Patent Publication Number: US-11046948-B2

Title: Engineered transcription activator-like effector (TALE) domains and uses thereof

Description:
RELATED APPLICATION 
     This application is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. application, U.S. Ser. No. 14/913,458, filed Feb. 22, 2016, which is a national stage filing under 35 U.S.C. § 371 of international PCT application, PCT/US2014/052231, filed Aug. 22, 2014, which claims priority under 35 U.S.C. § 365(c) to U.S. application, U.S. Ser. No. 14/320,519, filed Jun. 30, 2014, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S. Ser. No. 61/868,846, filed Aug. 22, 2013, and international PCT application, PCT/US2014/052231 also claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S. Ser. No. 61/868,846, filed Aug. 22, 2013, each of which is incorporated herein by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with U.S. Government support under grant HR0011-11-2-0003 and N66001-12-C-4207, awarded by the Defense Advanced Research Projects Agency; grant T32GM007753, awarded by the National Institute of General Medical Sciences; and grant DPI GM105378 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Transcription activator-like effector nucleases (TALENs) are fusions of the FokI restriction endonuclease cleavage domain with a DNA-binding transcription activator-like effector (TALE) repeat array. TALENs can be engineered to specifically bind and cleave a desired target DNA sequence, which is useful for the manipulation of nucleic acid molecules, genes, and genomes in vitro and in vivo. Engineered TALENs are useful in the context of many applications, including, but not limited to, basic research and therapeutic applications. For example, engineered TALENs can be employed to manipulate genomes in the context of the generation of gene knockouts or knock-ins via induction of DNA breaks at a target genomic site for targeted gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template, respectively. TALENs are thus useful in the generation of genetically engineered cells, tissues, and organisms. 
     TALENs can be designed to cleave any desired target DNA sequence, including naturally occurring and synthetic sequences. However, the ability of TALENs to distinguish target sequences from closely related off-target sequences has not been studied in depth. Understanding this ability and the parameters affecting it is of importance for the design of TALENs having the desired level of specificity and also for choosing unique target sequences to be cleaved, e.g., in order to minimize the chance of undesired off-target cleavage. 
     SUMMARY OF THE INVENTION 
     TALENs are versatile tools for the manipulation of genes and genomes in vitro and in vivo, as they can be designed to bind and cleave virtually any target sequence within a nucleic acid molecule. For example, TALENs can be used for the targeted deletion of a DNA sequence within a cellular genome via induction of DNA breaks that are then repaired by the cellular DNA repair machinery through non-homologous end joining (NHEJ). TALENs can also be used for targeted sequence replacement in the presence of a nucleic acid comprising a sequence to be inserted into a genomic sequence via homology-directed repair (HDR). As TALENs can be employed to manipulate the genomes of living cells, the resulting genetically modified cells can be used to generate transgenic cell or tissue cultures and organisms. 
     In scenarios where a TALEN is employed for the targeted cleavage of a DNA sequence in the context of a complex sample, e.g., in the context of a genome, it is often desirable for the TALEN to bind and cleave the specific target sequence only, with no or only minimal off-target cleavage activity (see, e.g., PCT Application Publication WO2013/066438 A2, the entire contents of which are incorporated herein by reference). In some embodiments, an ideal TALEN would specifically bind only its intended target sequence and have no off-target activity, thus allowing the targeted cleavage of a single sequence, e.g., a single allele of a gene of interest, in the context of a whole genome. 
     Some aspects of this disclosure are based on the recognition that the tendency of TALENs to cleave off-target sequences and the parameters affecting the propensity of off-target TALEN activity are poorly understood. The work presented here provides a better understanding of the structural parameters that result in TALEN off-target activity. Methods and systems for the generation of engineered TALENs having no or minimal off-target activity are provided herein, as are engineered TALENs having increased on-target cleavage efficiency and minimal off-target activity. It will be understood by those of skill in the art that the strategies, methods, and reagents provided herein for decreasing non-specific or off-target DNA binding by TALENs are applicable to other DNA-binding proteins as well. In particular, the strategies for modifying the amino acid sequence of DNA-binding proteins for reducing unspecific binding to DNA by substituting cationic amino acid residues with amino acid residues that are not cationic, are uncharged, or are anionic at physiological pH, can be used to decrease the specificity of, for example, other TALE effector proteins, engineered zinc finger proteins (including zinc finger nucleases), and Cas9 proteins. 
     Some aspects of this disclosure provide engineered isolated Transcription Activator-Like Effector (TALE) domains. In some embodiments, the isolated TALE domain is an N-terminal TALE domain and the net charge of the isolated N-terminal domain is less than the net charge of the canonical N-terminal domain (SEQ ID NO: 1) at physiological pH. In some embodiments, the isolated TALE domain is a C-terminal TALE domain and the net charge of the C-terminal domain is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22) at physiological pH. In some embodiments, the isolated TALE domain is an N-terminal TALE domain and the binding energy of the N-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1). In some embodiments, the isolated TALE domain is a C-terminal TALE domain and the binding energy of the C-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, the net charge of the C-terminal domain is less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to −1, less than or equal to −2, less than or equal to −3, less than or equal to −4, or less than or equal to −5. In some embodiments, the C-terminal domain comprises an amino acid sequence that differs from the canonical C-terminal domain sequence in that at least one cationic amino acid residue of the canonical C-terminal domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, the N-terminal domain comprises an amino acid sequence that differs from the canonical N-terminal domain sequence in that at least one cationic amino acid residue of the canonical N-terminal domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cationic amino acid(s) in the isolated TALE domain is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, the at least one cationic amino acid residue is arginine (R) or lysine (K). In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q) or glycine (G). In some embodiments, at least one lysine or arginine residue is replaced with a glutamine residue. In some embodiments, the C-terminal domain comprises one or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises a Q3 variant sequence (K788Q, R792Q, K801Q). In some embodiments, the C-terminal domain comprises a Q7 variant sequence (K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q). In some embodiments, the N-terminal domain is a truncated version of the canonical N-terminal domain. In some embodiments, wherein the C-terminal domain is a truncated version of the canonical C-terminal domain. In some embodiments, the truncated domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues. In some embodiments, the isolated TALE domain is comprised in a TALE molecule comprising the structure [N-terminal domain]-[TALE repeat array]-[C-terminal domain]-[effector domain]; or [effector domain]-[N-terminal domain]-[TALE repeat array]-[C-terminal domain]. In some embodiments, the effector domain comprises a nuclease domain, a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain. In some embodiments, the TALE molecule binds a target sequence within a gene known to be associated with a disease or disorder. 
     Some aspects of this disclosure provide Transcription Activator-Like Effector Nucleases (TALENs) having a modified net charge and/or a modified binding energy for binding their target nucleic acid sequence as compared to canonical TALENs. Typically, the inventive TALENs include (a) a nuclease cleavage domain; (b) a C-terminal domain conjugated to the nuclease cleavage domain; (c) a TALE repeat array conjugated to the C-terminal domain; and (d) an N-terminal domain conjugated to the TALE repeat array. In some embodiments, (i) the net charge on the N-terminal domain at physiological pH is less than the net charge on the canonical N-terminal domain (SEQ ID NO: 1) at physiological pH; and/or (ii) the net charge of the C-terminal domain at physiological pH is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22) at physiological pH. In some embodiments, (i) the binding energy of the N-terminal domain to a target nucleic acid molecule is less than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1); and/or (ii) the binding energy of the C-terminal domain to a target nucleic acid molecule is less than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, the net charge on the C-terminal domain at physiological pH is less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to −1, less than or equal to −2, less than or equal to −3, less than or equal to −4, or less than or equal to −5. In some embodiments, the N-terminal domain comprises an amino acid sequence that differs from the canonical N-terminal domain sequence in that at least one cationic amino acid residue of the canonical N-terminal domain sequence is replaced with an amino acid residue that does not have a cationic charge, has no charge, or has an anionic charge. In some embodiments, the C-terminal domain comprises an amino acid sequence that differs from the canonical C-terminal domain sequence in that at least one cationic amino acid residue of the canonical C-terminal domain sequence is replaced with an amino acid residue that does not have a cationic charge, has no charge, or has an anionic charge. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cationic amino acid(s) is/are replaced with an amino acid residue that does not have a cationic charge, has no charge, or has an anionic charge in the N-terminal domain and/or in the C-terminal domain. In some embodiments, the at least one cationic amino acid residue is arginine (R) or lysine (K). In some embodiments, the amino acid residue that replaces the cationic amino acid is glutamine (Q) or glycine (G). Positively charged residues in the C-terminal domain that can be replaced according to aspects of this disclosure include, but are not limited to, arginine (R) residues and lysine (K) residues, e.g., R747, R770, K777, K778, K788, R789, R792, R793, R797, and R801 in the C-terminal domain (see. e.g., SEQ ID NO: 22, the numbering refers to the position of the respective residue in the full-length TALEN protein, the equivalent positions for the C-terminal domain as provide in SEQ ID NO: 22 are R8, R30, K37, K38, K48, R49, R52, R53, R57, R61). Positively charged residues in the N-terminal domain that can be replaced according to aspects of this disclosure include, but are not limited to, arginine (R) residues and lysine (K) residues, e.g., K57, K78, R84, R97, K110, K113, and R114 (see, e.g., SEQ ID NO: 1). In some embodiments, at least one lysine or arginine residue is replaced with a glutamine residue. In some embodiments, the C-terminal domain comprises one or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises a Q3 variant sequence (K788Q, R792Q, R801Q). In some embodiments, the C-terminal domain comprises a Q7 variant sequence (K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q). In some embodiments, the N-terminal domain is a truncated version of the canonical N-terminal domain. In some embodiments, the C-terminal domain is a truncated version of the canonical C-terminal domain. In some embodiments, the truncated domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues. In some embodiments, the nuclease cleavage domain is a FokI nuclease domain. In some embodiments, the FokI nuclease domain comprises a sequence as provided in SEQ ID NOs: 26-30. In some embodiments, the TALEN is a monomer. In some embodiments, the TALEN monomer dimerizes with another TALEN monomer to form a TALEN dimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the TALEN binds a target sequence within a gene known to be associated with a disease or disorder. In some embodiments, the TALEN cleaves the target sequence upon dimerization. In some embodiments, the disease being treated or prevented is HIV infection or AIDS, or a proliferative disease. In some embodiments, the TALEN binds a CCR5 (CC chemokine receptor type 5) target sequence in the treatment or prevention of HIV infection or AIDS. In some embodiments, the TALEN binds an ATM (ataxia telangiectasia mutated) target sequence. In some embodiments, the TALEN binds a VEGFA (Vascular endothelial growth factor A) target sequence. 
     Some aspects of this disclosure provide compositions comprising a TALEN described herein, e.g., a TALEN monomer. In some embodiments, the composition comprises the inventive TALEN monomer and a different inventive TALEN monomer that form a heterodimer, wherein the dimer exhibits nuclease activity. In some embodiments, the composition is a pharmaceutical composition. 
     Some aspects of this disclosure provide a composition comprising a TALEN provided herein. In some embodiments, the composition is formulated to be suitable for contacting with a cell or tissue in vitro. In some embodiments, the pharmaceutical composition comprises an effective amount of the TALEN for cleaving a target sequence, e.g., in a cell or in a tissue in vitro or ex vivo. In some embodiments, the TALEN binds a target sequence within a gene of interest, e.g., a target sequence within a gene known to be associated with a disease or disorder, and the composition comprises an effective amount of the TALEN for alleviating a sign and/or symptom associated with the disease or disorder. Some aspects of this disclosure provide a pharmaceutical composition comprising a TALEN provided herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the pharmaceutical composition comprises an effective amount of the TALEN for cleaving a target sequence in a cell in the subject. In some embodiments, the TALEN binds a target sequence within a gene known to be associated with a disease or disorder, and the composition comprises an effective amount of the TALEN for alleviating a sign and/or symptom associated with the disease or disorder. 
     Some aspects of this disclosure provide methods of cleaving a target sequence in a nucleic acid molecule using a TALEN provided herein. In some embodiments, the method comprises contacting a nucleic acid molecule comprising the target sequence with an inventive TALEN binding the target sequence under conditions suitable for the TALEN to bind and cleave the target sequence. In some embodiments, the TALEN is provided as a monomer. In some embodiments, the inventive TALEN monomer is provided in a composition comprising a different TALEN monomer that can dimerize with the inventive TALEN monomer to form a heterodimer having nuclease activity. In some embodiments, the inventive TALEN is provided in a pharmaceutical composition. In some embodiments, the target sequence is in the genome of a cell. In some embodiments, the target sequence is in a subject. In some embodiments, the method comprises administering a composition, e.g., a pharmaceutical composition, comprising the TALEN to the subject in an amount sufficient for the TALEN to bind and cleave the target site. 
     Some aspects of this disclosure provide methods of preparing engineered TALENs. In some embodiments, the method comprises replacing at least one amino acid in the canonical N-terminal TALEN domain and/or the canonical C-terminal TALEN domain with an amino acid having no charge or a negative charge as compared to the amino acid being replaced at physiological pH; and/or truncating the N-terminal TALEN domain and/or the C-terminal TALEN domain to remove a positively charged fragment; thus generating an engineered TALEN having an N-terminal domain and/or a C-terminal domain of decreased net charge at physiological pH. In some embodiments, the at least one amino acid being replaced comprises a cationic amino acid or an amino acid having a positive charge at physiological pH. Positively charged residues in the C-terminal domain that can be replaced according to aspects of this disclosure include, but are not limited to, arginine (R) residues and lysine (K) residues, e.g., R747, R770, K777, K778, K788, R789, R792, R793, R797, and R801 in the C-terminal domain. Positively charged residues in the N-terminal domain that can be replaced according to aspects of this disclosure include, but are not limited to, arginine (R) residues and lysine (K) residues, e.g., K57, K78, R84, R97, K110, K113, and R114. In some embodiments, the amino acid replacing the at least one amino acid is a cationic amino acid or a neutral amino acid. In some embodiments, the truncated N-terminal TALEN domain and/or the truncated C-terminal TALEN domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the respective canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues. In some embodiments, the method comprises replacing at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in the canonical N-terminal TALEN domain and/or in the canonical C-terminal TALEN domain with an amino acid having no charge or a negative charge at physiological pH. In some embodiments, the amino acid being replaced is arginine (R) or lysine (K). In some embodiments, the amino acid residue having no charge or a negative charge at physiological pH is glutamine (Q) or glycine (G). In some embodiments, the method comprises replacing at least one lysine or arginine residue with a glutamine residue. 
     Some aspects of this disclosure provide kits comprising an engineered TALEN as provided herein, or a composition (e.g., a pharmaceutical composition) comprising such a TALEN. In some embodiments, the kit comprises an excipient and instructions for contacting the TALEN with the excipient to generate a composition suitable for contacting a nucleic acid with the TALEN. In some embodiments, the excipient is a pharmaceutically acceptable excipient. 
     The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1B . TALEN architecture and selection scheme. ( FIG. 1A ) Architecture of a TALEN. A TALEN monomer contains an N-terminal domain followed by an array of TALE repeats (brown), a C terminal domain (green), and a FokI nuclease cleavage domain (purple). The 12th and 13th amino acids (the RVD sequence LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 43), red) of each TALE repeat recognize a specific DNA base pair. Two different TALENs bind their corresponding half-sites, allowing FokI dimerization and DNA cleavage; ttcattacacctgcagct is SEQ ID NO: 44; agctgcaggtgtaatgaa is SEQ ID NO: 45; agtatcaattctggaaga is SEQ ID NO: 46; and tcttccagaattgatact is SEQ ID NO: 47. The C-terminal domain variants used in this study are shown in green (SEQ ID NOs: 48-50, and 25, from top to bottom, respectively). ( FIG. 1B ) A single-stranded library of DNA oligonucleotides containing partially randomized left half-site (L), spacer (S), right half-site (R) and constant region (thick black line) was circularized, then concatemerized by rolling circle amplification. The resulting DNA libraries were incubated with an in vitro-translated TALEN of interest. Cleaved library members were blunted and ligated to adapter #1. The ligation products were amplified by PCR using one primer consisting of adapter #1 and the other primer consisting of adapter #2-constant sequence, which anneals to the constant regions. Amplicons 1½ target-sequence cassettes in length were isolated by gel purification and subjected to high-throughput DNA sequencing and computational analysis. 
         FIGS. 2A-2G . In vitro selection results. The fraction of sequences surviving selection (green) and before selection (black) are shown for CCR5A TALENs ( FIG. 2A ) and ATM TALENs ( FIG. 2B ) as a function of the number of mutations in both half-sites. ( FIG. 2C ) Specificity scores for the L18+R18 CCR5A TALEN at all positions in the target half-sites plus a single flanking position. The colors range from a maximum specificity score of 1.0 to white (no specificity, score of 0) to a maximum negative score of −1.0. Boxed bases represent the intended target base. ( FIG. 2D ) Same as ( FIG. 2C ) for the L18+R18 ATM TALEN. ( FIG. 2E ) Enrichment values from the selection of L13+R13 CCR5B TALEN for 16 mutant DNA sequences (mutations in red) relative to on-target DNA (OnB). ( FIG. 2F ) Correspondence between discrete in vitro TALEN cleavage efficiency (cleaved DNA as a fraction of total DNA) for the sequences listed in ( FIG. 2E ) normalized to on-target cleavage (=1) versus their enrichment values in the selection normalized to the on-target enrichment value (=1). ( FIG. 2G ) Discrete assays of on-target and off-target sequences used in ( FIG. 2F ) as analyzed by PAGE. Sequences in  FIG. 2C  correspond, from left to right and top to bottom, to SEQ ID NOs: 51-52. Sequences in  FIG. 2D  correspond, from left to right and top to bottom, to SEQ ID NOs: 53-54. Left half-site sequences in  FIG. 2E  correspond to SEQ ID NOs: 55-71 and right half-site sequences correspond to SEQ ID NOs: 72-88. 
         FIGS. 3A-3C . Cellular modification induced by TALENs at on-target and predicted off-target genomic sites. ( FIG. 3A ) For cells treated with either no TALEN or CCR5A TALENs containing heterodimeric EL/KK, heterodimeric ELD/KKR, or the homodimeric (Homo) FokI variants, cellular modification rates are shown as the percentage of observed insertions or deletions (indels) consistent with TALEN cleavage relative to the total number of sequences for on-target (On) and predicted off-target sites (Off). ( FIG. 3B ) Same as ( FIG. 3A ) for ATM TALENs. ( FIG. 3C ) Examples of modified sequences at the on-target site and off-target sites for cells treated with CCR5A TALENs containing the ELD/KKR FokI domains (SEQ ID NOs: 89-109 from top to bottom). For each example shown, the unmodified genomic site is the first sequence, followed by the top three sequences containing deletions. The numbers in parentheses indicate sequencing counts and the half-sites are underlined and bolded. 
         FIGS. 4A-4C . Predicted off-target genomic cleavage as a function of TALEN length considering both TALEN specificity and off-target site abundance in the human genome. ( FIG. 4A ) The enrichment value of on-target (zero mutation) and off-target sequences containing one to six mutations are shown for CCR5B TALENs of varying TALE repeat array lengths. The TALENs targeted DNA sites of 32 bp (L16+R16), 29 bp (L16+R13 or L13+R16), 26 bp (L16+R10 or L13+R13 or L10+R16), 23 bp (L13+R10 or L10+R13) or 20 bp (L10+R10) in length. ( FIG. 4B ) Number of sites in the human genome related to each of the nine CCR5B on-target sequences (L10, L13, or L16 combined with R10, R13, or R16), allowing for a spacer length from 12 to 25 bps between the two half-sites. ( FIG. 4C ) For all nine CCR5B TALENs, overall genomic off-target cleavage frequency was predicted by multiplying the number of sites in the human genome containing a certain number of mutations by the enrichment value of off-target sequences containing that same number of mutations shown in ( FIG. 4A ). Because enrichment values level off at high mutation numbers likely due to the limit of sensitivity of the selection, it was necessary to extrapolate high-mutation enrichment values by fitting enrichment value as function of mutation number (Table 9). The overall predicted genomic cleavage was calculated only for mutation numbers with sites observed to occur more than once in the human genome. 
         FIGS. 5A-5F . In vitro specificity and discrete cleavage efficiencies of TALENs containing canonical or engineered C-terminal domains. ( FIG. 5A  and  FIG. 5B ) On-target enrichment values for selections of ( FIG. 5A ) CCR5A TALENs or ( FIG. 5B ) ATM TALENs containing canonical, Q3, Q7, or 28-aa C-terminal domains. ( FIG. 5C ) CCR5A on-target sequence (OnC) and double-mutant sequences with mutations in red. ( FIG. 5D ) ATM on-target sequence (OnA) and single-mutant sequences with mutations in red. ( FIG. 5E ) Discrete in vitro cleavage efficiency of DNA sequences listed in ( FIG. 5C ) with CCR5A TALENs containing either canonical or engineered Q7 C-terminal domains. ( FIG. 5F ) Same as ( FIG. 5E ) for ATM TALENs. Left half-site sequences in  FIG. 5C  correspond to SEQ ID NOs: 110-118 and right half-site sequences correspond to SEQ ID NOs: 119-127. Left half-site sequences in  FIG. 5D  correspond to SEQ ID NOs: 128-136 and right half-site sequences correspond to SEQ ID NOs: 137-145. 
         FIG. 6 . Specificity of engineered TALENs in human cells. The cellular modification efficiency of canonical and engineered TALENs expressed as a percentage of indels consistent with TALEN-induced modification out of total sequences is shown for the on-target CCR5A sequence and for CCR5A off-target site #5, the most highly cleaved off-target substrate tested. Cellular specificity, defined as the ratio of on-target to off-target modification, is shown below each pair of bars. 
         FIGS. 7A-7B . Target DNA sequences in human CCR5 and ATM genes. The target DNA sequences for the TALENs used in this study are shown in black. The N-terminal TALEN end recognizing the 5′ T for each half-site target is noted (5′) and TALENs are named according to number of base pairs targeted. TALENs targeting the CCR5 L18 and R18 shown are referred to as CCR5A TALENs while TALENs targeting the L10, L13, L16, R10, R13 or R16 half-sites shown are referred to as CCR5B TALENs. In  FIG. 7A , top to bottom, left to right the sequences correspond to SEQ ID NOs: 146-155. In  FIG. 7A , top to bottom, left to right the sequences correspond to SEQ ID NOs: 156-159. 
         FIGS. 8A-8B . Specificity profiles from all CCR5A TALEN selections as heat maps. Specificity scores for every targeted base pair in selections of CCR5A TALENs are shown. Specificity scores for the L18+R18 CCR5A TALEN at all positions in the target half-sites plus a single flanking position. The colors range from a maximum specificity score of 1.0 to white (score of 0, no specificity) to a maximum negative score of −1.0. Boxed bases represent the intended target base. The titles to the right indicate if the TALEN used in the selection differs from the canonical TALEN architecture, which contains a canonical C-terminal domain, wildtype N-terminal domain, and EL/KK FokI variant. Selections correspond to conditions listed in Table 2. ( FIG. 8A ) Specificity profiles of canonical, Q3, Q7, 28-aa, 32 nM canonical, 8 nM canonical, 4 nM canonical, 32 nM Q7 and 8 nM Q7 CCR5A TALEN selections. ( FIG. 8B ) Specificity profiles of 4 nM Q7, N1, N2, N3, canonical ELD/KKR, Q3 ELD/KKR, Q7 ELD/KKR and N2 ELD/KKR CCR5A TALEN selections. When not specified, TALEN concentration was 16 nM. Nttcattacacctgcagctn corresponds to SEQ ID NO: 51 and nagtatcaattctggaagan corresponds to SEQ ID NO: 52. 
         FIGS. 9A-9C . Specificity profiles from all CCR5A TALEN selections as bar graphs. Specificity scores for every targeted base pair in selections of CCR5A TALENs are shown. Positive specificity scores, up to complete specificity at a specificity score of 1.0, signify enrichment of that base pair over the other possibilities at that position. Negative specificity scores, down to complete antispecificity of −1.0, represents enrichment against that base pair. Specified positions were plotted as stacked bars above the X-axis (multiple specified base pairs at the same position were plotted over each other with the shortest bar in front, and not end-to-end) while anti-specified base pairs were plotted as narrow, grouped bars. The titles to the right indicate if the TALEN used in the selection differs from the canonical TALEN architecture, which contains a canonical C-terminal domain, wild-type N-terminal domain, and EL/KK FokI variant. Selections correspond to conditions listed in Table 2. ( FIG. 9A ) Specificity profiles of canonical, Q3, Q7, 28-aa, 32 nM canonical, and 8 nM canonical CCR5A TALEN selections. ( FIG. 9B ) Specificity profiles of 4 nM canonical, 32 nM Q7, 8 nM Q7, 4 nM Q7, N1, and N2 CCR5A TALEN selections. ( FIG. 9C ) Specificity profiles of N3, canonical ELD/KKR, Q3 ELD/KKR, Q7 ELD/KKR, and N2 ELD/KKR CCR5A TALEN selections. When not specified, TALEN concentration was 16 nM. nttcattacacctgcagctn corresponds to SEQ ID NO: 51, nagtatcaattctggaagan corresponds to SEQ ID NO: 52, ntgaattgggatgctgtttn corresponds to SEQ ID NO: 53; and ntttattttactgtctttan corresponds to SEQ ID NO: 54. 
         FIGS. 10A-10B . Specificity profiles from all ATM TALEN selections as heat maps. Specificity scores for every targeted base pair in selections of ATM TALENs are shown. Specificity scores for the L18+R18 ATM TALEN at all positions in the target half-sites plus a single flanking position. The colors range from a maximum specificity score of 1.0 to white (score of 0, no specificity) to a maximum negative score of −1.0. Boxed bases represent the intended target base. The titles to the right indicate if the TALEN used in the selection differs from the canonical TALEN architecture, which contains a canonical C-terminal domain, wild type N-terminal domain, and EL/KK FokI variant. Selections correspond to conditions listed in Table 2. ( FIG. 10A ) Specificity profiles of (12 nM) canonical, Q3, (12 nM) Q7, 24 nM canonical, 6 nM canonical, 3 nM canonical, 24 nM Q7, and 6 nM Q7 ATM TALEN selections. ( FIG. 10B ) Specificity profiles of N1, N2, N3, canonical ELD/KKR, Q3 ELD/KKR, Q7 ELD/KKR, and N2 ELD/KKR ATM TALEN selections. When not specified, TALEN concentration was 12 nM. ntgaattgggatgctgtttn corresponds to SEQ ID NO: 53; and ntttattttactgtctttan corresponds to SEQ ID NO: 54. 
         FIGS. 11A-11C . Specificity profiles from all ATM TALEN selections as bar graphs. Specificity scores for every targeted base pair in selections of ATM TALENs are shown. Positive specificity scores, up to complete specificity at a specificity score of 1.0, signify enrichment of that base pair over the other possibilities at that position. Negative specificity scores, down to complete antispecificity of −1.0, represents enrichment against that base pair. Specified positions were plotted as stacked bars above the X-axis (multiple specified base pairs at the same position were plotted over each other with the shortest bar in front, and not end-to-end) while anti-specified base pairs were plotted as narrow, grouped bars. The titles to the right indicate if the TALEN used in the selection differs from the canonical TALEN architecture, which contains a canonical C-terminal domain, wild-type N-terminal domain, and EL/KK FokI variant. Selections correspond to conditions listed in Table 2. ( FIG. 11A ) Specificity profiles of canonical, Q3, Q7, 32 nM canonical, and 8 nM canonical ATM TALEN selections. ( FIG. 11B ) Specificity profiles of 3 nM canonical, 24 nM Q7, 6 nM Q7, N1, N2, and N3 ATM TALEN selections. ( FIG. 11C ) Specificity profiles of canonical ELD/KKR, Q3 ELD/KKR, Q7 ELD/KKR, and N2 ELD/KKR ATM TALEN selections. When not specified, TALEN concentration was 12 nM. ntgaattgggatgctgtttn corresponds to SEQ ID NO: 53; and ntttattttactgtctttan corresponds to SEQ ID NO: 54. 
         FIG. 12 . Specificity profiles from all CCR5B TALEN selections as heat maps. Specificity scores for every targeted base pair in selections of CCR5B TALENs are shown. Specificity scores for CCR5B TALENs targeting all possible combinations of the left (L10, L13, L16) and right (R10, R13, R16) half-sites at all positions in the target half-sites plus a single flanking position. The colors range from a maximum specificity score of 1.0) to white (score of 0, no specificity) to a maximum negative score of −1.0. Boxed bases represent the intended target base. The titles to the right notes the targeted left (L) and right (R) target half-sites for the CCR5B TALEN used in the selection. Selections correspond to conditions listed in Table 2. Sequences in the left column correspond, from top to bottom, to SEQ ID NOs: 160, 160, 160, 161, 161, 161, 162, 162, and 162. Sequences in the right column correspond, from top to bottom, to SEQ ID NOs: 163, 164, 165, 163, 164, 165, 163, 164, and 165. 
         FIGS. 13A-13B . Specificity profiles from all CCR5B TALEN selections as bar graphs. Specificity scores for every targeted base pair in selections of CCR5B TALENs are shown. Positive specificity scores, up to complete specificity at a specificity score of 1.0, signify enrichment of that base pair over the other possibilities at that position. Negative specificity scores, down to complete antispecificity of −1.0, represents enrichment against that base pair. Specified positions were plotted as stacked bars above the X-axis (multiple specified base pairs at the same position were plotted over each other with the shortest bar in front, and not end-to-end) while anti-specified base pairs were plotted as narrow, grouped bars. The titles to the right notes the targeted left (L) and right (R) target half-sites for the CCR5B TALEN used in the selection. Selections correspond to conditions listed in Table 2. Sequences correspond to SEQ ID NO: 160 (left column) and SEQ ID NO: 163 (right column). 
         FIGS. 14A-14B . Observed versus predicted double-mutant sequence enrichment values. ( FIG. 14A ) For the L13+R13 CCR5A TALEN selection, the observed double-mutant enrichment values of individual sequences (post-selection sequence abundance÷pre-selection sequence abundance) were normalized to the on-target enrichment value (=1.0 by definition) and plotted against the corresponding predicted double-mutant enrichment values calculated by multiplying the enrichment value of the component single-mutants normalized to the on-target enrichment. 13The predicted double mutant enrichment values therefore assume independent contributions from each single mutation to the double-mutant&#39;s enrichment value. ( FIG. 14B ) The observed double-mutant sequence enrichment divided by the predicted double-mutant sequence enrichment plotted as a function of the distance (in base pairs) between the two mutations. Only sequences with two mutations in the same half-site were considered. 
         FIGS. 15A-15F . Effects of engineered TALEN domains and TALEN concentration on specificity. ( FIG. 15A ) The specificity score of the targeted base pair at each position of the CCR5A site was calculated for CCR5A TALENs containing the canonical, Q3, Q7, or 28-aa C-terminal domains. The specificity scores of the Q3, Q7, or 28-aa C-terminal domain TALENs subtracted by the specificity scores of the TALEN with the canonical C-terminal domain are shown. ( FIG. 15B ) Same as ( FIG. 15A ) but for CCR5A TALENs containing engineered N-terminal domains N1, N2, or N3. ( FIG. 15C ) Same as ( FIG. 15A ) but comparing specificity scores differences of the canonical CCR5A TALEN assayed at 16 nM, 8 nM, or 4 nM subtracted by the specificity scores of canonical CCR5A TALENs assayed at 32 nM. ( FIG. 15D-15F ) Same as ( FIG. 15A-15C ) but for ATM TALENs. Selections correspond to conditions listed in Table 2. ttcattacacctgcagct corresponds to SEQ ID NO: 44, agtatcaattctggaaga corresponds to SEQ ID NO: 46, tgaattgggatgctgttt corresponds to SEQ ID NO: 128 and tttattttactgtcttta corresponds to SEQ ID NO: 137. 
         FIGS. 16A-16B . Spacer-length preferences of TALENs. ( FIG. 16A ) For each selection with CCR5A TALENs containing various combinations of the canonical, Q3, Q7, or 28-aa C-terminal domains; N1, N2, or N3 N-terminal mutations; and the EL/KK or ELD/KKR FokI variants and at 4, 8, 16, or 32 nM, the DNA spacer-length enrichment values were calculated by dividing the abundance of DNA spacer lengths in post-selection sequences by the abundance of DNA spacer lengths in the preselection library sequences. ( FIG. 16B ) Same as ( FIG. 16A ) but for ATM TALENs. 
         FIGS. 17A-17B . DNA cleavage-site preferences of TALENs. ( FIG. 17A ) For each selection with CCR5A TALENs with various combinations of canonical, Q3, Q7, or 28-aa C-terminal domains; N1, N2, or N3 N-terminal mutations; and the EL/KK or ELD/KKR FokI variants and at 4, 8, 16, or 32 nM, histograms of the number of spacer DNA base pairs preceding the right half-site for each possible DNA spacer length, normalized to the total sequence counts of the entire selection, are shown. ( FIG. 17B ) Same as ( FIG. 17A ) for ATM TALENs. 
         FIG. 18 . DNA cleavage-site preferences of TALENs comprising N-terminal domains with different amino acid substitutions. Sequences in  FIG. 18 , from top to bottom, correspond to SEQ ID NOs: 31-41. 
         FIG. 19 . Exemplary TALEN plasmid construct. 
     
