypeptides. The fused or linked heterologous polypeptide can be located at the N-terminus, the C- terminus, or anywhere internally within the chimeric Cas protein. For example, a chimeric Cas protein can further include a nuclear localization signal. Examples of suitable nuclear localization signals and other modifications to Cas proteins are described in further detail herein. Chimeric Cas proteins can be provided in in the form of DNA encoding the chimeric Cas protein. Optionally, the nucleic acid encoding the chimeric Cas protein can be codon-optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid encoding the chimeric Cas protein can be modified to substitute codons having a higher frequency of usage in a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 a non-human mammalian cell, a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the chimeric Cas protein is introduced into the cell, the chimeric Cas protein can be transiently, conditionally, or constitutively expressed in the cell. Chimeric Cas proteins provided as mRNAs can be modified for improved stability and / or immunogenicity properties. The modifications may be made to one or more nucleosides within the mRNA. Examples of chemical modifications to mRNA nucleobases include pseudouridine, 1-methyl- pseudouridine, and 5-methyl-cytidine. mRNA encoding chimeric Cas proteins can also be capped. The cap can be, for example, a cap 1 structure in which the +1 ribonucleotide is methylated at the 2'O position of the ribose. The capping can, for example, give superior activity in vivo (e.g., by mimicking a natural cap), can result in a natural structure that reduces stimulation of the innate immune system of the host (e.g., can reduce activation of pattern recognition receptors in the innate immune system). mRNA encoding chimeric Cas proteins can also be polyadenylated (to include a poly(A) tail). mRNA encoding chimeric Cas proteins can also be modified to include pseudouridine (e.g., can be fully substituted with pseudouridine). For example, capped and polyadenylated chimeric Cas mRNA containing N1-methyl pseudouridine can be used. Likewise, chimeric Cas mRNAs can be modified by depletion of uridine using synonymous codons. Other possible modifications are described in more detail herein. Chimeric Cas mRNAs can include a modified uridine at at least one, a plurality of, or all uridine positions. The modified uridine can be a uridine modified at the 5 position (e.g., with a halogen, methyl, or ethyl). The modified uridine can be a pseudouridine modified at the 1 position (e.g., with a halogen, methyl, or ethyl). The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5- methoxyuridine, 5-iodouridine, or a combination thereof. In some examples, the modified uridine is 5- methoxyuridine. In some examples, the modified uridine is 5-iodouridine. In some examples, the modified uridine is pseudouridine. In some examples, the modified uridine is N1-methyl-pseudouridine. In some examples, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some examples, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some examples, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some examples, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some examples, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some examples, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine. Chimeric Cas mRNAs disclosed herein can also include a 5' cap, such as a Cap0, Cap1, or Cap2. A 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, e.g., with respect to ARCA) linked through a 5'-triphosphate to the 5' position of the first nucleotide of the 5'-to-3' chain of the mRNA (i.e., the first cap-proximal nucleotide). In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA include a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'- methoxy. See, e.g., Katibah et al. (2014) Proc. Natl. Acad. Sci. U.S.A.111(33):12025-30 and Abbas et al. (2017) Proc. Natl. Acad. Sci. U.S.A.114(11):E2106-E2115, each of which is herein incorporated byPATENT ATTORNEY DOCKET NO.: 51471-019WO2 reference in its entirety for all purposes. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, include Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as non-self by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA. A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog including a 7-methylguanine 3'-methoxy-5'- triphosphate linked to the 5' position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap in which the 2' position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al. (2001) RNA 7:1486-1495, herein incorporated by reference in its entirety for all purposes. CleanCap.TM. AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap.TM. GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally.3'-O- methylated versions of CleanCap.TM. AG and CleanCap.TM. GG are also available from TriLinkBiotechnologies as Cat. Nos. N-7413 and N- 7433, respectively. Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo and Moss (1990) Proc. Natl. Acad. Sci. U.S.A.87:4023-4027 and Mao and Shuman (1994) J. Biol. Chem.269:24472-24479, each of which is herein incorporated by reference in its entirety for all purposes. Chimeric Cas mRNAs can further include a poly-adenylated (poly-A) tail. The poly-A tail can, for example, include at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenines, and optionally up to 300 adenines. For example, the poly-A tail can include 95, 96, 97, 98, 99, or 100 adenine nucleotides. Nucleic acids encoding chimeric Cas proteins can be integrated into the genome of a cell and operably linked to a promoter active in the cell. Alternatively, nucleic acids encoding chimeric Cas proteins can be operably linked to a promoter in an expression construct. Expression constructs include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a chimeric Cas gene) and which can transfer such a nucleic acid sequence of interest to a target cell. For example, the nucleic acid encoding the chimeric Cas protein can be in a vector including a DNA encoding a gRNA. Alternatively, it can be in a vector or plasmid that is separate from the vector including the DNA encoding the gRNA. Promoters that can be used in an expression construct include promoters active, for example, in one or more of a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non- human cell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, an embryonic stem (ES) cell, an adultPATENT ATTORNEY DOCKET NO.: 51471-019WO2 stem cell, a developmentally restricted progenitor cell, an induced pluripotent stem (iPS) cell, or a one-cell stage embryo. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Optionally, the promoter can be a bidirectional promoter driving expression of both a chimeric Cas protein in one direction and a gRNA in the other direction. Such bidirectional promoters can consist of (1) a complete, conventional, unidirectional Pol III promoter that contains 3 external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basic Pol III promoter that includes a PSE and a TATA box fused to the 5' terminus of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and the promoter can be rendered bidirectional by creating a hybrid promoter in which transcription in the reverse direction is controlled by appending a PSE and TATA box derived from the U6 promoter. See, e.g., US 2016 / 0074535, herein incorporated by references in its entirety for all purposes. Use of a bidirectional promoter to express genes encoding a chimeric Cas protein and a gRNA simultaneously allow for the generation of compact expression cassettes to facilitate delivery. Cas Proteins Cas proteins generally include at least one RNA recognition or binding domain that can interact with gRNAs. A functional fragment or functional variant of a Cas protein is one that retains the ability to form a complex with a gRNA and to bind to a target sequence in a target gene (and, for example, activate transcription of the target gene). In addition to transcriptional activation domains as described herein, Cas proteins can also include nuclease domains (e.g., DNase domains or RNase domains), DNA-binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Some such domains (e.g., DNase domains) can be from a native Cas protein. Other such domains can be added to make a modified Cas protein. A nuclease domain possesses catalytic activity for nucleic acid cleavage, which includes the breakage of the covalent bonds of a nucleic acid molecule. Cleavage can produce blunt ends or staggered ends, and it can be single-stranded or double-stranded. For example, a wild type Cas9 protein will typically create a blunt cleavage product. Alternatively, a wild type Cpf1 protein (e.g., FnCpf1) can result in a cleavage product with a 5-nucleotide 5' overhang, with the cleavage occurring after the 18th base pair from the PAM sequence on the non-targeted strand and after the 23rd base on the targeted strand. A Cas protein can have full cleavage activity to create a double-strand break at a target genomic locus (e.g., a double-strand break with blunt ends), or it can be a nickase that creates a single-strand break at a target genomic locus. In one example, the Cas protein portions of the chimeric Cas proteins disclosed herein have been modified to have decreased nuclease activity (e.g., nuclease activity is diminished by at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to a wild type Cas protein) or to lack substantially all nuclease activity (i.e., nuclease activity is diminished by at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to a wild type Cas protein, or having no more than about 0%, 1%, 2%, 3%, 5%, or 10% of the nuclease activity of a wild type Cas protein). A nuclease-inactive Cas protein is a Cas protein having mutations known to be inactivatingPATENT ATTORNEY DOCKET NO.: 51471-019WO2 mutations in its catalytic (i.e., nuclease) domains (e.g., inactivating mutations in a RuvC-like endonuclease domain in a Cpf1 protein, or inactivating mutations in both an HNH endonuclease domain and a RuvC-like endonuclease domain in Cas9) or a Cas protein having nuclease activity diminished by at least about 97%, 98%, 99%, or 100% compared to a wild type Cas protein. Examples of different Cas protein mutations to reduce or substantially eliminate nuclease activity are disclosed below. Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. The Cas protein may be, e.g., a Cas9 protein or a protein derived from a Cas9 protein. Cas9 proteins are from a type II CRISPR / Cas system and typically share four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. Cas9 proteins may be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Additional examples of the Cas9 family members are described in WO 2014 / 131833, herein incorporated by reference in its entirety for all purposes. Cas9 from S. pyogenes (SpCas9) (assigned SwissProt accession number Q99ZW2) is an example Cas9 protein. Cas9 from S. aureus (SaCas9) (assigned UniProt accession number J7RUA5) is another example Cas9 protein. Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProt accession number Q0P897) is another example Cas9 protein. See, e.g., Kim et al. (2017) Nat. Commun.8:14500, herein incorporated by reference in its entirety for all purposes. SaCas9 is smaller than SpCas9, and CjCas9 is smaller than both SaCas9 and SpCas9. Cas9 from Neisseria meningitidis (Nme2Cas9) is another example Cas9 protein. See, e.g., Edraki et al. (2019) Mol. Cell 73(4):714-726, herein incorporated by reference in its entirety for all purposes. Cas9 proteins from Streptococcus thermophilus (e.g., Streptococcus thermophilus LMD-9 Cas9 encoded by the CRISPR1 locus (St1Cas9) or Streptococcus thermophilus Cas9 from the CRISPR3PATENT ATTORNEY DOCKET NO.: 51471-019WO2 locus (St3Cas9)) are other example Cas9 proteins. Cas9 from Francisella novicida (FnCas9) or the RHA Francisella novicida Cas9 variant that recognizes an alternative PAM (E1369R / E1449H / R1556A substitutions) are other example Cas9 proteins. These and other example Cas9 proteins are reviewed, e.g., in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, herein incorporated by reference in its entirety for all purposes. Examples of Cas9 coding sequences, Cas9 mRNAs, and Cas9 protein sequences are provided in WO 2013 / 176772, WO 2014 / 065596, WO 2016 / 106121, and WO 2019 / 067910, each of which is herein incorporated by reference in its entirety for all purposes. Specific examples of ORFs and Cas9 amino acid sequences are provided in Table 30 at paragraph

[0449] of WO 2019 / 067910, and specific examples of Cas9 mRNAs and ORFs are provided in paragraphs