    
    
     DEFINITIONS 
     As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of agents. 
     The term “canonical sequence,” as used herein, refers to a sequence of DNA, RNA, or amino acids that reflects the most common choice of base or amino acid at each position amongst known molecules of that type. For example, the canonical amino acid sequence of a protein domain may reflect the most common choice of amino acid resides at each position amongst all known domains of that type, or amongst the majority of known domains of that type. In some embodiments, a canonical sequence is a consensus sequence. 
     The terms “consensus sequence” and “consensus site,” as used herein in the context of nucleic acid sequences, refers to a calculated sequence representing the most frequent nucleotide residue found at each position in a plurality of similar sequences. Typically, a consensus sequence is determined by sequence alignment in which similar sequences are compared to each other and similar sequence motifs are calculated. In the context of nuclease target site sequences, a consensus sequence of a nuclease target site may, in some embodiments, be the sequence most frequently bound, bound with the highest affinity, and/or cleaved with the highest efficiency by a given nuclease. 
     The terms “conjugating,” “conjugated,” and “conjugation” refer to an association of two entities, for example, of two molecules such as two proteins, two domains (e.g., a binding domain and a cleavage domain), or a protein and an agent (e.g., a protein binding domain and a small molecule). The association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage or via non-covalent interactions. In some embodiments, the association is covalent. In some embodiments, two molecules are conjugated via a linker connecting both molecules. For example, in some embodiments where two proteins are conjugated to each other, e.g., a binding domain and a cleavage domain of an engineered nuclease, to form a protein fusion, the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein. 
     The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a TALE nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a target site specifically bound and cleaved by the nuclease, e.g., in a cell-free assay, or in a target cell, tissue, or organism. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a nuclease, a hybrid protein, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, the specific allele, genome, target site, cell, or tissue being targeted, and the agent being used. 
     The term “engineered,” as used herein refers to a molecule, complex, substance, or entity that has been designed, produced, prepared, synthesized, and/or manufactured by a human. Accordingly, an engineered product is a product that does not occur in nature. In some embodiments, an engineered molecule or complex, e.g., an engineered TALEN monomer, dimer, or multimer, is a TALEN that has been designed to meet particular requirements or to have particular desired features e.g., to specifically bind a target sequence of interest with minimal off-target binding, to have a specific minimal or maximal cleavage activity, and/or to have a specific stability. 
     As used herein, the term “isolated” refers to a molecule, complex, substance, or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, synthesized, and/or manufactured by a human. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. 
     The term “library,” as used herein in the context of nucleic acids or proteins, refers to a population of two or more different nucleic acids or proteins, respectively. For example, a library of nuclease target sites comprises at least two nucleic acid molecules comprising different nuclease target sites. In some embodiments, a library comprises at least 10 1 , at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , or at least 10 15  different nucleic acids or proteins. In some embodiments, the members of the library may comprise randomized sequences, for example, fully or partially randomized sequences. In some embodiments, the library comprises nucleic acid molecules that are unrelated to each other, e.g., nucleic acids comprising fully randomized sequences. In other embodiments, at least some members of the library may be related, for example, they may be variants or derivatives of a particular sequence, such as a consensus target site sequence. 
     The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. 
     The term “nuclease,” as used herein, refers to an agent, for example a protein or a small molecule, capable of cleaving a phosphodiester bond connecting nucleotide residues in a nucleic acid molecule. In some embodiments, a nuclease is a protein, e.g., an enzyme that can bind a nucleic acid molecule and cleave a phosphodiester bond connecting nucleotide residues within the nucleic acid molecule. A nuclease may be an endonuclease, cleaving a phosphodiester bonds within a polynucleotide chain, or an exonuclease, cleaving a phosphodiester bond at the end of the polynucleotide chain. In some embodiments, a nuclease is a site-specific nuclease, binding and/or cleaving a specific phosphodiester bond within a specific nucleotide sequence, which is also referred to herein as the “recognition sequence,” the “nuclease target site,” or the “target site.” In some embodiments, a nuclease recognizes a single stranded target site, while in other embodiments, a nuclease recognizes a double-stranded target site, for example a double-stranded DNA target site. The target sites of many naturally occurring nucleases, for example, many naturally occurring DNA restriction nucleases, are well known to those of skill in the art. In many cases, a DNA nuclease, such as EcoRI, HindIII, or BamHI, recognize a palindromic, double-stranded DNA target site of 4 to 10 base pairs in length, and cut each of the two DNA strands at a specific position within the target site. Some endonucleases cut a double-stranded nucleic acid target site symmetrically, i.e., cutting both strands at the same position so that the ends comprise base-paired nucleotides, also referred to herein as blunt ends. Other endonucleases cut a double-stranded nucleic acid target sites asymmetrically, i.e., cutting each strand at a different position so that the ends comprise unpaired nucleotides. Unpaired nucleotides at the end of a double-stranded DNA molecule are also referred to as “overhangs,” e.g., as “5′-overhang” or as “3′-overhang,” depending on whether the unpaired nucleotide(s) form(s) the 5′ or the 5′ end of the respective DNA strand. Double-stranded DNA molecule ends ending with unpaired nucleotide(s) are also referred to as sticky ends, as they can “stick to” other double-stranded DNA molecule ends comprising complementary unpaired nucleotide(s). A nuclease protein typically comprises a “binding domain” that mediates the interaction of the protein with the nucleic acid substrate, and a “cleavage domain” that catalyzes the cleavage of the phosphodiester bond within the nucleic acid backbone. In some embodiments, a nuclease protein can bind and cleave a nucleic acid molecule in a monomeric form, while, in other embodiments, a nuclease protein has to dimerize or multimerize in order to cleave a target nucleic acid molecule. Binding domains and cleavage domains of naturally occurring nucleases, as well as modular binding domains and cleavage domains that can be combined to create nucleases that bind specific target sites, are well known to those of skill in the art. For example, transcriptional activator like elements can be used as binding domains to specifically bind a desired target site, and fused or conjugated to a cleavage domain, for example, the cleavage domain of FokI, to create an engineered nuclease cleaving the desired target site. 
     The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications&#39; A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). 
     The term “pharmaceutical composition,” as used herein, refers to a composition that can be administrated to a subject in the context of treatment of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, e.g. a nuclease or a nucleic acid encoding a nuclease, and a pharmaceutically acceptable excipient. 
     The terms “prevention” or “prevent” refer to the prophylactic treatment of a subject who is at risk of developing a disease, disorder, or condition (e.g., at an elevated risk as compared to a control subject, or a control group of subject, or at an elevated risk as compared to the average risk of an age-matched and/or gender-matched subject), resulting in a decrease in the probability that the subject will develop the disease, disorder, or condition (as compared to the probability without prevention), and/or to the inhibition of further advancement of an already established disorder. 
     The term “proliferative disease,” as used herein, refers to any disease in which cell or tissue homeostasis is disturbed in that a cell or cell population exhibits an abnormally elevated proliferation rate. Proliferative diseases include hyperproliferative diseases, such as pre-neoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by an abnormal proliferation of cells and include both benign and malignant neoplasias. Malignant neoplasms are also referred to as cancers. 
     The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. 
     The term “randomized,” as used herein in the context of nucleic acid sequences, refers to a sequence or residue within a sequence that has been synthesized to incorporate a mixture of free nucleotides, for example, a mixture of all four nucleotides A, T, G, and C. Randomized residues are typically represented by the letter N within a nucleotide sequence. In some embodiments, a randomized sequence or residue is fully randomized, in which case the randomized residues are synthesized by adding equal amounts of the nucleotides to be incorporated (e.g., 25% T, 25% A, 25% G, and 25% C) during the synthesis step of the respective sequence residue. In some embodiments, a randomized sequence or residue is partially randomized, in which case the randomized residues are synthesized by adding non-equal amounts of the nucleotides to be incorporated (e.g., 79% T, 7% A, 7% G, and 7% C) during the synthesis step of the respective sequence residue. Partial randomization allows for the generation of sequences that are templated on a given sequence, but have incorporated mutations at a desired frequency. For example, if a known nuclease target site is used as a synthesis template, partial randomization in which at each step the nucleotide represented at the respective residue is added to the synthesis at 79%, and the other three nucleotides are added at 7% each, will result in a mixture of partially randomized target sites being synthesized, which still represent the consensus sequence of the original target site, but which differ from the original target site at each residue with a statistical frequency of 21% for each residue so synthesized (distributed binomially). In some embodiments, a partially randomized sequence differs from the consensus sequence by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, or more than 30% on average, distributed binomially. In some embodiments, a partially randomized sequence differs from the consensus site by no more than 10%, no more than 15%, no more than 20%, no more than 25%, nor more than 30%, no more than 40%, or no more than 50% on average, distributed binomially. 
     The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human of either sex at any stage of development. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. 
     The terms “target nucleic acid,” and “target genome,” as used herein in the context of nucleases, refer to a nucleic acid molecule or a genome, respectively, that comprises at least one target site of a given nuclease. 
     The term “target site,” used herein interchangeably with the term “nuclease target site,” refers to a sequence within a nucleic acid molecule that is bound and cleaved by a nuclease. A target site may be single-stranded or double-stranded. In the context of nucleases that dimerize, for example, nucleases comprising a FokI DNA cleavage domain, a target site typically comprises a left-half site (bound by one monomer of the nuclease), a right-half site (bound by the second monomer of the nuclease), and a spacer sequence between the half sites in which the cut is made. This structure ([left-half site]-[spacer sequence]-[right-half site]) is referred to herein as an LSR structure. In some embodiments, the left-half site and/or the right-half site is between 10-18 nucleotides long. In some embodiments, either or both half-sites are shorter or longer. In some embodiments, the left and right half sites comprise different nucleic acid sequences. 
     The term “Transcriptional Activator-Like Effector,” (TALE) as used herein, refers to proteins comprising a DNA binding domain, which contains a highly conserved 33-34 amino acid sequence comprising a highly variable two-amino acid motif (Repeat Variable Diresidue, RVD). The RVD motif determines binding specificity to a nucleic acid sequence, and can be engineered according to methods well known to those of skill in the art to specifically bind a desired DNA sequence (see, e.g., Miller, Jeffrey; et. al. (February 2011). “A TALE nuclease architecture for efficient genome editing”.  Nature Biotechnology  29 (2): 143-8; Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”.  Nature Biotechnology  29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”.  PLoS ONE  6 (5): e19509; Boch, Jens (February 2011). “TALEs of genome targeting”.  Nature Biotechnology  29 (2): 135-6; Boch, Jens; et. al. (December 2009). “Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”.  Science  326 (5959): 1509-12; and Moscou, Matthew J.; Adam J. Bogdanove (December 2009). “A Simple Cipher Governs DNA Recognition by TAL Effectors”.  Science  326 (5959): 1501; the entire contents of each of which are incorporated herein by reference). The simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs. 
     The term “Transcriptional Activator-Like Element Nuclease,” (TALEN) as used herein, refers to an artificial nuclease comprising a transcriptional activator like effector DNA binding domain to a DNA cleavage domain, for example, a FokI domain. A number of modular assembly schemes for generating engineered TALE constructs have been reported (Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”.  Nature Biotechnology  29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”.  PLoS ONE  6 (5): e19509; Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”.  Nucleic Acids Research ; Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”.  Nucleic Acids Research ; Li, T.; Huang, S.; Zhao, X.; Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B. (2011). “Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes”.  Nucleic Acids Research .; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). Bendahmane, Mohammed. ed. “Assembly of Designer TAL Effectors by Golden Gate Cloning”.  PLoS ONE  6 (5): e19722; the entire contents of each of which are incorporated herein by reference). 
     The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. 
     DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 
     Transcription activator-like effector nucleases (TALENs) are fusions of the FokI restriction endonuclease cleavage domain with a DNA-binding transcription activator-like effector (TALE) repeat array. TALENs can be engineered to reduce off-target cleavage activity and thus to specifically bind a target DNA sequence and can thus be used to cleave a target DNA sequence, e.g., in a genome, in vitro or in vivo. Such engineered TALENs can be used to manipulate genomes in vivo or in vitro, e.g., for gene knockouts or knock-ins via induction of DNA breaks at a target genomic site for targeted gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template. 
     TALENs can be designed to cleave any desired target DNA sequence, including naturally occurring and synthetic sequences. However, the ability of TALENs to distinguish target sequences from closely related off-target sequences has not been studied in depth. Understanding this ability and the parameters affecting it is of importance for the design of TALENs having the desired level of specificity for their therapeutic use and also for choosing unique target sequences to be cleaved in order to minimize the chance of off-target cleavage. 
     Some aspects of this disclosure are based on cleavage specificity data obtained from profiling 41 TALENs on 10 12  potential off-target sites through in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted off-target substrates in the human genome, thirteen of which were modified by TALENs in human cells. Some aspect of this disclosure are based on the surprising findings that (i) TALEN repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity. Based on these findings, optimized TALENs were engineered with mutations designed to reduce non-specific DNA binding. Some of these engineered TALENs exhibit improved specificity, e.g., 34- to &gt;116-fold greater specificity, in human cells compared to commonly used TALENs. 
     The ability to engineer site-specific changes in genomes represents a powerful research capability with significant therapeutic implications. TALENs are fusions of the FokI restriction endonuclease cleavage domain with a DNA-binding TALE repeat array ( FIG. 1A ). These arrays consist of multiple 34-amino acid TALE repeat sequences, each of which uses a repeat-variable di-residue (RVD), the amino acids at positions 12 and 13, to recognize a single DNA nucleotide. 1,2  Examples of RVDs that enable recognition of each of the four DNA base pairs are known, enabling arrays of TALE repeats to be constructed that can bind virtually any DNA sequence. TALENs can be engineered to be active only as heterodimers through the use of obligate heterodimeric FokI variants. 3,4  In this configuration, two distinct TALEN monomers are each designed to bind one target half-site and to cleave within the DNA spacer sequence between the two half-sites. 
     In cells, e.g., in mammalian cells, TALEN-induced double-strand breaks can result in targeted gene knockout through non-homologous end joining (NHEJ) 5  or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template. 6,7  TALENs have been successfully used to manipulate genomes in a variety of organisms 8-11  and cell lines. 7,12,13    
     TALEN-mediated DNA cleavage at off-target sites can result in unintended mutations at genomic loci. While SELEX experiments have characterized the DNA-binding specificities of monomeric TALE proteins, 5,7  the DNA cleavage specificities of active, dimeric nucleases can differ from the specificities of their component monomeric DNA-binding domains. 14  Full-genome sequencing of four TALEN-treated yeast strains 15  and two human cell lines 16  derived from a TALEN-treated cell revealed no evidence of TALE-induced genomic off-target mutations, consistent with other reports that observed no off-target genomic modification in  Xenopus   17  and human cell lines. 18  In contrast, TALENs were observed to cleave off-target sites containing two to eleven mutations relative to the on-target sequence in vivo in zebrafish, 13,19  rats, 9  human primary fibroblasts, 20  and embryonic stem cells. 7  A systematic and comprehensive profile of TALEN specificity generated from measurements of TALEN cleavage on a large set of related mutant target sites has not been described before. Such a broad specificity profile is fundamental to understand and improve the potential of TALENs as research tools and therapeutic agents. 
     Some of the work described herein relates to experiments performed to profile the ability of 41 TALEN pairs to cleave 10 12  off-target variants of each of their respective target sequences using a modified version of a previously described in vitro selection 14  for DNA cleavage specificity. These results from these experiments provide comprehensive profiles of TALEN cleavage specificities. The in vitro selection results were used to computationally predict off-target substrates in the human genome, 13 of which were confirmed to be cleaved by TALENs in human cells. 
     It was surprisingly found that, despite being less specific per base pair, TALENs designed to cleave longer target sites in general exhibit higher overall specificity than those that target shorter sites when considering the number of potential off-target sites in the human genome. The selection results also suggest a model in which excess non-specific TALEN binding energy gives rise to greater off-target cleavage relative to on-target cleavage. Based on this model, we engineered TALENs with substantially improved DNA cleavage specificity in vitro, and 30- to &gt;150-fold greater specificity in human cells, than currently used TALEN constructs. 
     Some aspects of this disclosure are based on data obtained from profiling the specificity of 41 heterodimeric TALENs designed to target one of three distinct sequence, as described in more detail elsewhere herein. The profiling was performed using an improved version of an in vitro selection method 14  (also described in PCT Application Publication WO2013/066438 A2, the entire contents of which are incorporated herein by reference) with modifications that increase the throughput and sensitivity of the selection ( FIG. 1B ). 
     Briefly, TALENs were profiled against libraries of &gt;10 12  DNA sequences and cleavage products were captured and analyzed to determine the specificity and off-target activity of each TALEN. The selection data accurately predicted the efficiency of off-target TALEN cleavage in vitro, and also indicated that TALENs are overall highly specific across the entire target sequence, but that some level of off-target cleavage occurs in conventional TALENs which can be undesirable in some scenarios of TALEN use. As a result of the experiments described herein, it was surprisingly found that that TALE repeats bind their respective DNA base pairs independently beyond a slightly increased tolerance for adjacent mismatches, which informed the recognition that TALEN specificity per base pair is independent of target-site length. It was experimentally validated that shorter TALENs have greater specificity per targeted base pair than longer TALENs, but that longer TALENs are more specific against the set of potential cleavage sites in the context of a whole genome than shorter TALENs for the tested TALEN lengths targeting 20- to 32-bp sites, as described in more detail elsewhere herein. 
     Some aspects of this disclosure are based on the surprising discovery that excess binding energy in longer TALENs reduces specificity by enabling the cleavage of off-target sequences without a corresponding increase in the efficiency of on-target cleavage efficiency. Some aspects of this disclosure are based on the surprising discovery that TALENs can be engineered to more specifically cleave their target sequences by reducing off-target binding energy without compromising on-target cleavage efficiency. The recognition that TALEN specificity can be improved by reducing non-specific DNA binding energy beyond what is required to enable efficient on-target cleavage served as the basis for the generation of engineered TALENs with improved target site specificity. 
     Typically, a TALEN monomer, e.g., a TALEN monomer as provided herein, comprises or is of the following structure: 
     [N-terminal domain]-[TALE repeat array]-[C-terminal domain]-[nuclease domain] 
     wherein each “-” individually indicates conjugation, either covalently or non-covalently, and wherein the conjugation can be direct, e.g., via direct bond, or indirect, e.g., via a linker domain. See also  FIG. 1 . 
     Some aspects of this disclosure provide TALENs with enhanced specificity as compared to TALENs that were previously used. In general, the sequence specificity of a TALEN is conferred by the TALE repeat array, which binds to a specific nucleotide sequence. TALE repeat arrays consist of multiple 34-amino acid TALE repeat sequences, each of which uses a repeat-variable di-residue (RVD), the amino acids at positions 12 and 13, to recognize a single DNA nucleotide. Some aspects of this disclosure provide that the specific binding of the TALE repeat array is sufficient for dimerization and nucleic acid cleavage, and that non-specific nucleic acid binding activity is due to the N-terminal and/or C-terminal domains of the TALEN. 
     Based on this recognition, improved TALENs have been engineered as provided herein. As it was discovered that non-specific binding via the N-terminal domain can occur through excess binding energy conferred by amino acid residues that are positively charged (cationic) at physiological pH, some of the improved TALENs provided herein have a decreased net charge and/or a decreased binding energy for binding their target nucleic acid sequence as compared to canonical TALENs. This decrease in charge leads to a decrease in off-target binding via the modified N-terminal and C-terminal domains. The portion of target recognition and binding, thus, is more narrowly confined to the specific recognition and binding activity of the TALE repeat array. The resulting TALENs, thus, exhibit an increase in the specificity of binding and, in turn, in the specificity of cleaving the target site by the improved TALEN as compared to a TALEN using non-modified domains. 
     In some embodiments, a TALEN is provided in which the net charge of the N-terminal domain is less than the net charge of the canonical N-terminal domain (SEQ ID NO: 1); and/or the net charge of the C-terminal domain is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, a TALEN is provided in which the binding energy of the N-terminal domain to a target nucleic acid molecule is less than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1); and/or the binding energy of the C-terminal domain to a target nucleic acid molecule is less than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, a modified TALEN N-terminal domain is provided the binding energy of which to the TALEN target nucleic acid molecule is less than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1). In some embodiments, a modified TALEN C-terminal domain is provided the binding energy of which to the TALEN target nucleic acid molecule is less than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, the binding energy of the N-terminal and/or of the C-terminal domain in the TALEN provided is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 98%, or at least 99%. 
     In some embodiments, the canonical N-terminal domain and/or the canonical C-terminal domain is modified to replace an amino acid residue that is positively charged at physiological pH with an amino acid residue that is not charged or is negatively charged. In some embodiments, the modification includes the replacement of a positively charged residue with a negatively charged residue. In some embodiments, the modification includes the replacement of a positively charged residue with a neutral (uncharged) residue. In some embodiments, the modification includes the replacement of a positively charged residue with a residue having no charge or a negative charge. In some embodiments, the net charge of the modified N-terminal domain and/or of the modified C-terminal domain is less than or equal to +10, less than or equal to +9, less than or equal to +8, less than or equal to +7, less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to −1, less than or equal to −2, less than or equal to −3, less than or equal to −4, or less than or equal to −5, or less than or equal to −10. In some embodiments, the net charge of the modified N-terminal domain and/or of the modified C-terminal domain is between +5 and −5, between +2 and −7, between 0 and −5, between 0 and −10, between −1 and −10, or between −2 and −15. In some embodiments, the net charge of the modified N-terminal domain and/or of the modified C-terminal domain is negative. In some embodiments, the net charge of the modified N-terminal domain and of the modified C-terminal domain, together, is negative. In some embodiments, the net charge of the modified N-terminal domain and/or of the modified C-terminal domain is neutral or slightly positive (e.g., less than +2 or less than +1). In some embodiments, the net charge of the modified N-terminal domain and of the modified C-terminal domain, together, is neutral or slightly positive (e.g., less than +2 or less than +1). 
     In some embodiments, the modified N-terminal domain and/or the modified C-terminal domain comprise(s) an amino acid sequence that differs from the respective canonical domain sequence in that at least one cationic amino acid residue of the canonical domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the modified N-terminal domain and/or in the modified C-terminal domain. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the modified N-terminal domain and/or in the modified C-terminal domain. 
     In some embodiments, the cationic amino acid residue is arginine (R), lysine (K), or histidine (H). In some embodiments, the cationic amino acid residue is R or H. In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q), Glycine (G), Asparagine (N), Threonine (T), Serine (S), Aspartic acid (D), or Glutamic Acid (E). In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is Q. In some embodiments, at least one lysine or arginine residue is replaced with a glutamine residue in the modified N-terminal domain and/or in the modified C-terminal domain. 
     In some embodiments, the C-terminal domain comprises one or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises two or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises three or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises four or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises five or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises six or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises all seven of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the C-terminal domain comprises a Q3 variant sequence (K788Q, R792Q, R801Q, see SEQ ID NO: 23). In some embodiments, the C-terminal domain comprises a Q7 variant sequence (K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q, see SEQ ID NO: 24). 
     In some embodiments, the N-terminal domain is a truncated version of the canonical N-terminal domain. In some embodiments, the C-terminal domain is a truncated version of the canonical C-terminal domain. In some embodiments, the truncated N-terminal domain and/or the truncated C-terminal domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues. In some embodiments, the modified N-terminal domain and/or the modified C-terminal domain is/are truncated and comprise one or more amino acid replacement(s). It will be apparent to those of skill in the art that it is desirable in some embodiments to adjust the DNA spacer length in TALENs using truncated domains, e.g., truncated C-terminal domains, in order to accommodate the truncation. 
     In some embodiments, the nuclease domain, also sometimes referred to as a nucleic acid cleavage domain is a non-specific cleavage domain, e.g., a FokI nuclease domain. In some embodiments, the nuclease domain is monomeric and must dimerize or multimerize in order to cleave a nucleic acid. Homo- or heterodimerization or multimerization of TALEN monomers typically occurs via binding of the monomers to binding sequences that are in sufficiently close proximity to allow dimerization, e.g., to sequences that are proximal to each other on the same nucleic acid molecule (e.g., the same double-stranded nucleic acid molecule). 
     The most commonly used domains, e.g., the most widely used N-terminal and C-terminal domains, are referred to herein as canonical domains. Exemplary sequences of a canonical N-terminal domain (SEQ ID NO: 1) and a canonical C-terminal domain (SEQ ID NO: 22) are provided herein. Exemplary sequences of FokI nuclease domains are also provided herein. In addition, exemplary sequences of TALE repeats forming a CCR5-binding TALE repeat array are provided. It will be understood that the sequences provided below are exemplary and provided for the purpose of illustrating some embodiments embraced by the present disclosure. They are not meant to be limiting and additional sequences useful according to aspects of this disclosure will be apparent to the skilled artisan based on this disclosure. 
     