[0214] -

[0234] of WO 2019 / 067910. Another example of a Cas protein is a Cpf1 (CRISPR from Prevotella and Francisella 1) protein. Cpf1 is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 along with a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacks the HNH nuclease domain that is present in Cas9 proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9 where it contains long inserts including the HNH domain. See, e.g., Zetsche et al. (2015) Cell 163(3):759-771, herein incorporated by reference in its entirety for all purposes. Cpf1 proteins may be, e.g., from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC20171, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 _GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae. Cpf1 from Francisella novicida U112 (FnCpf1; assigned UniProt accession number A0Q7Q2) is an example Cpf1 protein. Cas proteins can be wild type proteins (i.e., those that occur in nature), modified Cas proteins (i.e., Cas protein variants), or fragments of wild type or modified Cas proteins. Cas proteins can also be active variants or fragments with respect to catalytic activity of wild type or modified Cas proteins. Active variants or fragments with respect to catalytic activity can include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the wild type or modified Cas protein or a portion thereof, in which the active variants retain the ability to cut at a desired cleavage site and hence retain nick-inducing or double-strand-break-inducing activity. Assays for nick-inducing or double-strand-break-inducing activity are known and generally measure the overall activity and specificity of the Cas protein on DNA substrates containing the cleavage site. One example of a modified Cas protein is the modified SpCas9-HF1 protein, which is a high- fidelity variant of Streptococcus pyogenes Cas9 harboring alterations (N497A / R661A / Q695A / Q926A) designed to reduce non-specific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature 529(7587):490- 495, herein incorporated by reference in its entirety for all purposes. Another example of a modified Cas protein is the modified eSpCas9 variant (K848A / K1003A / R1060A) designed to reduce off-target effects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88, herein incorporated by reference in itsPATENT ATTORNEY DOCKET NO.: 51471-019WO2 entirety for all purposes. Other SpCas9 variants include K855A and K810A / K1003A / R1060A. These and other modified Cas proteins are reviewed, e.g., in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, herein incorporated by reference in its entirety for all purposes. Another example of a modified Cas9 protein is xCas9, which is a SpCas9 variant that can recognize an expanded range of PAM sequences. See, e.g., Hu et al. (2018) Nature 556:57-63, herein incorporated by reference in its entirety for all purposes. Cas proteins can be modified to increase or decrease one or more of nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of or a property of the Cas protein. Cas proteins can include at least one nuclease domain, such as a DNase domain. For example, a wild type Cpf1 protein generally includes a RuvC-like domain that cleaves both strands of target DNA, perhaps in a dimeric configuration. Cas proteins can also include at least two nuclease domains, such as DNase domains. For example, a wild type Cas9 protein generally includes a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cut a different strand of double-stranded DNA to make a double- stranded break in the DNA. See, e.g., Jinek et al. (2012) Science 337(6096):816-821, herein incorporated by reference in its entirety for all purposes. One or more or all of the nuclease domains can be deleted or mutated so that they are no longer functional or have reduced nuclease activity. For example, if one of the nuclease domains is deleted or mutated in a Cas9 protein, the resulting Cas9 protein can be referred to as a nickase and can generate a single-strand break within a double-stranded target DNA but not a double-strand break (i.e., it can cleave the complementary strand or the non-complementary strand, but not both). If both of the nuclease domains are deleted or mutated, the resulting Cas protein (e.g., Cas9) will have a reduced ability to cleave both strands of a double-stranded DNA (e.g., a nuclease-null or nuclease-inactive Cas protein, or a catalytically dead Cas protein (dCas)). An example of a mutation that converts Cas9 into a nickase is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. Likewise, H939A (histidine to alanine at amino acid position 839), H840A (histidine to alanine at amino acid position 840), or N863A (asparagine to alanine at amino acid position N863) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase. Other examples of mutations that convert Cas9 into a nickase include the corresponding mutations to Cas9 from S. thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic Acids Res.39(21):9275-9282 and WO 2013 / 141680, each of which is herein incorporated by reference in its entirety for all purposes. Such mutations can be generated using methods such as site-directed mutagenesis, PCR- mediated mutagenesis, or total gene synthesis. Examples of other mutations creating nickases can be found, for example, in WO 2013 / 176772 and WO 2013 / 142578, each of which is herein incorporated by reference in its entirety for all purposes. If all of the nuclease domains are deleted or mutated in a Cas protein (e.g., both of the nuclease domains are deleted or mutated in a Cas9 protein), the resulting Cas protein (e.g., Cas9) will have a reduced ability toPATENT ATTORNEY DOCKET NO.: 51471-019WO2 cleave both strands of a double-stranded DNA (e.g., a nuclease-null or nuclease-inactive Cas protein). One specific example is a D10A / H840A S. pyogenes Cas9 double mutant or a corresponding double mutant in a Cas9 from another species when optimally aligned with S. pyogenes Cas9. Another specific example is a D10A / N863A S. pyogenes Cas9 double mutant or a corresponding double mutant in a Cas9 from another species when optimally aligned with S. pyogenes Cas9. One example of a catalytically inactive Cas9 protein (dCas9) includes an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dCas9 protein sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is shown below: MKRPAATKKAGQAKKKKDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGD (SEQ ID NO: 2) Examples of inactivating mutations in the catalytic domains of xCas9 are the same as those described above for SpCas9. Examples of inactivating mutations in the catalytic domains of Staphylococcus aureus Cas9 proteins are also known. For example, the Staphylococcus aureus Cas9 enzyme (SaCas9) may include a substitution at position N580 (e.g., N580A substitution) and a substitution at position D10 (e.g., D10A substitution) to generate a nuclease-inactive Cas protein. See, e.g., WO 2016 / 106236, herein incorporated by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domains of Nme2Cas9 are also known (e.g., combination of D16A and H588A). Examples of inactivating mutations in the catalytic domains of St1Cas9 are also known (e.g.,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 combination of D9A, D598A, H599A, and N622A). Examples of inactivating mutations in the catalytic domains of St3Cas9 are also known (e.g., combination of D10A and N870A). Examples of inactivating mutations in the catalytic domains of CjCas9 are also known (e.g., combination of D8A and H559A). Examples of inactivating mutations in the catalytic domains of FnCas9 and RHA FnCas9 are also known (e.g., N995A). Examples of inactivating mutations in the catalytic domains of Cpf1 proteins are also known. With reference to Cpf1 proteins from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella bovoculi 237 (MbCpf1 Cpf1), such mutations can include mutations at positions 908, 993, or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, or positions 832, 925, 947, or 1180 of LbCpf1 or corresponding positions in Cpf1 orthologs. Such mutations can include, for example one or more of mutations D908A, E993A, and D1263A of AsCpf1 or corresponding mutations in Cpf1 orthologs, or D832A, E925A, D947A, and D1180A of LbCpf1 or corresponding mutations in Cpf1 orthologs. See, e.g., US 2016 / 0208243, herein incorporated by reference in its entirety for all purposes. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, in addition to transcriptional activation domains, a Cas protein can be fused to a cleavage domain or an epigenetic modification domain. See WO 2014 / 089290, herein incorporated by reference in its entirety for all purposes. Cas proteins can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N- terminus, the C-terminus, or internally within the Cas protein. As one example, a Cas protein can be fused to one or more heterologous polypeptides that provide for subcellular localization. Such heterologous polypeptides can include, for example, one or more nuclear localization signals (NLS) such as the monopartite SV40 NLS and / or a bipartite alpha- importin NLS for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, an ER retention signal, and the like. See, e.g., Lange et al. (2007) J. Biol. Chem. 282(8):5101-5105, herein incorporated by reference in its entirety for all purposes. Such subcellular localization signals can be located at the N-terminus, the C- terminus, or anywhere within the Cas protein. An NLS can include a stretch of basic amino acids and can be a monopartite sequence or a bipartite sequence. Optionally, a Cas protein can include two or more NLSs, including an NLS (e.g., an alpha- importin NLS or a monopartite NLS) at the N-terminus and an NLS (e.g., an SV40 NLS or a bipartite NLS) at the C-terminus. A Cas protein can also include two or more NLSs at the N-terminus and / or two or more NLSs at the C-terminus. In one example, a Cas protein may be fused with 1-10 NLSs, 1-5 NLSs, or one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the Cas sequence. It may also be inserted internally within the Cas sequence. In other examples, the Cas protein may be fused with more than one NLS. For example, the Cas protein may be fused with 2, 3, 4, or 5 NLSs or may fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. For example, the Cas protein may be fused to two SV40 NLS sequences linked at the carboxy terminus. In another example, the Cas protein may be fused with two NLSs, one linked at the N-terminus and onePATENT ATTORNEY DOCKET NO.: 51471-019WO2 at the C-terminus. In another example, the Cas protein may be fused with 3 NLSs. In another example, the Cas protein may be fused with no NLS. In some examples, the NLS may be a monopartite sequence, such as, for example, the SV40 NLS, PKKKRKV (SEQ ID NO: 3), or PKKKRRV (SEQ ID NO: 4). In some examples, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 5). In a specific example, a single PKKKRKV (SEQ ID NO: 3) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site. Cas proteins can also be operably linked to a cell-penetrating domain or protein transduction domain. For example, the cell-penetrating domain can be derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetrating peptide from Herpes simplex virus, or a polyarginine peptide sequence. See, e.g., WO 2014 / 089290 and WO 2013 / 176772, each of which is herein incorporated by reference in its entirety for all purposes. The cell- penetrating domain can be located at the N-terminus, the C-terminus, or anywhere within the Cas protein. Cas proteins can also be operably linked to a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu- Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin. Transcriptional Activation Domains The chimeric Cas proteins disclosed herein can include one or more transcriptional activation domains. Transcriptional activation domains include regions of a naturally occurring transcription factor which, in conjunction with a DNA-binding domain (e.g., a catalytically inactive Cas protein complexed with a gRNA), can activate transcription from a promoter by contacting transcriptional machinery either directly or through other proteins such as coactivators. Transcriptional activation domains also include functional fragments or variants of such regions of a transcription factor and engineered transcriptional activation domains that are derived from a native, naturally occurring transcriptional activation domain or that are artificially created or synthesized to activate transcription of a target gene. A functional fragment is a fragment that is capable of activating transcription of a target gene when operably linked to a suitablePATENT ATTORNEY DOCKET NO.: 51471-019WO2 DNA-binding domain. A functional variant is a variant that is capable of activating transcription of a target gene when operably linked to a suitable DNA-binding domain. A specific transcriptional activation domain for use in the chimeric Cas proteins disclosed herein includes a VP64 transcriptional activation domain or a functional fragment or variant thereof. VP64 is a tetrameric repeat of the minimal activation domain from the herpes simplex VP16 activation domain. For example, the transcriptional activation domain can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VP64 transcriptional activation domain protein sequence set forth in SEQ ID NO: 6. SEQ ID NO: 6 is shown below: AAAGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINCT Other examples of transcriptional activation domains include herpes simplex virus VP16 transactivation domain, VP64 (quadruple tandem repeat of the herpes simplex virus VP16), a NF-κB p65 (NF-κB trans-activating subunit p65) activation domain, a MyoD1 transactivation domain, an HSF1 transactivation domain (transactivation domain from human heat-shock factor 1), RTA (Epstein Barr virus R transactivator activation domain), a SETT / 9 transactivation domain, a p53 activation domain 1, a p53 activation domain 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, an NFAT (nuclear factor of activated T-cells) activation domain, and functional fragments and variants thereof. See, e.g., US 2016 / 0298125, US 2016 / 0281072, and WO 2016 / 049258, each of which is herein incorporated by reference in its entirety for all purposes. Other examples of transcriptional activation domains include Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, Leu3, and functional fragments and variants thereof. See, e.g., US 2016 / 0298125, herein incorporated by reference in its entirety for all purposes. Yet other examples of transcriptional activation domains include Sp1, Vax, GATA4, and functional fragments and variants thereof. See, e.g., WO 2016 / 149484, herein incorporated by reference in its entirety for all purposes. Other examples include activation domains from Oct1, Oct-2A, AP-2, CTF1, P300, CBP, PCAF, SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7, ARF-8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1PC4, and functional fragments and variants thereof. See, e.g., US 2016 / 0237456, EP3045537, and WO 2011 / 146121, each of which is incorporated by reference in its entirety for all purposes. Additional suitable transcriptional activation domains are also known. See, e.g., WO 2011 / 146121, herein incorporated by reference in its entirety for all purposes. Chimeric Adaptor Proteins Also provided are chimeric adaptor proteins that can bind to the gRNAs disclosed herein. The chimeric adaptor proteins disclosed herein are useful in dCas-SAM-like systems to increase the number and diversity of transcriptional activation domains being directed to a target sequence within a target gene to activate transcription of the target gene. Chimeric adaptor proteins include, for example: (a) an adaptor (i.e., adaptor domain or adaptor protein) that specifically binds to an adaptor-binding element within a gRNA; and (b) one or more transcriptional activation domains. For example, such fusion proteins can include 1, 2, 3, 4, 5, or morePATENT ATTORNEY DOCKET NO.: 51471-019WO2 transcriptional activation domains (e.g., two or more heterologous transcriptional activation domains or three or more heterologous transcriptional activation domains). In one example, such chimeric adaptor proteins can include: (a) an adaptor (i.e., an adaptor domain or adaptor protein) that specifically binds to an adaptor-binding element in a gRNA; and (b) two or more transcriptional activation domains. For example, the chimeric adaptor protein can include: (a) an MS2 coat protein adaptor that specifically binds to one or more MS2 aptamers in a gRNA (e.g., two MS2 aptamers in separate locations in a gRNA); and (b) one or more (e.g., two or more transcriptional activation domains). For example, the two transcriptional activation domains can be p65 and HSF1 transcriptional activation domains or functional fragments or variants thereof. However, chimeric adaptor proteins in which the transcriptional activation domains include other transcriptional activation domains or functional fragments or variants thereof are also provided. The one or more transcriptional activation domains can be fused directly to the adaptor. Alternatively, the one or more transcriptional activation domains can be linked to the adaptor via a linker or a combination of linkers or via one or more additional domains. Likewise, if two or more transcriptional activation domains are present, they can be fused directly to each other or can be linked to each other via a linker or a combination of linkers or via one or more additional domains. Linkers that can be used in these fusion proteins can include any sequence that does not interfere with the function of the fusion proteins. Example linkers are short (e.g., 2-20 amino acids) and are typically flexible (e.g., including amino acids with a high degree of freedom such as glycine, alanine, and serine). Some specific examples of linkers include one or more units consisting of GGGS (SEQ ID NO: 7) or GGGGS (SEQ ID NO: 8), such as two, three, four, or more repeats of GGGS (SEQ ID NO: 7) or GGGGS (SEQ ID NO: 8) in any combination. Other linker sequences that can be used include, for example, GGG, GGGG (SEQ ID NO: 145), GGGAG (SEQ ID NO: 146), GGGAGG (SEQ ID NO: 147), GGGAGGG (SEQ ID NO: 148), GGGGA (SEQ ID NO: 149), GGGGG (SEQ ID NO: 150), GGAG (SEQ ID NO: 151), GGSG (SEQ ID NO: 152), AGGG (SEQ ID NO: 153), SGGG (SEQ ID NO: 154), GAGA (SEQ ID NO: 155), GSGS (SEQ ID NO: 156), GAGAGA (SEQ ID NO: 157), GSGSGS (SEQ ID NO: 158), GAGAGAGA (SEQ ID NO: 159), GSGSGSGS (SEQ ID NO: 160), GAGAGAGAGA (SEQ ID NO: 161), GSGSGSGSGS (SEQ ID NO: 162), GAGAGAGAGAGA (SEQ ID NO: 163), GSGSGSGSGSGS (SEQ ID NO: 164), GGAGGA (SEQ ID NO: 165), GGSGGS (SEQ ID NO: 166), GGAGGAGGA (SEQ ID NO: 167), GGSGGSGGS (SEQ ID NO: 168), GGAGGAGGAGGA (SEQ ID NO: 169), GGSGGSGGSGGS (SEQ ID NO: 170), GGAGGGAG (SEQ ID NO: 171), GGSGGGSG (SEQ ID NO: 172), GGAGGGAGGGAG (SEQ ID NO: 173), GGSGGGSGGGSG (SEQ ID NO: 174), GGGGAGGGGAGGGGA (SEQ ID NO: 175), GGGGSGGGGSGGGGS (SEQ ID NO: 176), AAAL (SEQ ID NO: 177), AAAK (SEQ ID NO: 178), AAAR (SEQ ID NO: 179), EGKSSGSGSESKST (SEQ ID NO: 180), GSAGSAAGSGEF (SEQ ID NO: 181), AEAAAKEAAAKA (SEQ ID NO: 182), KESGSVSSEQLAQFRSLD (SEQ ID NO: 183), GENLYFQSGG (SEQ ID NO: 184), SACYCELS (SEQ ID NO: 185), RSIAT (SEQ ID NO: 186), RPACKIPNDLKQKVMNH (SEQ ID NO: 187), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 188), AAANSSIDLISVPVDSR (SEQ ID NO: 189), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 190), EAAAK (SEQ ID NO: 191), PAPAP (SEQ ID NO: 192), and GGGA (SEQ ID NO: 193).PATENT ATTORNEY DOCKET NO.: 51471-019WO2 The one or more transcriptional activation domains and the adaptor can be in any order within the chimeric adaptor protein. As one option, the one or more transcriptional activation domains can be C- terminal to the adaptor and the adaptor can be N-terminal to the one or more transcriptional activation domains. For example, the one or more transcriptional activation domains can be at the C-terminus of the chimeric adaptor protein, and the adaptor can be at the N-terminus of the chimeric adaptor protein. However, the one or more transcriptional activation domains can be C-terminal to the adaptor without being at the C-terminus of the chimeric adaptor protein (e.g., if a nuclear localization signal is at the C- terminus of the chimeric adaptor protein). Likewise, the adaptor can be N-terminal to the one or more transcriptional activation domains without being at the N-terminus of the chimeric adaptor protein (e.g., if a nuclear localization signal is at the N-terminus of the chimeric adaptor protein). As another option, the one or more transcriptional activation domains can be N-terminal to the adaptor and the adaptor can be C-terminal to the one or more transcriptional activation domains. For example, the one or more transcriptional activation domains can be at the N-terminus of the chimeric adaptor protein, and the adaptor can be at the C-terminus of the chimeric adaptor protein. As yet another option, if the chimeric adaptor protein includes two or more transcriptional activation domains, the two or more transcriptional activation domains can flank the adaptor. Chimeric adaptor proteins can also be operably linked or fused to additional heterologous polypeptides. The fused or linked heterologous polypeptide can be located at the N- terminus, the C- terminus, or anywhere internally within the chimeric adaptor protein. For example, a chimeric adaptor protein can further include a nuclear localization signal. A specific example of such a protein includes an MS2 coat protein (adaptor) linked (either directly or via an NLS) to a p65 transcriptional activation domain C-terminal to the MS2 coat protein (MCP), and HSF1 transcriptional activation domain C-terminal to the p65 transcriptional activation domain. Such a protein can include from N-terminus to C-terminus: an MCP; a nuclear localization signal; a p65 transcriptional activation domain; and an HSF1 transcriptional activation domain. For example, a chimeric adaptor protein can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the MCP-p65-HSF1 chimeric adaptor protein sequence set forth in SEQ ID NO: 9, shown below. MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEV PKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SAGGGGSGGGGSGGGGSGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQ PPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASV DNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFS SIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQ EPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPT ISLLTGSEPPKAKDPTVS (SEQ ID NO: 9) Chimeric adaptor proteins can also be fused or linked to one or more heterologous polypeptides that provide for subcellular localization. Such heterologous polypeptides can include, for example, one or more nuclear localization signals (NLS) such as the SV40 NLS and / or an alpha-importin NLS for targetingPATENT ATTORNEY DOCKET NO.: 51471-019WO2 to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, an ER retention signal, and the like. See, e.g., Lange et al. (2007) J. Biol. Chem.282(8):5101-5105, herein incorporated by reference in its entirety for all purposes. Such subcellular localization signals can be located at the N- terminus, the C-terminus, or anywhere within the chimeric adaptor protein (e.g., at the C-terminus or N- terminus of the adaptor protein component of the chimeric adaptor protein or at the C-terminus or N- terminus of a transcriptional activator domain component of the chimeric adaptor protein). An NLS can include, for example, a stretch of basic amino acids, and can be a monopartite sequence or a bipartite sequence. Optionally, the chimeric adaptor protein includes two or more NLSs, including an NLS (e.g., an alpha-importin NLS) at the N-terminus and / or an NLS (e.g., an SV40 NLS) at the C-terminus. A chimeric adaptor protein can also include two or more NLSs at the N-terminus and / or two or more NLSs at the C- terminus. In one example, a chimeric adaptor protein may be fused with 1-10 NLSs, 1-5 NLSs, or one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the chimeric adaptor protein sequence. It may also be inserted internally within the chimeric adaptor protein sequence. In other examples, the chimeric adaptor protein may be fused with more than one NLS. For example, the chimeric adaptor protein may be fused with 2, 3, 4, or 5 NLSs or may fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. For example, the chimeric adaptor protein may be fused to two SV40 NLS sequences linked at the carboxy terminus. In another example, the chimeric adaptor protein may be fused with two NLSs, one linked at the N-terminus and one at the C- terminus. In another example, the chimeric adaptor protein may be fused with 3 NLSs. In another example, the chimeric adaptor protein may be fused with no NLS. In some examples, the NLS may be a monopartite sequence, such as, for example, the SV40 NLS, PKKKRKV (SEQ ID NO: 3), or PKKKRRV (SEQ ID NO: 4). In some examples, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 5). In a specific example, a single PKKKRKV (SEQ ID NO: 3) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site. Chimeric adaptor proteins can also be operably linked to a cell-penetrating domain or protein transduction domain. For example, the cell-penetrating domain can be derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetrating peptide from Herpes simplex virus, or a polyarginine peptide sequence. See, e.g., WO 2014 / 089290 and WO2013 / l 76772, each of which is herein incorporated by reference in its entirety for all purposes. As another example, chimeric adaptor proteins can be fused or linked to a heterologous polypeptide providing increased or decreased stability. Chimeric adaptor proteins can also be operably linked to a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Cerulean, CyPet, AmCyan1, Midoriishi- Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcVS, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin. A chimeric adaptor protein can be provided in the form of DNA encoding the chimeric adaptor protein. Optionally, the nucleic acid encoding the chimeric adaptor protein can be codon-optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid encoding the chimeric adaptor protein can be modified to substitute codons having a higher frequency of usage in a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the chimeric adaptor protein is introduced into the cell, the chimeric adaptor protein can be transiently, conditionally, or constitutively expressed in the cell. Chimeric adaptor mRNAs can include a poly-adenylated (poly-A) tail. The poly-A tail can, for example, include at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenines, and optionally up to 300 adenines. For example, the poly-A tail can include 95, 96, 97, 98, 99, or 100 adenine nucleotides. Nucleic acids encoding chimeric adaptor proteins can be integrated into the genome of a cell and operably linked to a promoter active in the cell. Alternatively, nucleic acids encoding chimeric adaptor proteins can be operably linked to a promoter in an expression construct. Expression constructs include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a chimeric adaptor gene) and which can transfer such a nucleic acid sequence of interest to a target cell. For example, the nucleic acid encoding the chimeric adaptor protein can be in a vector including a DNA encoding a gRNA and / or a chimeric Cas protein. Alternatively, the nucleic acid encoding the chimeric adaptor protein can be in a vector or plasmid that is separate from the vector including the DNA encoding the gRNA or the DNA encoding the chimeric Cas protein. Promoters that can be used in an expression construct include promoters active, for example, in one or more of a eukaryotic cell, a non- human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, an embryonic stem (ES) cell, an adult stem cell, a developmentally restricted progenitor cell, an induced pluripotent stem (iPS) cell, or a one-cell stage embryo. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Optionally, the promoter can be a bidirectional promoter. Such bidirectional promoters can consist of (1) a complete, conventional, unidirectional Pol III promoter that contains 3 external control elements: a distal sequencePATENT ATTORNEY DOCKET NO.: 51471-019WO2 element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basic Pol III promoter that includes a PSE and a TATA box fused to the 5' terminus of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and the promoter can be rendered bidirectional by creating a hybrid promoter in which transcription in the reverse direction is controlled by appending a PSE and TATA box derived from the U6 promoter. See, e.g., US 2016 / 0074535, herein incorporated by reference in its entirety for all purposes. Adaptors Adaptors (i.e., adaptor domains or adaptor proteins) are nucleic-acid-binding domains (e.g., DNA- binding domains and / or RNA-binding domains) that specifically recognize and bind to distinct sequences (e.g., bind to distinct DNA and / or RNA sequences such as aptamers in a sequence-specific manner). Aptamers include nucleic acids that, through their ability to adopt a specific three-dimensional conformation, can bind to a target molecule with high affinity and specificity. Such adaptors can bind, for example, to a specific RNA sequence and secondary structure. These sequences (i.e., adaptor-binding elements) can be engineered into a gRNA. For example, an MS2 aptamer can be engineered into a gRNA to specifically bind an MS2 coat protein (MCP). For example, the adaptor can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the MCP sequence set forth in SEQ ID NO: 10, shown below. MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEV PKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 10) Some specific examples of adaptors and targets include RNA-binding protein / aptamer combinations that exist within the diversity of bacteriophage coat proteins. For example, the following adaptor proteins or functional fragments or variants thereof can be used: MS2 coat protein (MCP), PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, Fl, ID2, NL95, TW19, AP205, φCb5, Φ Cb8r, Φ Cb12r, ΦCb23r, 7s, and PRR1. See, e.g., WO 2016 / 049258, herein incorporated by reference in its entirety for all purposes. A functional fragment or functional variant of an adaptor protein is one that retains the ability to bind to a specific adaptor-binding element (e.g., ability to bind to a specific adaptor-binding sequence in a sequence-specific manner). For example, a PP7 Pseudomonas bacteriophage coat protein variant can be used in which amino acids 68-69 are mutated to SG and amino acids 70-75 are deleted from the wild type protein. See, e.g., Wu et al. (2012) Biophys. J. 102(12):2936-2944 and Chao et al. (2007) Nat. Struct. Mol. Biol.15(1):103-105, each of which is herein incorporated by reference in its entirety for all purposes. Likewise, an MCP variant may be used, such as a N55K mutant. See, e.g., Spingola and Peabody (1994) J. Biol. Chem.269(12):9006-9010, herein incorporated by reference in its entirety for all purposes. Other examples of adaptor proteins that can be used in a composition disclosed herein include all or part of (e.g., the DNA-binding from) endoribonuclease Csy4 or the lambda N protein. See, e.g., U S 2016 / 0312198, herein incorporated by reference in its entirety for all purposes.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Transcriptional Activation Domains The chimeric adaptor proteins disclosed herein can include one or more transcriptional activation domains. Such transcriptional activation domains can be naturally occurring transcriptional activation domains, can be functional fragments or functional variants of naturally occurring transcriptional activation domains, or can be engineered or synthetic transcriptional activation domains. Transcriptional activation domains that can be used include those described for use in chimeric Cas proteins herein. A specific transcriptional activation domain for use in the chimeric adaptor proteins disclosed herein includes p65 and / or HSF1 transcriptional activation domains or functional fragments or variants thereof. The HSF1 transcriptional activation domain can be a transcriptional activation domain of human heat shock factor 1 (HSF1). The p65 transcriptional activation domain can be a transcriptional activation domain of transcription factor p65, also known as nuclear factor NF-κB p65 subunit encoded by the RELA gene. As one example, a transcriptional activation domain can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the p65 transcriptional activation domain protein sequence set forth in SEQ ID NO: 11. As another example, a transcriptional activation domain can include an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HSF1 transcriptional activation domain protein sequence set forth in SEQ ID NO: 12. SEQ ID NO: 11 is shown below: PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQ AGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLM EYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISS SEQ ID NO: 12 is shown below: GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTA QPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS SAM guide RNAs Also provided are gRNAs that can bind to the chimeric Cas proteins and chimeric adaptor proteins disclosed herein to activate transcription of target genes. One or more gRNAs can be used in the methods or compositions disclosed herein. For example, two or more, three or more, four or more, or five or more gRNAs can be used. Two or more of the g RNAs can target a different target sequence in a single target gene. For example, two or more, three or more, four or more, or five or more gRNAs can each target a different target sequence in a single target gene. Similarly, the gRNAs can target multiple target genes (e.g., two or more, three or more, four or more, or five or more target genes). Examples of gRNA target sequences are disclosed herein.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Guide RNAs A “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein (e.g., Cas9 protein) and targets the Cas protein to a specific location within a target DNA. Guide RNAs can include two segments: a “DNA-targeting segment” (also called “guide sequence”) and a “protein-binding segment.” “Segment” includes a section or region of a molecule, such as a contiguous stretch of nucleotides in an RNA Some gRNAs, such as those for Cas9, can include two separate RNA molecules: an “activator-RNA” (e.g., tracrRNA) and a “targeter-RNA” (e.g., CRISPR RNA or crRNA). Other gRNAs are a single RNA molecule (single RNA polynucleotide), which can also be called a “single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.” See, e.g., WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833, each of which is herein incorporated by reference in its entirety for all purposes. A gRNA can refer to either a CRISPR RNA (crRNA) or the combination of a crRNA and a trans-activating CRISPR RNA (tracrRNA). The crRNA and tracrRNA can be associated as a single RNA molecule (single gRNA or sgRNA) or in two separate RNA molecules (dual guide RNA or dgRNA). For Cas9, for example, an sgRNA can include a crRNA fused to a tracrRNA (e.g., via a linker). For Cpf1, for example, only a crRNA is needed to achieve binding to a target sequence. The terms “guide RNA” and “gRNA” include both double-molecule (i.e., modular) gRNAs and single-molecule gRNAs. In some of the methods and compositions disclosed herein, a C5 gRNA is a S. pyogenes Cas9 gRNA or an equivalent thereof. In some of the methods and compositions disclosed herein, a C5 gRNA is a S. aureus Cas9 gRNA or an equivalent thereof. Example gRNA sequences of the disclosure are shown in Table 1 below. Table 1. gRNA sequences to the GJB2 regulatory elementPATENT ATTORNEY DOCKET NO.: 51471-019WO2PATENT ATTORNEY DOCKET NO.: 51471-019WO2In some embodiments, the gRNA includes a nucleic acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 62-102. In some embodiments, the gRNA includes the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO: 84. An example two-molecule gRNA includes a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-activating CRISPR RNA”PATENT ATTORNEY DOCKET NO.: 51471-019WO2 or “activator-RNA” or “tracrRNA”) molecule. A crRNA includes both the DNA-targeting segment (single- stranded) of the gRNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the gRNA. An example of a crRNA tail, located downstream (3') of the DNA- targeting segment, includes GUUUUAGAGCUAGGCCA (SEQ ID NO: 103). Any of the DNA-targeting segments disclosed herein can be joined to the 5' end of SEQ ID NO: 103 to form a crRNA. A corresponding tracrRNA (activator-RNA) includes a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. A stretch of nucleotides of a crRNA are complementary to and hybridize with a stretch of nucleotides of a tracrRNA to form the dsRNA duplex of the protein-binding domain of the gRNA. As such, each crRNA can be said to have a corresponding tracrRNA. Examples of tracrRNA sequences include any one of: AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU UU (SEQ ID NO: 104), AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCUUUU (SEQ ID NO: 105), or GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGC (SEQ ID NO: 106). In systems in which both a crRNA and a tracrRNA are needed, the crRNA and the corresponding tracrRNA hybridize to form a gRNA. In systems in which only a crRNA is needed, the crRNA can be the gRNA. The crRNA additionally provides the single-stranded DNA-targeting segment that hybridizes to the complementary strand of a target DNA If used for modification within a cell, the exact sequence of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used. See, e.g., Mali et al. (2013) Science 339(6121):823-826; Jinek et al. (2012) Science 337(6096):816-821; Hwang et al. (2013) Nat. Biotechnol.31(3):227-229; Jiang et al. (2013) Nat. Biotechnol.31(3):233-239; and Cong et al. (2013) Science 339(6121):819-823, each of which is herein incorporated by reference in its entirety for all purposes. The DNA-targeting segment (crRNA) of a given gRNA includes a nucleotide sequence that is complementary to a sequence on the complementary strand of the target DNA, as described in more detail below. The DNA-targeting segment of a gRNA interacts with the target DNA in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA-targeting segment may vary and determines the location within the target DNA with which the gRNA will interact. The DNA-targeting segment of a subject gRNA can be modified to hybridize to any desired sequence within a target DNA Naturally occurring crRNAs differ depending on the CRISPR / Cas system and organism but often contain a targeting segment of between 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides (see, e.g., WO 2014 / 131833, herein incorporated by reference in its entirety for all purposes). In the case of S. pyogenes, the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long. The 3' located DR is complementary to and hybridizes with the corresponding tracrRNA, which in tum binds to the Cas protein. The DNA-targeting segment can have, for example, a length of at least about 12, 15, 17, 18, 19, 20, 25, 30, 35, or 40 nucleotides. Such DNA-targeting segments can have, for example, a length fromPATENT ATTORNEY DOCKET NO.: 51471-019WO2 about 12 to about 100, from about 12 to about 80, from about 12 to about 50, from about 12 to about 40, from about 12 to about 30, from about 12 to about 25, or from about 12 to about 20 nucleotides. For example, the DNA targeting segment can be from about 15 to about 25 nucleotides (e.g., from about 17 to about 20 nucleotides, or about 17, 18, 19, or 20 nucleotides). See, e.g., US 2016 / 0024523, herein incorporated by reference in its entirety for all purposes. For Cas9 from S. pyogenes, a typical DNA- targeting segment is between 16 and 20 nucleotides in length or between 17 and 20 nucleotides in length. For Cas9 from S. aureus, a typical DNA- targeting segment is between 21 and 23 nucleotides in length. For Cpf1, a typical DNA-targeting segment is at least 16 nucleotides in length or at least 18 nucleotides in length. In one example, the DNA-targeting segment can be about 20 nucleotides in length. However, shorter and longer sequences can also be used for the targeting segment (e.g., 15-25 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The degree of identity between the DNA-targeting segment and the corresponding gRNA target sequence (or degree of complementarity between the DNA-targeting segment and the other strand of the gRNA target sequence) can be, for example, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. The DNA-targeting segment and the corresponding gRNA target sequence can contain one or more mismatches. For example, the DNA-targeting segment of the gRNA and the corresponding gRNA target sequence can contain 1-4, 1-3, 1-2, 1, 2, 3, or 4 mismatches (e.g., where the total length of the gRNA target sequence is at least 17, at least 18, at least 19, or at least 20 or more nucleotides). For example, the DNA-targeting segment of the gRNA and the corresponding gRNA target sequence can contain 1-4, 1-3, 1-2, 1, 2, 3, or 4 mismatches where the total length of the gRNA target sequence is 20 nucleotides. TracrRNAs can be in any form (e.g., full-length tracrRNAs or active partial tracrRNAs) and of varying lengths. They can include primary transcripts or processed forms. For example, tracrRNAs (as part of an sgRNA or as a separate molecule as part of a two-molecule gRNA) may include all or a portion of a wild type tracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild type tracrRNA sequence). Examples of wild type tracrRNA sequences from S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and 65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature 471(7340):602-607; WO 2014 / 093661, each of which is herein incorporated by reference in its entirety for all purposes. Examples of tracrRNAs within sgRNAs include the tracrRNA segments found within +48, +54, +67, and +85 versions of sgRNAs, where “+n” indicates that up to the +n nucleotide of wild type tracrRNA is included in the sgRNA. See U.S. Pat. No.8,697,359, herein incorporated by reference in its entirety for all purposes. The percent complementarity between the DNA-targeting segment of the gRNA and the complementary strand of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). The percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be at least 60% over about 20 contiguous nucleotides. As an example, the percent complementarity between the DNA-targeting segment and the complementaryPATENT ATTORNEY DOCKET NO.: 51471-019WO2 strand of the target DNA can be 100% over the 14 contiguous nucleotides at the 5' end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA- targeting segment can be considered to be 14 nucleotides in length. As another example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over the seven contiguous nucleotides at the 5' end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 7 nucleotides in length. In some gRNAs, at least 17 nucleotides within the DNA- targeting segment are complementary to the complementary strand of the target DNA For example, the DNA-targeting segment can be 20 nucleotides in length and can include 1, 2, or 3 mismatches with the complementary strand of the target DNA In one example, the mismatches are not adjacent to the region of the complementary strand corresponding to the protospacer adjacent motif (PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g., the mismatches are in the 5' end of the DNA-targeting segment of the gRNA, or the mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region of the complementary strand corresponding to the PAM sequence). The protein-binding segment of a gRNA can include two stretches of nucleotides that are complementary to one another. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of a subject gRNA interacts with a Cas protein, and the gRNA directs the bound Cas protein to a specific nucleotide sequence within target DNA via the DNA-targeting segment. Single-guide RNAs can include a DNA-targeting segment and a scaffold sequence (i.e., the protein-binding or Cas-binding sequence of the gRNA). For example, such gRNAs can have a 5' DNA- targeting segment joined to a 3' scaffold sequence. Example scaffold sequences include: (version 1; SEQ ID NO: 107) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU; (version 2; SEQ ID NO: 108) GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGC; (version 3; SEQ ID NO: 109) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (version 4; SEQ ID NO: 110) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC; (version 5; SEQ ID NO: 111)PATENT ATTORNEY DOCKET NO.: 51471-019WO2 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU UUUUUU; (version 6; SEQ ID NO: 112) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU UUU; and (version 7; SEQ ID NO: 113) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC. Guide RNAs targeting any of the gRNA target sequences disclosed herein can include, for example, a DNA-targeting segment on the 5' end of the gRNA fused to any of the example gRNA scaffold sequences on the 3' end of the gRNA. That is, any of the DNA-targeting segments disclosed herein can be joined to the 5' end of any one of the above scaffold sequences to form a single gRNA (chimeric gRNA). In some gRNAs (e.g., sgRNAs), at least one loop (e.g., two loops) of the gRNA is modified by insertion of a distinct RNA sequence that binds to one or more adaptors (i.e., adaptor proteins or domains). Such adaptor proteins can be used to further recruit one or more heterologous functional domains, such as transcriptional activation domains. Examples of fusion proteins including such adaptor proteins (i.e., chimeric adaptor proteins) are disclosed herein. For example, an MS2-binding loop ggccAACAUGAGGAUCACCCAUGUCUGCAGggcc (SEQ ID NO: 114) may replace nucleotides +13 to +16 and nucleotides +53 to +56 of the sgRNA scaffold (backbone) set forth in SEQ ID NO: 107, 109, 111, or 112 or the sgRNA backbone for the S. pyogenes CRISPR / Cas9 system described in WO 2016 / 049258 and Konermann et al. (2015) Nature 517(7536):583-588, each of which is herein incorporated by reference in its entirety for all purposes. The gRNA numbering used herein refers to the nucleotide numbering in the gRNA scaffold sequence (i.e., the sequence downstream of the DNA-targeting segment of the gRNA). For example, the first nucleotide of the gRNA scaffold is +1, the second nucleotide of the scaffold is +2, and so forth. Residues corresponding with nucleotides +13 to +16 in SEQ ID NO: 107, 109, 111, or 112 are the loop sequence in the region spanning nucleotides +9 to +21 in SEQ ID NO: 107, 109, 111, or 112, a region referred to herein as the tetraloop. Residues corresponding with nucleotides +53 to +56 in SEQ ID NO: 107, 109, 111, or 112 are the loop sequence in the region spanning nucleotides +48 to +61 in SEQ ID NO: 107, 109, 111, or 112, a region referred to herein as the stem loop 2. Other stem loop sequences in in SEQ ID NO: 107, 109, 111, or 112 include stem loop 1 (nucleotides +33 to +41) and stem loop 3 (nucleotides +63 to +75). The resulting structure is an sgRNA scaffold in which each of the tetraloop and stem loop 2 sequences have been replaced by an MS2 binding loop. The tetraloop and stem loop 2 protrude from the Cas9 protein in such a way that adding an MS2-binding loop should not interfere with any Cas9 residues. Additionally, the proximity of the tetraloop and stem loop 2 sites to the DNA indicates that localization to these locations could result in a high degree of interaction between thePATENT ATTORNEY DOCKET NO.: 51471-019WO2 DNA and any recruited protein, such as a transcriptional activator. Thus, in some sgRNAs, nucleotides corresponding to +13 to +16 and / or nucleotides corresponding to +53 to +56 of the gRNA scaffold set forth in SEQ ID NO: 107, 109, 111, or 112 or corresponding residues when optimally aligned with any of these scaffold / backbones are replaced by the distinct RNA sequences capable of binding to one or more adaptor proteins or domains. Alternatively or additionally, adaptor-binding sequences can be added to the 5' end or the 3' end of a gRNA. An example gRNA scaffold including MS2-binding loops in the tetraloop and stem loop 2 regions can include the sequence set forth in SEQ ID NO: 115. SEQ ID NO: 115 is shown below: GUUUUAGAGCUAGGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCCUAGCAAGUUAAAAUAAGGC UAGUCCGUUAUCAACUUGGCCAACAUGAGGAUCACCCAUGUCUGCAGGGCCAAGUGGCACCGAGU CGGUGCU The gRNA can also be provided in the form of DNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as one DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively. When a gRNA is provided in the form of DNA, the gRNA can be transiently, conditionally, or constitutively expressed in the cell. DNAs encoding gRNAs can be integrated into the genome of the cell and operably linked to a promoter active in the cell. Alternatively, DNAs encoding gRNAs can be operably linked to a promoter in an expression construct. For example, the DNA encoding the gRNA can be in a vector including a heterologous nucleic acid. Promoters that can be used in such expression constructs include promoters active, for example, in one or more of a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, an embryonic stem (ES) cell, an adult stem cell, a developmentally restricted progenitor cell, an induced pluripotent stem (iPS) cell, or a one-cell stage embryo. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Such promoters can also be, for example, bidirectional promoters. Specific examples of suitable promoters include an RNA polymerase III promoter, such as a human U6 promoter, a rat U6 polymerase III promoter, or a mouse U6 polymerase III promoter. guide RNA Target Sequences Target DNAs for gRNAs include nucleic acid sequences present in a DNA to which a DNA- targeting segment of a gRNA will bind, provided sufficient conditions for binding exist. Suitable DNA / RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), herein incorporated by reference in its entirety for all purposes). The strand of the target DNA that is complementary to and hybridizes with the gRNA can be called the “complementary strand,” and the strand of the target DNA that is complementary to the “complementary strand” (and is therefore notPATENT ATTORNEY DOCKET NO.: 51471-019WO2 complementary to the Cas protein or gRNA) can be called “noncomplementary strand” or “template strand.” The target DNA includes both the sequence on the complementary strand to which the gRNA hybridizes and the corresponding sequence on the non-complementary strand (e.g., adjacent to the protospacer adjacent motif (PAM)). The term “guide RNA target sequence” as used herein refers specifically to the sequence on the non-complementary strand corresponding to (i.e., the reverse complement of) the sequence to which the gRNA hybridizes on the complementary strand. That is, the gRNA target sequence refers to the sequence on the non-complementary strand adjacent to the PAM (e.g., upstream or 5' of the PAM in the case of Cas9). A gRNA target sequence is equivalent to the DNA- targeting segment of a gRNA, but with thymines instead of uracils. As one example, a gRNA target sequence for an SpCas9 enzyme can refer to the sequence upstream of the 5'-NGG-3' PAM on the non- complementary strand. A gRNA is designed to have complementarity to the complementary strand of a target DNA, where hybridization between the DNA-targeting segment of the gRNA and the complementary strand of the target DNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. If a gRNA is referred to herein as targeting a gRNA target sequence, what is meant is that the gRNA hybridizes to the complementary strand sequence of the target DNA that is the reverse complement of the gRNA target sequence on the non- complementary strand. A target DNA or gRNA target sequence can include any polynucleotide, and can be located, for example, in the nucleus or cytoplasm of a cell or within an organelle of a cell, such as a mitochondrion or chloroplast. A target DNA or gRNA target sequence can be any nucleic acid sequence endogenous or exogenous to a cell. The gRNA target sequence can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory sequence) or can include both. Preferably, the g RNA target sequence is a regulatory sequence such as a promoter exogenous to the cell of the present disclosure. Such promoter is preferably operably linked to a target gene according to the present disclosure. It can be preferable for the target sequence to be adjacent to the transcription start site of a gene. For example, the target sequence can be within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 base pair of the transcription start site, within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 base pair upstream of the transcription start site, or within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 base pair downstream of the transcription start site. Optionally, the target sequence is within the region 200 base pairs upstream of the transcription start site and 1 base pair downstream of the transcription start site (-200 to +I). The target sequence can be within any gene desired to be targeted for transcriptional activation. In some cases, a target gene may be one that is a non-expressing gene or a weakly expressing gene (e.g., only minimally expressed above background, such as 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold). The target gene may also be one that is expressed at low levels compared to a control gene. The target gene may also be one that is epigenetically silenced. The term “epigenetically silenced” refers to a gene that is not being transcribed or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a control sample (e.g., a corresponding control cell, such as a normal cell), due to a mechanism other than a genetic change such as a mutation. Epigenetic mechanisms of gene silencing are well known and include, for example, hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of a gene and structural changes in chromatin due, for example, to histone acetylation, such that gene transcription is reduced or inhibited. Target genes can include genes expressed in particular organs or tissues, such as the ear. Target genes can be any genes that can be encoded by a viral vector and can be transduced into a cell according to the present disclosure in order to measure the transduction ability and assess suitability of the viral vector to be used for in vivo therapeutic purposes. Target genes can include disease-associated genes. A disease-associated gene refers to any gene that yields transcription or translation products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene possessing a mutation or genetic variation that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown and may be at a normal or abnormal level. For example, target genes can be genes associated with protein aggregation diseases and disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, and amyloidoses such as transthyretin amyloidosis. Target genes can also be genes involved in pathways related to a disease or condition, such as hypercholesterolemia or atherosclerosis, or genes that when overexpressed can model such diseases or conditions. Target genes can also be genes expressed or overexpressed in one or more types of cancer. See, e.g., Santarius et al. (2010) Nat. Rev. Cancer 10(1):59-64, herein incorporated by reference in its entirety for all purposes. The GJB2 gene is an example of a target gene of the present disclosure. Gap junction protein beta 2 (Gjb2, also known as Connexin 26) is a protein encoded by the GJB2 gene and is a member of the connexin gene family. Connexins oligomerize into hexameric arrangements called connexons or hemichannels, which often dock with hemichannels from a contacting cell to form gap junctions. Nearly half of all hearing loss is attributed to mutations in one of four members of the connexin gene family, and GJB2 mutations are the most common. More than 100 different mutations in GJB2 have been identified that cause non-syndromic hearing loss, which is loss of hearing that is not associated with other signs and symptoms. One form of non-syndromic hearing loss that is associated with mutations in GJB2 is DFNB1, which is characterized by moderate to profound prelingual hearing loss and is inherited in an autosomal recessive pattern. DFNA3 is the other form of non-syndromic hearing loss that is associated with mutations in GJB2 and is moderate to severe prelingual or postlingual hearing loss that becomes more severe over time and is inherited in an autosomal dominant pattern. Other health conditionsPATENT ATTORNEY DOCKET NO.: 51471-019WO2 associated with mutations in GJB2 include Bart-Pumphrey syndrome, hystrix-like ichthyosis with deafness, keratitis-ichthyosis-deafness syndrome, palmoplantar keratoderma with deafness, and Vohwinkel syndrome, all of which are characterized by hearing loss and skin abnormalities. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a promoter or enhancer of GJB2. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 promoter having the sequence of SEQ ID NO: 120. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a GJB2 enhancer having the sequence of SEQ ID NO: 119. In some embodiments, the CRISPR SAM complex includes a gRNA that specifically targets a region bridging the GJB2 promoter and enhancer (e.g., targets a region including a 3’ portion of the enhancer sequence and a 5’ portion of the promoter sequence) in a GJB2 regulatory element having the sequence of SEQ ID NO: 144. SEQ ID NO: 119 is shown below: AAAAACTCTTACATAATTGTAACAGATTTGAGTTTCCTCTGGTTCAGGTTTTCTTGCCTCTTTGA TAATCAAATGATCTGAAGAAAGGCATAGAATTTCAAGGGAGAATCTGCATGACAGGATTACAATA AGGCTATTCATGGAGACTCTTTTATTAGCTTACACAGGATCTGCATCATTCTTCGTCTCTGGCTT TGCTACAAGGCTCCATTTAAACTTAACCCAACTTGCAGGCTTAACTGATCCAGGAATGACTCAAT ACAATGAGCCAGCTACTGTAGCTCTTTTTCCCCTTTTGATAAGGGGAGTTAACACAATGGGCTTT ACAGTTCTTAAATGA SEQ ID NO: 120 is shown below: ACAGAGGACAACGACCACAGCCATCCCTGAACCCCGCCCACGGCACAGCGCCGGAGCCGGGGTCT GGGGCGCCGCTTCCTGGGGGGTCCCGACTCTCAGCCGCCCCCGCTTCACCCGGGCCGCCAAGGGG CTGGGGGAGGCGGCGCTCGGGGTAACCGGGGGAGACTCAGGGCGCTGGGGGCACTTGGGGAACTC ATGGGGGCTCAAAGGAACTAGGAGATCGGGACCTCGAAGGGGACTTGGGGGGTTCGGGGCTTTCG GGGGCGGTCGGGGGTTCGCGGACCCGGGAAGCTCTGAGGACCCAGAGGCCGGGCGCGCTCCGCCC GCGGCGCCGCCCCCTCCGTAACTTTCCCAGTCTCCGAGGGAAGAGGCGGGGTGTGGGGTGCGGTT AAAAGGCGCCACGGCGGGAGACAGGTGTTGCGGCCCCGCAGCGCCCGCGCGCTCCTCTCCCCGAC TCGGAGCCCCTCGGCGGCGCCCGGCCCAGGACCCGCCTAGGAGCGCAGGAGCCCCAGCGCAGAGA CCCCAACGCCGAGACCCCCGCCCCGGCCCCGCCGCGCTTCCTCCCGACGCAGGTGAGCCCGCCGG CCCCGGACTGCCCGGCCAGGAACCTGGCGCGGGGAGGGACCGCGAGACCCAGAGCGGTTGCCCGG CCGCGTGGGTCTCGGGGAACCGGGGGGCTGGACCAACACACGTCCTTGGGCCGGGGGGCGGGGGC CGCCTTCTGGAGCGGGCGTTTCTGTTTATGATGTGTTTAAAGATTGGGTGAATTACTCAGGTGAA CAAGCTACTTTTTATCAGAGAACACCTAAAAACACGTTCAAGAGGGTTTGGGAACTATACATTTA ATCCTATGACAAACTAAGTTGGTTCTGTCTTCACCTGTTTTGGTGAGGTTGTGTAAGAGTTGGTG TTTGCTCAGGAAGAGATTTAAGCATGCTTGCTTACCCAGACTCAGAGAAGTCTCCCTGTTCTGTC CTAGCTAGTGATTCCTGTGTTGTGTGCATTCGTCTTTTCCAGAGCAAACCGCCCAGAGTAGAAG gRNAs that specifically target a GJB2 promoter include, for example, gRNA_1, gRNA_2, gRNA_3, gRNA_4, gRNA_5, gRNA_6, gRNA_7, gRNA_8, gRNA_9, gRNA_10, gRNA_11, gRNA_12,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 gRNA_13, gRNA_14, gRNA_15, gRNA_24, gRNA_25, gRNA_26, gRNA_27, gRNA_28, gRNA_29, gRNA_30, gRNA_31, gRNA_32, gRNA_33, gRNA_34, gRNA_35, gRNA_36, and gRNA_37, corresponding to SEQ ID NOs: 62-76 and SEQ ID NOs: 85-98, respectively. gRNAs that specifically target an enhancer of GJB2 include, for example, gRNA_20, gRNA_21, gRNA_22, gRNA_23, gRNA_38, gRNA_39, gRNA_40, and gRNA_41, corresponding to SEQ ID NOs: 81-84 and SEQ ID NOs: 99-102, respectively.Target site-specific binding and cleavage of a target DNA by a Cas protein can occur at locations determined by both (i) base-pairing complementarity between the gRNA and the complementary strand of the target DNA and (ii) a short motif, called the protospacer adjacent motif (PAM), in the non- complementary strand of the target DNA. The PAM can flank the gRNA target sequence. Optionally, the gRNA target sequence can be flanked on the 3' end by the PAM (e.g., for Cas9). Alternatively, the g RNA target sequence can be flanked on the 5' end by the PAM (e.g., for Cpf1). For example, the cleavage site of Cas proteins can be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence (e.g., within the gRNA target sequence). In the case of SpCas9, the PAM sequence (i.e., on the non-complementary strand) can be 5'-N1GG-3', where N1 is any DNA nucleotide, and where the PAM is immediately 3' of the gRNA target sequence on the non-complementary strand of the target DNA. As such, the sequence corresponding to the PAM on the complementary strand (i.e., the reverse complement) would be 5'-CCN2-3', where N2 is any DNA nucleotide and is immediately 5' of the sequence to which the DNA-targeting segment of the gRNA hybridizes on the complementary strand of the target DNA. In some such cases, N1 and N2 can be complementary and the N1--N2 base pair can be any base pair (e.g., N1=C and N2=G; N1=G and N2=C; N1=A and N2=T; or N1=T, and N2=A). In the case of Cas9 from S. aureus, the PAM can be NNGRRT or NNGRR, where N can A, G, C, or T, and R can be G or A. In the case of Cas9 from C. jejuni, the PAM can be, for example, NNNNACAC or NNNNRYAC, where N can be A, G, C, or T, and R can be G or A In some cases (e.g., for FnCpf1), the PAM sequence can be upstream of the 5' end and have the sequence 5'-TTN-3'. An example of a gRNA target sequence is a 20-nucleotide DNA sequence immediately preceding an NGG motif recognized by an SpCas9 protein. For example, two examples of gRNA target sequences plus PAMs are GN19NGG or N20NGG. See, e.g., WO 2014 / 165825, herein incorporated by reference in its entirety for all purposes. The guanine at the 5' end can facilitate transcription by RNA polymerase in cells. Other examples of gRNA target sequences plus PAMs can include two guanine nucleotides at the 5' end (e.g., GGN20NGG) to facilitate efficient transcription by T7 polymerase in vitro. See, e.g., WO 2014 / 065596, herein incorporated by reference in its entirety for all purposes. Other gRNA target sequences plus PAMs can have between 4-22 nucleotides in length of GN19NGG, N20NGG, or GGN20NGG, including the 5' G or GG and the 3' GG or NGG. Yet other g RNA target sequences plus PAMs can have between 14 and 20 nucleotides in length of GN19NGG, N20NGG, or GGN20NGG. Formation of a CRISPR complex hybridized to a target DNA can result in cleavage of one or both strands of the target DNA within or near the region corresponding to the gRNA target sequence (i.e., the gRNA target sequence on the non-complementary strand of the target DNA and the reverse complementPATENT ATTORNEY DOCKET NO.: 51471-019WO2 on the complementary strand to which the gRNA hybridizes). For example, the cleavage site can be within the gRNA target sequence (e.g., at a defined location relative to the PAM sequence). The “cleavage site” includes the position of a target DNA at which a Cas protein produces a single-strand break or a double-strand break. The cleavage site can be on only one strand (e.g., when a nickase is used) or on both strands of a double-stranded DNA. Cleavage sites can be at the same position on both strands (producing blunt ends, e.g., Cas9)) or can be at different sites on each strand (producing staggered ends (i.e., overhangs); e.g., Cpf1). Staggered ends can be produced, for example, by using two Cas proteins, each of which produces a single-strand break at a different cleavage site on a different strand, thereby producing a double-strand break. For example, a first nickase can create a single-strand break on the first strand of double-stranded DNA (dsDNA), and a second nickase can create a single- strand break on the second strand of dsDNA such that overhanging sequences are created. In some cases, the gRNA target sequence or cleavage site of the nickase on the first strand is separated from the gRNA target sequence or cleavage site of the nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000 base pairs. Nucleic Acids Encoding Chimeric Cas Protein, Chimeric Adaptor Protein, guide RNA, or Synergistic Activation Mediator The chimeric Cas protein, chimeric adaptor protein, and gRNAs described in detail herein can be provided in the form of DNA in the methods and compositions disclosed herein. For example, the nucleic acids can be chimeric Cas protein expression cassettes, chimeric adaptor protein expression cassettes, SAM expression cassettes including nucleic acids encoding both a chimeric Cas protein and a chimeric adaptor protein, gRNA expression cassettes, or any combination thereof. Such nucleic acids can, can be single-stranded or double-stranded, and can be linear or circular. DNA can be part of a vector, such as an expression vector or a targeting vector. The vector can also be a viral vector such as adenoviral, AAV, lentiviral, and retroviral vectors. When any of the nucleic acids disclosed herein is introduced into a cell of the present disclosure, the encoded chimeric DNA-targeting protein, chimeric adaptor protein, or gRNA can be transiently, conditionally, or preferably constitutively expressed in the cell. Optionally, the nucleic acids can be codon-optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid can be modified to substitute codons having a higher frequency of usage in a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. The Cas protein, chimeric adaptor protein, and gRNAs can be provided in the form of DNA. DNA or expression cassettes can be for integration into the genome (i.e., into a chromosome) of a cell or eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) or it can be for expression outside of a chromosome (e.g., extrachromosomally replicating DNA). The integrated expression cassettes or nucleic acids can be randomly integrated into the genome of the eukaryotic organism or cell line (e.g., animal, non-human animal, mammal, or non-human mammal) (i.e.,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 transgenic), or they can be integrated into a predetermined region of the genome of the eukaryotic organism or cell line (e.g., animal, non-human animal, mammal, or non-human mammal) (i.e., knock in). A nucleic acid or expression cassette described herein can be operably linked to any suitable promoter for expression in vivo within a eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) or ex vivo within a cell according to the present disclosure. The eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) can be any suitable eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) as described herein. As one example, a nucleic acid or expression cassette (e.g., a chimeric Cas protein expression cassette, a chimeric adaptor protein expression cassette, or a SAM cassette including nucleic acids encoding both a chimeric Cas protein and a chimeric adaptor protein) can be for operably linking to an endogenous promoter at a genomic locus. Alternatively, cassette nucleic acid or expression cassette can be operably linked to an exogenous promoter, such as a constitutively active promoter (e.g., a CAG promoter or a U6 promoter), a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell- specific or tissue-specific promoter). Promoters that can be used in an expression construct include promoters active, for example, in one or more of a eukaryotic cell, a non-human eukaryotic cell, an animal cell, a non-human animal cell, a mammalian cell, a non-human mammalian cell, a human cell, a non- human cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, a rabbit cell, a pluripotent cell, an embryonic stem (ES) cell, or a zygote. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. For example, a nucleic acid encoding a gRNA can be operably linked to a U6 promoter, such as a human U6 promoter or a mouse U6 promoter. Specific examples of suitable promoters (e.g., for expressing a gRNA) include an RNA polymerase III promoter, such as a human U6 promoter, a rat U6 polymerase III promoter, or a mouse U6 polymerase III promoter. Optionally, the promoter can be a bidirectional promoter driving expression of one gene (e.g., a gene encoding a chimeric Cas protein) and a second gene (e.g., a gene encoding a gRNA or a chimeric adaptor protein) in the other direction. Such bidirectional promoters can consist of (1) a complete, conventional, unidirectional Pol III promoter that contains 3 external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basic Pol III promoter that includes a PSE and a TATA box fused to the 5' terminus of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and the promoter can be rendered bidirectional by creating a hybrid promoter in which transcription in the reverse direction is controlled by appending a PSE and TATA box derived from the U6 promoter. See, e.g., US 2016 / 0074535, herein incorporated by references in its entirety for all purposes. Use of a bidirectional promoter to express two genes simultaneously allows for the generation of compact expression cassettes to facilitate delivery. One or more of the nucleic acids can be together in a multicistronic expression construct. For example, a nucleic acid encoding a chimeric Cas protein and a nucleic acid encoding a chimeric adaptor protein can be together in a bicistronic expression construct. Multicistronic expression vectorsPATENT ATTORNEY DOCKET NO.: 51471-019WO2 simultaneously express two or more separate proteins from the same mRNA (i.e., a transcript produced from the same promoter). Suitable strategies for multicistronic expression of proteins include, for example, the use of a 2A peptide and the use of an internal ribosome entry site (IRES). For example, such constructs can include: (1) nucleic acids encoding one or more chimeric Cas proteins and one or more chimeric adaptor proteins; (2) nucleic acids encoding two or more chimeric adaptor proteins; (3) nucleic acids encoding two or more chimeric Cas proteins; (4) nucleic acids encoding two or more gRNAs; (5) nucleic acids encoding one or more chimeric Cas proteins and one or more gRNAs; (6) nucleic acids encoding one or more chimeric adaptor proteins and one or more gRNAs; or (7) nucleic acids encoding one or more chimeric Cas proteins, one or more chimeric adaptor proteins, and one or more gRNAs. As one example, such multicistronic vectors can use one or more internal ribosome entry sites (IRES) to allow for initiation of translation from an internal region of an mRNA. As another example, such multicistronic vectors can use one or more 2A peptides. These peptides are small “self-cleaving” peptides, generally having a length of 18-22 amino acids and produce equimolar levels of multiple genes from the same mRNA. Ribosomes skip the synthesis of a glycyl-prolyl peptide bond at the C-terminus of a 2A peptide, leading to the “cleavage” between a 2A peptide and its immediate downstream peptide. See, e.g., Kim et al. (2011) PLoS One 6(4): e18556, herein incorporated by reference in its entirety for all purposes. The “cleavage” occurs between the glycine and praline residues found on the C-terminus, meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the praline. As a result, the “cleaved-off'' downstream peptide has praline at its N- terminus.2A-mediated cleavage is a universal phenomenon in all eukaryotic cells.2A peptides have been identified from picornaviruses, insect viruses and type C rotaviruses. See, e.g., Szymczak et al. (2005) Expert Opin. Biol. Ther.5(5):627-638, herein incorporated by reference in its entirety for all purposes. Examples of 2A peptides that can be used include Thoseaasigna virus 2A (T2A); porcine teschovirus-12A (P2A); equine rhinitis A virus (ERAV) 2A (E2A); and FMDV 2A (F2A). Example T2A, P2A, E2A, and F2A sequences include the following: T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 124); P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 125); E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 126); and F2A (VKQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 127). GSG residues can be added to the 5' end of any of these peptides to improve cleavage efficiency. Any of the nucleic acids or expression cassettes can also include a polyadenylation signal or transcription terminator upstream of a coding sequence. For example, a chimeric Cas protein expression cassette, a chimeric adaptor protein expression cassette, a SAM expression cassette, or a gRNA expression cassette can include a polyadenylation signal or transcription terminator upstream of the coding sequence(s) in the expression cassette. The polyadenylation signal or transcription terminator can be flanked by recombinase recognition sites recognized by a site-specific recombinase. The polyadenylation signal or transcription terminator prevents transcription and expression of the protein or RNA encoded by the coding sequence (e.g., chimeric Cas protein, chimeric adaptor protein, gRNA, or recombinase). However, upon exposure to the site-specific recombinase, the polyadenylation signal or transcription terminator will be excised, and the protein or RNA can be expressed.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Such a configuration for an expression cassette (e.g., a chimeric Cas protein expression cassette or a SAM expression cassette) can enable tissue-specific expression or developmental- stage-specific expression in eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) including the expression cassette if the polyadenylation signal or transcription terminator is excised in a tissue-specific or developmental-stage-specific manner. For example, in the case of the chimeric Cas protein, this may reduce toxicity due to prolonged expression of the chimeric Cas protein in a cell or eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) or expression of the chimeric Cas protein at undesired developmental stages or in undesired cell or tissue types within a eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal). See, e.g., Parikh et al. (2015) PLoS One 10(1):e0116484, herein incorporated by reference in its entirety for all purposes. Excision of the polyadenylation signal or transcription terminator in a tissue-specific or developmental-stage-specific manner can be achieved if a eukaryotic organism (e.g., animal, non-human animal, mammal, or non-human mammal) including the expression cassette further includes a coding sequence for the site-specific recombinase operably linked to a tissue- specific or developmental-stage- specific promoter. The polyadenylation signal or transcription terminator will then be excised only in those tissues or at those developmental stages, enabling tissue-specific expression or developmental-stage- specific expression. In one example, a chimeric Cas protein, a chimeric adaptor protein, a chimeric Cas protein and a chimeric adaptor protein, or a gRNA can be expressed in an inner ear-specific manner. Any transcription terminator or polyadenylation signal can be used. A “transcription terminator” as used herein refers to a DNA sequence that causes termination of transcription. In eukaryotes, transcription terminators are recognized by protein factors, and termination is followed by polyadenylation, a process of adding a poly(A) tail to the mRNA transcripts in presence of the poly(A) polymerase. The mammalian poly(A) signal typically includes a core sequence, about 45 nucleotides long, that may be flanked by diverse auxiliary sequences that serve to enhance cleavage and polyadenylation efficiency. The core sequence includes a highly conserved upstream element (AATAAA or AAUAAA) in the mRNA, referred to as a poly A recognition motif or poly A recognition sequence), recognized by cleavage and polyadenylation-specificity factor (CPSF), and a poorly defined downstream region (rich in Us or Gs and Us), bound by cleavage stimulation factor (CstF). Examples of transcription terminators that can be used include, for example, the human growth hormone (HGH) polyadenylation signal, the simian virus 40 (SV40) late polyadenylation signal, the rabbit beta-globin polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the phosphoglycerate kinase (PGK) polyadenylation signal, an AOXI transcription termination sequence, a CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. Site-specific recombinases include enzymes that can facilitate recombination between recombinase recognition sites, where the two recombination sites are physically separated within a single nucleic acid or on separate nucleic acids. Examples of recombinases include Cre, F1p, and Dre recombinases. One example of a Cre recombinase gene is Crei, in which two exons encoding the Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell. SuchPATENT ATTORNEY DOCKET NO.: 51471-019WO2 recombinases can further include a nuclear localization signal to facilitate localization to the nucleus (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by a site- specific recombinase and can serve as a substrate for a recombination event. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2, and lox5171. The expression cassettes disclosed herein can include other components as well. Such expression cassettes (e.g., chimeric Cas protein expression cassette, chimeric adaptor protein expression cassette, SAM expression cassette, gRNA expression cassette, or recombinase expression cassette) can further include a 3' splicing sequence at the 5' end of the expression cassette and / or a second polyadenylation signal following the coding sequence (e.g., encoding the chimeric Cas protein, the chimeric adaptor protein, or the gRNA). The term 3' splicing sequence refers to a nucleic acid sequence at a 3' intron / exon boundary that can be recognized and bound by splicing machinery. An expression cassette can further include a selection cassette including, for example, the coding sequence for a drug resistance protein. Examples of suitable selection markers include neomycin phosphotransferase (neo.sup.r), hygromycin B phosphotransferase (hyg.sup.r), puromycin-N-acetyltransferase (puro.sup.r), blasticidin S deaminase (bsr.sup.r), xanthine / guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-k). Optionally, the selection cassette can be flanked by recombinase recognition sites for a site-specific recombinase. If the expression cassette also includes recombinase recognition sites flanking a polyadenylation signal upstream of the coding sequence as described above, the selection cassette can be flanked by the same recombinase recognition sites or can be flanked by a different set of recombinase recognition sites recognized by a different recombinase. An expression cassette can also include a nucleic acid encoding one or more reporter proteins, such as a fluorescent protein (e.g., a green fluorescent protein). Any suitable reporter protein can be used. For example, a fluorescent reporter protein can be used, or a non-fluorescent reporter protein can be used. Examples of fluorescent reporter proteins are provided herein. Non-fluorescent reporter proteins include, for example, reporter proteins that can be used in histochemical or bioluminescent assays, such as beta-galactosidase, luciferase (e.g., Renilla luciferase, firefly luciferase, and NanoLuc luciferase), and beta-glucuronidase. An expression cassette can include a reporter protein that can be detected in a flow cytometry assay (e.g., a fluorescent reporter protein such as a green fluorescent protein) and / or a reporter protein that can be detected in a histochemical assay (e.g., beta-galactosidase protein). One example of such a histochemical assay is visualization of in situ beta-galactosidase expression histochemically through hydrolysis of X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside), which yields a blue precipitate, or using fluorogenic substrates such as beta-methyl umbelliferyl galactoside (MUG) and fluorescein digalactoside (FDG). The expression cassettes described herein can be in any form. For example, an expression cassette can be in a vector or plasmid. The expression cassette can be operably linked to a promoter in an expression construct capable of directing expression of a protein or RNA (e.g., upon removal of an upstream polyadenylation signal). Alternatively, an expression cassette can be in a targeting vector. ForPATENT ATTORNEY DOCKET NO.: 51471-019WO2 example, the targeting vector can include homology arms flanking the expression cassette, in which the homology arms are suitable for directing recombination with a desired target genomic locus to facilitate genomic integration and / or replacement of endogenous sequence. A specific example of a nucleic acid encoding a catalytically inactive Cas protein can include a nucleic acid encoding an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dCas9 protein sequence set forth in SEQ ID NO: 2. Optionally, the nucleic acid can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 128 (optionally in which the nucleic acid sequence encodes a protein at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dCas9 protein sequence set forth in SEQ ID NO: 2). SEQ ID NO: 128 is shown below: ATGAAAAGGC CGGCGGCCAC GAAAAAGGCC GGCCAGGCAA AAAAGAAAAA GGACAAGAAG TACAGCATCG GCCTGGCCAT CGGCACCAAC TCTGTGGGCT GGGCCGTGAT CACCGACGAG TACAAGGTGC CCAGCAAGAA ATTCAAGGTG CTGGGCAACA CCGACCGGCA CAGCATCAAG AAGAACCTGA TCGGAGCCCT GCTGTTCGAC AGCGGCGAAA CAGCCGAGGC CACCCGGCTG AAGAGAACCG CCAGAAGAAG ATACACCAGA CGGAAGAACC GGATCTGCTA TCTGCAAGAG ATCTTCAGCA ACGAGATGGC CAAGGTGGAC GACAGCTTCT TCCACAGACT GGAAGAGTCC TTCCTGGTGG AAGAGGATAA GAAGCACGAG CGGCACCCCA TCTTCGGCAA CATCGTGGAC GAGGTGGCCT ACCACGAGAA GTACCCCACC ATCTACCACC TGAGAAAGAA ACTGGTGGAC AGCACCGACA AGGCCGACCT GCGGCTGATC TATCTGGCCC TGGCCCACAT GATCAAGTTC CGGGGCCACT TCCTGATCGA GGGCGACCTG AACCCCGACA ACAGCGACGT GGACAAGCTG TTCATCCAGC TGGTGCAGAC CTACAACCAG CTGTTCGAGG AAAACCCCAT CAACGCCAGC GGCGTGGACG CCAAGGCCAT CCTGTCTGCC AGACTGAGCA AGAGCAGACG GCTGGAAAAT CTGATCGCCC AGCTGCCCGG CGAGAAGAAG AATGGCCTGT TCGGCAACCT GATTGCCCTG AGCCTGGGCC TGACCCCCAA CTTCAAGAGC AACTTCGACC TGGCCGAGGA TGCCAAACTG CAGCTGAGCA AGGACACCTA CGACGACGAC CTGGACAACC TGCTGGCCCA GATCGGCGAC