       
         
           
               
            
               
                 Canonical N-terminal domain: 
               
               
                 (SEQ ID NO: 1) 
               
               
                 VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA 
               
               
                   
               
               
                 LGTVAV   K   YQDMIAALPEATHEAIVGVG   K   QWSGA   R   ALEALLTVAGEL   R   GPP 
               
               
                   
               
               
                 LQLDTGQLL   K   IA   KR   GGVTAVEAVHAWRNALTGAPLN 
               
               
                   
               
               
                 Modified N-terminal domain: N1 
               
               
                 (SEQ ID NO: 2) 
               
               
                 VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA 
               
               
                   
               
               
                 LGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP 
               
               
                   
               
               
                 LQLDTGQLL   Q   IAKRGGVTAVEAVHAWRNALTGAPLN 
               
               
                   
               
               
                 Modified N-terminal domain: N2 
               
               
                 (SEQ ID NO: 3) 
               
               
                 VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA 
               
               
                   
               
               
                 LGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP 
               
               
                   
               
               
                 LQLDTGQLL   Q   IA   Q   RGGVTAVEAVHAWRNALTGAPLN 
               
               
                   
               
               
                 Modified N-terminal domain: N3 
               
               
                 (SEQ ID NO: 4) 
               
               
                 VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA 
               
               
                   
               
               
                 LGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP 
               
               
                   
               
               
                 LQLDTGQLL   Q   IA   QQ   GGVTAVEAVHAWRNALTGAPLN 
               
               
                   
               
               
                 TALE repeat array: L18 CCR5A 
               
               
                 (SEQ ID NO: 5) 
               
               
                 MTPDQVVAIAS NG GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 6) 
               
               
                 GLTPEQVVAIAS HD GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 7) 
               
               
                 GLTPDQVVAIAS NI GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 8) 
               
               
                 GLTPAQVVAIAS NG GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 9) 
               
               
                 GLTPDQVVAIAS NG GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 10) 
               
               
                 GLTPEQVVAIAS NI GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 11) 
               
               
                 GLTPDQVVAIAS HD GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 12) 
               
               
                 GLTPAQVVAIAS NI GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 13) 
               
               
                 GLTPDQVVAIAS HD GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 14) 
               
               
                 GLTPEQVVAIAS HD GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 15) 
               
               
                 GLTPDQVVAIAS NG GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 16) 
               
               
                 GLTPAQVVAIAN NN GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 17) 
               
               
                 GLTPDQVVAIAS HD GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 18) 
               
               
                 GLTPEQVVAIAS NI GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 19) 
               
               
                 GLTPDQVVAIAN NN GGKQALETVQRLLPVLCQAH 
               
               
                   
               
               
                 (SEQ ID NO: 20) 
               
               
                 GLTPAQVVAIAS HD GGKQALETVQRLLPVLCQDH 
               
               
                   
               
               
                 (SEQ ID NO: 21) 
               
               
                 GLTPEQVVAIAS NG GGRPALE 
               
               
                   
               
               
                 Canonical C-terminal domain: 
               
               
                 (SEQ ID NO: 22) 
               
               
                 SIVAQLS   R   PDPALAALTNDHLVALACLGG   R   PALDAV   KK   GLPHAPALI   KR   T 
               
               
                   
               
               
                 N   RR   IPE   R   TSH   R   VA 
               
               
                   
               
               
                 Modified C-terminal domain: Q3 
               
               
                 (SEQ ID NO: 23) 
               
               
                 SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALI   Q   RT 
               
               
                   
               
               
                 N   Q   RIPERTSH   Q   VA 
               
               
                   
               
               
                 Modified C-terminal domain: Q7 
               
               
                 (SEQ ID NO: 24) 
               
               
                 SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV   QQ   GLPHAPALI   QQ   T 
               
               
                   
               
               
                 N   QQ   IPERTSH   Q   VA 
               
               
                   
               
               
                 Modified C-terminal domain: 28-aa 
               
               
                 (SEQ ID NO: 25) 
               
               
                 SIVAQLSRPDPALAALTNDHLVALACLG 
               
               
                   
               
               
                 FokI: homodimeric 
               
               
                 (SEQ ID NO: 26) 
               
               
                 GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEF 
               
               
                   
               
               
                 FMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQ 
               
               
                   
               
               
                 ADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ 
               
               
                   
               
               
                 LTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF* 
               
               
                   
               
               
                 FokI: EL 
               
               
                 (SEQ ID NO: 27) 
               
               
                 GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEF 
               
               
                   
               
               
                 FMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQ 
               
               
                   
               
               
                 ADEM   E   RYVEENQTRNKH   L   NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ 
               
               
                   
               
               
                 LTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF* 
               
               
                   
               
               
                 FokI: KK 
               
               
                 (SEQ ID NO: 28) 
               
               
                 GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEF 
               
               
                   
               
               
                 FMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQ 
               
               
                   
               
               
                 ADEMQRYV   K   ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ 
               
               
                   
               
               
                 LTRLNH   K   TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF* 
               
               
                   
               
               
                 FokI: ELD 
               
               
                 (SEQ ID NO: 29) 
               
               
                 GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEF 
               
               
                   
               
               
                 FMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQ 
               
               
                   
               
               
                 ADEM   E   RYVEENQTR   D   KH   L   NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ 
               
               
                   
               
               
                 LTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF* 
               
               
                   
               
               
                 FokI: KKR 
               
               
                 (SEQ ID NO: 30) 
               
               
                 GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEF 
               
               
                   
               
               
                 FMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQ 
               
               
                   
               
               
                 ADEMQRYV   K   ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ 
               
               
                   
               
               
                 LTRLN   RK   TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF* 
               
            
           
         
       
     
     In some embodiments, a TALEN is provided herein that comprises a canonical N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain. In some embodiments, a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a canonical C-terminal domain, and a nuclease domain. In some embodiments, a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain. In some embodiments, the nuclease domain is a FokI nuclease domain. In some embodiments, the FokI nuclease domain is a homodimeric FokI domain, or a FokI-EL, FokI-KK, FokI-ELD, or FokI-KKR domain. 
     All possible combinations of the specific sequences of canonical and modified domains provided herein are embraced by this disclosure, including the following: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Exemplary TALENs embraced by the present disclosure. The respective 
               
               
                 TALE repeat array employed will depend on the specific target 
               
               
                 sequence. Those of skill in the art will be able to design 
               
               
                 such sequence-specific TALE repeat arrays based on the instant 
               
               
                 disclosure and the knowledge in the art. Sequences for the 
               
               
                 different N-terminal, C-terminal, and Nuclease domains are 
               
               
                 provided above (See, SEQ ID NOs 1-4 and 22-30). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 N-terminal 
                 TALE repeat 
                 C-terminal 
                 Nuclease 
               
               
                 TALEN 
                 domain 
                 array 
                 domain 
                 domain 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 Canonical 
                 Sequence-specific 
                 Q3 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 2 
                 Canonical 
                 Sequence-specific 
                 Q3 
                 EL 
               
               
                 3 
                 Canonical 
                 Sequence-specific 
                 Q3 
                 KK 
               
               
                 4 
                 Canonical 
                 Sequence-specific 
                 Q3 
                 ELD 
               
               
                 5 
                 Canonical 
                 Sequence-specific 
                 Q3 
                 KKR 
               
               
                 6 
                 Canonical 
                 Sequence-specific 
                 Q7 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 7 
                 Canonical 
                 Sequence-specific 
                 Q7 
                 EL 
               
               
                 8 
                 Canonical 
                 Sequence-specific 
                 Q7 
                 KK 
               
               
                 9 
                 Canonical 
                 Sequence-specific 
                 Q7 
                 ELD 
               
               
                 10 
                 Canonical 
                 Sequence-specific 
                 Q7 
                 KKR 
               
               
                 11 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 12 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 EL 
               
               
                 13 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 KK 
               
               
                 14 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 ELD 
               
               
                 15 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 KKR 
               
               
                 16 
                 N1 
                 Sequence-specific 
                 Canonical 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 17 
                 N1 
                 Sequence-specific 
                 Canonical 
                 EL 
               
               
                 18 
                 N1 
                 Sequence-specific 
                 Canonical 
                 KK 
               
               
                 19 
                 N1 
                 Sequence-specific 
                 Canonical 
                 ELD 
               
               
                 20 
                 N1 
                 Sequence-specific 
                 Canonical 
                 KKR 
               
               
                 21 
                 N1 
                 Sequence-specific 
                 Q3 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 22 
                 N1 
                 Sequence-specific 
                 Q3 
                 EL 
               
               
                 23 
                 N1 
                 Sequence-specific 
                 Q3 
                 KK 
               
               
                 24 
                 N1 
                 Sequence-specific 
                 Q3 
                 ELD 
               
               
                 25 
                 N1 
                 Sequence-specific 
                 Q3 
                 KKR 
               
               
                 26 
                 N1 
                 Sequence-specific 
                 Q7 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 27 
                 N1 
                 Sequence-specific 
                 Q7 
                 EL 
               
               
                 28 
                 N1 
                 Sequence-specific 
                 Q7 
                 KK 
               
               
                 29 
                 N1 
                 Sequence-specific 
                 Q7 
                 ELD 
               
               
                 30 
                 N1 
                 Sequence-specific 
                 Q7 
                 KKR 
               
               
                 31 
                 N1 
                 Sequence-specific 
                 Truncated (28aa) 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 32 
                 N1 
                 Sequence-specific 
                 Truncated (28aa) 
                 EL 
               
               
                 33 
                 N1 
                 Sequence-specific 
                 Truncated (28aa) 
                 KK 
               
               
                 34 
                 N1 
                 Sequence-specific 
                 Truncated (28aa) 
                 ELD 
               
               
                 35 
                 N1 
                 Sequence-specific 
                 Truncated (28aa) 
                 KKR 
               
               
                 36 
                 N2 
                 Sequence-specific 
                 Canonical 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 37 
                 N2 
                 Sequence-specific 
                 Canonical 
                 EL 
               
               
                 38 
                 N2 
                 Sequence-specific 
                 Canonical 
                 KK 
               
               
                 39 
                 N2 
                 Sequence-specific 
                 Canonical 
                 ELD 
               
               
                 40 
                 N2 
                 Sequence-specific 
                 Canonical 
                 KKR 
               
               
                 41 
                 N2 
                 Sequence-specific 
                 Q3 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 42 
                 N2 
                 Sequence-specific 
                 Q3 
                 EL 
               
               
                 43 
                 N2 
                 Sequence-specific 
                 Q3 
                 KK 
               
               
                 44 
                 N2 
                 Sequence-specific 
                 Q3 
                 ELD 
               
               
                 45 
                 N2 
                 Sequence-specific 
                 Q3 
                 KKR 
               
               
                 46 
                 N2 
                 Sequence-specific 
                 Q7 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 47 
                 N2 
                 Sequence-specific 
                 Q7 
                 EL 
               
               
                 48 
                 N2 
                 Sequence-specific 
                 Q7 
                 KK 
               
               
                 49 
                 N2 
                 Sequence-specific 
                 Q7 
                 ELD 
               
               
                 50 
                 N2 
                 Sequence-specific 
                 Q7 
                 KKR 
               
               
                 51 
                 N2 
                 Sequence-specific 
                 Truncated (28aa) 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 52 
                 N2 
                 Sequence-specific 
                 Truncated (28aa) 
                 EL 
               
               
                 53 
                 N2 
                 Sequence-specific 
                 Truncated (28aa) 
                 KK 
               
               
                 54 
                 N2 
                 Sequence-specific 
                 Truncated (28aa) 
                 ELD 
               
               
                 55 
                 N2 
                 Sequence-specific 
                 Truncated (28aa) 
                 KKR 
               
               
                 56 
                 N3 
                 Sequence-specific 
                 Canonical 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 57 
                 N3 
                 Sequence-specific 
                 Canonical 
                 EL 
               
               
                 58 
                 N3 
                 Sequence-specific 
                 Canonical 
                 KK 
               
               
                 59 
                 N3 
                 Sequence-specific 
                 Canonical 
                 ELD 
               
               
                 60 
                 N3 
                 Sequence-specific 
                 Canonical 
                 KKR 
               
               
                 61 
                 N3 
                 Sequence-specific 
                 Q3 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 62 
                 N3 
                 Sequence-specific 
                 Q3 
                 EL 
               
               
                 63 
                 N3 
                 Sequence-specific 
                 Q3 
                 KK 
               
               
                 64 
                 N3 
                 Sequence-specific 
                 Q3 
                 ELD 
               
               
                 65 
                 N3 
                 Sequence-specific 
                 Q3 
                 KKR 
               
               
                 66 
                 N3 
                 Sequence-specific 
                 Q7 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 67 
                 N3 
                 Sequence-specific 
                 Q7 
                 EL 
               
               
                 68 
                 N3 
                 Sequence-specific 
                 Q7 
                 KK 
               
               
                 69 
                 N3 
                 Sequence-specific 
                 Q7 
                 ELD 
               
               
                 70 
                 N3 
                 Sequence-specific 
                 Q7 
                 KKR 
               
               
                 71 
                 N3 
                 Sequence-specific 
                 Truncated (28aa) 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 72 
                 N3 
                 Sequence-specific 
                 Truncated (28aa) 
                 EL 
               
               
                 73 
                 N3 
                 Sequence-specific 
                 Truncated (28aa) 
                 KK 
               
               
                 74 
                 N3 
                 Sequence-specific 
                 Truncated (28aa) 
                 ELD 
               
               
                 75 
                 N3 
                 Sequence-specific 
                 Truncated (28aa) 
                 KKR 
               
               
                 76 
                 Canonical 
                 Sequence-specific 
                 Canonical 
                 EL 
               
               
                 77 
                 Canonical 
                 Sequence-specific 
                 Canonical 
                 KK 
               
               
                 78 
                 Canonical 
                 Sequence-specific 
                 Canonical 
                 ELD 
               
               
                 79 
                 Canonical 
                 Sequence-specific 
                 Canonical 
                 KKR 
               
               
                 80 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 Homo- 
               
               
                   
                   
                   
                   
                 dimeric 
               
               
                 81 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 EL 
               
               
                 82 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 KK 
               
               
                 83 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 ELD 
               
               
                 84 
                 Canonical 
                 Sequence-specific 
                 Truncated (28aa) 
                 KKR 
               
               
                   
               
            
           
         
       
     
     It will be understood by those of skill in the art that the exemplary sequences provided herein are for illustration purposes only and are not intended to limit the scope of the present disclosure. The disclosure also embraces the use of each of the inventive TALEN domains, e.g., the modified N-terminal domains, C-terminal domains, and nuclease domains described herein, in the context of other TALEN sequences, e.g., other modified or unmodified TALEN structures. Additional sequences satisfying the described principles and parameters that are useful in accordance to aspects of this disclosure will be apparent to the skilled artisan. 
     In some embodiments, the TALEN provided is a monomer. In some embodiments, the TALEN monomer can dimerize with another TALEN monomer to form a TALEN dimer. In some embodiments the formed dimer is a homodimer. In some embodiments, the dimer is a heterodimer. 
     In some embodiments, TALENs provided herein cleave their target sites with high specificity. For example, in some embodiments an improved TALEN is provided that has been engineered to cleave a desired target site within a genome while binding and/or cleaving less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 or less than 10 off-target sites at a concentration effective for the nuclease to cut its intended target site. In some embodiments, a TALEN is provided that has been engineered to cleave a desired unique target site that has been selected to differ from any other site within a genome by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotide residues. 
     Some aspects of this disclosure provide nucleic acids encoding the TALENs provided herein. For example, nucleic acids are provided herein that encode the TALENs described in Table 1. In some embodiments, the nucleic acids encoding the TALEN are under the control of a heterologous promoter. In some embodiments, the encoding nucleic acids are included in an expression construct, e.g., a plasmid, a viral vector, or a linear expression construct. In some embodiments, the nucleic acid or expression construct is in a cell, tissue, or organism. 
     The map of an exemplary nucleic acid encoding a TALEN provided herein is illustrated in  FIG. 19 . An exemplary sequence of such a nucleic acid is provided below. It will be understood by those of skill in the art that the maps and sequences provided herein are exemplary and do not limit the scope of this disclosure. 
     As described elsewhere herein, TALENs, including the improved TALENs provided by this disclosure, can be engineered to bind (and cleave) virtually any nucleic acid sequence based on the sequence-specific TALE repeat array employed. In some embodiments, an improved TALEN provided herein binds a target sequence within a gene known to be associated with a disease or disorder. In some embodiments, TALENs provided herein may be used for therapeutic purposes. For example, in some embodiments, TALENs provided herein may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g. Alzheimer&#39;s disease, Huntington&#39;s disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g. depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia); etc. In some embodiments, the TALEN cleaves the target sequence upon dimerization. In some embodiments, a TALEN provided herein cleaves a target site within an allele that is associated with a disease or disorder. In some embodiments, the TALEN cleaves a target site the cleavage of which results in the treatment or prevention of a disease or disorder. In some embodiments, the disease is HIV/AIDS. In some embodiments, the disease is a proliferative disease. In some embodiments, the TALEN binds a CCR5 target sequence (e.g., a CCR5 sequence associated with HIV). In some embodiments, the TALEN binds an ATM target sequence (e.g., an ATM target sequence associated with ataxia telangiectasia). In some embodiments, the TALEN binds a VEGFA target sequence (e.g., a VEGFA sequence associated with a proliferative disease). In some embodiments, the TALEN binds a CFTR target sequence (e.g., a CFTR sequence associated with cystic fibrosis). In some embodiments, the TALEN binds a dystrophin target sequence (e.g., a dystrophin gene sequence associated with Duchenne muscular dystrophy). In some embodiments, the TALEN binds a target sequence associated with haemochromatosis, haemophilia, Charcot-Marie-Tooth disease, neurofibromatosis, phenylketonuria, polycystic kidney disease, sickle-cell disease, or Tay-Sachs disease. Suitable target genes, e.g., genes causing the listed diseases, are known to those of skill in the art. Additional genes and gene sequences associated with a disease or disorder will be apparent to those of skill in the art. 
     Some aspects of this disclosure provide isolated TALE effector domains, e.g., N- and C-terminal TALE effector domains, with decreased non-specific nucleic acid binding activity as compared to previously used TALE effector domains. The isolated TALE effector domains provided herein can be used in the context of suitable TALE effector molecules, e.g., TALE nucleases, TALE transcriptional activators, TALE transcriptional repressors, TALE recombinases, and TALE epigenome modification enzymes. Additional suitable TALE effectors in the context of which the isolated TALE domains can be used will be apparent to those of skill in the art based on this disclosure. In general, the isolated N- and C-terminal domains provided herein are engineered to optimize, e.g., minimize, excess binding energy conferred by amino acid residues that are positively charged (cationic) at physiological pH. Some of the improved N-terminal or C-terminal TALE domains provided herein have a decreased net charge and/or a decreased binding energy for binding a target nucleic acid sequence as compared to the respective canonical TALE domains. When used as part of a TALE effector molecule, e.g., a TALE nuclease, TALE transcriptional activator, TALE transcriptional repressor, TALE recombinase, or TALE epigenome modification enzyme, this decrease in charge leads to a decrease in off-target binding via the modified N-terminal and C-terminal domain(s). The portion of target recognition and binding, thus, is more narrowly confined to the specific recognition and binding activity of the TALE repeat array, as explained in more detail elsewhere herein. The resulting TALE effector molecule, thus, exhibits an increase in the specificity of binding and, in turn, in the specificity of the respective effect of the TALE effector (e.g., cleaving the target site by a TALE nuclease, activation of a target gene by a TALE transcriptional activator, repression of expression of a target gene by a TALE transcriptional repressor, recombination of a target sequence by a TALE recombinase, or epigenetic modification of a target sequence by a TALE epigenome modification enzyme) as compared to TALE effector molecules using unmodified domains. 
     In some embodiments, an isolated N-terminal TALE domain is provided in which the net charge is less than the net charge of the canonical N-terminal domain (SEQ ID NO: 1). In some embodiments, an isolated C-terminal TALE domain is provided in which the net charge is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, an isolated N-terminal TALE domain is provided in which the binding energy to a target nucleic acid molecule is less than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1). In some embodiments, an isolated C-terminal TALE domain is provided in which the binding energy to a target nucleic acid molecule is less than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22). In some embodiments, the binding energy of the isolated N-terminal and/or of the isolated C-terminal TALE domain provided herein is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 98%, or at least 99%. 
     In some embodiments, the canonical N-terminal domain and/or the canonical C-terminal domain is modified to replace an amino acid residue that is positively charged at physiological pH with an amino acid residue that is not charged or is negatively charged to arrive at the isolated N-terminal and/or C-terminal domain provided herein. In some embodiments, the modification includes the replacement of a positively charged residue with a negatively charged residue. In some embodiments, the modification includes the replacement of a positively charged residue with a neutral (uncharged) residue. In some embodiments, the modification includes the replacement of a positively charged residue with a residue having no charge or a negative charge. In some embodiments, the net charge of the isolated N-terminal domain and/or of the isolated C-terminal domain provided herein is less than or equal to +10, less than or equal to +9, less than or equal to +8, less than or equal to +7, less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to −1, less than or equal to −2, less than or equal to −3, less than or equal to −4, or less than or equal to −5, or less than or equal to −10 at physiological pH. In some embodiments, the net charge of the isolated N-terminal domain and/or of the isolated C-terminal domain is between +5 and −5, between +2 and −7, between 0 and −5, between 0 and −10, between −1 and −10, or between −2 and −15 at physiological pH. In some embodiments, the net charge of the isolated N-terminal TALE domain and/or of the isolated C-terminal TALE domain is negative. In some embodiments, an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is negative. In some embodiments, the net charge of the isolated N-terminal TALE domain and/or of the isolated C-terminal TALE domain is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH). In some embodiments, an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided, and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH). 
     In some embodiments, the isolated N-terminal domain and/or the isolated C-terminal domain provided herein comprise(s) an amino acid sequence that differs from the respective canonical domain sequence in that at least one cationic amino acid residue of the canonical domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain provided. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain. 
     In some embodiments, the cationic amino acid residue is arginine (R), lysine (K), or histidine (H). In some embodiments, the cationic amino acid residue is R or H. In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q), glycine (G), asparagine (N), threonine (T), serine (S), aspartic acid (D), or glutamic acid (E). In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is Q. In some embodiments, at least one lysine or arginine residue is replaced with a glutamine residue in the isolated N-terminal domain and/or in the isolated C-terminal domain. 
     In some embodiments, an isolated C-terminal TALE domain is provided herein that comprises one or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises two or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises three or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises four or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises five or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises six or more of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises all seven of the following amino acid replacements: K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q. In some embodiments, the isolated C-terminal domain comprises a Q3 variant sequence (K788Q, R792Q, R801Q, see SEQ ID NO: 23). In some embodiments, the isolated C-terminal domain comprises a Q7 variant sequence (K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q, see SEQ ID NO: 24). 
     In some embodiments, an isolated N-terminal TALE domain is provided that is a truncated version of the canonical N-terminal domain. In some embodiments, an isolated C-terminal TALE domain is provided that is a truncated version of the canonical C-terminal domain. In some embodiments, the truncated N-terminal domain and/or the truncated C-terminal domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues. In some embodiments, an isolated N-terminal TALE domain and/or an isolated C-terminal domain is provided herein that is/are truncated and comprise(s) one or more amino acid replacement(s). In some embodiments, the isolated N-terminal TALE domains comprise an amino acid sequence as provided in any of SEQ ID NOs 2-5. In some embodiments, the isolated C-terminal TALE domains comprise an amino acid sequence as provided in any of SEQ ID NOs 23-25. 
     It will be apparent to those of skill in the art that the isolated C- and N-terminal TALE domains provided herein may be used in the context of any TALE effector molecule, e.g., as part of a TALE nuclease, a TALE transcriptional activator, a TALE transcriptional repressor, a TALE recombinase, a TALE epigenome modification enzyme, or any other suitable TALE effector molecule. In some embodiments, a TALE domain provided herein is used in the context of a TALE molecule comprising or consisting essentially of the following structure 
     [N-terminal domain]-[TALE repeat array]-[C-terminal domain]-[effector domain]
         or       

     [effector domain]-[N-terminal domain]-[TALE repeat array]-[C-terminal domain], 
     wherein the effector domain may, in some embodiments, be a nuclease domain, a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain. 
     It will also be apparent to those of skill in the art that it is desirable, in some embodiments, to adjust the DNA spacer length in TALE effector molecules comprising such a spacer, when using a truncated domain, e.g., truncated C-terminal domain as provided herein, in order to accommodate the truncation. 
     Some aspects of this disclosure provide compositions comprising a TALEN provided herein, e.g., a TALEN monomer. In some embodiments, the composition comprises the TALEN monomer and a different TALEN monomer that can form a heterodimer with the TALEN, wherein the dimer exhibits nuclease activity. 
     In some embodiments, the TALEN is provided in a composition formulated for administration to a subject, e.g., to a human subject. For example, in some embodiments, a pharmaceutical composition is provided that comprises the TALEN and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the pharmaceutical composition comprises an effective amount of the TALEN for cleaving a target sequence in a cell in the subject. In some embodiments, the TALEN binds a target sequence within a gene known to be associated with a disease or disorder and wherein the composition comprises an effective amount of the TALEN for alleviating a symptom associated with the disease or disorder. 
     For example, some embodiments provide pharmaceutical compositions comprising a TALEN as provided herein, or a nucleic acid encoding such a nuclease, and a pharmaceutically acceptable excipient. Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances. 
     Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. 
     Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington&#39;s  The Science and Practice of Pharmacy,  21 st  Edition, A. R. Gennaro (Lippincott, Williams &amp; Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. 
     In some embodiments, a composition provided herein is administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a nuclease or a nuclease-encoding nucleic acid ex vivo, and re-administered to the subject after the desired genomic modification has been effected or detected in the cells. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with no more than routine experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including, but not limited to, cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys. 
     The scope of this disclosure embraces methods of using the TALENs provided herein. It will be apparent to those of skill in the art that the TALENs provided herein can be used in any method suitable for the application of TALENs, including, but not limited to, those methods and applications known in the art. Such methods may include TALEN-mediated cleavage of DNA, e.g., in the context of genome manipulations such as, for example, targeted gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template, respectively. The improved features of the TALENs provided herein, e.g., the improved specificity of some of the TALENs provided herein, will typically allow for such methods and applications to be carried out with greater efficiency. All methods and applications suitable for the use of TALENs, and performed with the TALENs provided herein, are contemplated and are within the scope of this disclosure. For example, the instant disclosure provides the use of the TALENs provided herein in any method suitable for the use of TALENs as described in Boch, Jens (February 2011). “TALEs of genome targeting”. Nature Biotechnology 29 (2): 135-6. doi:10.1038/nbt.1767. PMID 21301438; Boch, Jens; et. al. (December 2009). “Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”. Science 326 (5959): 1509-12. Bibcode:2009Sci . . . 326.1509B. doi:10.1126/science.1178811. PMID 19933107; Moscou, Matthew J.; Adam J. Bogdanove (December 2009). “A Simple Cipher Governs DNA Recognition by TAL Effectors”. Science 326 (5959): 1501. Bibcode:2009Sci . . . 326.1501M. doi:10.1126/science.1178817. PMID 19933106; Christian, Michelle; et. al. (October 2010). “Targeting DNA Double-Strand Breaks with TAL Effector Nucleases”. Genetics 186 (2): 757-61. doi:10.1534/genetics.110.120717. PMC 2942870. PMID 20660643; Li, Ting; et. al. (August 2010). “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain”. Nucleic Acids Research 39: 1-14. doi:10.1093/nar/gkq704. PMC 3017587. PMID 20699274; Mahfouz, Magdy M.; et. al. (February 2010). “De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks”. PNAS 108 (6): 2623-8. Bibcode:2011PNAS . . . 108.2623M. doi:10.1073/pnas.1019533108. PMC 3038751. PMID 21262818; Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”. Nucleic Acids Research. doi:10.1093/nar/gkr218; Miller, Jeffrey; et. al. (February 2011). “A TALE nuclease architecture for efficient genome editing”. Nature Biotechnology 29 (2): 143-8. doi:10.1038/nbt.1755. PMID 21179091; Hockemeyer, D.; Wang, H.; Kiani, S.; Lai, C. S.; Gao, Q.; Cassady, J. P.; Cost, G. J.; Zhang, L. et al. (2011). “Genetic engineering of human pluripotent cells using TALE nucleases”. Nature Biotechnology 29 (8). doi:10.1038/nbt.1927; Wood, A. J.; Lo, T.-W.; Zeitler, B.; Pickle, C. S.; Ralston, E. J.; Lee, A. H.; Amora, R.; Miller, J. C. et al. (2011). “Targeted Genome Editing Across Species Using ZFNs and TALENs”. Science 333 (6040): 307. doi:10.1126/science.1207773. PMC 3489282. PMID 21700836; Tesson, L.; Usal, C.; Ménoret, S. V.; Leung, E.; Niles, B. J.; Remy, S. V.; Santiago, Y.; Vincent, A. I. et al. (2011). “Knockout rats generated by embryo microinjection of TALENs”. Nature Biotechnology 29 (8): 695. doi:10.1038/nbt.1940; Huang, P.; Xiao, A.; Zhou, M.; Zhu, Z.; Lin, S.; Zhang, B. (2011). “Heritable gene targeting in zebrafish using customized TALENs”. Nature Biotechnology 29 (8): 699. doi:10.1038/nbt.1939; Doyon, Y.; Vo, T. D.; Mendel, M. C.; Greenberg, S. G.; Wang, J.; Xia, D. F.; Miller, J. C.; Urnov, F. D. et al. (2010). “Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures”. Nature Methods 8 (1): 74-79. doi:10.1038/nmeth.1539. PMID 21131970; Szczepek, M.; Brondani, V.; Büchel, J.; Serrano, L.; Segal, D. J.; Cathomen, T. (2007). “Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases”. Nature Biotechnology 25 (7): 786. doi:10.1038/nbt1317. PMID 17603476; Guo, J.; Gaj, T.; Barbas Iii, C. F. (2010). “Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases”. Journal of Molecular Biology 400 (1): 96. doi:10.1016/j.jmb.2010.04.060. PMC 2885538. PMID 20447404; Mussolino, C.; Morbitzer, R.; Lutge, F.; Dannemann, N.; Lahaye, T.; Cathomen, T. (2011). “A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity”. Nucleic Acids Research. doi:10.1093/nar/gkr597; Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”. Nature Biotechnology 29 (2): 149-53. doi:10.1038/nbt.1775. PMC 3084533. PMID 21248753; Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”. Nucleic Acids Research. doi:10.1093/nar/gkr151; Li, T.; Huang, S.; Zhao, X.; Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B. (2011). “Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes”. Nucleic Acids Research. doi:10.1093/nar/gkr188; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011). “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”. In Shiu, Shin-Han. PLoS ONE 6 (5): e19509. doi:10.1371/journal.pone.0019509; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). “Assembly of Designer TAL Effectors by Golden Gate Cloning”. In Bendahmane, Mohammed. PLoS ONE 6 (5): e19722. doi:10.1371/journal.pone.0019722; Sander et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology Vol 29:697-98 (5 Aug. 2011) Sander, J. D.; Cade, L.; Khayter, C.; Reyon, D.; Peterson, R. T.; Joung, J. K.; Yeh, J. R. J. (2011). “Targeted gene disruption in somatic zebrafish cells using engineered TALENs”. Nature Biotechnology 29 (8): 697. doi:10.1038/nbt.1934; the entire contents of each of which are incorporated herein by reference. 
     In some embodiments, the TALENs, TALEN domains, TALEN-encoding or TALEN domain-encoding nucleic acids, compositions, and reagents described herein are isolated. In some embodiments, the TALENs, TALEN domains, TALEN-encoding or TALEN domain-encoding nucleic acids, compositions, and reagents described herein are purified, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% pure. 
     Some aspects of this disclosure provide methods of cleaving a target sequence in a nucleic acid molecule using an inventive TALEN as described herein. In some embodiments, the method comprises contacting a nucleic acid molecule comprising the target sequence with a TALEN binding the target sequence under conditions suitable for the TALEN to bind and cleave the target sequence. In some embodiments, the TALEN is provided as a monomer. In some embodiments, the inventive TALEN monomer is provided in a composition comprising a different TALEN monomer that can dimerize with the first inventive TALEN monomer to form a heterodimer having nuclease activity. In some embodiments, the inventive TALEN is provided in a pharmaceutical composition. In some embodiments, the target sequence is in a cell. In some embodiments, the target sequence is in the genome of a cell. In some embodiments, the target sequence is in a subject. In some embodiments, the method comprises administering a composition, e.g., a pharmaceutical composition, comprising the TALEN to the subject in an amount sufficient for the TALEN to bind and cleave the target site. 
     Some aspects of this disclosure provide methods of preparing engineered TALENs. In some embodiments, the method comprises replacing at least one amino acid in the canonical N-terminal TALEN domain and/or the canonical C-terminal TALEN domain with an amino acid having no charge or a negative charge at physiological pH; and/or truncating the N-terminal TALEN domain and/or the C-terminal TALEN domain to remove a positively charged fragment; thus generating an engineered TALEN having an N-terminal domain and/or a C-terminal domain of decreased net charge. In some embodiments, the at least one amino acid being replaced comprises a cationic amino acid or an amino acid having a positive charge at physiological pH. In some embodiments, the amino acid replacing the at least one amino acid is a cationic amino acid or a neutral amino acid. In some embodiments, the truncated N-terminal TALEN domain and/or the truncated C-terminal TALEN domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the respective canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. 
     In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues. In some embodiments, the method comprises replacing at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in the canonical N-terminal TALEN domain and/or in the canonical C-terminal TALEN domain with an amino acid having no charge or a negative charge at physiological pH. In some embodiments, the amino acid being replaced is arginine (R) or lysine (K). In some embodiments, the amino acid residue having no charge or a negative charge at physiological pH is glutamine (Q) or glycine (G). In some embodiments, the method comprises replacing at least one lysine or arginine residue with a glutamine residue. 
     In some embodiments, the improved TALENs provided herein are designed and/or generated by recombinant technology. In some embodiments, designing and/or generating comprises designing a TALE repeat array that specifically binds a desired target sequence, or a half-site thereof. 
     Some aspects of this disclosure provide kits comprising an engineered TALEN as provided herein, or a composition (e.g., a pharmaceutical composition) comprising such a TALEN. In some embodiments, the kit comprises an excipient and instructions for contacting the TALEN with the excipient to generate a composition suitable for contacting a nucleic acid with the TALEN. In some embodiments, the excipient is a pharmaceutically acceptable excipient. 
     Typically, the kit will comprise a container housing the components of the kit, as well as written instructions stating how the components of the kit should be stored and used. 
     The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention. 
     EXAMPLES 
     Example 1 
     Materials and Methods 
     Oligonucleotides, PCR and DNA Purification 
     All oligonucleotides were purchased from Integrated DNA Technologies (IDT). Oligonucleotide sequences are listed in Table 10. PCR was performed with 0.4 μL of 2 U/μL Phusion Hot Start II DNA polymerase (Thermo-Fisher) in 50 μL with 1×HF Buffer, 0.2 mM dNTP mix (0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP) (NEB), 0.5 μM to 1 μM of each primer and a program of: 98° C., 1 min; 35 cycles of [98° C., 15 s; 62° C., 15 s; 72° C., 1 min] unless otherwise noted. Many DNA reactions were purified with a QIAquick PCR Purification Kit (Qiagen) referred to below as Q-column purification or MinElute PCR Purification Kit (Qiagen) referred to below as M-column purification. 
     TALEN Construction 
     The canonical TALEN plasmids were constructed by the FLASH method 12  with each TALEN targeting 10-18 base pairs. N-terminal mutations were cloned by PCR with Q5 Hot Start Master Mix (NEB) [98° C., 22 s; 62° C., 15 s; 72° C., 7 min]) using phosphorylated TAL-N1fwd (for N1), phosphorylated TAL-N2fwd (for N2), or phosphorylated TAL-N3fwd (for N3) and phosphorylated TALNrev as primers. 1 μL DpnI (NEB) was added and the reaction was incubated at 37° C. for 30 min then M-column purified. ˜25 ng of eluted DNA was blunt-end ligated intramolecularly in 10 μL 2× Quick Ligase Buffer, 1 μL of Quick Ligase (NEB) in a total volume of 20 μL at room temperature (˜21° C.) for 15 min. 1 μL of this ligation reaction was transformed into Top10 chemically competent cells (Invitrogen). C-terminal domain mutations were cloned by PCR using TAL-Cifwd and TAL-Cirev primers, then Q-column purified. ˜1 ng of this eluted DNA was used as the template for PCR with TALCifwd and either TAL-Q3 (for Q3) or TAL-Q7 (for Q7) for primers, then Q-column purified. ˜1 ng of this eluted DNA was used as the template for PCR with TAL-Cifwd and TAL-Ciirev for primers, then Qcolumn purified. ˜1 μg of this DNA fragment was digested with HpaI and BamHI in 1×NEBuffer 4 and cloned into ˜2 μg of desired TALEN plasmid pre-digested with HpaI and BamHI. 
     In Vitro TALEN Expression 
     TALEN proteins, all containing a 3×FLAG tag, were expressed by in vitro transcription/translation. 800 ng of TALEN-encoding plasmid or no plasmid (“empty lysate” control) was added to an in vitro transcription/translation reaction using the TNT® Quick Coupled Transcription/Translation System, T7 Variant (Promega) in a final volume of 20 μL at 30° C. for 1.5 h. Western blots were used to visualize protein using the anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). TALEN concentrations were calculated by comparison to standard curve of 1 ng to 16 ng N-terminally FLAG-tagged bacterial alkaline phosphatase (Sigma-Aldrich). 
     In Vitro Selection for DNA Cleavage 
     Pre-selection libraries were prepared with 10 pmol of oligo libraries containing partially randomized target half-site sequences (CCR5A, ATM, or CCR5B) and fully randomized 10- to 24-bp spacer sequences (Table 10). Oligonucleotide libraries were separately circularized by incubation with 100 units of CircLigase II ssDNA Ligase (Epicentre) in 1× CircLigase II Reaction Buffer (33 mM Tris-acetate, 66 mM potassium acetate, 0.5 mM dithiothreitol, pH 7.5) supplemented with 2.5 mM MnCl2 in 20 μL total for 16 h at 60° C. then incubated at 80° C. for 10 min. 2.5 μL of each circularization reaction was used as a substrate for rolling-circle amplification at 30° C. for 16 h in a 50-μL reaction using the Illustra TempliPhi 100 Amplification Kit (GE Healthcare). The resulting concatemerized libraries were quantified with Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen) and libraries with different spacer lengths were combined in an equimolar ratio. 
     For selections on the CCR5B sequence libraries, 500 ng of pre-selection library was digested for 2 h at 37° C. in 1×NEBuffer 3 with in vitro transcribed/translated TALEN plus empty lysate (30 μL total). For all CCR5B TALENs, in vitro transcribed/translated TALEN concentrations were quantified by Western blot (during the blot, TALENs were stored for 16 h at 4° C.) and then TALEN was added to 40 nM final concentration per monomer. For selections on CCR5A and ATM sequence libraries, the combined pre-selection library was further purified in a 300,000 MWCO spin column (Sartorius) with three 500-μL washes in 1×NEBuffer 3. 125 ng pre-selection library was digested for 30 min at 37° C. in 1×NEBuffer 3 with a total 24 μL of fresh in vitro transcribed/translated TALENs and empty lysate. For all CCR5A and ATM TALENs, 6 μL of in vitro transcription/translation left TALEN and 6 μL of right TALEN were used, corresponding to a final concentration in a cleavage reaction of 16 nM±2 nM or 12 nM±1.5 nM for CC5A or ATM TALENs, respectively. These TALEN concentrations were quantified by Western blot performed in parallel with digestion. 
     For all selections, the TALEN-digested library was incubated with 1 μL of 100 μg/μL RNase A (Qiagen) for 2 min and then Q-column purified. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10×NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. 50 μL of the eluted DNA was ligated with 2 pmol of heated and cooled #1 adapters containing barcodes corresponding to each sample (selections with different TALEN concentrations or constructs) (Table 10A). Ligation was performed in 1×T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5) with 1 μL of 400 U/μL T4 DNA ligase (NEB) in 60 μL total volume for 16 h at room temperature, then Q-column purified. 
     6 μL of the eluted DNA was amplified by PCR in 150 μL total reaction volume (divided into 3×50 μL reactions) for 14 to 22 cycles using the #2A adapter primers in Table 10A. The PCR products were purified by Q-column. Each DNA sample was quantified with Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen) and then pooled into an equimolar mixture. 500 ng of pooled DNA was run a 5% TBE 18-well Criterion PAGE gel (BioRad) for 30 min at 200 V and DNAs of length ˜230 bp (corresponding to 1.5 target site repeats plus adapter sequences) were isolated and purified by Qcolumn. ˜2 ng of eluted DNA was amplified by PCR for 5 to 8 cycles with #2B adapter primers (Table 10A) and purified by M-column. 
     10 μL of eluted DNA was purified using 12 μL of AMPure XP beads (Agencourt) and quantified with an Illumina/Universal Library Quantification Kit (Kapa Biosystems). DNA was prepared for high-throughput DNA sequencing according to Illumina instructions and sequenced using a MiSeq DNA Sequencer (Illumina) using a 12 pM final solution and 156-bp paired-end reads. To prepare the preselection library for sequencing, the pre-selection library was digested with 1 μL to 4 μL of appropriate restriction enzyme (CCR5A=Tsp45I, ATM=Acc65I, CCR5B=AvaI (NEB)) for 1 h at 37° C. then ligated as described above with 2 pmol of heated and cooled #1 library adapters (Table 10A). Pre-selection library DNA was prepared as described above using #2A library adapter primers and #2B library adapter primers in place of #2A adapter primers and #2B adapter primers, respectively (Table 10A). The resulting pre-selection library DNA was sequenced together with the TALEN-digested samples. 
     Discrete In Vitro TALEN Cleavage Assays 
     Discrete DNA substrates for TALEN digestion were constructed by combining pairs of oligonucleotides as specified in Table 9B with restriction cloning 14 into pUC19 (NEB). Corresponding cloned plasmids were amplified by PCR (59° C. annealing for 15 s) for 24 cycles with pUC19Ofwd and pUC19Orev primers (Table 10B) and Q-column purified. 50 ng of amplified DNAs were digested in 1×NEBuffer 3 with 3 μL each of in vitro transcribed/translated TALEN left and right monomers (corresponding to a ˜16 nM to ˜12 nM final TALEN concentration), and 6 μL of empty lysate in a total reaction volume of 120 μL. The digestion reaction was incubated for 30 min at 37° C., then incubated with 1 μL of 100 μg/μL RNase A (Qiagen) for 2 min and purified by M-column. The entire 10 μL of eluted DNA with glycerol added to 15% was analyzed on a 5% TBE 18-well Criterion PAGE gel (Bio-Rad) for 45 min at 200 V, then stained with 1×SYBR Gold (Invitrogen) for 10 min. Bands were visualized and quantified on an AlphaImager HP (Alpha Innotech). 
     Cellular TALEN Cleavage Assays 
     TALENs were cloned into mammalian expression vectors 12 and the resulting TALEN vectors transfected into U2OS-EGFP cells as previously described. 12  Genomic DNA was isolated after 2 days as previously described. 12  For each assay, 50 ng of isolated genomic DNA was amplified by PCR [98° C., 15 s 67.5° C., 15 s; 72° C., 22 s] for 35 cycles with pairs of primers with or without 4% DMSO as specified in Table 10C. The relative DNA content of the PCR reaction for each genomic site was quantified with Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen) and then pooled into an equimolar mixture, keeping no-TALEN and all TALEN-treated samples separate. DNA corresponding to 150 to 350 bp was purified by PAGE as described above. 
     44 μL of eluted DNA was incubated with 5 μL of 1×T4 DNA Ligase Buffer and 1 μL of 10 U/μL Polynucleotide kinase (NEB) for 30 min at 37° C. and Q-column purified. 43 μL of eluted DNA was incubated with 1 μL of 10 mM dATP (NEB), 5 μL of 10×NEBuffer 2, and 1 μL of 5 U/μL DNA Klenow Fragment (3′→5′ exo−) (NEB) for 30 min at 37° C. and purified by M-column. 10 μL of eluted DNA was ligated as above with 10 pmol of heated and cooled G (genomic) adapters (Table 10A). 8 μL of eluted DNA was amplified by PCR for 6 to 8 cycles with G-B primers containing barcodes corresponding to each sample. Each sample DNA was quantified with Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen) and then pooled into an equimolar mixture. The combined DNA was subjected to high throughput sequencing using a MiSeq as described above. 
     Data Analysis 
     Illumina sequencing reads were filtered and parsed with scripts written in Unix Bash as outlined in the Algorithms section. The source code is available upon request. Specificity scores were calculated as previously described. 14  Statistical analysis on the distribution of number of mutations in various TALEN selections in Table 3 was performed as previously described. 14  Statistical analysis of modified sites in Table 7 was performed as previously described. 14    
     Algorithms 
     All scripts were written in bash or MATLAB. 
     Computational Filtering of Pre-Selection Sequences and Selected Sequences 
     For Pre-Selection Sequences
     1) Search for 16 bp constant sequence (CCR5A=CGTCACGCTCACCACT (SEQ ID NO: 166), CCR5B=CCTCGGGACTCCACGCT (SEQ ID NO: 167), ATM=GGTACCCCACTCCGCGT (SEQ ID NO: 168)) immediately after first 4 bases read (random bases), accepting only sequences with the 16 bp constant sequence allowing for one mutation.   2) Search for 9 bp final sequence at a position at least the minimum possible full site length away and up to the max full site length away from constant sequence to confirm the presence of a full site, accept only sequences with this 9 bp final sequence. (Final sequence: CCR5A=CGTCACGCT, CCR5B=CCTCGGGAC, ATM=GGTACGTGC)   3) Search for best instances of each half site in the full site, accept any sequences with proper left and right half-site order of left then right.   4) Determine DNA spacer sequence between the two half sites, the single flanking nucleotide to left of the left half-site and single flanking nucleotide to right of the right half-site (sequence between half sites and constant sequences).   5) Filter by sequencing read quality scores, accepting sequences with quality scores of A or better across three fourths of the half site positions.   

     For Selected Sequences
     1) Output to separate files all sequence reads and position quality scores of all sequences starting with correct 5 bp barcodes corresponding to different selection conditions.   2) Search for the initial 16 bp sequence immediately after the 5 bp barcode repeated at a position at least the minimum possible full site length away and up to the max full site length away from initial sequence to confirm the presence of a full site with repeated sequence, accept only sequences with a 16 bp repeat allowing for 1 mutation.   3) Search for 16 bp constant sequence within the full site, accept only sequences with a constant sequence allowing for one mutation. Parse sequence to start with constant sequence plus 5′ sequence to second instance of repeated sequence then initial sequence after barcode to constant sequence resulting in constant sequences sandwiching the equivalent of one full site: CONSTANT-LFLANK-LHS-SPACER-RHS-RFLANK-CONSTANT
 
LFLANK=Left Flank Sequence (designed as a single random base)
 
LHS=Left Half Site Sequence
 
RHS=Right Half Site Sequence
 
RFLANK=Right Flank Sequence (designed as a single random base)
 
CONSTANT=Constant Sequence (CCR5A=CGTCACGCTCACCACT (SEQ ID NO: 166), CCR5B=CCTCGGGACTCCACGCT (SEQ ID NO: 167), ATM=GGTACCCCACTCCGCGT (SEQ ID NO: 168))
   4) Search for best instances of each half site in the full site, accept any sequences with proper left and right half-site order of left then right.   5) With half site positions determine corresponding spacer (sequence between the two half sites), left flank and right flank sequences (sequence between half sites and constant sequences).   6) Determine sequence end by taking sequence from the start of read after the 5 bp barcode sequence to the beginning of the constant sequence.
 
SEQUENCESTART-RHS-RFLANK-CONSTANT
   7) Filter by sequencing read quality scores, accepting sequences with quality scores of A or better across three fourths of the half site positions.   8) Selected sequences were filtered by sequence end, by accepting only sequences with sequence ends in the spacer that were 2.5-fold more abundant than the amount of sequence end background calculated as the mean of the number of sequences with ends zero to five base pairs into each half-site from the spacer side (sequence end background number was calculated for both half sites with the closest half site to the sequence end utilized as sequence end background for comparison).   

     Computational Search for Genomic Off-Target Sites Related to the CCR5B Target Site
     1) The Patmatch program 39  was used to search the human genome (GRCh37/hg19 build) for pattern sequences as follows: CCR5B left half-site sequence (L16, L13 or L10) NNNNNNNNN . . . CCR5B right half-site sequence (R16, R13 or R10)[M,0,0] where number of Ns varied from 12 to 25 and M (indicating mutations allowed) varied from 0 to 14.   2) The number of output off-target sites were de-cumulated since the program outputs all sequences with X or fewer mutations, resulting in the number of off-target sites in the human genome that are a specific number of mutations away from the target site.   