CAGTACGCCG ACCTGTTTCT GGCCGCCAAG AACCTGTCCG ACGCCATCCT GCTGAGCGAC ATCCTGAGAG TGAACACCGA GATCACCAAG GCCCCCCTGA GCGCCTCTAT GATCAAGAGA TACGACGAGC ACCACCAGGA CCTGACCCTG CTGAAAGCTC TCGTGCGGCA GCAGCTGCCT GAGAAGTACA AAGAGATTTT CTTCGACCAG AGCAAGAACG GCTACGCCGG CTACATTGAC GGCGGAGCCA GCCAGGAAGA GTTCTACAAG TTCATCAAGC CCATCCTGGA AAAGATGGAC GGCACCGAGG AACTGCTCGT GAAGCTGAAC AGAGAGGACC TGCTGCGGAA GCAGCGGACC TTCGACAACG GCAGCATCCC CCACCAGATC CACCTGGGAG AGCTGCACGC CATTCTGCGG CGGCAGGAAG ATTTTTACCC ATTCCTGAAG GACAACCGGG AAAAGATCGA GAAGATCCTG ACCTTCCGCA TCCCCTACTA CGTGGGCCCT CTGGCCAGGG GAAACAGCAG ATTCGCCTGG ATGACCAGAA AGAGCGAGGA AACCATCACC CCCTGGAACT TCGAGGAAGT GGTGGACAAG GGCGCTTCCG CCCAGAGCTT CATCGAGCGG ATGACCAACT TCGATAAGAA CCTGCCCAAC GAGAAGGTGC TGCCCAAGCA CAGCCTGCTG TACGAGTACT TCACCGTGTA TAACGAGCTG ACCAAAGTGA AATACGTGAC CGAGGGAATG AGAAAGCCCG CCTTCCTGAG CGGCGAGCAGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 AAAAAGGCCA TCGTGGACCT GCTGTTCAAG ACCAACCGGA AAGTGACCGT GAAGCAGCTG AAAGAGGACT ACTTCAAGAA AATCGAGTGC TTCGACTCCG TGGAAATCTC CGGCGTGGAA GATCGGTTCA ACGCCTCCCT GGGCACATAC CACGATCTGC TGAAAATTAT CAAGGACAAG GACTTCCTGG ACAATGAGGA AAACGAGGAC ATTCTGGAAG ATATCGTGCT GACCCTGACA CTGTTTGAGG ACAGAGAGAT GATCGAGGAA CGGCTGAAAA CCTATGCCCA CCTGTTCGAC GACAAAGTGA TGAAGCAGCT GAAGCGGCGG AGATACACCG GCTGGGGCAG GCTGAGCCGG AAGCTGATCA ACGGCATCCG GGACAAGCAG TCCGGCAAGA CAATCCTGGA TTTCCTGAAG TCCGACGGCT TCGCCAACAG AAACTTCATG CAGCTGATCC ACGACGACAG CCTGACCTTT AAAGAGGACA TCCAGAAAGC CCAGGTGTCC GGCCAGGGCG ATAGCCTGCA CGAGCACATT GCCAATCTGG CCGGCAGCCC CGCCATTAAG AAGGGCATCC TGCAGACAGT GAAGGTGGTG GACGAGCTCG TGAAAGTGAT GGGCCGGCAC AAGCCCGAGA ACATCGTGAT CGAAATGGCC AGAGAGAACC AGACCACCCA GAAGGGACAG AAGAACAGCC GCGAGAGAAT GAAGCGGATC GAAGAGGGCA TCAAAGAGCT GGGCAGCCAG ATCCTGAAAG AACACCCCGT GGAAAACACC CAGCTGCAGA ACGAGAAGCT GTACCTGTAC TACCTGCAGA ATGGGCGGGA TATGTACGTG GACCAGGAAC TGGACATCAA CCGGCTGTCC GACTACGATG TGGACCACAT CGTGCCTCAG AGCTTTCTGA AGGACGACTC CATCGACAAC AAGGTGCTGA CCAGAAGCGA CAAGGCCCGG GGCAAGAGCG ACAACGTGCC CTCCGAAGAG GTCGTGAAGA AGATGAAGAA CTACTGGCGG CAGCTGCTGA ACGCCAAGCT GATTACCCAG AGAAAGTTCG ACAATCTGAC CAAGGCCGAG AGAGGCGGCC TGAGCGAACT GGATAAGGCC GGCTTCATCA AGAGACAGCT GGTGGAAACC CGGCAGATCA CAAAGCACGT GGCACAGATC CTGGACTCCC GGATGAACAC TAAGTACGAC GAGAATGACA AGCTGATCCG GGAAGTGAAA GTGATCACCC TGAAGTCCAA GCTGGTGTCC GATTTCCGGA AGGATTTCCA GTTTTACAAA GTGCGCGAGA TCAACAACTA CCACCACGCC CACGACGCCT ACCTGAACGC CGTCGTGGGA ACCGCCCTGA TCAAAAAGTA CCCTAAGCTG GAAAGCGAGT TCGTGTACGG CGACTACAAG GTGTACGACG TGCGGAAGAT GATCGCCAAG AGCGAGCAGG AAATCGGCAA GGCTACCGCC AAGTACTTCT TCTACAGCAA CATCATGAAC TTTTTCAAGA CCGAGATTAC CCTGGCCAAC GGCGAGATCC GGAAGCGGCC TCTGATCGAG ACAAACGGCG AAACCGGGGA GATCGTGTGG GATAAGGGCC GGGATTTTGC CACCGTGCGG AAAGTGCTGA GCATGCCCCA AGTGAATATC GTGAAAAAGA CCGAGGTGCA GACAGGCGGC TTCAGCAAAG AGTCTATCCT GCCCAAGAGG AACAGCGATA AGCTGATCGC CAGAAAGAAG GACTGGGACC CTAAGAAGTA CGGCGGCTTC GACAGCCCCA CCGTGGCCTA TTCTGTGCTG GTGGTGGCCA AAGTGGAAAA GGGCAAGTCC AAGAAACTGA AGAGTGTGAA AGAGCTGCTG GGGATCACCA TCATGGAAAG AAGCAGCTTC GAGAAGAATC CCATCGACTT TCTGGAAGCC AAGGGCTACA AAGAAGTGAA AAAGGACCTG ATCATCAAGC TGCCTAAGTA CTCCCTGTTC GAGCTGGAAA ACGGCCGGAA GAGAATGCTG GCCTCTGCCG GCGAACTGCA GAAGGGAAAC GAACTGGCCC TGCCCTCCAA ATATGTGAAC TTCCTGTACC TGGCCAGCCA CTATGAGAAG CTGAAGGGCT CCCCCGAGGA TAATGAGCAG AAACAGCTGT TTGTGGAACA GCACAAGCAC TACCTGGACG AGATCATCGA GCAGATCAGC GAGTTCTCCA AGAGAGTGAT CCTGGCCGAC GCTAATCTGG ACAAAGTGCT GTCCGCCTAC AACAAGCACC GGGATAAGCC CATCAGAGAG CAGGCCGAGA ATATCATCCA CCTGTTTACC CTGACCAATC TGGGAGCCCC TGCCGCCTTCPATENT ATTORNEY DOCKET NO.: 51471-019WO2 AAGTACTTTG ACACCACCAT CGACCGGAAG AGGTACACCA GCACCAAAGA GGTGCTGGAC GCCACCCTGA TCCACCAGAG CATCACCGGC CTGTACGAGA CACGGATCGA CCTGTCTCAG CTGGGAGGCG AC A specific example of a nucleic acid encoding a chimeric Cas protein can include a nucleic acid encoding an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric Cas protein sequence set forth in SEQ ID NO: 1. Optionally, the nucleic acid can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 129 (optionally in which the nucleic acid sequence encodes a protein at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric Cas protein sequence set forth in SEQ ID NO: 1). SEQ ID NO: 129 is shown below: ATGAAAAGGC CGGCGGCCAC GAAAAAGGCC GGCCAGGCAA AAAAGAAAAA GGACAAGAAG TACAGCATCG GCCTGGCCAT CGGCACCAAC TCTGTGGGCT GGGCCGTGAT CACCGACGAG TACAAGGTGC CCAGCAAGAA ATTCAAGGTG CTGGGCAACA CCGACCGGCA CAGCATCAAG AAGAACCTGA TCGGAGCCCT GCTGTTCGAC AGCGGCGAAA CAGCCGAGGC CACCCGGCTG AAGAGAACCG CCAGAAGAAG ATACACCAGA CGGAAGAACC GGATCTGCTA TCTGCAAGAG ATCTTCAGCA ACGAGATGGC CAAGGTGGAC GACAGCTTCT TCCACAGACT GGAAGAGTCC TTCCTGGTGG AAGAGGATAA GAAGCACGAG CGGCACCCCA TCTTCGGCAA CATCGTGGAC GAGGTGGCCT ACCACGAGAA GTACCCCACC ATCTACCACC TGAGAAAGAA ACTGGTGGAC AGCACCGACA AGGCCGACCT GCGGCTGATC TATCTGGCCC TGGCCCACAT GATCAAGTTC CGGGGCCACT TCCTGATCGA GGGCGACCTG AACCCCGACA ACAGCGACGT GGACAAGCTG TTCATCCAGC TGGTGCAGAC CTACAACCAG CTGTTCGAGG AAAACCCCAT CAACGCCAGC GGCGTGGACG CCAAGGCCAT CCTGTCTGCC AGACTGAGCA AGAGCAGACG GCTGGAAAAT CTGATCGCCC AGCTGCCCGG CGAGAAGAAG AATGGCCTGT TCGGCAACCT GATTGCCCTG AGCCTGGGCC TGACCCCCAA CTTCAAGAGC AACTTCGACC TGGCCGAGGA TGCCAAACTG CAGCTGAGCA AGGACACCTA CGACGACGAC CTGGACAACC TGCTGGCCCA GATCGGCGAC CAGTACGCCG ACCTGTTTCT GGCCGCCAAG AACCTGTCCG ACGCCATCCT GCTGAGCGAC ATCCTGAGAG TGAACACCGA GATCACCAAG GCCCCCCTGA GCGCCTCTAT GATCAAGAGA TACGACGAGC ACCACCAGGA CCTGACCCTG CTGAAAGCTC TCGTGCGGCA GCAGCTGCCT GAGAAGTACA AAGAGATTTT CTTCGACCAG AGCAAGAACG GCTACGCCGG CTACATTGAC GGCGGAGCCA GCCAGGAAGA GTTCTACAAG TTCATCAAGC CCATCCTGGA AAAGATGGAC GGCACCGAGG AACTGCTCGT GAAGCTGAAC AGAGAGGACC TGCTGCGGAA GCAGCGGACC TTCGACAACG GCAGCATCCC CCACCAGATC CACCTGGGAG AGCTGCACGC CATTCTGCGG CGGCAGGAAG ATTTTTACCC ATTCCTGAAG GACAACCGGG AAAAGATCGA GAAGATCCTG ACCTTCCGCA TCCCCTACTA CGTGGGCCCT CTGGCCAGGG GAAACAGCAG ATTCGCCTGG ATGACCAGAA AGAGCGAGGA AACCATCACC CCCTGGAACT TCGAGGAAGT GGTGGACAAG GGCGCTTCCG CCCAGAGCTT CATCGAGCGG ATGACCAACT TCGATAAGAA CCTGCCCAAC GAGAAGGTGC TGCCCAAGCA CAGCCTGCTG TACGAGTACT TCACCGTGTA TAACGAGCTGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 ACCAAAGTGA AATACGTGAC CGAGGGAATG AGAAAGCCCG CCTTCCTGAG CGGCGAGCAG AAAAAGGCCA TCGTGGACCT GCTGTTCAAG ACCAACCGGA AAGTGACCGT GAAGCAGCTG AAAGAGGACT ACTTCAAGAA AATCGAGTGC TTCGACTCCG TGGAAATCTC CGGCGTGGAA GATCGGTTCA ACGCCTCCCT GGGCACATAC CACGATCTGC TGAAAATTAT CAAGGACAAG GACTTCCTGG ACAATGAGGA AAACGAGGAC ATTCTGGAAG ATATCGTGCT GACCCTGACA CTGTTTGAGG ACAGAGAGAT GATCGAGGAA CGGCTGAAAA CCTATGCCCA CCTGTTCGAC GACAAAGTGA TGAAGCAGCT GAAGCGGCGG AGATACACCG GCTGGGGCAG GCTGAGCCGG AAGCTGATCA ACGGCATCCG GGACAAGCAG TCCGGCAAGA CAATCCTGGA TTTCCTGAAG TCCGACGGCT TCGCCAACAG AAACTTCATG CAGCTGATCC ACGACGACAG CCTGACCTTT AAAGAGGACA TCCAGAAAGC CCAGGTGTCC GGCCAGGGCG ATAGCCTGCA CGAGCACATT GCCAATCTGG CCGGCAGCCC CGCCATTAAG AAGGGCATCC TGCAGACAGT GAAGGTGGTG GACGAGCTCG TGAAAGTGAT GGGCCGGCAC AAGCCCGAGA ACATCGTGAT CGAAATGGCC AGAGAGAACC AGACCACCCA GAAGGGACAG AAGAACAGCC GCGAGAGAAT GAAGCGGATC GAAGAGGGCA TCAAAGAGCT GGGCAGCCAG ATCCTGAAAG AACACCCCGT GGAAAACACC CAGCTGCAGA ACGAGAAGCT GTACCTGTAC TACCTGCAGA ATGGGCGGGA TATGTACGTG GACCAGGAAC TGGACATCAA CCGGCTGTCC GACTACGATG TGGACCACAT CGTGCCTCAG AGCTTTCTGA AGGACGACTC CATCGACAAC AAGGTGCTGA CCAGAAGCGA CAAGGCCCGG GGCAAGAGCG ACAACGTGCC CTCCGAAGAG GTCGTGAAGA AGATGAAGAA CTACTGGCGG CAGCTGCTGA ACGCCAAGCT GATTACCCAG AGAAAGTTCG ACAATCTGAC CAAGGCCGAG AGAGGCGGCC TGAGCGAACT GGATAAGGCC GGCTTCATCA AGAGACAGCT GGTGGAAACC CGGCAGATCA CAAAGCACGT GGCACAGATC CTGGACTCCC GGATGAACAC TAAGTACGAC GAGAATGACA AGCTGATCCG GGAAGTGAAA GTGATCACCC TGAAGTCCAA GCTGGTGTCC GATTTCCGGA AGGATTTCCA GTTTTACAAA GTGCGCGAGA TCAACAACTA CCACCACGCC CACGACGCCT ACCTGAACGC CGTCGTGGGA ACCGCCCTGA TCAAAAAGTA CCCTAAGCTG GAAAGCGAGT TCGTGTACGG CGACTACAAG GTGTACGACG TGCGGAAGAT GATCGCCAAG AGCGAGCAGG AAATCGGCAA GGCTACCGCC AAGTACTTCT TCTACAGCAA CATCATGAAC TTTTTCAAGA CCGAGATTAC CCTGGCCAAC GGCGAGATCC GGAAGCGGCC TCTGATCGAG ACAAACGGCG AAACCGGGGA GATCGTGTGG GATAAGGGCC GGGATTTTGC CACCGTGCGG AAAGTGCTGA GCATGCCCCA AGTGAATATC GTGAAAAAGA CCGAGGTGCA GACAGGCGGC TTCAGCAAAG AGTCTATCCT GCCCAAGAGG AACAGCGATA AGCTGATCGC CAGAAAGAAG GACTGGGACC CTAAGAAGTA CGGCGGCTTC GACAGCCCCA CCGTGGCCTA TTCTGTGCTG GTGGTGGCCA AAGTGGAAAA GGGCAAGTCC AAGAAACTGA AGAGTGTGAA AGAGCTGCTG GGGATCACCA TCATGGAAAG AAGCAGCTTC GAGAAGAATC CCATCGACTT TCTGGAAGCC AAGGGCTACA AAGAAGTGAA AAAGGACCTG ATCATCAAGC TGCCTAAGTA CTCCCTGTTC GAGCTGGAAA ACGGCCGGAA GAGAATGCTG GCCTCTGCCG GCGAACTGCA GAAGGGAAAC GAACTGGCCC TGCCCTCCAA ATATGTGAAC TTCCTGTACC TGGCCAGCCA CTATGAGAAG CTGAAGGGCT CCCCCGAGGA TAATGAGCAG AAACAGCTGT TTGTGGAACA GCACAAGCAC TACCTGGACG AGATCATCGA GCAGATCAGC GAGTTCTCCA AGAGAGTGAT CCTGGCCGAC GCTAATCTGG ACAAAGTGCT GTCCGCCTAC AACAAGCACC GGGATAAGCC CATCAGAGAGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 CAGGCCGAGA ATATCATCCA CCTGTTTACC CTGACCAATC TGGGAGCCCC TGCCGCCTTC AAGTACTTTG ACACCACCAT CGACCGGAAG AGGTACACCA GCACCAAAGA GGTGCTGGAC GCCACCCTGA TCCACCAGAG CATCACCGGC CTGTACGAGA CACGGATCGA CCTGTCTCAG CTGGGAGGCG ACAGCGCTGG AGGAGGTGGA AGCGGAGGAG GAGGAAGCGG AGGAGGAGGT AGCGGACCTA AGAAAAAGAG GAAGGTGGCG GCCGCTGGAT CCGGACGGGC TGACGCATTG GACGATTTTG ATCTGGATAT GCTGGGAAGT GACGCCCTCG ATGATTTTGA CCTTGACATG CTTGGTTCGG ATGCCCTTGA TGACTTTGAC CTCGACATGC TCGGCAGTGA CGCCCTTGAT GATTTCGACC TGGACATGCT GATTAACTGT ACAG A specific example of a nucleic acid encoding an adaptor can include a nucleic acid encoding an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MCP sequence set forth in SEQ ID NO: 10. Optionally, the nucleic acid can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 130 (optionally in which the nucleic acid sequence encodes a protein at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the MCP sequence set forth in SEQ ID NO: 10). SEQ ID NO: 130 is shown below: ATGGCTTCAA ACTTTACTCA GTTCGTGCTC GTGGACAATG GTGGGACAGG GGATGTGACA GTGGCTCCTT CTAATTTCGC TAATGGGGTG GCAGAGTGGA TCAGCTCCAA CTCACGGAGC CAGGCCTACA AGGTGACATG CAGCGTCAGG CAGTCTAGTG CCCAGAAGAG AAAGTATACC ATCAAGGTGG AGGTCCCCAA AGTGGCTACC CAGACAGTGG GCGGAGTCGA ACTGCCTGTC GCCGCTTGGA GGTCCTACCT GAACATGGAG CTCACTATCC CAATTTTCGC TACCAATTCT GACTGTGAAC TCATCGTGAA GGCAATGCAG GGGCTCCTCA AAGACGGTAA TCCTATCCCT TCCGCCATCG CCGCTAACTC AGGTATCTAC A specific example of a nucleic acid encoding a chimeric adaptor protein can include a nucleic acid encoding an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric adaptor protein sequence set forth in SEQ ID NO: 9. Optionally, the nucleic acid can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 131 (optionally in which the nucleic acid sequence encodes a protein at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric adaptor protein sequence set forth in SEQ ID NO: 9). SEQ ID NO: 131 is shown below: ATGGCTTCAA ACTTTACTCA GTTCGTGCTC GTGGACAATG GTGGGACAGG GGATGTGACA GTGGCTCCTT CTAATTTCGC TAATGGGGTG GCAGAGTGGA TCAGCTCCAA CTCACGGAGC CAGGCCTACA AGGTGACATG CAGCGTCAGG CAGTCTAGTG CCCAGAAGAG AAAGTATACC ATCAAGGTGG AGGTCCCCAA AGTGGCTACC CAGACAGTGG GCGGAGTCGA ACTGCCTGTC GCCGCTTGGA GGTCCTACCT GAACATGGAG CTCACTATCC CAATTTTCGC TACCAATTCT GACTGTGAAC TCATCGTGAA GGCAATGCAG GGGCTCCTCA AAGACGGTAA TCCTATCCCTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 TCCGCCATCG CCGCTAACTC AGGTATCTAC AGCGCTGGAG GAGGTGGAAG CGGAGGAGGA GGAAGCGGAG GAGGAGGTAG CGGACCTAAG AAAAAGAGGA AGGTGGCGGC CGCTGGATCC CCTTCAGGGC AGATCAGCAA CCAGGCCCTG GCTCTGGCCC CTAGCTCCGC TCCAGTGCTG GCCCAGACTA TGGTGCCCTC TAGTGCTATG GTGCCTCTGG CCCAGCCACC TGCTCCAGCC CCTGTGCTGA CCCCAGGACC ACCCCAGTCA CTGAGCGCTC CAGTGCCCAA GTCTACACAG GCCGGCGAGG GGACTCTGAG TGAAGCTCTG CTGCACCTGC AGTTCGACGC TGATGAGGAC CTGGGAGCTC TGCTGGGGAA CAGCACCGAT CCCGGAGTGT TCACAGATCT GGCCTCCGTG GACAACTCTG AGTTTCAGCA GCTGCTGAAT CAGGGCGTGT CCATGTCTCA TAGTACAGCC GAACCAATGC TGATGGAGTA CCCCGAAGCC ATTACCCGGC TGGTGACCGG CAGCCAGCGG CCCCCCGACC CCGCTCCAAC TCCCCTGGGA ACCAGCGGCC TGCCTAATGG GCTGTCCGGA GATGAAGACT TCTCAAGCAT CGCTGATATG GACTTTAGTG CCCTGCTGTC ACAGATTTCC TCTAGTGGGC AGGGAGGAGG TGGAAGCGGC TTCAGCGTGG ACACCAGTGC CCTGCTGGAC CTGTTCAGCC CCTCGGTGAC CGTGCCCGAC ATGAGCCTGC CTGACCTTGA CAGCAGCCTG GCCAGTATCC AAGAGCTCCT GTCTCCCCAG GAGCCCCCCA GGCCTCCCGA GGCAGAGAAC AGCAGCCCGG ATTCAGGGAA GCAGCTGGTG CACTACACAG CGCAGCCGCT GTTCCTGCTG GACCCCGGCT CCGTGGACAC CGGGAGCAAC GACCTGCCGG TGCTGTTTGA GCTGGGAGAG GGCTCCTACT TCTCCGAAGG GGACGGCTTC GCCGAGGACC CCACCATCTC CCTGCTGACA GGCTCGGAGC CTCCCAAAGC CAAGGACCCC ACTGTCTCC Specific examples of nucleic acids encoding transcriptional activation domains can include a nucleic acid encoding an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VP64, p65, or HSF1 sequences set forth in SEQ ID NO: 6, 11, or 12, respectively. Optionally, the nucleic acid can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 132, 133, or 134, respectively (optionally in which the nucleic acid sequence encodes a protein at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VP64, p65, or HSF1 sequences set forth in SEQ ID NO: 6, 11, or 12, respectively). SEQ ID NO: 132 is shown below: GCGGCCGCTG GATCCGGACG GGCTGACGCA TTGGACGATT TTGATCTGGA TATGCTGGGA AGTGACGCCC TCGATGATTT TGACCTTGAC ATGCTTGGTT CGGATGCCCT TGATGACTTT GACCTCGACA TGCTCGGCAG TGACGCCCTT GATGATTTCG ACCTGGACAT GCTGATTAAC TGTACAG SEQ ID NO: 133 is shown below: CCTTCAGGGC AGATCAGCAA CCAGGCCCTG GCTCTGGCCC CTAGCTCCGC TCCAGTGCTG GCCCAGACTA TGGTGCCCTC TAGTGCTATG GTGCCTCTGG CCCAGCCACC TGCTCCAGCC CCTGTGCTGA CCCCAGGACC ACCCCAGTCA CTGAGCGCTC CAGTGCCCAA GTCTACACAG GCCGGCGAGG GGACTCTGAG TGAAGCTCTG CTGCACCTGC AGTTCGACGC TGATGAGGAC CTGGGAGCTC TGCTGGGGAA CAGCACCGAT CCCGGAGTGT TCACAGATCT GGCCTCCGTGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GACAACTCTG AGTTTCAGCA GCTGCTGAAT CAGGGCGTGT CCATGTCTCA TAGTACAGCC GAACCAATGC TGATGGAGTA CCCCGAAGCC ATTACCCGGC TGGTGACCGG CAGCCAGCGG CCCCCCGACC CCGCTCCAAC TCCCCTGGGA ACCAGCGGCC TGCCTAATGG GCTGTCCGGA GATGAAGACT TCTCAAGCAT CGCTGATATG GACTTTAGTG CCCTGCTGTC ACAGATTTCC TCT SEQ ID NO: 134 is shown below: GGCTTCAGCG TGGACACCAG TGCCCTGCTG GACCTGTTCA GCCCCTCGGT GACCGTGCCC GACATGAGCC TGCCTGACCT TGACAGCAGC CTGGCCAGTA TCCAAGAGCT CCTGTCTCCC CAGGAGCCCC CCAGGCCTCC CGAGGCAGAG AACAGCAGCC CGGATTCAGG GAAGCAGCTG GTGCACTACA CAGCGCAGCC GCTGTTCCTG CTGGACCCCG GCTCCGTGGA CACCGGGAGC AACGACCTGC CGGTGCTGTT TGAGCTGGGA GAGGGCTCCT ACTTCTCCGA AGGGGACGGC TTCGCCGAGG ACCCCACCAT CTCCCTGCTG ACAGGCTCGG AGCCTCCCAA AGCCAAGGAC CCCACTGTCT CC One example of a SAM expression cassette includes from 5' to 3': (a) a 3' splicing sequence; (b) a first recombinase recognition site (e.g., loxP site); (c) a coding sequence for a drug resistance gene (e.g., neomycin phosphotransferase (neon) coding sequence); (d) a polyadenylation signal; (e) a second recombinase recognition site (e.g., loxP site); (f) a chimeric Cas protein coding sequence (e.g., dCas9- NLS-VP64 fusion protein); (g) a 2A protein coding sequence (e.g., a T2A coding sequence); and (e) a chimeric adaptor protein coding sequence (e.g., MCP-NLS-p65-HSF1). See, e.g., SEQ ID NO: 135 (coding sequence set forth in SEQ ID NO: 136 and encoding protein set forth in SEQ ID NO: 137, with the mRNA sequence set forth in SEQ ID NO: 138). SEQ ID NO: 135 is shown below: ATAACTTCGT ATAATGTATG CTATACGAAG TTATTAGGTC CCTCGACCTG CAGGAATTGT TGACAATTAA TCATCGGCAT AGTATATCGG CATAGTATAA TACGACAAGG TGAGGAACTA AACCATGGGA TCGGCCATTG AACAAGATGG ATTGCACGCA GGTTCTCCGG CCGCTTGGGT GGAGAGGCTA TTCGGCTATG ACTGGGCACA ACAGACAATC GGCTGCTCTG ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT TCTTTTTGTC AAGACCGACC TGTCCGGTGC CCTGAATGAA CTGCAGGACG AGGCAGCGCG GCTATCGTGG CTGGCCACGA CGGGCGTTCC TTGCGCAGCT GTGCTCGACG TTGTCACTGA AGCGGGAAGG GACTGGCTGC TATTGGGCGA AGTGCCGGGG CAGGATCTCC TGTCATCTCA CCTTGCTCCT GCCGAGAAAG TATCCATCAT GGCTGATGCA ATGCGGCGGC TGCATACGCT TGATCCGGCT ACCTGCCCAT TCGACCACCA AGCGAAACAT CGCATCGAGC GAGCACGTAC TCGGATGGAA GCCGGTCTTG TCGATCAGGA TGATCTGGAC GAAGAGCATC AGGGGCTCGC GCCAGCCGAA CTGTTCGCCA GGCTCAAGGC GCGCATGCCC GACGGCGATG ATCTCGTCGT GACCCATGGC GATGCCTGCT TGCCGAATAT CATGGTGGAA AATGGCCGCT TTTCTGGATT CATCGACTGT GGCCGGCTGG GTGTGGCGGA CCGCTATCAG GACATAGCGT TGGCTACCCG TGATATTGCT GAAGAGCTTG GCGGCGAATG GGCTGACCGC TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 CTATCGCCTT CTTGACGAGT TCTTCTGAGG GGATCCGCTG TAAGTCTGCA GAAATTGATG ATCTATTAAA CAATAAAGAT GTCCACTAAA ATGGAAGTTT TTCCTGTCAT ACTTTGTTAA GAAGGGTGAG AACAGAGTAC CTACATTTTG AATGGAAGGA TTGGAGCTAC GGGGGTGGGG GTGGGGTGGG ATTAGATAAA TGCCTGCTCT TTACTGAAGG CTCTTTACTA TTGCTTTATG ATAATGTTTC ATAGTTGGAT ATCATAATTT AAACAAGCAA AACCAAATTA AGGGCCAGCT CATTCCTCCC ACTCATGATC TATAGATCTA TAGATCTCTC GTGGGATCAT TGTTTTTCTC TTGATTCCCA CTTTGTGGTT CTAAGTACTG TGGTTTCCAA ATGTGTCAGT TTCATAGCCT GAAGAACGAG ATCAGCAGCC TCTGTTCCAC ATACACTTCA TTCTCAGTAT TGTTTTGCCA AGTTCTAATT CCATCAGAAG CTTGCAGATC TGCGACTCTA GAGGATCTGC GACTCTAGAG GATCATAATC AGCCATACCA CATTTGTAGA GGTTTTACTT GCTTTAAAAA ACCTCCCACA CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTATC ATGTCTGGAT CTGCGACTCT AGAGGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT GGATCTGCGA CTCTAGAGGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGATCC CCATCAAGCT GATCCGGAAC CCTTAATATA ACTTCGTATA ATGTATGCTA TACGAAGTTA TTAGGTCCCT CGACCTGCAG CCCAAGCTAG TGCCCGGGAA TTCGCTAGGG CCACCATGAA AAGGCCGGCG GCCACGAAAA AGGCCGGCCA GGCAAAAAAG AAAAAGGACA AGAAGTACAG CATCGGCCTG GCCATCGGCA CCAACTCTGT GGGCTGGGCC GTGATCACCG ACGAGTACAA GGTGCCCAGC AAGAAATTCA AGGTGCTGGG CAACACCGAC CGGCACAGCA TCAAGAAGAA CCTGATCGGA GCCCTGCTGT TCGACAGCGG CGAAACAGCC GAGGCCACCC GGCTGAAGAG AACCGCCAGA AGAAGATACA CCAGACGGAA GAACCGGATC TGCTATCTGC AAGAGATCTT CAGCAACGAG ATGGCCAAGG TGGACGACAG CTTCTTCCAC AGACTGGAAG AGTCCTTCCT GGTGGAAGAG GATAAGAAGC ACGAGCGGCA CCCCATCTTC GGCAACATCG TGGACGAGGT GGCCTACCAC GAGAAGTACC CCACCATCTA CCACCTGAGA AAGAAACTGG TGGACAGCAC CGACAAGGCC GACCTGCGGC TGATCTATCT GGCCCTGGCC CACATGATCA AGTTCCGGGG CCACTTCCTG ATCGAGGGCG ACCTGAACCC CGACAACAGC GACGTGGACA AGCTGTTCAT CCAGCTGGTG CAGACCTACA ACCAGCTGTT CGAGGAAAAC CCCATCAACG CCAGCGGCGT GGACGCCAAG GCCATCCTGT CTGCCAGACT GAGCAAGAGC AGACGGCTGG AAAATCTGAT CGCCCAGCTG CCCGGCGAGA AGAAGAATGG CCTGTTCGGC AACCTGATTG CCCTGAGCCT GGGCCTGACC CCCAACTTCA AGAGCAACTT CGACCTGGCC GAGGATGCCA AACTGCAGCT GAGCAAGGAC ACCTACGACG ACGACCTGGA CAACCTGCTG GCCCAGATCG GCGACCAGTA CGCCGACCTG TTTCTGGCCG CCAAGAACCTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GTCCGACGCC ATCCTGCTGA GCGACATCCT GAGAGTGAAC ACCGAGATCA CCAAGGCCCC CCTGAGCGCC TCTATGATCA AGAGATACGA CGAGCACCAC CAGGACCTGA CCCTGCTGAA AGCTCTCGTG CGGCAGCAGC TGCCTGAGAA GTACAAAGAG ATTTTCTTCG ACCAGAGCAA GAACGGCTAC GCCGGCTACA TTGACGGCGG AGCCAGCCAG GAAGAGTTCT ACAAGTTCAT CAAGCCCATC CTGGAAAAGA TGGACGGCAC CGAGGAACTG CTCGTGAAGC TGAACAGAGA GGACCTGCTG CGGAAGCAGC GGACCTTCGA CAACGGCAGC ATCCCCCACC AGATCCACCT GGGAGAGCTG CACGCCATTC TGCGGCGGCA GGAAGATTTT TACCCATTCC TGAAGGACAA CCGGGAAAAG ATCGAGAAGA TCCTGACCTT CCGCATCCCC TACTACGTGG GCCCTCTGGC CAGGGGAAAC AGCAGATTCG CCTGGATGAC CAGAAAGAGC GAGGAAACCA TCACCCCCTG GAACTTCGAG GAAGTGGTGG ACAAGGGCGC TTCCGCCCAG AGCTTCATCG AGCGGATGAC CAACTTCGAT AAGAACCTGC CCAACGAGAA GGTGCTGCCC AAGCACAGCC TGCTGTACGA GTACTTCACC GTGTATAACG AGCTGACCAA AGTGAAATAC GTGACCGAGG GAATGAGAAA GCCCGCCTTC CTGAGCGGCG AGCAGAAAAA GGCCATCGTG GACCTGCTGT TCAAGACCAA CCGGAAAGTG ACCGTGAAGC AGCTGAAAGA GGACTACTTC AAGAAAATCG AGTGCTTCGA CTCCGTGGAA ATCTCCGGCG TGGAAGATCG GTTCAACGCC TCCCTGGGCA CATACCACGA TCTGCTGAAA ATTATCAAGG ACAAGGACTT CCTGGACAAT GAGGAAAACG AGGACATTCT GGAAGATATC GTGCTGACCC TGACACTGTT TGAGGACAGA GAGATGATCG AGGAACGGCT GAAAACCTAT GCCCACCTGT TCGACGACAA AGTGATGAAG CAGCTGAAGC GGCGGAGATA CACCGGCTGG GGCAGGCTGA GCCGGAAGCT GATCAACGGC ATCCGGGACA AGCAGTCCGG CAAGACAATC CTGGATTTCC TGAAGTCCGA CGGCTTCGCC AACAGAAACT TCATGCAGCT GATCCACGAC GACAGCCTGA CCTTTAAAGA GGACATCCAG AAAGCCCAGG TGTCCGGCCA GGGCGATAGC CTGCACGAGC ACATTGCCAA TCTGGCCGGC AGCCCCGCCA TTAAGAAGGG CATCCTGCAG ACAGTGAAGG TGGTGGACGA GCTCGTGAAA GTGATGGGCC GGCACAAGCC CGAGAACATC GTGATCGAAA TGGCCAGAGA GAACCAGACC ACCCAGAAGG GACAGAAGAA CAGCCGCGAG AGAATGAAGC GGATCGAAGA GGGCATCAAA GAGCTGGGCA GCCAGATCCT GAAAGAACAC CCCGTGGAAA ACACCCAGCT GCAGAACGAG AAGCTGTACC TGTACTACCT GCAGAATGGG CGGGATATGT ACGTGGACCA GGAACTGGAC ATCAACCGGC TGTCCGACTA CGATGTGGAC CACATCGTGC CTCAGAGCTT TCTGAAGGAC GACTCCATCG ACAACAAGGT GCTGACCAGA AGCGACAAGG CCCGGGGCAA GAGCGACAAC GTGCCCTCCG AAGAGGTCGT GAAGAAGATG AAGAACTACT GGCGGCAGCT GCTGAACGCC AAGCTGATTA CCCAGAGAAA GTTCGACAAT CTGACCAAGG CCGAGAGAGG CGGCCTGAGC GAACTGGATA AGGCCGGCTT CATCAAGAGA CAGCTGGTGG AAACCCGGCA GATCACAAAG CACGTGGCAC AGATCCTGGA CTCCCGGATG AACACTAAGT ACGACGAGAA TGACAAGCTG ATCCGGGAAG TGAAAGTGAT CACCCTGAAG TCCAAGCTGG TGTCCGATTT CCGGAAGGAT TTCCAGTTTT ACAAAGTGCG CGAGATCAAC AACTACCACC ACGCCCACGA CGCCTACCTG AACGCCGTCG TGGGAACCGC CCTGATCAAA AAGTACCCTA AGCTGGAAAG CGAGTTCGTG TACGGCGACT ACAAGGTGTA CGACGTGCGG AAGATGATCG CCAAGAGCGA GCAGGAAATC GGCAAGGCTA CCGCCAAGTA CTTCTTCTAC AGCAACATCA TGAACTTTTT CAAGACCGAG ATTACCCTGG CCAACGGCGA GATCCGGAAG CGGCCTCTGA TCGAGACAAA CGGCGAAACC GGGGAGATCG TGTGGGATAAPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GGGCCGGGAT TTTGCCACCG TGCGGAAAGT GCTGAGCATG CCCCAAGTGA ATATCGTGAA AAAGACCGAG GTGCAGACAG GCGGCTTCAG CAAAGAGTCT ATCCTGCCCA AGAGGAACAG CGATAAGCTG ATCGCCAGAA AGAAGGACTG GGACCCTAAG AAGTACGGCG GCTTCGACAG CCCCACCGTG GCCTATTCTG TGCTGGTGGT GGCCAAAGTG GAAAAGGGCA AGTCCAAGAA ACTGAAGAGT GTGAAAGAGC TGCTGGGGAT CACCATCATG GAAAGAAGCA GCTTCGAGAA GAATCCCATC GACTTTCTGG AAGCCAAGGG CTACAAAGAA GTGAAAAAGG ACCTGATCAT CAAGCTGCCT AAGTACTCCC TGTTCGAGCT GGAAAACGGC CGGAAGAGAA TGCTGGCCTC TGCCGGCGAA CTGCAGAAGG GAAACGAACT GGCCCTGCCC TCCAAATATG TGAACTTCCT GTACCTGGCC AGCCACTATG AGAAGCTGAA GGGCTCCCCC GAGGATAATG AGCAGAAACA GCTGTTTGTG GAACAGCACA AGCACTACCT GGACGAGATC ATCGAGCAGA TCAGCGAGTT CTCCAAGAGA GTGATCCTGG CCGACGCTAA TCTGGACAAA GTGCTGTCCG CCTACAACAA GCACCGGGAT AAGCCCATCA GAGAGCAGGC CGAGAATATC ATCCACCTGT TTACCCTGAC CAATCTGGGA GCCCCTGCCG CCTTCAAGTA CTTTGACACC ACCATCGACC GGAAGAGGTA CACCAGCACC AAAGAGGTGC TGGACGCCAC CCTGATCCAC CAGAGCATCA CCGGCCTGTA CGAGACACGG ATCGACCTGT CTCAGCTGGG AGGCGACAGC GCTGGAGGAG GTGGAAGCGG AGGAGGAGGA AGCGGAGGAG GAGGTAGCGG ACCTAAGAAA AAGAGGAAGG TGGCGGCCGC TGGATCCGGA CGGGCTGACG CATTGGACGA TTTTGATCTG GATATGCTGG GAAGTGACGC CCTCGATGAT TTTGACCTTG ACATGCTTGG TTCGGATGCC CTTGATGACT TTGACCTCGA CATGCTCGGC AGTGACGCCC TTGATGATTT CGACCTGGAC ATGCTGATTA ACTGTACAGG CAGTGGAGAG GGCAGAGGAA GTCTGCTAAC ATGCGGTGAC GTCGAGGAGA ATCCTGGCCC AATGGCTTCA AACTTTACTC AGTTCGTGCT CGTGGACAAT GGTGGGACAG GGGATGTGAC AGTGGCTCCT TCTAATTTCG CTAATGGGGT GGCAGAGTGG ATCAGCTCCA ACTCACGGAG CCAGGCCTAC AAGGTGACAT GCAGCGTCAG GCAGTCTAGT GCCCAGAAGA GAAAGTATAC CATCAAGGTG GAGGTCCCCA AAGTGGCTAC CCAGACAGTG GGCGGAGTCG AACTGCCTGT CGCCGCTTGG AGGTCCTACC TGAACATGGA GCTCACTATC CCAATTTTCG CTACCAATTC TGACTGTGAA CTCATCGTGA AGGCAATGCA GGGGCTCCTC AAAGACGGTA ATCCTATCCC TTCCGCCATC GCCGCTAACT CAGGTATCTA CAGCGCTGGA GGAGGTGGAA GCGGAGGAGG AGGAAGCGGA GGAGGAGGTA GCGGACCTAA GAAAAAGAGG AAGGTGGCGG CCGCTGGATC CCCTTCAGGG CAGATCAGCA ACCAGGCCCT GGCTCTGGCC CCTAGCTCCG CTCCAGTGCT GGCCCAGACT ATGGTGCCCT CTAGTGCTAT GGTGCCTCTG GCCCAGCCAC CTGCTCCAGC CCCTGTGCTG ACCCCAGGAC CACCCCAGTC ACTGAGCGCT CCAGTGCCCA AGTCTACACA GGCCGGCGAG GGGACTCTGA GTGAAGCTCT GCTGCACCTG CAGTTCGACG CTGATGAGGA CCTGGGAGCT CTGCTGGGGA ACAGCACCGA TCCCGGAGTG TTCACAGATC TGGCCTCCGT GGACAACTCT GAGTTTCAGC AGCTGCTGAA TCAGGGCGTG TCCATGTCTC ATAGTACAGC CGAACCAATG CTGATGGAGT ACCCCGAAGC CATTACCCGG CTGGTGACCG GCAGCCAGCG GCCCCCCGAC CCCGCTCCAA CTCCCCTGGG AACCAGCGGC CTGCCTAATG GGCTGTCCGG AGATGAAGAC TTCTCAAGCA TCGCTGATAT GGACTTTAGT GCCCTGCTGT CACAGATTTC CTCTAGTGGG CAGGGAGGAG GTGGAAGCGG CTTCAGCGTG GACACCAGTG CCCTGCTGGA CCTGTTCAGC CCCTCGGTGA CCGTGCCCGA CATGAGCCTG CCTGACCTTG ACAGCAGCCTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GGCCAGTATC CAAGAGCTCC TGTCTCCCCA GGAGCCCCCC AGGCCTCCCG AGGCAGAGAA CAGCAGCCCG GATTCAGGGA AGCAGCTGGT GCACTACACA GCGCAGCCGC TGTTCCTGCT GGACCCCGGC TCCGTGGACA CCGGGAGCAA CGACCTGCCG GTGCTGTTTG AGCTGGGAGA GGGCTCCTAC TTCTCCGAAG GGGACGGCTT CGCCGAGGAC CCCACCATCT CCCTGCTGAC AGGCTCGGAG CCTCCCAAAG CCAAGGACCC CACTGTCTCC TGAGAATTCG ATATCAAGCT TATCGATAAT CAACCTCTGG ATTACAAAAT TTGTGAAAGA TTGACTGGTA TTCTTAACTA TGTTGCTCCT TTTACGCTAT GTGGATACGC TGCTTTAATG CCTTTGTATC ATGCTATTGC TTCCCGTATG GCTTTCATTT TCTCCTCCTT GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCA GGCAACGTGG CGTGGTGTGC ACTGTGTTTG CTGACGCAAC CCCCACTGGT TGGGGCATTG CCACCACCTG TCAGCTCCTT TCCGGGACTT TCGCTTTCCC CCTCCCTATT GCCACGGCGG AACTCATCGC CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TCGGCTGTTG GGCACTGACA ATTCCGTGGT GTTGTCGGGG AAATCATCGT CCTTTCCTTG GCTGCTCGCC TGTGTTGCCA CCTGGATTCT GCGCGGGACG TCCTTCTGCT ACGTCCCTTC GGCCCTCAAT CCAGCGGACC TTCCTTCCCG CGGCCTGCTG CCGGCTCTGC GGCCTCTTCC GCGTCTTCGC CTTCGCCCTC AGACGAGTCG GATCTCCCTT TGGGCCGCCT CCCCGCATCG ATACCGTCGA CCTCGACCTC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC AATGGCAGGC ATG SEQ ID NO: 136 is shown below: ATGAAAAGGC CGGCGGCCAC GAAAAAGGCC GGCCAGGCAA AAAAGAAAAA GGACAAGAAG TACAGCATCG GCCTGGCCAT CGGCACCAAC TCTGTGGGCT GGGCCGTGAT CACCGACGAG TACAAGGTGC CCAGCAAGAA ATTCAAGGTG CTGGGCAACA CCGACCGGCA CAGCATCAAG AAGAACCTGA TCGGAGCCCT GCTGTTCGAC AGCGGCGAAA CAGCCGAGGC CACCCGGCTG AAGAGAACCG CCAGAAGAAG ATACACCAGA CGGAAGAACC GGATCTGCTA TCTGCAAGAG ATCTTCAGCA ACGAGATGGC CAAGGTGGAC GACAGCTTCT TCCACAGACT GGAAGAGTCC TTCCTGGTGG AAGAGGATAA GAAGCACGAG CGGCACCCCA TCTTCGGCAA CATCGTGGAC GAGGTGGCCT ACCACGAGAA GTACCCCACC ATCTACCACC TGAGAAAGAA ACTGGTGGAC AGCACCGACA AGGCCGACCT GCGGCTGATC TATCTGGCCC TGGCCCACAT GATCAAGTTC CGGGGCCACT TCCTGATCGA GGGCGACCTG AACCCCGACA ACAGCGACGT GGACAAGCTG TTCATCCAGC TGGTGCAGAC CTACAACCAG CTGTTCGAGG AAAACCCCAT CAACGCCAGC GGCGTGGACG CCAAGGCCAT CCTGTCTGCC AGACTGAGCA AGAGCAGACG GCTGGAAAAT CTGATCGCCC AGCTGCCCGG CGAGAAGAAG AATGGCCTGT TCGGCAACCT GATTGCCCTG AGCCTGGGCC TGACCCCCAA CTTCAAGAGC AACTTCGACC TGGCCGAGGA TGCCAAACTG CAGCTGAGCA AGGACACCTA CGACGACGAC CTGGACAACC TGCTGGCCCA GATCGGCGAC CAGTACGCCG ACCTGTTTCT GGCCGCCAAG AACCTGTCCG ACGCCATCCT GCTGAGCGAC ATCCTGAGAG TGAACACCGA GATCACCAAG GCCCCCCTGA GCGCCTCTAT GATCAAGAGA TACGACGAGC ACCACCAGGA CCTGACCCTG CTGAAAGCTC TCGTGCGGCA GCAGCTGCCTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GAGAAGTACA AAGAGATTTT CTTCGACCAG AGCAAGAACG GCTACGCCGG CTACATTGAC GGCGGAGCCA GCCAGGAAGA GTTCTACAAG TTCATCAAGC CCATCCTGGA AAAGATGGAC GGCACCGAGG AACTGCTCGT GAAGCTGAAC AGAGAGGACC TGCTGCGGAA GCAGCGGACC TTCGACAACG GCAGCATCCC CCACCAGATC CACCTGGGAG AGCTGCACGC CATTCTGCGG CGGCAGGAAG ATTTTTACCC ATTCCTGAAG GACAACCGGG AAAAGATCGA GAAGATCCTG ACCTTCCGCA TCCCCTACTA CGTGGGCCCT CTGGCCAGGG GAAACAGCAG ATTCGCCTGG ATGACCAGAA AGAGCGAGGA AACCATCACC CCCTGGAACT TCGAGGAAGT GGTGGACAAG GGCGCTTCCG CCCAGAGCTT CATCGAGCGG ATGACCAACT TCGATAAGAA CCTGCCCAAC GAGAAGGTGC TGCCCAAGCA CAGCCTGCTG TACGAGTACT TCACCGTGTA TAACGAGCTG ACCAAAGTGA AATACGTGAC CGAGGGAATG AGAAAGCCCG CCTTCCTGAG CGGCGAGCAG AAAAAGGCCA TCGTGGACCT GCTGTTCAAG ACCAACCGGA AAGTGACCGT GAAGCAGCTG AAAGAGGACT ACTTCAAGAA AATCGAGTGC TTCGACTCCG TGGAAATCTC CGGCGTGGAA GATCGGTTCA ACGCCTCCCT GGGCACATAC CACGATCTGC TGAAAATTAT CAAGGACAAG GACTTCCTGG ACAATGAGGA AAACGAGGAC ATTCTGGAAG ATATCGTGCT GACCCTGACA CTGTTTGAGG ACAGAGAGAT GATCGAGGAA CGGCTGAAAA CCTATGCCCA CCTGTTCGAC GACAAAGTGA TGAAGCAGCT GAAGCGGCGG AGATACACCG GCTGGGGCAG GCTGAGCCGG AAGCTGATCA ACGGCATCCG GGACAAGCAG TCCGGCAAGA CAATCCTGGA TTTCCTGAAG TCCGACGGCT TCGCCAACAG AAACTTCATG CAGCTGATCC ACGACGACAG CCTGACCTTT AAAGAGGACA TCCAGAAAGC CCAGGTGTCC GGCCAGGGCG ATAGCCTGCA CGAGCACATT GCCAATCTGG CCGGCAGCCC CGCCATTAAG AAGGGCATCC TGCAGACAGT GAAGGTGGTG GACGAGCTCG TGAAAGTGAT GGGCCGGCAC AAGCCCGAGA ACATCGTGAT CGAAATGGCC AGAGAGAACC AGACCACCCA GAAGGGACAG AAGAACAGCC GCGAGAGAAT GAAGCGGATC GAAGAGGGCA TCAAAGAGCT GGGCAGCCAG ATCCTGAAAG AACACCCCGT GGAAAACACC CAGCTGCAGA ACGAGAAGCT GTACCTGTAC TACCTGCAGA ATGGGCGGGA TATGTACGTG GACCAGGAAC TGGACATCAA CCGGCTGTCC GACTACGATG TGGACCACAT CGTGCCTCAG AGCTTTCTGA AGGACGACTC CATCGACAAC AAGGTGCTGA CCAGAAGCGA CAAGGCCCGG GGCAAGAGCG ACAACGTGCC CTCCGAAGAG GTCGTGAAGA AGATGAAGAA CTACTGGCGG CAGCTGCTGA ACGCCAAGCT GATTACCCAG AGAAAGTTCG ACAATCTGAC CAAGGCCGAG AGAGGCGGCC TGAGCGAACT GGATAAGGCC GGCTTCATCA AGAGACAGCT GGTGGAAACC CGGCAGATCA CAAAGCACGT GGCACAGATC CTGGACTCCC GGATGAACAC TAAGTACGAC GAGAATGACA AGCTGATCCG GGAAGTGAAA GTGATCACCC TGAAGTCCAA GCTGGTGTCC GATTTCCGGA AGGATTTCCA GTTTTACAAA GTGCGCGAGA