     Identification of Indels in Sequences of Genomic Sites
     1) For each sequence the primer sequence was used to identify the genomic site.   2) Sequences containing the reference genomic sequence corresponding to 8 bp to the left of the target site and reference genomic sequence 8 bp (or 6 bp for genomic sites at the very end of sequencing reads) to the right of the full target site were considered target site sequences.   3) Any target site sequences corresponding to the same size as the reference genomic site were considered unmodified and any sequences not the reference size were aligned with ClustalW 40  to the reference genomic site.   4) Aligned sequences with more than two insertions or two deletions in the DNA spacer sequence between the two half-site sequences were considered indels.   

     Results 
     Specificity Profiling of TALENs Targeting CCR5 and ATM 
     We profiled the specificity of 41 heterodimeric TALEN pairs (hereafter referred to as TALENs) in total, comprising TALENs targeting left and right half-sites of various lengths and TALENs with different domain variants. Each of the 41 TALENs was designed to target one of three distinct sequences, which we refer to as CCR5A, CCR5B, or ATM, in two different human genes, CCR5 and ATM ( FIG. 7 ). We used an improved version of a previously described in vitro selection method 14  with modifications that increase the throughput and sensitivity of the selection ( FIG. 1B ). 
     Briefly, preselection libraries of &gt;10 12  DNA sequences each were digested with 3 nM to 40 nM of an in vitro translated TALEN. These concentrations correspond to ˜20 to ˜200 dimeric TALEN molecules per human cell nucleus, 21  a relatively low level of cellular protein expression. 22,23  Cleaved library members contained a free 5′ monophosphate that was captured by adapter ligation and isolated by gel purification ( FIG. 1B ). In the control sample, all members of the pre-selection library were cleaved by a restriction endonuclease at a constant sequence to enable them to be captured by adapter ligation and isolated by gel purification. High-throughput sequencing of TALEN-treated or control samples surviving this selection process and computational analysis revealed the abundance of all TALEN-cleaved sequences as well as the abundance of the corresponding sequences before selection. The enrichment value for each library member surviving selection was calculated by dividing its post-selection sequence abundance by its preselection abundance. The pre-selection DNA libraries were sufficiently large that they each contain, in theory, at least ten copies of all possible DNA sequences with six or fewer mutations relative to the on-target sequence. 
     For all 41 TALENs tested, the DNA that survived the selection contained significantly fewer mean mutations in the targeted half-sites than were present in the pre-selection libraries (Table 3 and 4). For example, the mean number of mutations in DNA sequences surviving selection after treatment with TALENs targeting 18-bp left and right half-sites was 4.06 for CCR5A and 3.18 for ATM sequences, respectively, compared to 7.54 and 6.82 mutations in the corresponding pre-selection libraries ( FIGS. 2A and 2B ). For all selections, the on-target sequences were enriched by 8- to 640-fold (Table 5). To validate our selection results in vitro, we assayed the ability of the CCR5B TALENs targeting 13-bp left and right half-sites (L13+R13) to cleave each of 16 diverse off-target substrates ( FIGS. 2E and 2F ). The resulting discrete in vitro cleavage efficiencies correlated well with the observed enrichment values ( FIG. 2G ). 
     To determine the specificity at each position in the TALEN target site for all four possible base pairs, a specificity score was calculated as the difference between pre-selection and post-selection base pair frequencies, normalized to the maximum possible change of the pre-selection frequency from complete specificity (defined as 1.0) to complete anti-specificity (defined as −1.0). For all TALENs tested, the targeted base pair at every position in both half-sites is preferred, with the sole exception of the base pair closest to the spacer for some ATM TALENs at the right-half site ( FIG. 2C, 2D  and  FIGS. 8 through 13 ). The 5′ T nucleotide recognized by the N-terminal domain is highly specified, and the 5′ DNA end (the N-terminal TALEN end) generally exhibits higher specificity than the 3′ DNA end; both observations are consistent with previous reports. 24,25  Taken together, these results show that the selection data accurately predicts the efficiency of off-target TALEN cleavage in vitro, and that TALENs are overall highly specific across the entire target sequence. 
     TALEN Off-Target Cleavage in Cells 
     To test if off-target cleavage activities reported by the selection are relevant to off-target cleavage in cells, we used the in vitro selection results to train a machine-learning algorithm to generate potential TALEN off-target sites in the human genome. 26  This computational step was necessary because the preselection libraries cover all sequences with six or fewer mutations, while almost all potential off-target sites in the human genome for CCR5 and ATM sequences differ at more than six positions relative to the target sequence. The algorithm calculates the posterior probability of each nucleotide in each position of a target to occur in a sequence that was cleaved by the TALENs in opposition to sequences from the target library that were not observed to be cleaved. 27  These posterior probabilities were then used to score the likelihood that the TALEN used to train the algorithm would cleave every possible target sequence in the human genome with monomer spacing of 10 to 30 bps. Using the machine-learning algorithm, we identified 36 CCR5A and 36 ATM TALEN off-target sites that differ from the on-target sequence at seven to fourteen positions (Table 6). 
     The 72 best-scoring genomic off-target sites for CCR5A and ATM TALENs were amplified from genomic DNA purified from human U2OS-EGFP cells12 expressing either CCR5A or ATM TALENs. 3  Sequences containing insertions or deletions of three or more base pairs in the DNA spacer of the potential genomic off-target sites and present in significantly greater numbers in the TALEN-treated samples versus the untreated control sample were considered TALEN-induced modifications. Of the 35 CCR5A off-target sites that we successfully amplified, we identified six off-target sites with TALEN-induced modifications; likewise, of the 31 ATM off-target sites that we successfully amplified, we observed seven off-target sites with TALEN-induced modifications ( FIG. 3  and Table 7). The inspection of modified on-target and off-target sites yielded a prevalence of deletions ranging from three to dozens of base pairs ( FIG. 3 ), consistent with previously described characteristics of TALEN-induced genomic modification. 28    
     These results collectively indicate that the in vitro selection data, processed through a machine-learning algorithm, can predict bona fide off-target substrates that undergo TALEN-induced modification in human cells. TALE Repeats Productively Bind Base Pairs with Relative Independence The extensive number of quantitatively characterized off-target substrates in the selection data enabled us to assess whether mutations at one position in the target sequence affect the ability of TALEN repeats to productively bind other positions. We generated an expected enrichment value for every possible double-mutant sequence for the L13+R13 CCR5B TALENs assuming independent contributions from the two corresponding single-mutation enrichments. In general, the predicted enrichment values closely resembled the actual observed enrichment values for each double-mutant sequence ( FIG. 14A ), suggesting that component single mutations independently contributed to the overall cleavability of double-mutant sequences. The difference between the observed and predicted double-mutant enrichment values was relatively independent of the distance between the two mutations, except that two neighboring mismatches were slightly better tolerated than would be expected ( FIG. 14B ). 
     To determine the potential interdependence of more than two mutations, we evaluated the relationship between selection enrichment values and the number of mutations in the post-selection target for the L13+R13 CCR5B TALEN ( FIG. 4A , black line). For 0 to 5 mutations, enrichment values closely followed a simple exponential function of the mean number of mutations (m) (Table 8). This relationship is consistent with a model in which each successive mutation reduces the binding energy by a constant amount (ΔG), resulting in an exponential decrease in TALEN binding (Keq(m)) such that Keq(m)˜eΔG*m. The observed exponential relationship therefore suggests that the mean reduction in binding energy from a typical mismatch is independent of the number of mismatches already present in the TALEN:DNA interaction. Collectively, these results indicate that TALE repeats bind their respective DNA base pairs independently beyond a slightly increased tolerance for adjacent mismatches. 
     Longer TALENs are Less Specific Per Recognized Base Pair 
     The independent binding of TALE repeats simplistically predicts that TALEN specificity per base pair is independent of target-site length. To experimentally characterize the relationship between TALE array length and off-target cleavage, we constructed TALENs targeting 10, 13, or 16 bps (including the 5′ T) for both the left (L10, L13, L16) and right (R10, R13, R16) half-sites. TALENs representing all nine possible combinations of left and right CCR5B TALENs were subjected to in vitro selection. The results revealed that shorter TALENs have greater specificity per targeted base pair than longer TALENs (Table 3). For example, sequences cleaved by the L10+R10 TALEN contained a mean of 0.032 mutations per recognized base pair, while those cleaved by the L16+R16 TALEN contained a mean of 0.067 mutations per recognized base pair. For selections with the longest CCR5B TALENs targeting 16+16 base pairs or CCR5A and ATM TALENs targeting 18+18 bp, the mean selection enrichment values do not follow a simple exponential decrease as function of mutation number ( FIG. 4A  and Table 8). 
     We hypothesized that excess binding energy from the larger number of TALE repeats in longer TALENs reduces specificity by enabling the cleavage of sequences with more mutations, without a corresponding increase in the cleavage of sequences with fewer mutations, because the latter are already nearly completely cleaved. Indeed, the in vitro cleavage efficiencies of discrete DNA sequences for these longer TALENs are independent of the presence of a small number of mutations in the target site ( FIGS. 5C-5F ), suggesting there is nearly complete binding and cleavage of sequences containing few mutations. Likewise, higher TALEN concentrations also result in decreased enrichment values of sequences with few mutations while increasing the enrichment values of sequences with many mutations (Table 5). These results together support a model in which excessive TALEN binding arising from either long TALE arrays or high TALEN concentrations decreases observed TALEN DNA cleavage specificity of each recognized base pair. 
     Longer TALENs Induce Less Off-Target Cleavage in a Genomic Context 
     Although longer TALENs are more tolerant of mismatched sequences ( FIG. 4A ) than shorter TALENs, in the human genome there are far fewer closely related off-target sites for a longer target site than for a shorter target site ( FIG. 4B ). Since off-target site abundance and cleavage efficiency both contribute to the number of off-target cleavage events in a genomic context, we calculated overall genome cleavage specificity as a function of TALEN length by multiplying the extrapolated mean enrichment value of mutant sequences of a given length with the number of corresponding mutant sequences in the human genome. The decrease in potential off-target site abundance resulting from the longer target site length is large enough to outweigh the decrease in specificity per recognized base pair observed for longer TALENs ( FIG. 4C ). As a result, longer TALENs are predicted to be more specific against the set of potential cleavage sites in the human genome than shorter TALENs for the tested TALEN lengths targeting 20- to 32-bp sites. 
     Engineering TALENs with Improved Specificity 
     The findings above suggest that TALEN specificity can be improved by reducing non-specific DNA binding energy beyond what is needed to enable efficient on-target cleavage. The most widely used 63-aa C-terminal domain between the TALE repeat array and the FokI nuclease domain contains ten cationic residues. We speculated that reducing the cationic charge of the canonical TALE C-terminal domain would decrease non-specific DNA binding 29  and improve TALEN specificity. 
     We constructed two C-terminal domain variants in which three (“Q3”, K788Q, R792Q, R801Q) or seven (“Q7”, K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q) cationic Arg or Lys residues in the canonical 63-aa C-terminal domain were mutated to Gln. We performed in vitro selections on CCR5A and ATM TALENs containing the canonical, engineered Q3, and engineered Q7 C-terminal domains, as well as a previously reported 28-aa truncated C-terminal domain 5  with a theoretical net charge identical to that of the Q7 C-terminal domain (−1). 
     The on-target sequence enrichment values for the CCR5A and ATM selections increased substantially as the net charge of the C-terminal domain decreased ( FIGS. 5A and 5B ). For example, the ATM selections resulted in on-target enrichment values of 510, 50, and 20 for the Q7, Q3, and canonical 63-aa C-terminal variants, respectively. These results suggest that the TALEN variants in which cationic residues in the C-terminal domain have been partially replaced by neutral residues or completely removed are substantially more specific in vitro than the TALENs that containing the canonical 63-aa C-terminal domain. Similarly, mutating one, two, or three cationic residues in the TALEN N-terminus to Gln also increased cleavage specificity (Table 5, and  FIGS. 8-11 ). 
     In order to confirm the greater DNA cleavage specificity of Q7 over canonical 63-aa C-terminal domains in vitro, a representative collection of 16 off-target DNA substrates were digested in vitro with TALENs containing either canonical or engineered Q7 C-terminal domains. ATM and CCR5A TALENs with the canonical 63-aa C-terminal domain TALEN demonstrate comparable in vitro cleavage activity on target sites with zero, one, or two mutations ( FIGS. 5C-5F ). In contrast, for 11 of the 16 off-target substrates tested, the engineered Q7 TALEN variants showed substantially higher (˜4-fold or greater) discrimination against off-target DNA substrates with one or two mutations than the canonical 63-aa C-terminal domain TALENs, even though the Q7 TALENs cleaved their respective on-target sequences with comparable or greater efficiency than TALENs with the canonical 63-aa C-terminal domains ( FIGS. 5C-5F ). Overall, the discrete cleavage assays are consistent with the selection results and indicate that TALENs with engineered Q7 C-terminal domains are substantially more specific than TALENs with canonical 63-aa C-terminal domains in vitro. 
     Improved Specificity of Engineered TALENs in Human Cells 
     To determine if the increased specificity of the engineered TALENs observed in vitro also applies in human cells, TALEN-induced modification rates of the on-target and top 36 predicted off-target sites were measured for CCR5A and ATM TALENs containing all six possible combinations of the canonical 63-aa, Q3, or Q7 C-terminal domains and the EL/KK or ELD/KKR FokI domains (12 TALENs total). 
     For both FokI variants, the TALENs with Q3 C-terminal domains demonstrate significant on-target activities ranging from 8% to 24% modification, comparable to the activity of TALENs with the canonical 63-aa C-terminal domains. TALENs with canonical 63-aa or Q3 C-terminal domains and the ELD/KKR FokI domain are both more active in modifying the CCR5A and ATM on-target site in cells than the corresponding TALENs with the Q7 C-terminal domain by ˜5-fold ( FIG. 6  and Table 7). 
     Consistent with the improved specificity observed in vitro, the engineered Q7 TALENs are more specific than the Q3 variants, which in turn are more specific than the canonical 63-aa C-terminal domain TALENs. Compared to the canonical 63-aa C-terminal domains, TALENs with Q3 C-terminal domains demonstrate a mean increase in cellular specificity (defined as the ratio of the cellular modification percentage for on-target to off-target sites) of more than 13-fold and more than 9-fold for CCR5A and ATM sites, respectively, with the ELD/KKR FokI domain (Table 7). These mean improvements can only be expressed as lower limits due to the absence or near-absence of observed cleavage events by the engineered TALENs for many off-target sequences. For the most abundantly cleaved off-target site (CCR5A off-target site #5), the Q3 C-terminal domain is 34-fold more specific ( FIG. 6 ), and the Q7 C-terminal domain is &gt;116-fold more specific, than the canonical 63-aa C-terminal domain. 
     Together, these results reveal that for targeting the CCR5 and ATM sequences, replacing the canonical 63-aa C-terminal domain with the engineered Q3 C-terminal domain results in comparable activity for the on-target site in cells, a 34-fold improvement in specificity in cells for the most readily cleaved off-target site, and a consistent increase in specificity for other off-target sites. When less activity is required, the engineered Q7 C-terminal domain offers additional gains in specificity. 
     Engineering N-Terminal Domains for Improved TALEN DNA Cleavage Specificity 
     The model of TALEN binding and specificity described herein predicts that reducing excess TALEN binding energy will increase TALEN DNA cleavage specificity. To further test this prediction and potentially further augment TALEN specificity, we mutated one (“N1”, K150Q), two (“N2”, K150Q and K153Q), or three (“N3”, K150Q, K153Q, and R154Q) Lys or Arg residues to Gln in the N-terminal domain of TALENs targeting CCR5A and ATM. These N-terminal residues have been shown in previous studies to bind non-specifically to DNA, and mutations at these specific residues to neutralize the cationic charge decrease non-specific DNA binding energy. 33  We hypothesized the reduction in non-specific binding energy from these N-terminal mutations would decrease excess TALEN binding energy resulting in increased specificity. In vitro selections on these three TALEN variants revealed that the less cationic N-terminal TALENs indeed exhibit greater enrichment values of on-target cleavage (Table 5). 
     Effects of N-Terminal and C-Terminal Domains and TALEN Concentration on Specificity 
     All TALEN constructs tested specifically recognize the intended base pair across both half-sites ( FIGS. 8 to 13 ), except that some of the ATM TALENs do not specifically interact with the base pair adjacent to the spacer (targeted by the most C-terminal TALE repeat) ( FIGS. 10 and 11 ). To compare the broad specificity profiles of canonical TALENs with those containing engineered C-terminal or N-terminal domains, the specificity scores of each target base pair from selections using CCR5A and ATM TALENs with the canonical, Q3, or Q7 C-terminal domains and N1, N2, or N3 N-terminal domains were subtracted by the corresponding specificity scores from selections on the canonical TALEN (canonical 63-aa C-terminal domain, wild-type N-terminal domain). 
     The results are shown in  FIG. 15 . Mutations in the C-terminal domain that increase specificity did so most strongly in the middle and at the C-terminal end of each half-site. Likewise, the specificity-increasing mutations in the N-terminus tended to increase specificity most strongly at positions near the TALEN N-terminus (5′ DNA end) although mutations in the N-terminus of ATM TALEN targeting the right half-site did not significantly alter specificity. These results are consistent with a local binding compensation model in which weaker binding at either terminus demands increased specificity in the TALE repeats near this terminus. To characterize the effects of TALEN concentration on specificity, the specificity scores from selections of ATM and CCR5A TALENs performed at three different concentrations ranging from 3 nM to 16 nM were each subtracted by the specificity scores of corresponding selections performed at the highest TALEN concentration assayed, 24 nM for ATM, or 32 nM for CCR5A. The results ( FIG. 15 ) indicate that specificity scores increase fairly uniformly across the half-sites as the concentration of TALEN is decreased. 
     DNA Spacer-Length and Cut-Site Preferences 
     To assess the spacer-length preference of various TALEN architectures (C-terminal mutations, N-terminal mutations, and FokI variants) and various TALEN concentrations, the enrichment values of library members with 10- to 24-base pair spacer lengths in each of the selections with CCR5A and ATM TALEN with various combinations of the canonical, Q3, Q7, or 28-aa C-terminal domains; N1, N2, or N3 N-terminal mutations; and the EL/KK or ELD/KKR FokI variants at 4 nM to 32 nM CCR5A and ATM TALEN were calculated ( FIG. 16 ). All of the tested concentrations, N-terminal variants, C-terminal variants, and FokI variants demonstrated a broad DNA spacer-length preference ranging from 14- to 24-base pairs with three notable exceptions. First, the CCR5A 28-aa C-terminal domain exhibited a much narrower DNA spacer-length preference than the broader DNA spacer-length preference of the canonical C-terminal domain, consistent with previous reports. 34-36  Second, the CCR5A TALENs containing Q7 C-terminal domains showed an increased tolerance for 12-base spacers compared to the canonical C-terminal domain variant ( FIG. 16 ). This slightly broadened spacer-length preference may reflect greater conformational flexibility in the Q7 C-terminal domain, perhaps resulting from a smaller number of non-specific protein:DNA interactions along the TALEN:DNA interface. Third, the ATM TALENs with Q7 C-terminal domains and the ATM TALENs with N3 mutant N-terminal domains showed a narrowed spacer preference. 
     These more specific TALENs (Table 5) with lower DNA-binding affinity may have faster off-rates that are competitive with the rate of cleavage of non-optimal DNA spacer lengths, altering the observed spacer-length preference. While previous reports have focused on the length of the TALEN C-terminal domain as a primary determinant of DNA spacer-length preference, these results suggest the net charge of the C-terminal domain as well as overall DNA-binding affinity can also affect TALEN spacer-length preference. 
     We also characterized the location of TALEN DNA cleavage within the spacer. We created histograms reporting the number of spacer DNA bases observed preceding the right half-site in each of the sequences from the selections with CCR5A and ATM TALEN with various combinations of the canonical, Q3, Q7, or 28-aa C-terminal domains; N1, N2, or N3 N-terminal mutations; and the EL/KK or ELD/KKR FokI variants ( FIG. 17 ). The peaks in the histogram were interpreted to represent the most likely locations of DNA cleavage within the spacer. The cleavage positions are dependent on the length of the DNA spacer between the TALEN binding half-sites, as might be expected from conformational constraints imposed by the TALEN C-terminal domain and DNA spacer lengths. 
     Discussion 
     The in vitro selection of 41 TALENs challenged with 10 12  closed related off-target sequences and subsequent analysis inform our understanding of TALEN specificity through four key findings: (i) TALENs are highly specific for their intended target base pair at all positions with specificity increasing near the N-terminal TALEN end of each TALE repeat array (corresponding to the 5′ end of the bound DNA); (ii) longer TALENs are more specific in a genomic context while shorter TALENs have higher specificity per nucleotide; (iii) TALE repeats each bind their respective base pair relatively independently; and (iv) excess DNA-binding affinity leads to increased TALEN activity against off-target sites and therefore decreased specificity. 
     The observed decrease in specificity for TALENs with more TALE repeats or more cationic residues in the C-terminal domain or N-terminus are consistent with a model in which excess TALEN binding affinity leads to increased promiscuity. Excess binding energy could also explain the previously reported promiscuity at the 5′ terminal T of TALENs with longer C-terminal domains 30  and is also consistent with a report of higher TALEN protein concentrations resulting in more off-target site cleavage in vivo. 9  While decreasing TALEN protein expression in cells in theory could reduce off-target cleavage, the Kd values of some TALEN constructs for their target DNA sequences are likely already comparable to, or below, the theoretical minimum protein concentration in a human cell nucleus, ˜0.2 nM. 21    
     The difficulty of improving the specificity of such TALENs by lowering their expression levels, coupled with the need to maintain sufficient TALEN concentrations to effect desired levels of on-target cleavage, highlight the value of engineering TALENs with higher intrinsic specificity such as those described in this work. Our findings suggest that mutant C-terminal domains with reduced non-specific DNA binding may be used to fine-tune the DNA-binding affinity of TALENs such that on-target sequences are cleaved efficiently but with minimal excess binding energy, resulting in better discrimination between on-target and off-target sites. Since TALENs targeting up to 46 total base pairs have been shown to be active in cells, 15  the results presented here are consistent with the notion that specificity may be even further improved by engineering TALENs with a combination of mutant N-terminal and C-terminal domains that impart reduced non-specific DNA binding, a greater number of TALE repeats to contribute additional on-target DNA binding, and the more specific (but lower-affinity) NK RVD to recognize G. 25,31    
     Our study has identified more bona fide TALEN genomic off-target sites than other studies using methods such as SELEX or integrase-deficient lentiviral vectors (IDLVs). 32  Our model and the resulting improved TALENs would have been difficult to derive from cellular off-target cleavage methods, which are intrinsically limited by the small number of sequences closely related to a target sequence of interest that are present in a genome, or from SELEX experiments with monomeric TALE repeat arrays, 5  which do not measure DNA cleavage activity and therefore does not characterize active, dimeric TALENs. In contrast, each TALEN in this study was evaluated for its ability to cleave any of 10 12  close variants of its on-target sequence, a library size several orders of magnitude greater than the number of different sequences in a mammalian genome. This dense coverage of off-target sequence space enabled the elucidation of detailed relationships between DNA-cleavage specificity and target base pair position, TALE repeat length, TALEN concentration, mismatch location, and engineered TALEN domain composition. 
     Example 2 
     A number of TALENs were generated in which at least one cationic amino acid residue of the canonical N-terminal domain sequence was replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. The TALENs comprised substitutions of glycine (G) and/or glutamine (Q) in their N-terminal domains (see  FIG. 18 ). An evaluation of the cutting preferences of the engineered TALENs demonstrated that mutations to glycine (G) are equivalent to glutamine (Q). Mutating the positively charged amino acids in the TALEN N-terminal domain (K150Q, K153Q, and R154Q) result in similar decreases in binding affinity and off-target cleavage for mutations to either Q or G. For example, TALENs comprising the M3 and M4 N-terminus, which comprises the same amino acid (R154) mutated to either Q or G, respectively, demonstrated roughly equivalent amounts of cleavage. Similarly TALENs comprising the M6 and M8 N-terminus, varying only in whether Q or G substitutions were introduced at positions K150 and R154, and TALENs comprising the M9 and M10 N-terminus, varying only in whether Q or G substitutions were introduced at positions K150, K153, and R154, showed similar cleavage activity. 
     Example 3 
     A plasmid was generated for cloning and expression of engineered TALENs as provided herein. A map of the plasmid is shown in  FIG. 19 . The plasmid allows for the modular cloning of N-terminal and C-terminal domains, e.g., engineered domains as provided herein, and for TALE repeats, thus generating a recombinant nucleic acid encoding the desired engineered TALEN. The plasmid also encodes amino acid tags, e.g., an N-terminal FLAG tag and a C-terminal V5 tag, which can, optionally be utilized for purification or detection of the encoded TALEN. Use of these tags is optional and one of skill in the art will understand that the TALEN-encoding sequences will have to be cloned in-frame with the tag-encoding sequences in order to result in a tagged TALEN protein being encoded. 
     An exemplary sequence of a cloning vector as illustrated in  FIG. 19  is provided below. Those of skill in the art will understand that the sequence below is illustrative of an exemplary embodiment and does not limit this disclosure. 
     