TCAACAACTA CCACCACGCC CACGACGCCT ACCTGAACGC CGTCGTGGGA ACCGCCCTGA TCAAAAAGTA CCCTAAGCTG GAAAGCGAGT TCGTGTACGG CGACTACAAG GTGTACGACG TGCGGAAGAT GATCGCCAAG AGCGAGCAGG AAATCGGCAA GGCTACCGCC AAGTACTTCT TCTACAGCAA CATCATGAAC TTTTTCAAGA CCGAGATTAC CCTGGCCAAC GGCGAGATCC GGAAGCGGCC TCTGATCGAG ACAAACGGCG AAACCGGGGA GATCGTGTGG GATAAGGGCC GGGATTTTGC CACCGTGCGG AAAGTGCTGA GCATGCCCCA AGTGAATATC GTGAAAAAGA CCGAGGTGCA GACAGGCGGC TTCAGCAAAG AGTCTATCCT GCCCAAGAGG AACAGCGATA AGCTGATCGC CAGAAAGAAGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GACTGGGACC CTAAGAAGTA CGGCGGCTTC GACAGCCCCA CCGTGGCCTA TTCTGTGCTG GTGGTGGCCA AAGTGGAAAA GGGCAAGTCC AAGAAACTGA AGAGTGTGAA AGAGCTGCTG GGGATCACCA TCATGGAAAG AAGCAGCTTC GAGAAGAATC CCATCGACTT TCTGGAAGCC AAGGGCTACA AAGAAGTGAA AAAGGACCTG ATCATCAAGC TGCCTAAGTA CTCCCTGTTC GAGCTGGAAA ACGGCCGGAA GAGAATGCTG GCCTCTGCCG GCGAACTGCA GAAGGGAAAC GAACTGGCCC TGCCCTCCAA ATATGTGAAC TTCCTGTACC TGGCCAGCCA CTATGAGAAG CTGAAGGGCT CCCCCGAGGA TAATGAGCAG AAACAGCTGT TTGTGGAACA GCACAAGCAC TACCTGGACG AGATCATCGA GCAGATCAGC GAGTTCTCCA AGAGAGTGAT CCTGGCCGAC GCTAATCTGG ACAAAGTGCT GTCCGCCTAC AACAAGCACC GGGATAAGCC CATCAGAGAG CAGGCCGAGA ATATCATCCA CCTGTTTACC CTGACCAATC TGGGAGCCCC TGCCGCCTTC AAGTACTTTG ACACCACCAT CGACCGGAAG AGGTACACCA GCACCAAAGA GGTGCTGGAC GCCACCCTGA TCCACCAGAG CATCACCGGC CTGTACGAGA CACGGATCGA CCTGTCTCAG CTGGGAGGCG ACAGCGCTGG AGGAGGTGGA AGCGGAGGAG GAGGAAGCGG AGGAGGAGGT AGCGGACCTA AGAAAAAGAG GAAGGTGGCG GCCGCTGGAT CCGGACGGGC TGACGCATTG GACGATTTTG ATCTGGATAT GCTGGGAAGT GACGCCCTCG ATGATTTTGA CCTTGACATG CTTGGTTCGG ATGCCCTTGA TGACTTTGAC CTCGACATGC TCGGCAGTGA CGCCCTTGAT GATTTCGACC TGGACATGCT GATTAACTGT ACAGGCAGTG GAGAGGGCAG AGGAAGTCTG CTAACATGCG GTGACGTCGA GGAGAATCCT GGCCCAATGG CTTCAAACTT TACTCAGTTC GTGCTCGTGG ACAATGGTGG GACAGGGGAT GTGACAGTGG CTCCTTCTAA TTTCGCTAAT GGGGTGGCAG AGTGGATCAG CTCCAACTCA CGGAGCCAGG CCTACAAGGT GACATGCAGC GTCAGGCAGT CTAGTGCCCA GAAGAGAAAG TATACCATCA AGGTGGAGGT CCCCAAAGTG GCTACCCAGA CAGTGGGCGG AGTCGAACTG CCTGTCGCCG CTTGGAGGTC CTACCTGAAC ATGGAGCTCA CTATCCCAAT TTTCGCTACC AATTCTGACT GTGAACTCAT CGTGAAGGCA ATGCAGGGGC TCCTCAAAGA CGGTAATCCT ATCCCTTCCG CCATCGCCGC TAACTCAGGT ATCTACAGCG CTGGAGGAGG TGGAAGCGGA GGAGGAGGAA GCGGAGGAGG AGGTAGCGGA CCTAAGAAAA AGAGGAAGGT GGCGGCCGCT GGATCCCCTT CAGGGCAGAT CAGCAACCAG GCCCTGGCTC TGGCCCCTAG CTCCGCTCCA GTGCTGGCCC AGACTATGGT GCCCTCTAGT GCTATGGTGC CTCTGGCCCA GCCACCTGCT CCAGCCCCTG TGCTGACCCC AGGACCACCC CAGTCACTGA GCGCTCCAGT GCCCAAGTCT ACACAGGCCG GCGAGGGGAC TCTGAGTGAA GCTCTGCTGC ACCTGCAGTT CGACGCTGAT GAGGACCTGG GAGCTCTGCT GGGGAACAGC ACCGATCCCG GAGTGTTCAC AGATCTGGCC TCCGTGGACA ACTCTGAGTT TCAGCAGCTG CTGAATCAGG GCGTGTCCAT GTCTCATAGT ACAGCCGAAC CAATGCTGAT GGAGTACCCC GAAGCCATTA CCCGGCTGGT GACCGGCAGC CAGCGGCCCC CCGACCCCGC TCCAACTCCC CTGGGAACCA GCGGCCTGCC TAATGGGCTG TCCGGAGATG AAGACTTCTC AAGCATCGCT GATATGGACT TTAGTGCCCT GCTGTCACAG ATTTCCTCTA GTGGGCAGGG AGGAGGTGGA AGCGGCTTCA GCGTGGACAC CAGTGCCCTG CTGGACCTGT TCAGCCCCTC GGTGACCGTG CCCGACATGA GCCTGCCTGA CCTTGACAGC AGCCTGGCCA GTATCCAAGA GCTCCTGTCT CCCCAGGAGC CCCCCAGGCC TCCCGAGGCA GAGAACAGCA GCCCGGATTC AGGGAAGCAG CTGGTGCACT ACACAGCGCA GCCGCTGTTC CTGCTGGACC CCGGCTCCGT GGACACCGGGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 AGCAACGACC TGCCGGTGCT GTTTGAGCTG GGAGAGGGCT CCTACTTCTC CGAAGGGGAC GGCTTCGCCG AGGACCCCAC CATCTCCCTG CTGACAGGCT CGGAGCCTCC CAAAGCCAAG GACCCCACTG TCTCC SEQ ID NO: 137 is shown below: MKRPAATKKAGQAKKKKDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNEL TKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ LGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSGRADALDDFDLDMLGSDALDDFDLDM LGSDALDDFDLDMLGSDALDDFDLDMLINCTGSGEGRGSLLTCGDVEENPGPMASNFTQF VLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKV ATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSG IYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSS AMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNS TDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTP LGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTV PDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTG SNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVSPATENT ATTORNEY DOCKET NO.: 51471-019WO2 SEQ ID NO: 138 is shown below: AUGAAAAGGC CGGCGGCCAC GAAAAAGGCC GGCCAGGCAA AAAAGAAAAA GGACAAGAAG UACAGCAUCG GCCUGGCCAU CGGCACCAAC UCUGUGGGCU GGGCCGUGAU CACCGACGAG UACAAGGUGC CCAGCAAGAA AUUCAAGGUG CUGGGCAACA CCGACCGGCA CAGCAUCAAG AAGAACCUGA UCGGAGCCCU GCUGUUCGAC AGCGGCGAAA CAGCCGAGGC CACCCGGCUG AAGAGAACCG CCAGAAGAAG AUACACCAGA CGGAAGAACC GGAUCUGCUA UCUGCAAGAG AUCUUCAGCA ACGAGAUGGC CAAGGUGGAC GACAGCUUCU UCCACAGACU GGAAGAGUCC UUCCUGGUGG AAGAGGAUAA GAAGCACGAG CGGCACCCCA UCUUCGGCAA CAUCGUGGAC GAGGUGGCCU ACCACGAGAA GUACCCCACC AUCUACCACC UGAGAAAGAA ACUGGUGGAC AGCACCGACA AGGCCGACCU GCGGCUGAUC UAUCUGGCCC UGGCCCACAU GAUCAAGUUC CGGGGCCACU UCCUGAUCGA GGGCGACCUG AACCCCGACA ACAGCGACGU GGACAAGCUG UUCAUCCAGC UGGUGCAGAC CUACAACCAG CUGUUCGAGG AAAACCCCAU CAACGCCAGC GGCGUGGACG CCAAGGCCAU CCUGUCUGCC AGACUGAGCA AGAGCAGACG GCUGGAAAAU CUGAUCGCCC AGCUGCCCGG CGAGAAGAAG AAUGGCCUGU UCGGCAACCU GAUUGCCCUG AGCCUGGGCC UGACCCCCAA CUUCAAGAGC AACUUCGACC UGGCCGAGGA UGCCAAACUG CAGCUGAGCA AGGACACCUA CGACGACGAC CUGGACAACC UGCUGGCCCA GAUCGGCGAC CAGUACGCCG ACCUGUUUCU GGCCGCCAAG AACCUGUCCG ACGCCAUCCU GCUGAGCGAC AUCCUGAGAG UGAACACCGA GAUCACCAAG GCCCCCCUGA GCGCCUCUAU GAUCAAGAGA UACGACGAGC ACCACCAGGA CCUGACCCUG CUGAAAGCUC UCGUGCGGCA GCAGCUGCCU GAGAAGUACA AAGAGAUUUU CUUCGACCAG AGCAAGAACG GCUACGCCGG CUACAUUGAC GGCGGAGCCA GCCAGGAAGA GUUCUACAAG UUCAUCAAGC CCAUCCUGGA AAAGAUGGAC GGCACCGAGG AACUGCUCGU GAAGCUGAAC AGAGAGGACC UGCUGCGGAA GCAGCGGACC UUCGACAACG GCAGCAUCCC CCACCAGAUC CACCUGGGAG AGCUGCACGC CAUUCUGCGG CGGCAGGAAG AUUUUUACCC AUUCCUGAAG GACAACCGGG AAAAGAUCGA GAAGAUCCUG ACCUUCCGCA UCCCCUACUA CGUGGGCCCU CUGGCCAGGG GAAACAGCAG AUUCGCCUGG AUGACCAGAA AGAGCGAGGA AACCAUCACC CCCUGGAACU UCGAGGAAGU GGUGGACAAG GGCGCUUCCG CCCAGAGCUU CAUCGAGCGG AUGACCAACU UCGAUAAGAA CCUGCCCAAC GAGAAGGUGC UGCCCAAGCA CAGCCUGCUG UACGAGUACU UCACCGUGUA UAACGAGCUG ACCAAAGUGA AAUACGUGAC CGAGGGAAUG AGAAAGCCCG CCUUCCUGAG CGGCGAGCAG AAAAAGGCCA UCGUGGACCU GCUGUUCAAG ACCAACCGGA AAGUGACCGU GAAGCAGCUG AAAGAGGACU ACUUCAAGAA AAUCGAGUGC UUCGACUCCG UGGAAAUCUC CGGCGUGGAA GAUCGGUUCA ACGCCUCCCU GGGCACAUAC CACGAUCUGC UGAAAAUUAU CAAGGACAAG GACUUCCUGG ACAAUGAGGA AAACGAGGAC AUUCUGGAAG AUAUCGUGCU GACCCUGACA CUGUUUGAGG ACAGAGAGAU GAUCGAGGAA CGGCUGAAAA CCUAUGCCCA CCUGUUCGAC GACAAAGUGA UGAAGCAGCU GAAGCGGCGG AGAUACACCG GCUGGGGCAG GCUGAGCCGG AAGCUGAUCA ACGGCAUCCG GGACAAGCAG UCCGGCAAGA CAAUCCUGGA UUUCCUGAAG UCCGACGGCU UCGCCAACAG AAACUUCAUG CAGCUGAUCC ACGACGACAG CCUGACCUUU AAAGAGGACA UCCAGAAAGC CCAGGUGUCC GGCCAGGGCG AUAGCCUGCA CGAGCACAUU GCCAAUCUGG CCGGCAGCCC CGCCAUUAAG AAGGGCAUCC UGCAGACAGU GAAGGUGGUGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GACGAGCUCG UGAAAGUGAU GGGCCGGCAC AAGCCCGAGA ACAUCGUGAU CGAAAUGGCC AGAGAGAACC AGACCACCCA GAAGGGACAG AAGAACAGCC GCGAGAGAAU GAAGCGGAUC GAAGAGGGCA UCAAAGAGCU GGGCAGCCAG AUCCUGAAAG AACACCCCGU GGAAAACACC CAGCUGCAGA ACGAGAAGCU GUACCUGUAC UACCUGCAGA AUGGGCGGGA UAUGUACGUG GACCAGGAAC UGGACAUCAA CCGGCUGUCC GACUACGAUG UGGACCACAU CGUGCCUCAG AGCUUUCUGA AGGACGACUC CAUCGACAAC AAGGUGCUGA CCAGAAGCGA CAAGGCCCGG GGCAAGAGCG ACAACGUGCC CUCCGAAGAG GUCGUGAAGA AGAUGAAGAA CUACUGGCGG CAGCUGCUGA ACGCCAAGCU GAUUACCCAG AGAAAGUUCG ACAAUCUGAC CAAGGCCGAG AGAGGCGGCC UGAGCGAACU GGAUAAGGCC GGCUUCAUCA AGAGACAGCU GGUGGAAACC CGGCAGAUCA CAAAGCACGU GGCACAGAUC CUGGACUCCC GGAUGAACAC UAAGUACGAC GAGAAUGACA AGCUGAUCCG GGAAGUGAAA GUGAUCACCC UGAAGUCCAA GCUGGUGUCC GAUUUCCGGA AGGAUUUCCA GUUUUACAAA GUGCGCGAGA UCAACAACUA CCACCACGCC CACGACGCCU ACCUGAACGC CGUCGUGGGA ACCGCCCUGA UCAAAAAGUA CCCUAAGCUG GAAAGCGAGU UCGUGUACGG CGACUACAAG GUGUACGACG UGCGGAAGAU GAUCGCCAAG AGCGAGCAGG AAAUCGGCAA GGCUACCGCC AAGUACUUCU UCUACAGCAA CAUCAUGAAC UUUUUCAAGA CCGAGAUUAC CCUGGCCAAC GGCGAGAUCC GGAAGCGGCC UCUGAUCGAG ACAAACGGCG AAACCGGGGA GAUCGUGUGG GAUAAGGGCC GGGAUUUUGC CACCGUGCGG AAAGUGCUGA GCAUGCCCCA AGUGAAUAUC GUGAAAAAGA CCGAGGUGCA GACAGGCGGC UUCAGCAAAG AGUCUAUCCU GCCCAAGAGG AACAGCGAUA AGCUGAUCGC CAGAAAGAAG GACUGGGACC CUAAGAAGUA CGGCGGCUUC GACAGCCCCA CCGUGGCCUA UUCUGUGCUG GUGGUGGCCA AAGUGGAAAA GGGCAAGUCC AAGAAACUGA AGAGUGUGAA AGAGCUGCUG GGGAUCACCA UCAUGGAAAG AAGCAGCUUC GAGAAGAAUC CCAUCGACUU UCUGGAAGCC AAGGGCUACA AAGAAGUGAA AAAGGACCUG AUCAUCAAGC UGCCUAAGUA CUCCCUGUUC GAGCUGGAAA ACGGCCGGAA GAGAAUGCUG GCCUCUGCCG GCGAACUGCA GAAGGGAAAC GAACUGGCCC UGCCCUCCAA AUAUGUGAAC UUCCUGUACC UGGCCAGCCA CUAUGAGAAG CUGAAGGGCU CCCCCGAGGA UAAUGAGCAG AAACAGCUGU UUGUGGAACA GCACAAGCAC UACCUGGACG AGAUCAUCGA GCAGAUCAGC GAGUUCUCCA AGAGAGUGAU CCUGGCCGAC GCUAAUCUGG ACAAAGUGCU GUCCGCCUAC AACAAGCACC GGGAUAAGCC CAUCAGAGAG CAGGCCGAGA AUAUCAUCCA CCUGUUUACC CUGACCAAUC UGGGAGCCCC UGCCGCCUUC AAGUACUUUG ACACCACCAU CGACCGGAAG AGGUACACCA GCACCAAAGA GGUGCUGGAC GCCACCCUGA UCCACCAGAG CAUCACCGGC CUGUACGAGA CACGGAUCGA CCUGUCUCAG CUGGGAGGCG ACAGCGCUGG AGGAGGUGGA AGCGGAGGAG GAGGAAGCGG AGGAGGAGGU AGCGGACCUA AGAAAAAGAG GAAGGUGGCG GCCGCUGGAU CCGGACGGGC UGACGCAUUG GACGAUUUUG AUCUGGAUAU GCUGGGAAGU GACGCCCUCG AUGAUUUUGA CCUUGACAUG CUUGGUUCGG AUGCCCUUGA UGACUUUGAC CUCGACAUGC UCGGCAGUGA CGCCCUUGAU GAUUUCGACC UGGACAUGCU GAUUAACUGU ACAGGCAGUG GAGAGGGCAG AGGAAGUCUG CUAACAUGCG GUGACGUCGA GGAGAAUCCU GGCCCAAUGG CUUCAAACUU UACUCAGUUC GUGCUCGUGG ACAAUGGUGG GACAGGGGAU GUGACAGUGG CUCCUUCUAA UUUCGCUAAU GGGGUGGCAG AGUGGAUCAG CUCCAACUCA CGGAGCCAGG CCUACAAGGU GACAUGCAGCPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GUCAGGCAGU CUAGUGCCCA GAAGAGAAAG UAUACCAUCA AGGUGGAGGU CCCCAAAGUG GCUACCCAGA CAGUGGGCGG AGUCGAACUG CCUGUCGCCG CUUGGAGGUC CUACCUGAAC AUGGAGCUCA CUAUCCCAAU UUUCGCUACC AAUUCUGACU GUGAACUCAU CGUGAAGGCA AUGCAGGGGC UCCUCAAAGA CGGUAAUCCU AUCCCUUCCG CCAUCGCCGC UAACUCAGGU AUCUACAGCG CUGGAGGAGG UGGAAGCGGA GGAGGAGGAA GCGGAGGAGG AGGUAGCGGA CCUAAGAAAA AGAGGAAGGU GGCGGCCGCU GGAUCCCCUU CAGGGCAGAU CAGCAACCAG GCCCUGGCUC UGGCCCCUAG CUCCGCUCCA GUGCUGGCCC AGACUAUGGU GCCCUCUAGU GCUAUGGUGC CUCUGGCCCA GCCACCUGCU CCAGCCCCUG UGCUGACCCC AGGACCACCC CAGUCACUGA GCGCUCCAGU GCCCAAGUCU ACACAGGCCG GCGAGGGGAC UCUGAGUGAA GCUCUGCUGC ACCUGCAGUU CGACGCUGAU GAGGACCUGG GAGCUCUGCU GGGGAACAGC ACCGAUCCCG GAGUGUUCAC AGAUCUGGCC UCCGUGGACA ACUCUGAGUU UCAGCAGCUG CUGAAUCAGG GCGUGUCCAU GUCUCAUAGU ACAGCCGAAC CAAUGCUGAU GGAGUACCCC GAAGCCAUUA CCCGGCUGGU GACCGGCAGC CAGCGGCCCC CCGACCCCGC UCCAACUCCC CUGGGAACCA GCGGCCUGCC UAAUGGGCUG UCCGGAGAUG AAGACUUCUC AAGCAUCGCU GAUAUGGACU UUAGUGCCCU GCUGUCACAG AUUUCCUCUA GUGGGCAGGG AGGAGGUGGA AGCGGCUUCA GCGUGGACAC CAGUGCCCUG CUGGACCUGU UCAGCCCCUC GGUGACCGUG CCCGACAUGA GCCUGCCUGA CCUUGACAGC AGCCUGGCCA GUAUCCAAGA GCUCCUGUCU CCCCAGGAGC CCCCCAGGCC UCCCGAGGCA GAGAACAGCA GCCCGGAUUC AGGGAAGCAG CUGGUGCACU ACACAGCGCA GCCGCUGUUC CUGCUGGACC CCGGCUCCGU GGACACCGGG AGCAACGACC UGCCGGUGCU GUUUGAGCUG GGAGAGGGCU CCUACUUCUC CGAAGGGGAC GGCUUCGCCG AGGACCCCAC CAUCUCCCUG CUGACAGGCU CGGAGCCUCC CAAAGCCAAG GACCCCACUG UCUCC One example of a generic gRNA array expression cassette includes from 5' to 3': (a) a 3' splicing sequence; (b) a first recombinase recognition site (e.g., rox site); (c) a coding sequence for a drug resistance gene (e.g., puromycin-N-acetyltransferase (puro.r) coding sequence or bleomycin (Bleo) resistance gene coding sequence); (d) a polyadenylation signal; (e) a second recombinase recognition site (e.g., rox site); (f) a gRNA including one or more gRNA genes (e.g., a first U6 promoter followed by a first gRNA coding sequence, a second U6 promoter followed by a second gRNA coding sequence, and a third U6 promoter followed by a third gRNA coding sequence). The full sequence of another example gRNA array expression cassette is set forth in SEQ ID NO: 194. The gRNA array expression cassette includes the U6 promoter, gRNA sequence shown as “NNN”, crRNA, tracr, EF1a promoter, Bleo resistance gene, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), and bGH polyA. SEQ ID NO: 194 is shown below: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGAT AATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGT AATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTT ACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC CNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTGCAPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCAACATGAGGATCACCCA TGTCTGCAGGGCCAAGTGGCACCGAGTCGGTGCTTTTTTTGGATCCTGCAAAGATGGATAAA GTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAA TTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGG GGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAG TCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGT GGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCAC TGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGG GCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA GCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAA TGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC GTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGA CGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGC CCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGC TTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCAC GGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTT TAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAA GTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATC TTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGT GATGTACAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGA GCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGC CGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGG ACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAG GTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCC GTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGG AGCAGGACTGAGAATTCGATATCAAGCTTATCGGTAATCAACCTCTGGATTACAAAATTTGT GAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTT AATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAT CCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGC ACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTC CGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCC GCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCA TCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTG CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGC GGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCC CCGCATCGATACCGTCGACCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACPATENT ATTORNEY DOCKET NO.: 51471-019WO2 AGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTC CAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC TTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCT TGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGGGC CAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCAAGAG AAGGTAGAAGAAGCCAATGAAGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGG GATGGATGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATC ACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCC TGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCT TTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAA TAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG The sequence of the U6 promoter and gRNA coding sequence is set forth in SEQ ID NO: 195. The gRNA sequence is shown as “NNN”, where N could be any one of A, C, G, or T. In some embodiments, the gRNA sequence is a gRNA sequence from Table 1. SEQ ID NO: 195 is shown below: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGAT AATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGT AATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTT ACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC CNNNNNNNNNNNNNNNNNNNN The sequence of the EF1a promoter is set forth in SEQ ID NO: 196. SEQ ID NO: 196 is shown below: GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGG AGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATG TCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTC GCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGG TTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTG GCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGC CTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGC CGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGC CATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATG CGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCC CGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTT CCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGA GTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGG AGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTA GGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGT TAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTT GGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA The sequence of the Bleo resistance gene is set forth in SEQ ID NO: 197. SEQ ID NO: 197 is shown below: ATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGA GTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGG TCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACC CTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTC CACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGC GGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGAC TGA The sequence of the WPRE is set forth in SEQ ID NO: 198. SEQ ID NO: 198 is shown below: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCC TTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCC GTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGG CATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGG CGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGAC AATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAC CTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTC CTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACG AGTCGGATCTCCCTTTGGGCCGCCTCCCCGC The sequence of the bGH polyA is set forth in SEQ ID NO: 199. SEQ ID NO: 199 is shown below: CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAG TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 AAV Virus AAV is a small, replication-deficient parvovirus. AAV is about 20-24 nm long, with a density of about 1.40-1.41 g / cc. AAV contains a single-stranded linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either a plus strand, or a minus strand. AAV contains two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). AAVs contain a single intron. Cis-acting sequences directing viral DNA replication (Rep), encapsidation / packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The p5 and p19 are the rep promoters. When coupled with the differential splicing of the single AAV intron, the two rep promoters result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties that are responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter, and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single polyadenylation site is located at map position 95 of the AAV genome. Muzyczka reviews the life cycle and genetics of AAV (Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)). AAV infection is non-cytopathic in cultured cells. Natural infection of humans and other animals is silent and asymptomatic (does not cause disease). Because AAV infects many mammalian cells, there is the possibility of targeting many different tissues in vivo. In addition to dividing cells, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (i.e. extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids, which makes construction of recombinant genomes possible. Moreover, because the signals directing AAV replication, genome encapsidation, and integration are all contained with the ITRs of the AAV genome, some or all of the approximately 4.3 kb of the genome, encoding replication and structural capsid proteins (rep-cap) are contained within the ITRs of the AAV genome, can be replaced with heterologous DNA, such as a gene cassette containing a promoter, a DNA of interest, and a polyadenylation signal. The rep and cap proteins may be provided in trans. Several AAV serotypes have been identified, differing in their tropism (type of cell that they infect). Serotype AAV1 shows tropism to the following tissues: central nervous system (CNS); heart; retinal pigment epithelium (RPE); and skeletal muscle. Serotype AAV2 shows tropism to the following tissues: CNS; kidney; photoreceptor cells; and RPE. Serotype AAV4 shows tropism to the following tissues: CNS; lung; and RPE. Serotype AAV5 shows tropism to the following tissues: CNS; lung; photoreceptor cells; and RPE. Serotype AAV6 shows tropism to the following tissues: lung; and skeletal muscle. Serotype AAV7 shows tropism to the following tissues: liver; and skeletal muscle. Serotype AAV8 shows tropism to the following tissues: CNS; heart; liver; pancreas; photoreceptor cells; RPE; and skeletal muscle. Serotype AAV9 shows tropism for the following tissues: CNS; heart; liver; lung; and skeletal muscle. The tropism of AAV viruses may be related to the variability of the amino acid sequences of the capsid protein, which may bind to different functional receptors present on different types of cells.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 For example, it has recently been shown that including a human rhodopsin kinase (hGRK1) promoter in an AAV5 vector results in rod- and cone-specific expression in the primate retina (Boye, et al., Human Gene Therapy, 23:1101-1115 (October 2012) (DOI: 10.1089 / hum.2012.125)). It has also recently been shown that AAV virions with altered capsid proteins may impart greater tissue specific infectivity. For example, AAV6 with a variant capsid protein shows increased infectivity of retinal cells, compared to wild-type AAV capsid protein (US 8,663,624). A variant capsid protein including a peptide insertion between two adjacent amino acids corresponding to amino acids 570 ad 611 of VP1 of AAV2, or the corresponding position in a capsid protein of another AAV serotype, confers increased infectivity of retinal cells, compared to wild-type AAV (US 9,193,956). Lentivirus Lentivirus is a genus of retrovirus that causes chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species. The best known lentivirus is the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses are also hosted in apes, cows, goats, horses, cats, and sheep. Recently, lentiviruses have been found in monkeys, lemurs, Malayan flying lemur (neither a true lemur nor a primate), rabbits, and ferrets. Lentiviruses and their hosts have worldwide distribution. Lentiviruses can integrate a significant amount of viral cDNA into the DNA of the host cell and can efficiently infect non-dividing cells, so they are one of the most efficient methods of gene delivery. Lentiviruses can become endogenous (ERV), integrating their genome into the host germline genome, so that the virus is henceforth inherited by the host's descendants. Lentivirus is primarily a research tool used to introduce a gene product into in vitro systems or animal models. Conversely, lentivirus is also used to stably overexpress certain genes, thus allowing researchers to examine the effect of increased gene expression in a model system. Another common application is to use a lentivirus to introduce a new gene into human or animal cells. For example, a model of mouse hemophilia is corrected by expressing wild-type platelet-factor VIII, the gene that is mutated in human hemophilia. Lentiviral infection has advantages over other gene- therapy methods including high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity. Lentiviruses have also been successfully used for transduction of diabetic mice with the gene encoding PDGF (platelet-derived growth factor), a therapy being considered for use in humans. Finally, lentiviruses have been also used to elicit an immune response against tumor antigens. These treatments, like most current gene therapy experiments, show promise but are yet to be established as safe and effective in controlled human studies. Gammaretroviral and lentiviral vectors have so far been used in more than 300 clinical trials, addressing treatment options for various diseases. Lipid Nanoparticles Lipid nanoparticles (“LNPs”) are examples of vectors according to the present disclosure. LNPs are particles including a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g.,PATENT ATTORNEY DOCKET NO.: 51471-019WO2 liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016 / 010840 A1 and WO 2017 / 173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. An example lipid nanoparticle can include a cationic lipid and one or more other components. In one example, the other component can include a helper lipid such as cholesterol. In another example, the other components can include a helper lipid such as cholesterol and a neutral lipid such as DSPC. In another example, the other components can include a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033. The LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) a helper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235 and WO 2017 / 173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. In certain LNPs, the cargo can include a gRNA or a nucleic acid encoding a gRNA. In certain LNPs, the cargo can include a SAM mRNA and a g RNA or a nucleic acid encoding a gRNA. The lipid for encapsulation and endosomal escape can be a cationic lipid. The lipid can also be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is Lipid A or LP01, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy- )carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- ((((3-(diethylamino)propoxy)carbonyl- )oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235 and WO 2017 / 173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. Another example of a suitable lipid is Lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bi- s(decanoate), also called ((5- ((dimethyl amino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bi- s(decanoate). Another example of a suitable lipid is Lipid C, which is 2-((4-(((3- (dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1- ,3-diyl(9Z,9'Z, 12Z,127)-bis(octadeca-9, 12-dienoate). Another example of a suitable lipid is Lipid D, which is 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy )tridecyl 3-octylundecanoate. Other suitable lipids include heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (also known as [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate or Dlin-MC3-DMA (MC3))). Some such lipids suitable for use in the LNPs described herein are biodegradable in vivo. For example, LNPs including such a lipid include those where at least 75% of the lipid is cleared from thePATENT ATTORNEY DOCKET NO.: 51471-019WO2 plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. As another example, at least 50% of the LNP is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. Such lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipids may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the lipids may not be protonated and thus bear no charge. In some embodiments, the lipids may be protonated at a pH of at least about 9, 9.5, or 10. The ability of such a lipid to bear a charge is related to its intrinsic pKa. For example, the lipid may, independently, have a pKa in the range of from about 5.8 to about 6.2. Neutral lipids function to stabilize and improve processing of the LNPs. Examples of suitable neutral lipids include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5- heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-diarachidonoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl- 2- palmitoyl phosphatidyl choline (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC), and combinations thereof. For example, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). Helper lipids include lipids that enhance transfection. The mechanism by which the helper lipid enhances transfection can include enhancing particle stability. In certain cases, the helper lipid can enhance membrane fusogenicity. Helper lipids include steroids, sterols, and alkyl resorcinols. Examples of suitable helper lipids suitable include cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one example, the helper lipid may be cholesterol or cholesterol hemisuccinate. Stealth lipids include lipids that alter the length of time the nanoparticles can exist in vivo. Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids may modulate pharmacokinetic properties of the LNP. Suitable stealth lipids include lipids having a hydrophilic head group linked to a lipid moiety. The hydrophilic head group of stealth lipid can include, for example, a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids, and poly N-(2- hydroxypropyl)methacrylamide. The term PEG means any polyethylene glycol or other polyalkylene etherPATENT ATTORNEY DOCKET NO.: 51471-019WO2 polymer. In certain LNP formulations, the PEG, is a PEG-2K, also termed PEG 2000, which has an average molecular weight of about 2,000 daltons. See, e.g., WO 2017 / 173054 A1, herein incorporated by reference in its entirety for all purposes. The lipid moiety of the stealth lipid may be derived, for example, from diacylglycerol or diacylglycamide, including those including a dialkylglycerol or dialkylglycamide group having alkyl chain length independently including from about C4 to about C40 saturated or unsaturated carbon atoms, in which the chain may include one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further include one or more substituted alkyl groups. As one example, the stealth lipid may be selected from PEG-dilauroylglycerol, PEG- dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG- dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- -[omega]- methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one particular example, the stealth lipid may be PEG2k-DMG. The LNPs can include different respective molar ratios of the component lipids in the formulation. The mol-% of the CCD lipid may be, for example, from about 30 mol-% to about 60 mol-%, from about 35 mol-% to about 55 mol-%, from about 40 mol-% to about 50 mol-%, from about 42 mol-% to about 47 mol-%, or about 45%. The mol-% of the helper lipid may be, for example, from about 30 mol-% to about 60 mol-%, from about 35 mol-% to about 55 mol-%, from about 40 mol-% to about 50 mol-%, from about 41 mol-% to about 46 mol-%, or about 44 mol-%. The mol-% of the neutral lipid may be, for example, from about 1 mol-% to about 20 mol-%, from about 5 mol-% to about 15 mol-%, from about 7 mol-% to about 12 mol-%, or about 9 mol-%. The mol-% of the stealth lipid may be, for example, from about 1 mol- % to about 10 mol-%, from about 1 mol-% to about 5 mol-%, from about 1 mol-% to about 3 mol-%, about 2 mol-%, or about 1 mol-%. The LNPs can have different ratios between the positively charged amine groups of the biodegradable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N / P. For example, the N / P ratio may be from about 0.5 to about 100, from about 1 to about 50, from about 1 to about 25, from about 1 to about 10, from about 1 to about 7, from about 3 to about 5, from about 4 to about 5, about 4, about 4.5, or about 5. The N / P ratio can also be from about 4 to about 7 or from about 4.5 to about 6. In specific examples, the N / P ratio can be 4.5 or can be 6. A specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 4.5 and contains biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2 molar ratio. The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate, also calledPATENT ATTORNEY DOCKET NO.: 51471-019WO2 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)- octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235, herein incorporated by reference in its entirety for all purposes. Another specific example of a suitable LNP contains Dlin-MC3- DMA (MC3), cholesterol, DSPC, and PEG-DMG in a 50:38.5:10:1.5 molar ratio. Another specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 6 and contains biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio. The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca- 9,12-dienoate. The Cas9 mRNA / SAM mRNA can be in a 1:2 ratio by weight to the gRNA. Another specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 3 and contains a cationic lipid, a structural lipid, cholesterol (e.g., cholesterol (ovine) (Avanti 700000)), and PEG2k-DMG (e.g., PEG-DMG 2000 (NOF America-STJNBRIGHT.RTM. GM-020(DMG-PEG)) in a 50:10:38.5:1.5 ratio or a 47:10:42:1 ratio. The structural lipid can be, for example, DSPC (e.g., DSPC (Avanti 850365)), SOPC, DOPC, or DOPE. The cationic / ionizable lipid can be, for example, Dlin-MC3- DMA (e.g., Dlin-MC3-DMA (Biofine International)). Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and a PEG lipid in a 45:9:44:2 ratio. Another specific example of a suitable LNP contains Dlin-MC3-DMA, DOPE, cholesterol, and PEG lipid or PEG DMG in a 50:10:39:1 ratio. Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG2k-DMG at a 55:10:32.5:2.5 ratio. Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a 50:10:38.5:1.5 ratio. Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a 50:10:38.5:1.5 ratio. According to the present disclosure, and in order to overcome the hurdle of not having an immortalized cell line useful for in vitro potency assays and measurements of the ability of a vector to transfer a nucleic acid molecule into a cell, a CRISPR-SAM complex was employed to drive expression from cell-type specific regulatory elements in immortalized cell lines such as for example, the Henrietta Lacks (HeLa) cell line. The HeLa cell line is a permanent and immortal line established from cervical cancer cells. Several variants or strains of the HeLa cell line may be used, including those adapted for high density suspension culture in serum-free media. In one embodiment, preferably for auditory gene therapy in hair cells, a proprietary human GJB2 regulatory element containing a promoter and enhancer is used to drive expression of a transgene (i.e., a therapeutic target or reporter protein) specifically in GJB2-expressing cells of the inner ear. Not all immortalized cell lines are able to express GJB2 from a proprietary human GJB2 regulatory element containing a promoter and enhancer. This makes it challenging to test or validate gene therapy (such as an AAV gene therapy) in cells prior to moving into an in vivo system. Therefore, CRISPR-SAM with an activating gRNA against the human GJB2 promoter and / or enhancer is used.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 In some embodiments, the CRISPR-SAM components are first introduced using lentivirus into HeLa cells. Next, an eGFP reporter is introduced under the control of a human GJB2 regulatory element containing a GJB2 promoter and enhancer. Activating gRNAs are then designed across the GJB2 regulatory element (e.g., designed to target the promoter and / or enhancer) and then the activating gRNAs are tested in the CRISPR-SAM GJB2 regulatory element-eGFP reporter cell line. In some embodiments, the gRNAs tested induce expression of the GFP reporter gene to different extents. Because the GFP reporter is only useful for identifying the best activating gRNA, further experiments are performed in the HeLa CRISPR-SAM cell line in which the best GJB2 regulatory element activating gRNA is introduced. Integration of both the activating gRNA and the CRISPR-SAM components is beneficial for eliminating cell-to-cell variability in expression. The result is a HeLa cell line that expresses consistent levels of the CRISPR-SAM machinery and an activating gRNA against the GJB2 regulatory element. It is shown that the engineered CRISPR-SAM GJB2 regulatory element gRNA cell line is capable of promoting expression of the transgene of interest when introduced via AAV transduction. Examples The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Development of a cell-based potency assay for a gene therapy program A cell-based potency assay was developed to assess the ability of an engineered cell type- specific regulatory element containing a GJB2 promoter and enhancer, which is also referred to as the AAV GJB2 regulatory element (SEQ ID NO: 144), to induce expression of a transgene (e.g., a transgene encoding Gjb2 or reporter protein) in cells prior to moving into an in vivo system. SEQ ID NO: 144 is shown below. This regulatory element was designed for specific expression in GJB2-expressing cells of the cochlea (e.g., cochlear supporting cells). The GJB2 regulatory element has low-level expression in HeLa cells, which do not natively express GJB2. To induce and increase expression from this regulatory element, HeLa cells were engineered with a CRISPR activation system, CRISPR SAM, and gRNAs to the GJB2 regulatory element were generated. HeLa cells were selected for this study as these cells are used in the field to examine functions of gap junctions via dye transfer and uptake assays. HeLa SAM Cloning and Validation CRISPR SAM components (as described in Konermann et al. Nature 517, 583-588 (2015)) were integrated into HeLa cells by lentivirus infection and selection. A clonal population was achieved by serially diluting the selected population to single cells. Clones were picked and evaluated for expression of CRISPR SAM components; MS2, p65, dCas9, and VP64. Clones were validated for gene activation by lentivirus packaged gRNA infection and qRT-PCR. HeLa SAM D73 clone was selected based on expression and activity.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 The sequence of the EF1a promoter used for both dCas9-2A-VP64 and MS2-p65- HSF1 is set forth in SEQ ID NO: 196. SEQ ID NO: 196 is shown below: GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGG GGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAAC GTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCC TGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGAT TCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCC CTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGG CACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGC TGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTT CGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGG GGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGT GCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAG TTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGG CGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGT CGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTT GGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGG GTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGA GTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGG TGTCGTGA GJB2 gRNA Design and Screening gRNAs to the GJB2 regulatory element sequence were identified using UCSC Genome Browser CRISPR target sites track and ordered as synthetic gRNA. The gRNA sequences to the GJB2 regulatory element are shown in Table 3 below. Guide activity was evaluated by transfecting gRNA in a reporter cell line containing the GJB2 regulatory element operably linked to GFP. Subsequent analysis of GFP expression by qRT-PCR identified several active gRNAs. The positive control was HeLa SAM cell line containing Myo15-eGFP transfected with Myo15-gRNA11. This gRNA was used previously in potency assays. GJB2 gRNA1, gRNA5, gRNA21, and gRNA23 were selected for further study (FIG.1). GJB2 gRNA Cloning and Validation GJB2 gRNA1, gRNA5, gRNA21, and gRNA23 were synthesized in a pLenti backbone with puromycin selection and packaged in lentivirus (Table 4, below). HeLa SAM cells were infected with each gRNA vector. Clonal populations were achieved by serially diluting the selected population to single cells. Clones were evaluated for 1x106MOI AAV1-GJB2 regulatory element-hGJB2 expression level by qRT- PCR (FIG.2). Sensitivity of expression at multiple MOIs were tested with a subset of clones (FIG.3).PATENT ATTORNEY DOCKET NO.: 51471-019WO2 AAV GJB2 regulatory element sequence containing the enhancer of SEQ ID NO: 119 and the promoter of SEQ ID NO: 120 (SEQ ID NO: 144) AAAAACTCTTACATAATTGTAACAGATTTGAGTTTCCTCTGGTTCAGGTTTTCTTGCCTCTTTGA TAATCAAATGATCTGAAGAAAGGCATAGAATTTCAAGGGAGAATCTGCATGACAGGATTACAATA AGGCTATTCATGGAGACTCTTTTATTAGCTTACACAGGATCTGCATCATTCTTCGTCTCTGGCTT TGCTACAAGGCTCCATTTAAACTTAACCCAACTTGCAGGCTTAACTGATCCAGGAATGACTCAAT ACAATGAGCCAGCTACTGTAGCTCTTTTTCCCCTTTTGATAAGGGGAGTTAACACAATGGGCTTT ACAGTTCTTAAATGAACGCGTACAGAGGACAACGACCACAGCCATCCCTGAACCCCGCCCACGGC ACAGCGCCGGAGCCGGGGTCTGGGGCGCCGCTTCCTGGGGGGTCCCGACTCTCAGCCGCCCCCGC TTCACCCGGGCCGCCAAGGGGCTGGGGGAGGCGGCGCTCGGGGTAACCGGGGGAGACTCAGGGCG CTGGGGGCACTTGGGGAACTCATGGGGGCTCAAAGGAACTAGGAGATCGGGACCTCGAAGGGGAC TTGGGGGGTTCGGGGCTTTCGGGGGCGGTCGGGGGTTCGCGGACCCGGGAAGCTCTGAGGACCCA GAGGCCGGGCGCGCTCCGCCCGCGGCGCCGCCCCCTCCGTAACTTTCCCAGTCTCCGAGGGAAGA GGCGGGGTGTGGGGTGCGGTTAAAAGGCGCCACGGCGGGAGACAGGTGTTGCGGCCCCGCAGCGC CCGCGCGCTCCTCTCCCCGACTCGGAGCCCCTCGGCGGCGCCCGGCCCAGGACCCGCCTAGGAGC GCAGGAGCCCCAGCGCAGAGACCCCAACGCCGAGACCCCCGCCCCGGCCCCGCCGCGCTTCCTCC CGACGCAGGTGAGCCCGCCGGCCCCGGACTGCCCGGCCAGGAACCTGGCGCGGGGAGGGACCGCG AGACCCAGAGCGGTTGCCCGGCCGCGTGGGTCTCGGGGAACCGGGGGGCTGGACCAACACACGTC CTTGGGCCGGGGGGCGGGGGCCGCCTTCTGGAGCGGGCGTTTCTGTTTATGATGTGTTTAAAGAT TGGGTGAATTACTCAGGTGAACAAGCTACTTTTTATCAGAGAACACCTAAAAACACGTTCAAGAG GGTTTGGGAACTATACATTTAATCCTATGACAAACTAAGTTGGTTCTGTCTTCACCTGTTTTGGT GAGGTTGTGTAAGAGTTGGTGTTTGCTCAGGAAGAGATTTAAGCATGCTTGCTTACCCAGACTCA GAGAAGTCTCCCTGTTCTGTCCTAGCTAGTGATTCCTGTGTTGTGTGCATTCGTCTTTTCCAGAG CAAACCGCCCAGAGTAGAAG Table 2. gRNA sequences for GJB2 promoter knockoutPATENT ATTORNEY DOCKET NO.: 51471-019WO2 Table 3. gRNA sequences to the GJB2 regulatory elementPATENT ATTORNEY DOCKET NO.: 51471-019WO2PATENT ATTORNEY DOCKET NO.: 51471-019WO2An example pLenti gRNA plasmid sequence is set forth in SEQ ID NO: 201. The full pLenti gRNA plasmid sequence can include any of the gRNA sequences described herein, such as one of the following gRNAs: gRNA1, gRNA5, gRNA21, and gRNA23. The gRNA sequence is shown as “NNN”. The backbone in every plasmid is the same ~10kb sequence but the gRNA is different. The annotation of the elements contained within the full pLenti gRNA plasmid are as follows: U6 promoter from nucleotides 2607-2847, gRNA from nucleotides 2856-2875, MS2-MS2 from nucleotides 2893-2981, EF1a promoter from nucleotides 3105-4282, BleoR from nucleotides 4289-4663, WPRE from nucleotides 4688-5276, and bGH polyA from nucleotides 6013-6237. The map of the full pLenti gRNA plasmid including gRNA23 is shown in FIG.4. The full plasmid sequence is set forth in SEQ ID NO: 201. SEQ ID NO: 201 is shown below: GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGAT GCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGC GAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTA GGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTA TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAG TACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTG ATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGCGCGTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGG AGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTT CAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAPATENT ATTORNEY DOCKET NO.: 51471-019WO2 GTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACC AGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGC GGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGC GAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCC AGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGAT TCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTA CAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCT CTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGG AAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGG AGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAAC CATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTG GGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTC AATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATT TGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAG CTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTG GGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATA AATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAAT TACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACA AGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGC TGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTT GCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCA CCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAG ACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACA AATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGTTAATTAGCTA GCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAA GTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGC TTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAAC ACCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTG CAGGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCAACATGAGGATCACC CATGTCTGCAGGGCCAAGTGGCACCGAGTCGGTGCTTTTTTTGGATCCTGCAAAGATGGATA AAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGG AATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG GGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGT GATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGT GTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCPATENT ATTORNEY DOCKET NO.: 51471-019WO2 ACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCG AGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTG GGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTC TAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTA AATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCG GACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATC GCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCC GCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGG GTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCC ACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTC TTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTG AAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGA TCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTC GTGATGTACAATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCG GAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTC GCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCC GGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGG AGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAG CCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGA GGAGCAGGACTGAGAATTCGATATCAAGCTTATCGGTAATCAACCTCTGGATTACAAAATTT GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCT TTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAA ATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGT GCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTT TCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAAT CATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTC TGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCT GCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCT CCCCGCATCGATACCGTCGACCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAAT ACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTT TCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCC ACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATC CTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG GCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCAAG AGAAGGTAGAAGAAGCCAATGAAGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCAT GGGATGGATGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCA TCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGPATENT ATTORNEY DOCKET NO.: 51471-019WO2 CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGA GTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC CTTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACT GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA AGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA GGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC AATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG GGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTT AGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTC TTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTT TGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAA AATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCT CCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAG TCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAT AGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC CCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTA TTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGC TTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATAT CGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTC CGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTC TCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCAT CAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCC TGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGG CCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGC CGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATT CCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATG ATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGC TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC TGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCG ACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG TCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT CAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTPATENT ATTORNEY DOCKET NO.: 51471-019WO2 CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA GAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCA GATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGC AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC AACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATA CTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAA AAGTGCCACCTGAC Results The relative fold change of eGFP expression for several gRNAs as compared to an un- transfected control reporter cell line (a cell line in which GFP is under the control of the GJB2 regulatory element) is shown in FIG.1. The relative fold change was used to identify active gRNAs. The arrows above the bars indicate guides selected for further study. n=4 replicate wells. The expression of human GJB2 under the control of the GJB2 regulatory element in an AAV1 vector in gRNA clones was evaluated as shown in FIG.2. The relative fold change of hGJB2 expression in HeLa SAM gRNA clones as compared to HeLa SAM D73 infected cells is shown in the graph. OrdinaryPATENT ATTORNEY DOCKET NO.: 51471-019WO2 one-way ANOVA, p<0.0001 with Dunnett's multiple comparisons test was used to statistically analyze the data in this experiment. n=3 replicate wells. The sensitivity of the expression of human GJB2 under the control of the GJB2 regulatory element in an AAV1 vector in gRNA clones was evaluated as shown in FIG.3. The relative fold change of hGJB2 expression in HeLa SAM gRNA_1 (left graph) and gRNA_23 (right graph) clones at multiple MOIs; 2x106, 1x106, 5x105, 2.5x105, 1.25x105, 6.25x104, 3.13x104, and 0 vg / cell is shown in the graphs. n=4 replicate wells. Example 2. Dose dependent GJB2 expression in G31 cell line mRNA Expression Engineered HeLa cells (HeLa / SAM / GJB2-g23; clone G31) were transduced with human GJB2 under the control of the GJB2 regulatory element in an AAV1 vector at various multiplicities of infection (MOIs). Five days post-transduction, total RNA was extracted from the cells, and quantitative reverse transcription PCR (qRT-PCR) was performed to quantify the expression levels of the payload mRNA, normalized to the housekeeping gene PPIA. Protein Expression Engineered HeLa cells (HeLa / SAM / GJB2-g23; clone G31) were transduced with human GJB2 under the control of the GJB2 regulatory element in an AAV1 vector at various MOIs, in the presence or absence of 250 nM etoposide. Seven days post-transduction, cells were fixed, stained with an anti-GJB2 primary antibody and a fluorophore-conjugated secondary antibody, and analyzed using flow cytometry. Cell Culture and Maintenance G31 clonal cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin (Pen / Strep), 100 µg / mL hygromycin, 10 µg / mL Blasticidin, and 30 µg / mL zeocin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. Cell Seeding and Transduction On the day of transduction, cells were resuspended in DMEM supplemented with 10% FBS and 1% Pen / Strep. A total of 50,000 cells per well were seeded in a 96-well plate in 100 µL of media. AAV vials were thawed at room temperature, and the required AAV stock volume (based on titer) was diluted in DMEM to a final volume of 50 µL. This mixture was added to each well to achieve the desired MOI. For etoposide treatment, etoposide was diluted in DMEM to achieve a final working concentration of 250 nM. RNA Extraction Three days post-transduction, cells were lysed, and total RNA was extracted using the ThermoFisher RNA extraction kit (Catalog # AM1830) according to the manufacturer’s protocol.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Quantitative PCR (qPCR) qPCR was performed using the QuantiNova Multiplex RT-PCR Kit (Catalog # 208552). The following primers and probes were used for amplification of the target gene (GJB2) and normalization against the housekeeping gene (PPIA): GJB2 Forward Primer: CCAGCGCAGAGACCCCAA (SEQ ID NO: 202) GJB2 Reverse Primer: ATGCTGGTGGAGTGTTTGTTC (SEQ ID NO: 203) GJB2 Probe (5’FAM): CTCCCGACGCAGAGCAAACCGC (SEQ ID NO: 204) Expression levels of the payload mRNA were quantified and normalized to PPIA using standard qPCR protocols. Flow Cytometry Seven days post-infection, cells were washed with 1X phosphate-buffered saline (PBS), followed by incubation with 4% paraformaldehyde (PFA) in 1X PBS for fifteen minutes at room temperature (RT). After fixation, PFA was removed, and cells were washed three times with PBS. Fixed samples were stored at 4°C overnight in 1X PBS. The next day, permeabilization was performed using 0.1% Triton X- 100 in PBS for 10 minutes with gentle shaking at RT, followed by three additional PBS washes. Blocking was carried out in a solution containing 5% donkey serum and 1% bovine serum albumin (BSA) in PBS for one hour with gentle shaking at RT. The primary antibody (ThermoFisher, Cat# 71-0500) (1:2000 dilution) and isotype control (at the same final concentration as the primary antibody) were prepared in antibody blocking solution and applied to the cells. Incubation was carried out overnight with gentle shaking at 4°C. The following day, cells were washed three times for five minutes each with PBS at RT. Secondary antibody incubation was performed using donkey anti-rabbit Alexa Fluor Plus 647 (1:500 dilution) prepared in blocking solution. Cells were incubated with the secondary antibody for one hour with gentle shaking at RT. Following staining, cells were washed three times for ten minutes each with PBS, trypsinized, and analyzed on a flow cytometer using the APC channel. Results The normalized fold change in payload mRNA expression across the tested MOI range of 1.5 × 10⁴ to 1.5 × 10⁶ MOI is shown in FIG.5. The mean fluorescence intensity (MFI) across the tested MOI range of 4 × 10⁴ to 1 × 10⁷ MOI is shown in FIG.6. Error bars represent the standard deviation (SD) of n=3. Presence or absence of 250 nM etoposide is represented by open and closed circles, respectively. Collectively, these experimental data demonstrate that AAV dose-dependent GJB2 expression, driven by the GJB2 regulatory element, can be detected at both the mRNA and protein levels in the G31 clonal cell line. Other Embodiments All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