       
         
           
               
               
            
               
                 &gt;pExpCCR5A-L18_(63 aa) 
                   
               
               
                 (SEQ ID NO: 42) 
                   
               
               
                 GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAG 
                   
               
               
                   
               
               
                 TATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTT 
               
               
                   
               
               
                 GACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGC 
               
               
                   
               
               
                 GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTC 
               
               
                   
               
               
                 CGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGAC 
               
               
                   
               
               
                 GTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACT 
               
               
                   
               
               
                 TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT 
               
               
                   
               
               
                 TATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT 
               
               
                   
               
               
                 GATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG 
               
               
                   
               
               
                 ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG 
               
               
                   
               
               
                 CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTA 
               
               
                   
               
               
                 CTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCACCATGGACTACAAAGACCATGAC 
               
               
                   
               
               
                 GGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGG 
               
               
                   
               
               
                 CATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTA 
               
               
                   
               
               
                 AGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTT 
               
               
                   
               
               
                 TCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCA 
               
               
                   
               
               
                 CGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGC 
               
               
                   
               
               
                 TTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAG 
               
               
                   
               
               
                 GCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTC 
               
               
                   
               
               
                 AAACGGAGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACAC 
               
               
                   
               
               
                 CGGAGCAAGTCGTGGCCATTGCATCCCACGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTT 
               
               
                   
               
               
                 CTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGTCGAACATTGGAGGGAAACAAGCATTGGA 
               
               
                   
               
               
                 GACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCGA 
               
               
                   
               
               
                 ATGGCGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTGACCCCA 
               
               
                   
               
               
                 GACCAGGTAGTCGCAATCGCGTCAAACGGAGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCT 
               
               
                   
               
               
                 TTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCAAGCAACATCGGTGGCAAACAGGCTCTTGAGA 
               
               
                   
               
               
                 CGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGTCGCAT 
               
               
                   
               
               
                 GACGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGC 
               
               
                   
               
               
                 ACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGT 
               
               
                   
               
               
                 GCCAGGATCATGGACTGACCCCAGACCAGGTAGTCGCAATCGCGTCACATGACGGGGGAAAGCAAGCCCTGGAAACC 
               
               
                   
               
               
                 GTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCCCACGA 
               
               
                   
               
               
                 CGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATC 
               
               
                   
               
               
                 AAGTTGTAGCGATTGCGTCCAACGGTGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGT 
               
               
                   
               
               
                 CAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGT 
               
               
                   
               
               
                 ACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTGACCCCAGACCAGGTAGTCGCAATCGCGTCACATGACG 
               
               
                   
               
               
                 GGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAA 
               
               
                   
               
               
                 GTCGTGGCCATTGCAAGCAACATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCA 
               
               
                   
               
               
                 AGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCC 
               
               
                   
               
               
                 AACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAGCCATGATGGC 
               
               
                   
               
               
                 GGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTGACACCCGAACAGGT 
               
               
                   
               
               
                 GGTCGCCATTGCTTCTAATGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCG 
               
               
                   
               
               
                 CGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAA 
               
               
                   
               
               
                 AAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGC 
               
               
                   
               
               
                 GGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATG 
               
               
                   
               
               
                 AATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTT 
               
               
                   
               
               
                 ATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATC 
               
               
                   
               
               
                 TCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATG 
               
               
                   
               
               
                 AAATGGAGCGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCA 
               
               
                   
               
               
                 TCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATT 
               
               
                   
               
               
                 AAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCG 
               
               
                   
               
               
                 GCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTTAAGGGCCCTTCGAAGGTAAG 
               
               
                   
               
               
                 CCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCG 
               
               
                   
               
               
                 CTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGG 
               
               
                   
               
               
                 AAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATT 
               
               
                   
               
               
                 CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG 
               
               
                   
               
               
                 CTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAA 
               
               
                   
               
               
                 GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC 
               
               
                   
               
               
                 TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATT 
               
               
                   
               
               
                 TAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA 
               
               
                   
               
               
                 CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAAC 
               
               
                   
               
               
                 CCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA 
               
               
                   
               
               
                 ACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAG 
               
               
                   
               
               
                 GCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGA 
               
               
                   
               
               
                 AGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCC 
               
               
                   
               
               
                 GCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCT 
               
               
                   
               
               
                 CTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATA 
               
               
                   
               
               
                 TCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAG 
               
               
                   
               
               
                 GTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAA 
               
               
                   
               
               
                 CAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCA 
               
               
                   
               
               
                 ATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAAC 
               
               
                   
               
               
                 CTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGTCGACAGGTGCTTCT 
               
               
                   
               
               
                 CGATCTGCATCCTGGGATCAAAGCGATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGC 
               
               
                   
               
               
                 TGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGA 
               
               
                   
               
               
                 TTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCG 
               
               
                   
               
               
                 GGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGC 
               
               
                   
               
               
                 ATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA 
               
               
                   
               
               
                 TCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTT 
               
               
                   
               
               
                 ATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA 
               
               
                   
               
               
                 CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG 
               
               
                   
               
               
                 CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG 
               
               
                   
               
               
                 TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA 
               
               
                   
               
               
                 AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT 
               
               
                   
               
               
                 CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC 
               
               
                   
               
               
                 AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT 
               
               
                   
               
               
                 CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAA 
               
               
                   
               
               
                 GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACC 
               
               
                   
               
               
                 CGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT 
               
               
                   
               
               
                 ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCC 
               
               
                   
               
               
                 AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT 
               
               
                   
               
               
                 GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG 
               
               
                   
               
               
                 TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA 
               
               
                   
               
               
                 AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG 
               
               
                   
               
               
                 CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGG 
               
               
                   
               
               
                 GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT 
               
               
                   
               
               
                 AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT 
               
               
                   
               
               
                 GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG 
               
               
                   
               
               
                 TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT 
               
               
                   
               
               
                 GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG 
               
               
                   
               
               
                 TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC 
               
               
                   
               
               
                 AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACA 
               
               
                   
               
               
                 TAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA 
               
               
                   
               
               
                 GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA 
               
               
                   
               
               
                 GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCT 
               
               
                   
               
               
                 TTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA 
               
               
                   
               
               
                 AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC 
               
            
           
         
       
     
     REFERENCES 
     
         
         1. Moscou, M. J. &amp; Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009). 
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     Nat Biotechnol 29, 731-734 (2011).
     8. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82 (2011).   9. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat   

     Biotechnol 29, 695-696 (2011).
     10. Moore, F. E. et al. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs). PLoS One 7, e37877 (2012).   11. Wood, A. J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).   12. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat   

     Biotechnol 30, 460-465 (2012).
     13. Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39, 9283-9293 (2011).   14. Pattanayak, V., Ramirez, C. L., Joung, J. K. &amp; Liu, D. R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8, 765-770 (2011).   15. Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39, 6315-6325 (2011).   16. Ding, Q. et al. A TALEN Genome-Editing System for Generating Human Stem Cell-Based   

     Disease Models. Cell Stem Cell (2012).
     17. Lei, Y. et al. Efficient targeted gene disruption in  Xenopus  embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci USA 109, 17484-17489 (2012).   18. Kim, Y. et al. A library of TAL effector nucleases spanning the human genome. Nat   

     Biotechnol 31, 251-258 (2013).
     19. Dahlem, T. J. et al. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 8, e1002861 (2012).   20. Osborn, M. J. et al. TALEN-based Gene Correction for Epidermolysis Bullosa. Molecular   

     Therapy (2013).
     21. Maul, G. G. &amp; Deaven, L. Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content. J Cell Biol 73, 748-760 (1977).   22. Huang, B. et al. Counting low-copy number proteins in a single cell. Science 315, 81-84 (2007).   23. Beck, M. et al. The quantitative proteome of a human cell line. Mol Syst Biol 7, 549 (2011).   24. Meckler, J. F. et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Res (2013).   25. Christian, M. L. et al. Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues. PLoS One 7, e45383 (2012).   26. Sander, J. D. et al. Abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target mutations. Submitted (2013).   27. Witten, I. H. &amp; Frank, E. Data mining: practical machine learning tools and techniques, Edn. 2nd. (Morgan Kaufman, San Francisco; 2005).   28. Kim, Y., Kweon, J. &amp; Kim, J. S. TALENs and ZFNs are associated with different mutation signatures. Nat Methods 10, 185 (2013).   29. McNaughton, B. R., Cronican, J. J., Thompson, D. B. &amp; Liu, D. R. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc Natl Acad Sci USA 106, 6111-6116 (2009).   30. Sun, N., Liang, J., Abil, Z. &amp; Zhao, H. Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease. Mol Biosyst 8, 1255-1263 (2012).   31. Cong, L., Zhou, R., Kuo, Y. C., Cunniff, M. &amp; Zhang, F. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun 3, 968 (2012).   32. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29, 816-823 (2011).   33. Gao, H., Wu, X., Chai, J. &amp; Han, Z. Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region. Cell Res 22, 1716-1720 (2012).   34. Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39, 6315-6325 (2011).   35. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29, 143-148 (2011).   36. Mahfouz, M. M. et al. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108, 2623-2628 (2011).   37. Pattanayak, V., Ramirez, C. L., Joung, J. K. &amp; Liu, D. R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8, 765-770 (2011).   38. Sander, J. D. et al. Abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target mutations. Submitted (2013).   39. Yan, T. et al. PatMatch: a program for finding patterns in peptide and nucleotide sequences. Nucleic Acids Res 33, W262-266 (2005).   40. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007).   

     All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control. 
     EQUIVALENTS AND SCOPE 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. 
     In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. 
     Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. 
     Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc. 
     Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. 
     In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein. 
     TABLES 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 TALEN constructs and concentrations used in the selections. For each selection using TALENs 
               
               
                 targeting the CCR5A target sequence (A), ATM target sequence (B) and CCR5B target sequence 
               
               
                 (C), the selection name, the target DNA site, the TALEN N-terminal domain, the TALEN C-terminal 
               
               
                 domain, the TALEN FokI domain, and the TALEN concentration (conc.) are shown. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Target 
                 Left + Right 
                 Site 
                 N-terminal 
                 C-terminal 
                 FokI 
                 TALEN 
               
               
                 Selection name 
                 site 
                 half-site 
                 length 
                 domain 
                 domain 
                 domain 
                 conc. (nM) 
               
               
                   
               
            
           
           
               
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CCR5A 32 nM 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 32 
               
               
                 canonical 
               
               
                 CCR5A 16 nM 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 16 
               
               
                 canonical 
               
               
                 (or CCR5A 32 
               
               
                 canonical) 
               
               
                 CCR5A 8 nM 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 8 
               
               
                 canonical 
               
               
                 CCR5A 4 nM 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 4 
               
               
                 canonical 
               
               
                 CCR5A Q3 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q3 
                 EL/KK 
                 16 
               
               
                 CCR5A 32 nM Q7 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 32 
               
               
                 CCR5A 16 nM Q7 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 16 
               
               
                 (or CCR5A Q7) 
               
               
                 CCR5A 8 nM Q7 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 8 
               
               
                 CCR5A 4 nM Q7 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 4 
               
               
                 CCR5A 26-aa 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 26-aa 
                 EL/KK 
                 16 
               
               
                 CCR5A N1 
                 CCR5A 
                 L18 + R18 
                 36 
                 N1 
                 Canonical 
                 EL/KK 
                 16 
               
               
                 CCR5A N2 
                 CCR5A 
                 L18 + R18 
                 36 
                 N2 
                 Canonical 
                 EL/KK 
                 16 
               
               
                 CCR5A N3 
                 CCR5A 
                 L18 + R18 
                 36 
                 N3 
                 Canonical 
                 EL/KK 
                 16 
               
               
                 CCR5A canonical 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 ELD/KKR 
                 16 
               
               
                 ELD/KKR 
               
               
                 CCR5A Q3 ELD/KKR 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q3 
                 ELD/KKR 
                 16 
               
               
                 CCR5A Q7 ELD/KKR 
                 CCR5A 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 ELD/KKR 
                 16 
               
               
                 CCR5A N2 ELD/KKR 
                 CCR5A 
                 L18 + R18 
                 36 
                 N2 
                 Canonical 
                 ELD/KKR 
                 16 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 ATM 32 nM 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 24 
               
               
                 canonical 
               
               
                 ATM 16 nM 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 12 
               
               
                 canonical 
               
               
                 (or ATM 
               
               
                 canonical) 
               
               
                 ATM 8 nM 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 6 
               
               
                 canonical 
               
               
                 ATM 4 nM 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 EL/KK 
                 3 
               
               
                 canonical 
               
               
                 ATM Q3 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q3 
                 EL/KK 
                 12 
               
               
                 ATM 32 nM Q7 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 24 
               
               
                 ATM 16 nM Q7 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 12 
               
               
                 (or ATM Q7) 
               
               
                 ATM 6 nM Q7 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 6 
               
               
                 ATM 4 nM Q7 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 EL/KK 
                 3 
               
               
                 ATM 26-aa 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 26aa 
                 EL/KK 
                 12 
               
               
                 ATM N1 
                 ATM 
                 L18 + R18 
                 36 
                 N1 
                 Canonical 
                 EL/KK 
                 12 
               
               
                 ATM N2 
                 ATM 
                 L18 + R18 
                 36 
                 N2 
                 Canonical 
                 EL/KK 
                 12 
               
               
                 ATM N3 
                 ATM 
                 L18 + R18 
                 36 
                 N3 
                 Canonical 
                 EL/KK 
                 12 
               
               
                 ATM canonical 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Canonical 
                 ELD/KKR 
                 12 
               
               
                 ELD/KKR 
               
               
                 ATM Q3 ELD/KKR 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q3 
                 ELD/KKR 
                 12 
               
               
                 ATM Q7 ELD/KKR 
                 ATM 
                 L18 + R18 
                 36 
                 canonical 
                 Q7 
                 ELD/KKR 
                 12 
               
               
                 ATM N2 ELD/KKR 
                 ATM 
                 L18 + R18 
                 36 
                 N2 
                 Canonical 
                 ELD/KKR 
                 12 
               
            
           
           
               
            
               
                 C 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 L16 + R16 CCR5B 
                 CCR5B 
                 L16 + R16 
                 32 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L16 + R13 CCR5B 
                 CCR5B 
                 L16 + R13 
                 29 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L16 + R10 CCR5B 
                 CCR5B 
                 L16 + R10 
                 26 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L13 + R16 CCR5B 
                 CCR5B 
                 L13 + R16 
                 29 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L13 + R13 CCR5B 
                 CCR5B 
                 L13 + R13 
                 26 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L13 + R10 CCR5B 
                 CCR5B 
                 L13 + R10 
                 23 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L10 + R16 CCR5B 
                 CCR5B 
                 L10 + R16 
                 26 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L10 + R13 CCR5B 
                 CCR5B 
                 L10 + R13 
                 23 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                 L10 + R10 CCR5B 
                 CCR5B 
                 L10 + R10 
                 20 
                 canonical 
                 Canonical 
                 EL/KK 
                 10 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Statistics of sequences selected by TALEN digestion. Statistics are shown for each 
               
               
                 TALEN selection on the CCR5A target sequence (A), ATM target sequence (B), and CCR5B 
               
               
                 target sequences (C). Seq. counts: total counts of high-throughput sequenced and 
               
               
                 computationally filtered selection sequences. Mean mut.: mean mutations in selected 
               
               
                 sequences. Stdev. mut.: standard deviation of mutations in selected sequences. Mut./bp: 
               
               
                 mean mutation normalized to target site length (bp). P-value vs. library: P-values 
               
               
                 between the TALEN selection sequence distributions to the corresponding pre-selection 
               
               
                 library sequence distributions (Supplementary Table 4) were determined as previously 
               
               
                 reported. 5 P-value vs. other TALENs: all pair-wise comparisons between all TALEN 
               
               
                 digestions were calculated and P-values between 0.01 and 0.5 are shown. Note that 
               
               
                 for the 3 nM Q7 ATM and the 28-aa ATM selection not enough sequences were obtained 
               
               
                 to interpret, although these selections were performed. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Seq. 
                 Mean 
                 Stdev 
                   
                 P-value 
                 P-value 
               
               
                 Selection name 
                 count 
                 mut. 
                 mut. 
                 Mut./bp 
                 vs. library 
                 vs. other TALENs 
               
               
                   
               
            
           
           
               
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 CCR5A 32 nM 
                 53883 
                 4.327 
                 1.463 
                 0.120 
                 3.3E−10 
                 vs. CCR5A canonical 
               
               
                 canonical 
                   
                   
                   
                   
                   
                 ELD/KKR = 0.260 
               
               
                 CCR5A 16 nM 
                 28940 
                 4.061 
                 1.436 
                 0.113 
                 5.4E−10 
                 vs. CCR5A Q3 
               
               
                 canonical 
                   
                   
                   
                   
                   
                 ELD/KKR = 0.026 
               
               
                 CCR5A 8 nM 
                 29568 
                 3.751 
                 1.394 
                 0.104 
                 3.3E−10 
               
               
                 canonical 
               
               
                 CCR5A 4 nM 
                 34355 
                 3.347 
                 1.355 
                 0.093 
                 1.5E−10 
               
               
                 canonical 
               
               
                 CCR5A Q3 
                 51694 
                 3.841 
                 1.380 
                 0.107 
                 1.7E−10 
               
               
                 CCR5A 32 nM Q7 
                 48473 
                 2.718 
                 1.197 
                 0.076 
                 4.4E−11 
               
               
                 CCR5A 16 nM Q7 
                 56593 
                 2.559 
                 1.154 
                 0.071 
                 3.1E−11 
               
               
                 CCR5A 8 nM Q7 
                 43895 
                 2.303 
                 1.157 
                 0.064 
                 3.0E−11 
               
               
                 CCR5A 4 nM Q7 
                 43737 
                 2.018 
                 1.234 
                 0.056 
                 2.1E−11 
               
               
                 CCR5A 28-aa 
                 47395 
                 2.614 
                 1.203 
                 0.073 
                 4.0E−11 
               
               
                 CCR5A N1 
                 64257 
                 3.721 
                 1.379 
                 0.103 
                 1.1E−10 
                 vs. CCR5A 8 nM 
               
               
                   
                   
                   
                   
                   
                   
                 canonical = 0.039 
               
               
                 CCR5A N2 
                 45467 
                 3.148 
                 1.306 
                 0.087 
                 8.2E−11 
               
               
                 CCR5A N3 
                 24064 
                 2.474 
                 1.493 
                 0.069 
                 8.1E−11 
               
               
                 CCR5A 
                 46998 
                 4.336 
                 1.491 
                 0.120 
                 4.0E−10 
               
               
                 canonical 
               
               
                 ELD/KKR 
               
               
                 CCR5A Q3 
                 56978 
                 4.098 
                 1.415 
                 0.114 
                 2.2E−10 
               
               
                 ELD/KKR 
               
               
                 CCR5A Q7 
                 54903 
                 3.234 
                 1.330 
                 0.090 
                 7.3E−11 
               
               
                 ELD/KKR 
               
               
                 CCR5A N2 
                 79632 
                 3.286 
                 1.341 
                 0.091 
                 5.2E−11 
               
               
                 ELD/KKR 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ATM 24 nM 
                 89571 
                 3.262 
                 1.360 
                 0.091 
                 6.54E−11  
                 vs. ATM canonical 
               
               
                 canonical 
                   
                   
                   
                   
                   
                 ELD/KKR = 0.012 
               
               
                 ATM 12 nM 
                 96703 
                 3.181 
                 1.307 
                 0.088 
                 5.36E−11  
               
               
                 canonical 
               
               
                 (or ATM 
               
               
                 canonical) 
               
               
                 ATM 6 nM 
                 78652 
                 2.736 
                 1.259 
                 0.076 
                 3.63E−11  
               
               
                 canonical 
               
               
                 ATM 3 nM 
                 82527 
                 2.552 
                 1.258 
                 0.071 
                 2.71E−11  
               
               
                 canonical 
               
               
                 ATM Q3 
                 96582 
                 2.551 
                 1.248 
                 0.071 
                 2.31E−11  
                 vs. ATM 4 nM 
               
               
                   
                   
                   
                   
                   
                   
                 canonical = 0.222 
               
               
                 ATM 24 nM Q7 
                 10166 
                 1.885 
                 2.125 
                 0.052 
                 2.06E−10  
               
               
                 ATM 12 nM Q7 
                 4662 
                 1.626 
                 2.083 
                 0.045 
                 5.31E−10  
               
               
                 (or ATM Q7) 
               
               
                 ATM 6 nM Q7 
                 1290 
                 1.700 
                 2.376 
                 0.047 
                 7.16E−09  
                 vs. ATM 16 nM 
               
               
                   
                   
                   
                   
                   
                   
                 Q7 = 0.035 
               
               
                 ATM N1 
                 84402 
                 2.627 
                 1.318 
                 0.073 
                 2.92E−11  
               
               
                 ATM N2 
                 62470 
                 2.317 
                 1.516 
                 0.064 
                 2.69E−11  
               
               
                 ATM N3 
                 1605 
                 2.720 
                 2.363 
                 0.076 
                 2.69E−08  
               
               
                 ATM 
                 107970 
                 3.279 
                 1.329 
                 0.091 
                 5.48E−11  
               
               
                 canonical 
               
               
                 ELD/KKR 
               
               
                 ATM Q3 
                 104099 
                 2.846 
                 1.244 
                 0.079 
                 3.15E−11  
               
               
                 ELD/KKR 
               
               
                 ATM Q7 ELD/KKR 
                 21108 
                 1.444 
                 1.56 
                 0.040 
                 3.02E−11  
               
               
                 ATM N2 ELD/KKR 
                 70185 
                 2.45 
                 1.444 
                 0.06805 
                 2.82E−11  
               
            
           
           
               
            
               
                 C 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 L16 + R16 CCR5B 
                 34904 
                 2.134 
                 1.168 
                 0.067 
                 4.7E−11 
                   
               
               
                 L16 + R13 CCR5B 
                 38229 
                 1.581 
                 1.142 
                 0.055 
                 2.7E−11 
               
               
                 L16 + R10 CCR5B 
                 37801 
                 1.187 
                 0.949 
                 0.046 
                 2.2E−11 
               
               
                 L13 + R16 CCR5B 
                 46608 
                 1.505 
                 1.090 
                 0.052 
                 1.7E−11 
               
               
                 L13 + R13 CCR5B 
                 53973 
                 0.996 
                 1.025 
                 0.038 
                 8.8E−12 
               
               
                 L13 + R10 CCR5B 
                 60550 
                 0.737 
                 0.684 
                 0.032 
                 7.4E−12 
               
               
                 L10 + R16 CCR5B 
                 36927 
                 1.387 
                 0.971 
                 0.053 
                 3.0E−11 
               
               
                 L10 + R13 CCR5B 
                 58170 
                 0.839 
                 0.882 
                 0.036 
                 9.1E−12 
               
               
                 L10 + R10 CCR5B 
                 57331 
                 0.646 
                 0.779 
                 0.032 
                 1.0E−11 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Statistics of sequences from pre-selection libraries. For each preselection library 
               
               
                 containing a distribution of mutant sequences of the CCR5A target sequence, ATM 
               
               
                 target sequence and CCR5B target sequences. Seq. counts: total counts of high- 
               
               
                 throughput sequenced and the computationally filtered selection sequences. Mean 
               
               
                 mut.: mean mutations of sequences. Stdev. mut.: standard deviation of sequences. 
               
               
                 Mut./bp: mean mutation normalized to target site length (bp). 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Target 
                 Left + Right 
                 Site 
                 Seq. 
                 Mean 
                 Stdev 
                   
               
               
                 Library name 
                 site 
                 half-site 
                 length 
                 count 
                 mut. 
                 mut. 
                 Mut./bp 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CCR5A Library 
                 CCR5A 
                 L18 + R18 
                 36 
                 158643 
                 7.539 
                 2.475 
                 0.209 
               
               
                 ATM Library 
                 ATM 
                 L18 + R18 
                 36 
                 212661 
                 6.820 
                 2.327 
                 0.189 
               
               
                 CCR5B Library 
                 CCR5B 
                 L16 + R16 
                 32 
                 280223 
                 6.500 
                 2.441 
                 0.203 
               
               
                 CCR5B Library 
                 CCR5B 
                 L16 + R13 
                 29 
                 280223 
                 5.914 
                 2.336 
                 0.204 
               
               
                 CCR5B Library 
                 CCR5B 
                 L16 + R10 
                 26 
                 280223 
                 5.273 
                 2.218 
                 0.203 
               
               
                 CCR5B Library 
                 CCR5B 
                 L13 + R16 
                 29 
                 280223 
                 5.969 
                 2.340 
                 0.206 
               
               
                 CCR5B Library 
                 CCR5B 
                 L13 + R13 
                 26 
                 280223 
                 5.383 
                 2.230 
                 0.207 
               
               
                 CCR5B Library 
                 CCR5B 
                 L13 + R10 
                 23 
                 280223 
                 4.742 
                 2.106 
                 0.206 
               
               
                 CCR5B Library 
                 CCR5B 
                 L10 + R16 
                 26 
                 280223 
                 5.396 
                 2.217 
                 0.208 
               
               
                 CCR5B Library 
                 CCR5B 
                 L10 + R13 
                 23 
                 280223 
                 4.810 
                 2.100 
                 0.209 
               
               
                 CCR5B Library 
                 CCR5B 
                 L10 + R10 
                 20 
                 280223 
                 4.169 
                 1.971 
                 0.208 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Enrichment values of sequences as a function of number of mutations. For each TALEN 
               
               
                 selection on the CCR5A target sequence (A), ATM target sequence (B) and CCR5B target 
               
               
                 sequence (C), enrichment values calculated by dividing the fractional abundance of 
               
               
                 post-selection sequences from a TALEN digestion by the fractional abundance of pre- 
               
               
                 selection sequences as a function of total mutations (Mut.) in the half-sites. 
               
            
           
           
               
               
            
               
                   
                 Enrichment value 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Selection 
                 0 Mut. 
                 1 Mut. 
                 2 Mut. 
                 3 Mut. 
                 4 Mut. 
                 5 Mut. 
                 6 Mut. 
                 7 Mut. 
                 8 Mut. 
               
               
                   
               
            
           
           
               
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 CCR5A 32 nM 
                 9.879 
                 9.191 
                 8.335 
                 6.149 
                 4.205 
                 2.269 
                 1.005 
                 0.325 
                 0.085 
               
               
                 canonical 
               
               
                 CCR5A 16 nM 
                 12.182 
                 13.200 
                 10.322 
                 7.195 
                 4.442 
                 2.127 
                 0.748 
                 0.216 
                 0.052 
               
               
                 canonical 
               
               
                 CCR5A 8 nM 
                 19.673 
                 17.935 
                 13.731 
                 8.505 
                 4.512 
                 1.756 
                 0.531 
                 0.116 
                 0.028 
               
               
                 canonical 
               
               
                 CCR5A 4 nM 
                 36.737 
                 29.407 
                 19.224 
                 9.958 
                 4.047 
                 1.242 
                 0.302 
                 0.058 
                 0.014 
               
               
                 canonical 
               
               
                 CCR5A Q3 
                 18.550 
                 16.466 
                 12.024 
                 8.070 
                 4.632 
                 1.938 
                 0.572 
                 0.126 
                 0.026 
               
               
                 CCR5A 32 nM Q7 
                 60.583 
                 54.117 
                 31.082 
                 11.031 
                 2.640 
                 0.469 
                 0.073 
                 0.013 
                 0.006 
               
               
                 CCR5A 16 nM Q7 
                 62.294 
                 64.689 
                 35.036 
                 10.538 
                 2.163 
                 0.322 
                 0.046 
                 0.010 
                 0.006 
               
               
                 CCR5A 8 nM Q7 
                 97.020 
                 91.633 
                 38.634 
                 8.974 
                 1.485 
                 0.189 
                 0.029 
                 0.010 
                 0.007 
               
               
                 CCR5A 4 nM Q7 
                 197.239 
                 130.497 
                 38.361 
                 6.535 
                 0.896 
                 0.120 
                 0.025 
                 0.019 
                 0.017 
               
               
                 CCR5A 28-aa 
                 70.441 
                 62.213 
                 33.481 
                 10.486 
                 2.317 
                 0.402 
                 0.064 
                 0.012 
                 0.006 
               
               
                 CCR5A N1 
                 19.038 
                 16.052 
                 13.858 
                 8.788 
                 4.546 
                 1.697 
                 0.499 
                 0.115 
                 0.025 
               
               
                 CCR5A N2 
                 41.715 
                 35.752 
                 22.638 
                 10.424 
                 3.777 
                 0.989 
                 0.194 
                 0.038 
                 0.007 
               
               
                 CCR5A N3 
                 173.897 
                 86.392 
                 31.503 
                 8.770 
                 1.853 
                 0.350 
                 0.089 
                 0.036 
                 0.027 
               
               
                 CCR5A 
                 8.101 
                 10.012 
                 8.220 
                 6.147 
                 4.119 
                 2.291 
                 1.019 
                 0.330 
                 0.083 
               
               
                 canonical 
               
               
                 ELD/KKR 
               
               
                 CCR5A Q3 
                 14.664 
                 12.975 
                 9.409 
                 6.819 
                 4.544 
                 2.235 
                 0.797 
                 0.198 
                 0.041 
               
               
                 ELD/KKR 
               
               
                 CCR5A Q7 
                 37.435 
                 32.922 
                 21.033 
                 10.397 
                 3.867 
                 1.087 
                 0.238 
                 0.046 
                 0.010 
               
               
                 ELD/KKR 
               
               
                 CCR5A N2 
                 35.860 
                 31.459 
                 20.135 
                 10.189 
                 3.983 
                 1.155 
                 0.260 
                 0.050 
                 0.013 
               
               
                 ELD/KKR 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 ATM 24 nM 
                 19.900 
                 16.881 
                 12.162 
                 6.318 
                 2.629 
                 0.884 
                 0.226 
                 0.057 
                 0.015 
               
               
                 canonical 
               
               
                 ATM 2 nM 
                 20.472 
                 17.645 
                 12.724 
                 6.549 
                 2.606 
                 0.803 
                 0.189 
                 0.039 
                 0.007 
               
               
                 canonical 
               
               
                 ATM 6 nM 
                 41.141 
                 29.522 
                 17.153 
                 6.551 
                 1.872 
                 0.431 
                 0.062 
                 0.017 
                 0.006 
               
               
                 canonical 
               
               
                 ATM 3 nM 
                 56.152 
                 37.152 
                 18.530 
                 6.196 
                 1.562 
                 0.308 
                 0.056 
                 0.015 
                 0.008 
               
               
                 canonical 
               
               
                 ATM Q3 
                 50.403 
                 36.687 
                 19.031 
                 6.245 
                 1.513 
                 0.294 
                 0.057 
                 0.016 
                 0.010 
               
               
                 ATM 24 nM Q7 
                 353.148 
                 90.350 
                 13.475 
                 1.531 
                 0.186 
                 0.128 
                 0.116 
                 0.118 
                 0.103 
               
               
                 ATM 12 nM Q7 
                 513.385 
                 89.962 
                 11.310 
                 0.860 
                 0.190 
                 0.093 
                 0.115 
                 0.092 
                 0.111 
               
               
                 ATM 6 nM Q7 
                 644.427 
                 82.074 
                 7.650 
                 0.677 
                 0.170 
                 0.205 
                 0.163 
                 0.164 
                 0.071 
               
               
                 ATM N1 
                 57.218 
                 35.388 
                 17.808 
                 6.124 
                 1.644 
                 0.383 
                 0.076 
                 0.023 
                 0.011 
               
               
                 ATM N2 
                 119.240 
                 53.618 
                 18.977 
                 4.742 
                 0.992 
                 0.233 
                 0.076 
                 0.044 
                 0.037 
               
               
                 ATM N3 
                 201.158 
                 55.468 
                 15.244 
                 3.187 
                 0.764 
                 0.307 
                 0.154 
                 0.173 
                 0.267 
               
               
                 ATM 
                 19.356 
                 15.692 
                 11.855 
                 6.403 
                 2.706 
                 0.899 
                 0.224 
                 0.054 
                 0.011 
               
               
                 canonical 
               
               
                 ELD/KKR 
               
               
                 ATM Q3 
                 32.816 
                 25.151 
                 16.172 
                 6.727 
                 2.095 
                 0.506 
                 0.095 
                 0.018 
                 0.004 
               
               
                 ELD/KKR 
               
               
                 ATM Q7 
                 447.509 
                 93.166 
                 13.505 
                 1.543 
                 0.170 
                 0.053 
                 0.049 
                 0.045 
                 0.045 
               
               
                 ELD/KKR 
               
               
                 ATM N2 
                 90.625 
                 45.525 
                 18.683 
                 5.369 
                 1.267 
                 0.274 
                 0.075 
                 0.035 
                 0.027 
               
               
                 ELD/KKR 
               
            
           
           
               
            
               
                 C 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 L16 + R16 CCR5B 
                 59.422 
                 35.499 
                 13.719 
                 3.770 
                 0.737 
                 0.132 
                 0.024 
                 0.011 
                 0.008 
               
               
                 L16 + R13 CCR5B 
                 80.852 
                 31.434 
                 7.754 
                 1.380 
                 0.218 
                 0.040 
                 0.022 
                 0.016 
                 0.017 
               
               
                 L16 + R10 CCR5B 
                 64.944 
                 20.056 
                 3.867 
                 0.515 
                 0.056 
                 0.010 
                 0.006 
                 0.006 
                 0.007 
               
               
                 L13 + R16 CCR5B 
                 101.929 
                 34.255 
                 8.131 
                 1.299 
                 0.167 
                 0.033 
                 0.016 
                 0.011 
                 0.014 
               
               
                 L13 + R13 CCR5B 
                 113.102 
                 22.582 
                 3.037 
                 0.315 
                 0.044 
                 0.022 
                 0.017 
                 0.017 
                 0.016 
               
               
                 L13 + R10 CCR5B 
                 74.085 
                 11.483 
                 1.270 
                 0.121 
                 0.022 
                 0.013 
                 0.011 
                 0.013 
                 0.008 
               
               
                 L10 + R16 CCR5B 
                 60.186 
                 22.393 
                 5.286 
                 0.777 
                 0.084 
                 0.012 
                 0.006 
                 0.006 
                 0.008 
               
               
                 L10 + R13 CCR5B 
                 74.204 
                 13.696 
                 1.673 
                 0.152 
                 0.021 
                 0.011 
                 0.010 
                 0.009 
                 0.010 
               
               
                 L10 + R10 CCR5B 
                 43.983 
                 7.018 
                 0.740 
                 0.061 
                 0.013 
                 0.007 
                 0.007 
                 0.008 
                 0.005 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Predicted off target sites in the human genome. (A) Using a machine learning 
               
               
                 “classifier” algorithm trained on the output of the in Vitro CCR5A TALEN selection, 6 mutant 
               
               
                 sequences of the target site allowing for spacer lengths of 10 to 30 base pairs were scored. The 
               
               
                 resulting 36 predicted off targets sites with the best scores for the CCR5A TALENs are shown 
               
               
                 with classifier scores, mutation numbers, left and right half site sequences (mutations from on  
               
               
                 target in lower case), the length of the spacer between half sites in base pairs, and the gene 
               
               
                 (including introns) in which the predicted off target sites occurs, if it lies within a gene. (B) 
               
               
                 Same as (A) for ATM TALENs. Sequences correspond to SEQ ID NOs: 44, 169 204 (left half  
               
               
                 site column of Table 6A); SEQ ID NOs: 46, 205 240 (right half site column of Table 6A); SEQ 
               
               
                 ID NOs: 128, 242 276 (left half site column of Table 6B); and SEQ ID NOs: 137, 277 312 (right 
               
               
                 half site column of Table 6B). 
               
               
                   
               
             
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 CCR5A 
                   
                   
                   
                 Spacer 
                   
                   
               
               
                 Site 
                 Score 
                 Mut. 
                 Left half-site 
                 length 
                 Right half-site 
                 Gene 
               
               
                   
               
               
                 OnCCR5A 
                 0.008 
                 0 
                 TTCATTACACCTGCAGCT 
                 18 
                 AGTATCAATTCTGGAAGA 
                 CCR5 
               
               
                   
               
               
                 OffC-1 
                 0.747 
                 9 
                 TaCATcACAtaTGCAaaT 
                 29 
                 tGTATCAtTTCTGGgAGA 
                 ARL17A 
               
               
                   
                   
                   
                   
                   
                   
                 &amp; LRRC37A 
               
               
                   
               
               
                 OffC-2 
                 0.747 
                 9 
                 TaCATcACAtaTGCAaaT 
                 29 
                 tGTATCAtTTCTGGgAGA 
                 ARL17A 
               
               
                   
                   
                   
                   
                   
                   
                 &amp; LRRC37A 
               
               
                   
               
               
                 OffC-3 
                 0.747 
                 9 
                 TaCATcACAtaTGCAaaT 
                 29 
                 tGTATCAtTTCTGGgAGA 
                 ARL17A 
               
               
                   
                   
                   
                   
                   
                   
                 &amp; LRRC37A 
               
               
                   
               
               
                 OffC-4 
                 0.747 
                 11 
                 TcCATaACACaTctttCT 
                 10 
                 tGcATCAtTcCTGGAAGA 
                 ZSCAN51 
               
               
                   
               
               
                 OffC-5 
                 0.804 
                 11 
                 TcCAaTACctCTGCcACa 
                 14 
                 AGgAgCAAcTCTGGgAGA 
                   
               
               
                   
               
               
                 OffC-6 
                 0.818 
                 10 
                 TTCAgTcCAtCTGaAaaC 
                 16 
                 gGTATCAtTTCTGGAgGA 
                 KL 
               
               
                   
               
               
                 OffC-7 
                 0.834 
                 14 
                 TaCAaaACcCtTGCCaaa 
                 27 
                 taTATCAATTtgGGgAGA 
                   
               
               
                   
               
               
                 OffC-8 
                 0.837 
                 12 
                 TcCAagACACCTGCttac 
                 26 
                 tcTATCAATTtgGGgAGA 
                   
               
               
                   
               
               
                 OffC-9 
                 0.874 
                 10 
                 TTCATaACAtCTtaAaaT 
                 27 
                 AaTAcCAAcTCTGGAtGA 
                 ZEB2 
               
               
                   
               
               
                 OffC-10 
                 0.89 
                 12 
                 TcCAaaACAtCTGaAaaT 
                 25 
                 tGgATCAAaTtgGGAAGA 
                   
               
               
                   
               
               
                 OffC-11 
                 0.896 
                 12 
                 TTCAgaACACaTGactac 
                 21 
                 tGTATCAgTTaTGGAtGA 
                 GABPA 
               
               
                   
               
               
                 OffC-12 
                 0.904 
                 13 
                 TcCATaAtAtCTtCctCT 
                 28 
                 gFgATtAATTtgGGAgGA 
                   
               
               
                   
               
               
                 OffC-13 
                 0.905 
                 11 
                 TgCAaTAtACCTGttGaT 
                 16 
                 ctcATCAATTCTGGgtGA 
                   
               
               
                   
               
               
                 OffC-14 
                 0.906 
                 12 
                 TTCATaACACtccacctT 
                 16 
                 gGTATCAAaTCTGGggGA 
                 SYN3 
               
               
                   
               
               
                 OffC-15 
                 0.906 
                 12 
                 TcCATgACACaaaagaCT 
                 26 
                 gGTATCtATcCTGGAAtA 
                 SPOCK3 
               
               
                   
               
               
                 OffC-16 
                 0.906 
                 9 
                 TTCcTTcCACCaGtgtCc 
                 28 
                 AGcATCAATcCTGGAAGA 
                   
               
               
                   
               
               
                 OffC-17 
                 0.907 
                 10 
                 TTaATaACAtCTcCAaCT 
                 24 
                 gGcAcCAAaTCTGGAtGA 
                 ATP13A5 
               
               
                   
               
               
                 OffC-18 
                 0.909 
                 13 
                 TcCATcACcCCTcCcTCc 
                 10 
                 gGTgcCAgcTCTGGAgGA 
                 TBC1D7 
               
               
                   
               
               
                 OffC-19 
                 0.909 
                 8 
                 TTCATTACtCCTcCttCT 
                 30 
                 ctTATCAcTTtTGGAAGA 
                   
               
               
                   
               
               
                 OffC-20 
                 0.912 
                 10 
                 TgCATTACACaTtatGtg 
                 17 
                 AGcAgCAcTTCTGGAAGA 
                   
               
               
                   
               
               
                 OffC-21 
                 0.913 
                 11 
                 TTCAaaACACaTaCAtCT 
                 28 
                 AacAaCAtTcCTGtAAGA 
                 PRKAG2 
               
               
                   
               
               
                 OffC-22 
                 0.913 
                 10 
                 TcCATTACcaCTGCAGaT 
                 25 
                 gacATCAgTTaTGGAtGA 
                   
               
               
                   
               
               
                 OffC-23 
                 0.925 
                 13 
                 TTCcagACcCCTtCctCa 
                 13 
                 gacATCAAaTCTGGgAGA 
                   
               
               
                   
               
               
                 OffC-24 
                 0.927 
                 12 
                 TTCcaaACACCcGCttCc 
                 26 
                 taTATCcTTTCTGGAAtA 
                   
               
               
                   
               
               
                 OffC-25 
                 0.93 
                 12 
                 TgaAaTACACCTGCctaT 
                 13 
                 gGCCTCAAGGCTGGAtGA 
                 IL15 
               
               
                   
               
               
                 OffC-26 
                 0.93 
                 12 
                 TgCcaaACctCTGtcaCc 
                 22 
                 AGgATCAcTTCTGGAAGA 
                   
               
               
                   
               
               
                 OffC-27 
                 0.931 
                 12 
                 TgCcaaACctCTGtcaCc 
                 22 
                 AGgATCAcTTCTGGAAGA 
                   
               
               
                   
               
               
                 OffC-28 
                 0.931 
                 8 
                 TTtATTACACtTcCAGaT 
                 19 
                 gaTATCctTTCTGGAAGA 
                 ADIPOR2 
               
               
                   
               
               
                 OffC-29 
                 0.932 
                 13 
                 TaCAaaAaACtTtCtGag 
                 27 
                 tGTATCAATTtgGGgAGA 
                 FBXL17 
               
               
                   
               
               
                 OffC-30 
                 0.932 
                 11 
                 TcCAaaACACCcaCAGac 
                 19 
                 gGTATagATTgTGGAAGA 
                 ZNF365 
               
               
                   
               
               
                 OffC-31 
                 0.934 
                 13 
                 TTCATTcCACaTcCccac 
                 25 
                 gtTATCAAcatgGGAAGA 
                 MYO18B 
               
               
                   
               
               
                 OffC-32 
                 0.934 
                 11 
                 TTCAaTAtgCCaaCAGCT 
                 11 
                 AGctTCAATctgGGAgGA 
                   
               
               
                   
               
               
                 OffC-33 
                 0.934 
                 12 
                 TTCAaTACACtTGtctaT 
                 12 
                 tGTgTCAtTTCTGGgttA 
                   
               
               
                   
               
               
                 OffC-34 
                 0.935 
                 11 
                 TTCAacACACCTtCAaaa 
                 12 
                 tGTgTCAtTaaTGGAAGA 
                   
               
               
                   
               
               
                 OffC-35 
                 0.935 
                 10 
                 TTCAaaACAtCTGacatT 
                 10 
                 AaTAgaAATTCTGGAAGA 
                   
               
               
                   
               
               
                 OffC-36 
                 0.935 
                 11 
                 cTCcTaAtACCTGCAaaT 
                 21 
                 gaTATtAtTTCTGGAgGA 
               
               
                   
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Spacer 
                   
                   
               
               
                 ATM Site 
                 Score 
                 Mut 
                 Left half-site 
                 length 
                 Right half-site 
                 Gene 
               
               
                   
               
               
                 OnATM 
                 0.000 
                 0 
                 TGAATTGGGATGCTGTTT 
                 18 
                 TTTATTTTACTGTCTTTA 
                 ATM 
               
               
                   
               
               
                 OffA-1 
                 0.595 
                 7 
                 TGAATaGGaAataTaTTT 
                 20 
                 TTTATTTTACTGTtTTTA 
                   
               
               
                   
               
               
                 OffA-2 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-3 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-4 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-5 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-6 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-7 
                 0.697 
                 9 
                 TGgATTcaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-8 
                 0.7 
                 8 
                 TGcATaGGaATGCTaaTT 
                 10 
                 TTTATTTTACTaTtTaTA 
                 MGAT4C 
               
               
                   
               
               
                 OffA-9 
                 0.708 
                 10 
                 TGAATTaaaATcCTGcTT 
                 19 
                 gTTATaTgACTaTtTTTA 
                 BRCA2 
               
               
                   
               
               
                 OffA-10 
                 0.711 
                 10 
                 TccATTaaaATaCTaTTT 
                 18 
                 TTTATTTTAtTaTtTTTA 
                 CPNE4 
               
               
                   
               
               
                 OffA-11 
                 0.715 
                 10 
                 TGAATTGaGAgaagcaTT 
                 16 
                 TTTATTTTAtTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-12 
                 0.725 
                 10 
                 TGAAgTGGGATaCTGTTa 
                 29 
                 ggTATaTTAtaaTtTTTA 
                   
               
               
                   
               
               
                 OffA-13 
                 0.729 
                 9 
                 TGAATTatGAaGCTacTT 
                 17 
                 TTTATTgTAaTaTtTTTA 
                 NAALADL2 
               
               
                   
               
               
                 OffA-14 
                 0.731 
                 9 
                 TGAATaaGGATGCTaTTa 
                 25 
                 TTTATTTattTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-15 
                 0.744 
                 10 
                 TGAATgGGGAcaCaGCCA 
                 29 
                 TTTATTTTAtTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-16 
                 0.752 
                 9 
                 TaAATgGaaATGCTGTTc 
                 24 
                 aTTATTTTAtTGTtTTTt 
                   
               
               
                   
               
               
                 OffA-17 
                 0.761 
                 9 
                 gGAAaTGGGATaCTGagT 
                 15 
                 TTTATgTTACTaTtTcTA 
                   
               
               
                   
               
               
                 OffA-18 
                 0.781 
                 11 
                 TGgATcGaagTGaTtaTT 
                 23 
                 TTTATTTTAtTaTtTTTA 
                 CIDEC 
               
               
                   
               
               
                 OffA-19 
                 0.792 
                 11 
                 TGAATTGaGATtCacagc 
                 23 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-20 
                 0.803 
                 8 
                 TGAATTaGGAatCTGaTT 
                 10 
                 TTTATTTTAtTaTtaTTA 
                 THSD7B 
               
               
                   
               
               
                 OffA-21 
                 0.807 
                 12 
                 TaAATTaaaATaCTccag 
                 23 
                 aTTATTTTAaTGTtTTTA 
                 ARID1B 
               
               
                   
               
               
                 OffA-22 
                 0.811 
                 10 
                 TGAATaGGaATatTcTTT 
                 12 
                 TTTATTTattTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-23 
                 0.811 
                 9 
                 TagATTGaaATGCTGTTT 
                 15 
                 TTTtTaTTAtTaTtTTTA 
                 KLHL4 
               
               
                   
               
               
                 OffA-24 
                 0.816 
                 10 
                 TGAcTaGaaATGaTGaTT 
                 25 
                 TTTATTTTctTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-25 
                 0.817 
                 12 
                 TGAATTtaaAaaaTGTcc 
                 13 
                 aTTATTTTAtTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-26 
                 0.817 
                 12 
                 TGAATTtaaAaaaTGTcc 
                 13 
                 aTTATTTTAtTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-27 
                 0.817 
                 10 
                 TGgATccaGATaCTcTTT 
                 10 
                 TTTATTTTttTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-28 
                 0.819 
                 7 
                 TGgAgTGaGATcCTGTTT 
                 21 
                 TTTATTTTAtTGTtaTTA 
                   
               
               
                   
               
               
                 OffA-29 
                 0.824 
                 8 
                 TGAAcTtGGATGaTaTaT 
                 24 
                 TTTATTTgAtTaTCTTTA 
                   
               
               
                   
               
               
                 OffA-30 
                 0.832 
                 9 
                 TGtATTGGGATaCcaTTT 
                 26 
                 TcTATTTTAtTaTtTTTt 
                   
               
               
                   
               
               
                 OffA-31 
                 0.833 
                 9 
                 TcAATTGGGATGaTcaTa 
                 23 
                 TTTATTcTAtTtTtTTTA 
                   
               
               
                   
               
               
                 OffA-32 
                 0.835 
                 9 
                 TGAAagGGaAaGtTGgaT 
                 23 
                 TTTATTTTACTaTtTTTA 
                   
               
               
                   
               
               
                 OffA-33 
                 0.841 
                 9 
                 TGgtTTGGGATcCTGTgT 
                 27 
                 TTTATgTTttTaTtTTTA 
                 PTCHD2 
               
               
                   
               
               
                 OffA-34 
                 0.841 
                 9 
                 TGAAaTGGGATGagcTTg 
                 28 
                 TTTATTTTAtTaTtTTaA 
                   
               
               
                   
               
               
                 OffA-35 
                 0.844 
                 10 
                 TGAATTGGGATaCTGTag 
                 29 
                 cTTAAAtaAaTaTtTTTA 
                 ST6GALNAC3 
               
               
                   
               
               
                 OffA-36 
                 0.844 
                 10 
                 TGAATTGtGgTatTGccT 
                 18 
                 TTTATggTttTGTCTTTA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Cellular modification induced by TALENs at on-target and predicted off-target genomic sites. (A) Results from sequencing CCR5A 
               
               
                 on-target and each predicted genomic off-target site that amplified from genomic DNA isolated from human cells treated with either 
               
               
                 no TALEN or TALENs containing canonical, Q3 or Q7 C-terminal domains, and either EL/KK heterodimeric, ELD/KKR heterodimeric, or 
               
               
                 homodimeric (Homo) FokI domains. Indels: the number of observed sequences containing insertions or deletions consistent with TALEN- 
               
               
                 induced cleavage. Total: total number of sequence counts. Modified: number of indels divided by total number of sequences as percentages. 
               
               
                 Upper limits of potential modification were calculated for sites with no observed indels by assuming there is less than one indel 
               
               
                 then dividing by the total sequence count to arrive at an upper limit modification percentage, or taking the theoretical limit 
               
               
                 of detection (1/16,400), whichever value was more conservative (larger). P-values: calculated as previously reported5 between each 
               
               
                 TALEN-treated sample and the untreated control sample. P-values less than 0.05 are shown. Specificity: the ratio of ontarget to 
               
               
                 off-target genomic modification frequency for each site. (B) Same as (A) for the ATM target sites. 
               
            
           
           
               
               
            
               
                   
                 C-terminal domain 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 No TALEN 
                 Q7 
                 Q7 
                 Q3 
                 Q3 
                 Canonical 
                 Canonical 
                 Canonical 
               
               
                 FokI domain 
                 No TALEN 
                 EL/KK 
                 ELD/KKR 
                 EL/KK 
                 ELD/KKR 
                 EL/KK 
                 ELD/KKR 
                 Homo 
               
               
                   
               
            
           
           
               
            
               
                 A 
               
               
                 CCR5A Sites 
               
               
                 OnC 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 5 
                 147 
                 705 
                 1430 
                 3731 
                 641 
                 2004 
                 3943 
               
               
                 Total 
                 23644 
                 7192 
                 12667 
                 16843 
                 15381 
                 8546 
                 7267 
                 8422 
               
               
                 % Modified 
                 0.021% 
                 2.044% 
                 5.566% 
                 8.490% 
                 24.257% 
                 9.841% 
                 27.577% 
                 46.818% 
               
               
                 P-value 
                   
                 1.3E−33 
                 2.5E−160 
                 &lt;1.0E−200 
                 &lt;1.0E−200 
                 5.9E−200 
                 &lt;1.0E−200 
                 &lt;1.0E−200 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-1 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
               
               
                 Total 
                 51248 
                 38975 
                 79858 
                 35491 
                 77804 
                 34227 
                 87497 
                 42498 
               
               
                 % Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-2 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 10 
                 0 
               
               
                 Total 
                 124356 
                 96280 
                 157387 
                 93337 
                 159817 
                 85603 
                 163332 
                 114663 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.006% 
                 &lt;0.006% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 1.6E−03 
               
               
                 Specificity 
                   
                 &gt;307 
                 &gt;835 
                 &gt;1274 
                 &gt;3639 
                 &gt;1476 
                 &gt;4137 
                 &gt;7023 
               
            
           
           
               
            
               
                 OffC-3 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 5 
                 0 
                 4 
                 1 
                 0 
                 0 
                 6 
                 3 
               
               
                 Total 
                 93085 
                 75958 
                 130027 
                 72919 
                 131132 
                 57192 
                 136796 
                 90039 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-4 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 45377 
                 44674 
                 52876 
                 35133 
                 53909 
                 26034 
                 42284 
                 40452 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-5 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 3 
                 22 
                 134 
                 385 
                 395 
               
               
                 Total 
                 27009 
                 28172 
                 26035 
                 22432 
                 25800 
                 25273 
                 17045 
                 17077 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.013% 
                 0.085% 
                 0.527% 
                 2.209% 
                 2.261% 
               
               
                 P-value 
                   
                   
                   
                   
                 2.7E−06 
                 4.5E−31  
                 4.9E−87 
                 2.8E−89 
               
               
                 Specificity 
                   
                 &gt;576 
                 &gt;1450 
                 635 
                 285 
                 19 
                 12 
                 21 
               
            
           
           
               
            
               
                 OffC-6 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 10766 
                 12309 
                 10886 
                 9240 
                 10558 
                 10500 
                 5943 
                 6560 
               
               
                 Modified 
                 &lt;0.009% 
                 &lt;0.008% 
                 &lt;0.009% 
                 &lt;0.011% 
                 &lt;0.009% 
                 &lt;0.010% 
                 &lt;0.017% 
                 &lt;0.015% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-7 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Total 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Modified 
                 15526 
                 28825 
                 22138 
                 31742 
                 19577 
                 11902 
                 33200 
                 15400 
               
               
                 P-value 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.008% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-9 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 40603 
                 39765 
                 47974 
                 51595 
                 44002 
                 34520 
                 25211 
                 30771 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-10 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 4142 
                 9591 
                 5187 
                 1413 
                 7975 
                 4378 
                 2215 
                 3779 
               
               
                 Modified 
                 &lt;0.024% 
                 &lt;0.010% 
                 &lt;0.019% 
                 &lt;0.071% 
                 &lt;0.013% 
                 &lt;0.023% 
                 &lt;0.045% 
                 &lt;0.026% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-11 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 71180 
                 55455 
                 65015 
                 44847 
                 70907 
                 50967 
                 65257 
                 60191 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-12 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 3242 
                 1784 
                 30274 
                 14006 
                 4897 
                 19830 
                 9747 
                 12910 
               