PATENT ATTORNEY DOCKET NO.: 51471-019WO2 Claims 1. A cell that expresses a CRISPR / Cas9 Synergistic Activation Mediator (CRISPR SAM) complex, wherein: the CRISPR SAM complex comprises (a) a chimeric dead Cas9 (dCas9) protein with reduced or eliminated nuclease activity; (b) a guide RNA (gRNA) that specifically targets a promoter and / or an enhancer of GJB2, wherein the gRNA comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102; and (c) a chimeric adaptor protein.

2. The cell of claim 1, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a promoter of GJB2.

3. The cell of claim 1, wherein the CRISPR SAM complex comprises a gRNA that specifically targets an enhancer of GJB2.

4. The cell of claim 1, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a promoter and an enhancer of GJB2.

5. The cell of any one of claims 1-4, wherein the chimeric dCas9 protein has a nuclease activity that is reduced or eliminated by at least about 70% as compared to a wild-type Cas protein.

6. The cell of any one of claims 1-5, wherein the chimeric dCas9 protein with reduced or eliminated nuclease activity is fused to one or more transcriptional activation domains.

7. The cell of claim 6, wherein the one or more transcriptional activation domains in the CRISPR SAM complex are different from each other.

8. The cell of any one of claims 1-6, wherein the chimeric dCas9 protein with reduced or eliminated nuclease activity is a dCas9-VP64 fusion protein.

9. The cell of any one of claims 1-8, wherein the gRNA is a single gRNA (sgRNA).

10. The cell of any one of claims 1-9, wherein the gRNA further comprises an MS2 bacteriophage protein (MS2) aptamer.

11. The cell of claim 10, wherein the gRNA comprises two MS2 RNA aptamers.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 12. The cell of any one of claims 1-11, wherein the chimeric adaptor protein comprises an MS2 coat protein (MCP) fused to a NF-κB trans-activating subunit p65 and an activation domain of Heat Shock Factor 1 (MCP-p65-HSF1) 13. The cell of any one of claims 1-12, wherein the cell is a mammalian cell.

14. The cell of claim 13, wherein the mammalian cell is a human cell.

15. The cell of claim 14, wherein the human cell is a HeLa cell.

16. The cell of claim 1, wherein the gRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 62-102.

17. The cell of claim 16, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO:

84.

18. The cell of any one of claims 1-17, wherein the promoter is a human GJB2 (hGJB2) promoter, or the enhancer is an hGJB2 enhancer.

19. A HeLa cell line that expresses a CRISPR SAM complex, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a GJB2 promoter or enhancer, wherein: a) the CRISPR SAM complex comprises a chimeric dCas9 protein with reduced or eliminated nuclease activity and a chimeric adaptor protein; and b) the gRNA comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102.

20. The HeLa cell line of claim 19, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a GJB2 promoter.

21. The HeLa cell line of claim 19, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a GJB2 enhancer.

22. The HeLa cell line of claim 19, wherein the CRISPR SAM complex comprises a gRNA that specifically targets a GJB2 promoter and enhancer.

23. The HeLa cell line of any one of claims 19-22, wherein the gRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 62-102.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 24. The HeLa cell line of claim 23, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO:

84.

25. The HeLa cell line of any one of claims 19-24, wherein the chimeric dCas9 protein with reduced or eliminated nuclease activity is a dCas9-VP64 fusion protein.

26. The HeLa cell line of any one of claims 19-25, wherein the gRNA further comprises an MS2 aptamer.

27. The HeLa cell line of any one of claims 19-26, wherein the chimeric adaptor protein comprises MCP- p65-HSF1.

28. A gRNA comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 62-102.

29. The gRNA of claim 28, wherein the gRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 62-102.

30. The gRNA of claim 29, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 82, or SEQ ID NO:

84.

31. A method of measuring the ability of a vector to transfer a nucleic acid molecule into a cell comprising: a) introducing the nucleic acid molecule using the vector into the cell of any one of claims 1-17, wherein the nucleic acid molecule encodes a gene, or a fragment thereof operably linked to the GJB2 promoter or enhancer that binds the gRNA expressed by the cell; and b) measuring the expression of the gene.

32. The method of claim 31, wherein the vector is a viral vector.

33. The method of claim 32, wherein the viral vector is an adeno-associated viral vector.

34. The method of claim 32, wherein the viral vector is a retroviral vector.

35. The method of claim 32, wherein the viral vector is a lentiviral vector.

36. The method of claim 32, wherein the viral vector is an adenoviral vector.

37. The method of claim 31, wherein the vector is a lipid nanoparticle.

38. The method of claim 31, wherein the gene is a reporter gene.PATENT ATTORNEY DOCKET NO.: 51471-019WO2 39. The method of claim 38, wherein the reporter gene is selected from the group consisting of: a gene encoding beta-galactosidase, a bacterial chloramphenicol acetyltransferase gene, a firefly luciferase gene, a gene encoding beta-glucuronidase, and a gene encoding a fluorescent protein.

40. The method of claim 38 or 39, wherein the reporter gene encodes an enhanced green fluorescent protein.

41. The method of claim 31, wherein the gene is GJB2.