               
                 Modified 
                 &lt;0.031% 
                 &lt;0.056% 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.020% 
                 &lt;0.006% 
                 &lt;0.010% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-13 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 65518 
                 52459 
                 53413 
                 38156 
                 61600 
                 47922 
                 57211 
                 78546 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-14 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
               
               
                 Total 
                 34607 
                 7217 
                 26301 
                 8339 
                 29845 
                 1081 
                 9471 
                 19026 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.014% 
                 &lt;0.008% 
                 &lt;0.012% 
                 &lt;0.006% 
                 &lt;0.093% 
                 0.021% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-15 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 16 
                 2 
               
               
                 Total 
                 4989 
                 4880 
                 6026 
                 9370 
                 9156 
                 7371 
                 6967 
                 4662 
               
               
                 Modified 
                 &lt;0.020% 
                 &lt;0.020% 
                 &lt;0.017% 
                 &lt;0.011% 
                 &lt;0.011% 
                 &lt;0.014% 
                 0.230% 
                 0.043% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 6.3E−05 
               
               
                 Specificity 
                   
                 &gt;100 
                 &gt;335 
                 &gt;796 
                 &gt;2221 
                 &gt;725 
                 120 
                 1091 
               
            
           
           
               
            
               
                 OffC-16 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 1 
                 1 
                 1 
                 14 
                 1 
                 12 
                 0 
               
               
                 Total 
                 36228 
                 34728 
                 34403 
                 34866 
                 44362 
                 38384 
                 38536 
                 32636 
               
               
                 Modified 
                 &lt;0.06% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.032% 
                 &lt;0.006% 
                 0.031% 
                 &lt;0.006% 
               
               
                 P-value 
                   
                   
                   
                   
                 1.8E−04 
                   
                 5.3E−04 
               
               
                 Specificity 
                   
                 &gt;307 
                 &gt;835 
                 &gt;1274 
                 769 
                 &gt;1476 
                 886 
                 &gt;7023 
               
            
           
           
               
            
               
                 OffC-17 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 32112 
                 23901 
                 31273 
                 33968 
                 27437 
                 29670 
                 27133 
                 31299 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-18 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 9437 
                 9661 
                 13505 
                 14900 
                 13848 
                 12720 
                 6624 
                 12804 
               
               
                 Modified 
                 &lt;0.011% 
                 &lt;0.010% 
                 &lt;0.007% 
                 &lt;0.007% 
                 &lt;0.007% 
                 &lt;0.008% 
                 &lt;0.015% 
                 &lt;0.008% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-19 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 1 
                 1 
                 2 
                 2 
                 2 
                 1 
                 0 
               
               
                 Total 
                 22869 
                 11479 
                 22702 
                 15258 
                 20733 
                 17449 
                 14638 
                 28478 
               
               
                 Modified 
                 &lt;0.006% 
                 0.009% 
                 &lt;0.006% 
                 0.013% 
                 0.010% 
                 0.011% 
                 0.007% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-20 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 Total 
                 23335 
                 26164 
                 30782 
                 15261 
                 20231 
                 21184 
                 14144 
                 18972 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-21 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 34302 
                 27573 
                 31694 
                 24451 
                 25826 
                 27192 
                 18110 
                 21161 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-22 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 81037 
                 86687 
                 74274 
                 79004 
                 93477 
                 92089 
                 75359 
                 104857 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-23 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 18812 
                 19337 
                 23034 
                 25603 
                 25023 
                 28615 
                 17172 
                 21033 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-24 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                 Total 
                 23538 
                 21673 
                 24594 
                 27687 
                 18343 
                 29113 
                 21709 
                 26610 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-25 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 28941 
                 25326 
                 25871 
                 10641 
                 21422 
                 20171 
                 18946 
                 18711 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.009% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-26 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 71831 
                 48494 
                 62650 
                 45801 
                 60175 
                 65137 
                 28795 
                 64632 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-27 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 12181 
                 2423 
                 11258 
                 7188 
                 5126 
                 4003 
                 2116 
                 4503 
               
               
                 % Modified 
                 &lt;0.008% 
                 &lt;0.041% 
                 &lt;0.009% 
                 &lt;0.014% 
                 &lt;0.020% 
                 &lt;0.025% 
                 &lt;0.047% 
                 &lt;0.022% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-28 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 6 
                 1 
                 12 
                 5 
               
               
                 Total 
                 10651 
                 6410 
                 16179 
                 13980 
                 13022 
                 7232 
                 7379 
                 8998 
               
               
                 % Modified 
                 &lt;0.009% 
                 &lt;0.016% 
                 &lt;0.006% 
                 &lt;0.007% 
                 0.046% 
                 0.014% 
                 0.163% 
                 0.056% 
               
               
                 P-value 
                   
                   
                   
                   
                 1.4E−02 
                   
                 5.3E−04 
               
               
                 Specificity 
                   
                 &gt;131 
                 &gt;835 
                 &gt;1187 
                 526 
                 712 
                 170 
                 843 
               
            
           
           
               
            
               
                 OffC-29 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 4262 
                 3766 
                 4228 
                 6960 
                 3234 
                 1516 
                 2466 
                 1810 
               
               
                 % Modified 
                 &lt;0.023% 
                 &lt;0.027% 
                 &lt;0.024% 
                 &lt;0.014% 
                 &lt;0.031% 
                 &lt;0.066% 
                 &lt;0.041% 
                 &lt;0.055% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-30 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 11840 
                 12257 
                 9617 
                 34097 
                 20507 
                 5029 
                 22248 
                 6285 
               
               
                 % Modified 
                 &lt;0.008% 
                 &lt;0.008% 
                 &lt;0.010% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.020% 
                 &lt;0.006% 
                 &lt;0.016% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-31 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 64522 
                 67791 
                 50085 
                 50056 
                 56241 
                 48287 
                 72230 
                 100410 
               
               
                 % Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-32 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 1944 
                 6888 
                 9330 
                 3207 
                 4591 
                 6699 
                 13607 
                 19115 
               
               
                 % Modified 
                 &lt;0.051% 
                 &lt;0.015% 
                 &lt;0.011% 
                 &lt;0.031% 
                 &lt;0.022% 
                 &lt;0.015% 
                 &lt;0.007% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-33 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 34475 
                 27039 
                 18547 
                 33467 
                 15745 
                 17075 
                 4 
                 18844 
               
               
                 % Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;25.000% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-34 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 9052 
                 18858 
                 13647 
                 11796 
                 6945 
                 6114 
                 4979 
                 9072 
               
               
                 % Modified 
                 &lt;0.011% 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.014% 
                 &lt;0.016% 
                 &lt;0.020% 
                 &lt;0.011% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-35 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 23839 
                 22290 
                 25133 
                 24190 
                 10 
                 10459 
                 22554 
                 11897 
               
               
                 % Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;10.000% 
                 &lt;0.010% 
                 &lt;0.006% 
                 &lt;0.008% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffC-36 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 0 
                 0 
                 1 
                 2 
                 1 
                 19 
                 5 
               
               
                 Total 
                 23412 
                 24394 
                 23427 
                 24132 
                 19723 
                 28369 
                 12461 
                 18052 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.010% 
                 &lt;0.006% 
                 0.152% 
                 0.028% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 2.6E−05 
               
               
                 Specificity 
                   
                 &gt;307 
                 &gt;835 
                 &gt;1274 
                 2392 
                 &gt;1476 
                 181 
                 1690 
               
            
           
           
               
            
               
                 B 
               
               
                 ATM Sites 
               
               
                 On-A 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 3 
                 0 
                 46 
                 104 
                 309 
                 1289 
                 410 
                 909 
               
               
                 Total 
                 6886 
                 1869 
                 2520 
                 1198 
                 1808 
                 19025 
                 2533 
                 5003 
               
               
                 Modified 
                 0.03% 
                 0.00% 
                 1.83% 
                 8.68% 
                 17.09% 
                 6.78% 
                 16.19% 
                 18.17% 
               
               
                 P-value 
                 0 
                   
                 2.2E−11  
                 3.2E−26 
                 4.9E−81 
                 6.4E−276 
                  4.5E−105 
                  1.5E−228 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-1 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 13 
                 34 
               
               
                 Total 
                 52490 
                 45383 
                 34195 
                 32325 
                 47589 
                 39704 
                 50349 
                 44056 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.0026% 
                 0.077% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 3.1E−04 
                 5.5E−09 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;274 
                 &gt;1302 
                 &gt;2564 
                 &gt;1016 
                 627 
                 235 
               
            
           
           
               
            
               
                 OffA-2 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 6777 
                 11846 
                 11362 
                 12273 
                 20704 
                 3776 
                 5650 
                 5025 
               
               
                 Modified 
                 &lt;0.011% 
                 &lt;0.006% 
                 &lt;0.009% 
                 &lt;0.008% 
                 &lt;0.006% 
                 &lt;0.026% 
                 &lt;0.018% 
                 &lt;0.020% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-3 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 Total 
                 47338 
                 14352 
                 21253 
                 17777 
                 26512 
                 19483 
                 43728 
                 29469 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-4 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 12292 
                 532 
                 1383 
                 2597 
                 861 
                 2598 
                 1356 
                 3573 
               
               
                 Modified 
                 &lt;0.008% 
                 &lt;0.188% 
                 &lt;0.072% 
                 &lt;0.039% 
                 &lt;0.116% 
                 &lt;0.038% 
                 0.074% 
                 &lt;0.028% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-5 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 60859 
                 22846 
                 25573 
                 19054 
                 25315 
                 31754 
                 66622 
                 60925 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-6 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 60859 
                 22846 
                 25573 
                 19054 
                 25315 
                 31754 
                 66622 
                 60925 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-7 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 60859 
                 22846 
                 25573 
                 19054 
                 25315 
                 31754 
                 66622 
                 60925 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-8 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 9170 
                 1614 
                 5934 
                 3215 
                 2450 
                 12750 
                 10120 
                 13003 
               
               
                 Modified 
                 &lt;0.011% 
                 &lt;0.062% 
                 &lt;0.017% 
                 &lt;0.031% 
                 &lt;0.041% 
                 &lt;0.008% 
                 &lt;0.010% 
                 &lt;0.008% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-9 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
               
               
                 Total 
                 8753 
                 12766 
                 9504 
                 10114 
                 11086 
                 10676 
                 9013 
                 11110 
               
               
                 Modified 
                 &lt;0.011% 
                 &lt;0.008% 
                 &lt;0.011% 
                 &lt;0.010% 
                 &lt;0.009% 
                 &lt;0.009% 
                 &lt;0.011% 
                 0.027% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-10 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 0 
                 0 
                 2 
                 2 
                 3 
                 5 
                 7 
               
               
                 Total 
                 8151 
                 16888 
                 8804 
                 7061 
                 6891 
                 32138 
                 14889 
                 40120 
               
               
                 Modified 
                 0.012% 
                 &lt;0.006% 
                 &lt;0.011% 
                 0.028% 
                 0.022% 
                 0.009% 
                 0.034% 
                 0.017% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-11 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 9 
                 76 
               
               
                 Total 
                 41343 
                 32352 
                 26834 
                 28709 
                 26188 
                 32519 
                 24894 
                 19586 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.036% 
                 0.388% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 2.7E−03 
                 2.5E−18 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;274 
                 &gt;1302 
                 &gt;2564 
                 &gt;1016 
                 448 
                 47 
               
            
           
           
               
            
               
                 OffA-12 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 13186 
                 2326 
                 13961 
                 12911 
                 21134 
                 9220 
                 7792 
                 8068 
               
               
                 Modified 
                 &lt;0.008% 
                 &lt;0.043% 
                 &lt;0.007% 
                 &lt;0.008% 
                 &lt;0.006% 
                 &lt;0.011% 
                 &lt;0.013% 
                 &lt;0.012% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-13 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 9 
                 0 
               
               
                 Total 
                 32704 
                 32015 
                 12312 
                 23645 
                 26315 
                 24078 
                 36111 
                 22364 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.008% 
                 0.025% 
                 &lt;0.006% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 2.7E−03 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;225 
                 &gt;1302 
                 &gt;2564 
                 616 
                 649 
                 &gt;2725 
               
            
           
           
               
            
               
                 OffA-15 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 Total 
                 14654 
                 15934 
                 12313 
                 6581 
                 13053 
                 18996 
                 10916 
                 21519 
               
               
                 Modified 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.008% 
                 &lt;0.015% 
                 0.008% 
                 &lt;0.006% 
                 &lt;0.009% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-16 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 12 
               
               
                 Total 
                 65190 
                 35633 
                 37252 
                 30378 
                 31469 
                 22590 
                 13594 
                 20922 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.007% 
                 0.057% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                   
                 7.9E−04 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;274 
                 &gt;1302 
                 &gt;2564 
                 &gt;1016 
                 &gt;2200 
                 317 
               
            
           
           
               
            
               
                 OffA-17 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
               
               
                 Total 
                 1972 
                 606 
                 1439 
                 2113 
                 2862 
                 728 
                 597 
                 636 
               
               
                 Modified 
                 &lt;0.051% 
                 &lt;0.165% 
                 &lt;0.069% 
                 &lt;0.047% 
                 &lt;0.035% 
                 &lt;0.137% 
                 &lt;0.168% 
                 0.943% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                   
                 1.4E−02 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;26 
                 &gt;183 
                 &gt;489 
                 &gt;49 
                 &gt;97 
                 19 
               
            
           
           
               
            
               
                 OffA-18 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 5425 
                 995 
                 1453 
                 1831 
                 3132 
                 1934 
                 1534 
                 5816 
               
               
                 Modified 
                 &lt;0.018% 
                 &lt;0.101% 
                 &lt;0.069% 
                 &lt;0.055% 
                 &lt;0.032% 
                 &lt;0.052% 
                 &lt;0.065% 
                 &lt;0.017% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-19 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 2 
                 0 
                 1 
                 1 
                 1 
                 1 
                 3 
               
               
                 Total 
                 31094 
                 41252 
                 33213 
                 29518 
                 32337 
                 25904 
                 27575 
                 38711 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.008% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-21 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 15297 
                 9710 
                 16719 
                 12119 
                 15483 
                 21692 
                 16558 
                 15418 
               
               
                 Modified 
                 &lt;0.007% 
                 &lt;0.010% 
                 &lt;0.006% 
                 &lt;0.008% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-22 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 27 
                 41 
                 38 
                 46 
                 32 
                 50 
                 55 
                 57 
               
               
                 Total 
                 9406 
                 11150 
                 11516 
                 10269 
                 13814 
                 14057 
                 11685 
                 14291 
               
               
                 Modified 
                 0.267% 
                 0.368% 
                 0.330% 
                 0.448% 
                 0.232% 
                 0.356% 
                 0.471% 
                 0.399% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-23 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 10 
                 20 
               
               
                 Total 
                 5671 
                 9363 
                 2203 
                 7011 
                 7078 
                 12068 
                 3484 
                 8619 
               
               
                 Modified 
                 0.018% 
                 &lt;0.011% 
                 &lt;0.045% 
                 &lt;0.014% 
                 &lt;0.014% 
                 &lt;0.008% 
                 0.287% 
                 0.232% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                 3.5E−03 
                 9.1E−05 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;40 
                 &gt;609 
                 &gt;1210 
                 &gt;818 
                 56 
                 78 
               
            
           
           
               
            
               
                 OffA-24 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 4 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 2 
               
               
                 Total 
                 17288 
                 7909 
                 14261 
                 29936 
                 6943 
                 6333 
                 14973 
                 19953 
               
               
                 Modified 
                 0.023% 
                 &lt;0.013% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.014% 
                 0.016% 
                 &lt;0.007% 
                 0.010% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-25 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 20089 
                 45320 
                 50758 
                 108581 
                 11574 
                 20948 
                 123827 
                 74151 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.009% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-27 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 Total 
                 47338 
                 14352 
                 21253 
                 17777 
                 26512 
                 19483 
                 43728 
                 29469 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-29 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 5174 
                 12618 
                 36909 
                 18063 
                 16486 
                 17934 
                 9999 
                 35072 
               
               
                 Modified 
                 &lt;0.019% 
                 &lt;0.008% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.010% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-30 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 4 
                 4 
                 0 
                 7 
                 4 
                 4 
                 0 
                 3 
               
               
                 Total 
                 45082 
                 56531 
                 35333 
                 88651 
                 69652 
                 20362 
                 29180 
                 21350 
               
               
                 Modified 
                 0.009% 
                 0.007% 
                 &lt;0.006% 
                 0.008% 
                 &lt;0.006% 
                 0.020% 
                 &lt;0.006% 
                 0.014% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-32 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 13405 
                 6721 
                 14013 
                 7513 
                 14135 
                 22376 
                 6407 
                 13720 
               
               
                 Modified 
                 &lt;0.007% 
                 &lt;0.015% 
                 &lt;0.007% 
                 &lt;0.013% 
                 &lt;0.007% 
                 &lt;0.006% 
                 &lt;0.016% 
                 &lt;0.007% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-33 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 4 
               
               
                 Total 
                 106222 
                 46866 
                 157329 
                 48611 
                 92559 
                 152094 
                 201408 
                 225805 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-34 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
               
               
                 Total 
                 3889 
                 3158 
                 2903 
                 2235 
                 2112 
                 3022 
                 2322 
                 2481 
               
               
                 Modified 
                 &lt;0.0026% 
                 &lt;0.032% 
                 &lt;0.034% 
                 &lt;0.045% 
                 &lt;0.047% 
                 &lt;0.033% 
                 &lt;0.043% 
                 0.061% 
               
               
                 P-value 
               
               
                 Specificity 
               
            
           
           
               
            
               
                 OffA-35 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 33 
               
               
                 Total 
                 46462 
                 37431 
                 38043 
                 31033 
                 44803 
                 37257 
                 41073 
                 47273 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 0.070% 
               
               
                 P-value 
                   
                   
                   
                   
                   
                   
                   
                 9.2E−09 
               
               
                 Specificity 
                   
                 &gt;0 
                 &gt;274 
                 &gt;1302 
                 &gt;2564 
                 &gt;1016 
                 &gt;2428 
                 260 
               
            
           
           
               
            
               
                 OffA-36 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Indels 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 27115 
                 17075 
                 45425 
                 35059 
                 22298 
                 19610 
                 12620 
                 27170 
               
               
                 Modified 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.006% 
                 &lt;0.008% 
                 &lt;0.006% 
               
               
                 P-value 
               
               
                 Specificity 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Exponential fitting of enrichment values as function of mutation 
               
               
                 number. Enrichment values of post-selection sequences as function 
               
               
                 of mutation were normalized relative to on-target enrichment (=1.0 
               
               
                 by definition). Normalized enrichment values of sequences with zero 
               
               
                 to four mutations were fit to an exponential function, a*eb, with 
               
               
                 R2 reported using the non-linear least squares method. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 TALEN selectors 
                 a 
                 b 
                 R 2   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 L13 + R10 CCR5B 
                 1.00 
                 −1.88 
                 0.999937 
               
               
                   
                 L10 + R10 CCR5B 
                 1.00 
                 −1.85 
                 0.999901 
               
               
                   
                 L10 + R13 CCR5B 
                 1.00 
                 −1.71 
                 0.999822 
               
               
                   
                 L13 + R13 CCR5B 
                 1.00 
                 −1.64 
                 0.999771 
               
               
                   
                 L13 + R16 CCR5B 
                 1.00 
                 −1.15 
                 0.998286 
               
               
                   
                 L16 + R10 CCR5B 
                 1.00 
                 −1.24 
                 0.998252 
               
               
                   
                 L10 + R16 CCR5B 
                 1.01 
                 −1.08 
                 0.996343 
               
               
                   
                 L16 + R13 CCR5B 
                 1.01 
                 −1.04 
                 0.995844 
               
               
                   
                 L16 + R16 CCR5B 
                 1.03 
                 −0.70 
                 0.977880 
               
               
                   
                 L18 + R18 ATM 
                 1.08 
                 −0.36 
                 0.913087 
               
               
                   
                 L18 + R18 CCR5A 
                 1.13 
                 −0.21 
                 0.798923 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Exponential fitting and extrapolation of enrichment values as 
               
               
                 function of mutation number. Enrichment values of all sequences 
               
               
                 from all nine of the CCR5B selections as function of mutation 
               
               
                 number were normalized relative to enrichment values of sequences 
               
               
                 with the lowest mutation number in the range shown (=1.0 by definition). 
               
               
                 Normalized enrichment values of sequences from the range of mutations 
               
               
                 specified were fit to an exponential function, a*e b , with R 2  reported 
               
               
                 utilizing the non-linear least squares method. These exponential 
               
               
                 decrease, b, were used to extrapolate all mean enrichment values 
               
               
                 beyond five mutations. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 TALEN selection 
                 Range 
                 a 
                 b 
                 R 2   
               
               
                   
                   
               
               
                   
                 L16 + R16 CCR5B 
                 3-5 
                 1.00 
                 −1.638 
                 0.99998 
               
               
                   
                 L16 + R13 CCR5B 
                 2-4 
                 1.00 
                 −1.733 
                 0.99998 
               
               
                   
                 L16 + R10 CCR5B 
                 2-4 
                 1.00 
                 −2.023 
                 0.99999 
               
               
                   
                 L13 + R16 CCR5B 
                 2-4 
                 1.00 
                 −1.844 
                 0.99997 
               
               
                   
                 L13 + R13 CCR5B 
                 1-3 
                 1.00 
                 −2.014 
                 0.99998 
               
               
                   
                 L13 + R10 CCR5B 
                 1-3 
                 1.00 
                 −2.205 
                 0.99999 
               
               
                   
                 L10 + R16 CCR5B 
                 2-4 
                 1.00 
                 −1.929 
                 0.99995 
               
               
                   
                 L10 + R13 CCR5B 
                 1-3 
                 1.00 
                 −2.110 
                 0.99998 
               
               
                   
                 L10 + R10 CCR5B 
                 1-3 
                 1.00 
                 −2.254 
                 0.99999 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Oligonucleotides used in this study. (A) All oligonucleotides were purchased from 
               
               
                 Integrated DNA Technologies (SEQ ID NO: 313 447 from top to bottom). ‘/5Phos/’ indicates 5′ 
               
               
                 phosphorylated oligonucleotides. A % symbol indicates that the preceding nucleotide was 
               
               
                 incorporated as a mixture of phosphoramidites consisting of 79 mol % of the phosphoramidite 
               
               
                 corresponding to the preceding nucleotide and 7 mol % of each of the other three canonical 
               
               
                 phosphoramidites. An (*) indicates that the oligonucleotide primer was specific to a selection 
               
               
                 sequence (either CCR5A, ATM or CCR5B). An (**) indicates that the oligonucleotide adapter or 
               
               
                 primer had a unique sequence identifier to distinguish between different samples (selection 
               
               
                 conditions or cellular TALEN treatment). (B) Combinations of oligonucleotides used to 
               
               
                 construct discrete DNA substrates used in TALEN digestion assays. (C) Primer pairs for PCR 
               
               
                 amplifying on target and off target genomic sites. +DMSO: DMSO was used in the PCR; ND: 
               
               
                 no correct DNA product was detected from the PCR reaction. Sequences correspond to SEQ ID 
               
               
                 NOs: 472 545 (Fwd primers from top to bottom); and SEQ ID NOs: 546 619 (Rev primers from 
               
               
                 top to bottom). 
               
               
                   
               
             
            
               
                 A 
               
            
           
           
               
               
            
               
                 oligonucleotide 
                   
               
               
                 name 
                 oligonucleotide sequence (5′→3′) 
               
               
                   
               
               
                 TAL-Nrev 
                 5Phos/CAGCAGCTGCCCGGT 
               
               
                   
               
               
                 TAL-N1fwd 
                 5Phos/cAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAG 
               
               
                   
               
               
                 TAL-N2fwd 
                 5Phos/cAGATCGCGcAGAGAGGGGGAGTAACAGCGGTAG 
               
               
                   
               
               
                 TAL-N3fwd 
                 5Phos/cAGATCGCGcAGcagGGGGGAGTAACAGCGGTAG 
               
               
                   
               
               
                 TAL-CIfwd 
                 ATC GTA GCC CAA TTG TCC A 
               
               
                   
               
               
                 TAL-CIrev 
                 GTTGGTTCTTTGGATCAATGCG 
               
               
                   
               
               
                 TAL-Q3 
                 AAGTTCTCTCGGGAATCCGTTGGTTGGTTCTTTGGATCA 
               
               
                   
               
               
                 TAL-Q7 
                 GAAGTTCTCTCGGGAATTTGTTGGTTGGTTTGTTGGATCAATGCGGGAGCATGAGGCAGACCTT 
               
               
                   
                 GTTGGACTGCATC 
               
               
                   
               
               
                 TAL-CIIrev 
                 CTTTTGACTAGTTGGGATCCCGCGACTTGATGGGAAGTTCTCTCGGGAAT 
               
               
                   
               
               
                 CCR5A Library10 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%T%C%A%T%C%T%NNNNNNNNNN 
               
               
                   
                 A%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library12 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%T%C%A%T%C%T%NNNNNNNNNN 
               
               
                   
                 NNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library14 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library16 
                 5Phos/CACCACTCT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library18 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNNNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library20 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNNNNNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library22 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNNNNNNNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACGCT 
               
               
                   
               
               
                 CCR5A Library24 
                 5Phos/CACCACTNT%T%C%A%T%T%A%C%A%C%C%T%G%C%A%G%C%T%NNNNNNNNNN 
               
               
                   
                 NNNNNNNNNNNNA%G%T%A%T%C%A%A%T%T%C%T%G%G%A%A%G%A%NCGTCACG 
               
               
                   
                 CT 
               
               
                   
               
               
                 CCR5B Library10 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNC%A% 
               
               
                   
                 T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library12 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNC% 
               
               
                   
                 A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library14 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 C%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library16 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 NNC%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library18 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 NNNNC%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library20 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 NNNNNNC%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library22 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 NNNNNNNNC%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 CCR5B Library24 
                 5Phos/CCACGCTNT%C%T%T%C%A%T%T%A%C%A%C%C%T%G%C%NNNNNNNNNNNNNN 
               
               
                   
                 NNNNNNNNNNC%A%T%A%C%A%G%T%C%A%G%T%A%T%C%A%NCCTCGGGACT 
               
               
                   
               
               
                 ATM Library10 
                 Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNNN 
               
               
                   
                 T%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library12 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNT]T]T]A]T]T]T]T]A]C]T]G]T]C]T]T]T]A]GGTACCCCA 
               
               
                   
               
               
                 ATM Library14 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library16 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library18 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library20 
                 5Phos/CTCCGCGTCT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNNNNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library22 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNNNNNNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCCA 
               
               
                   
               
               
                 ATM Library24 
                 5Phos/CTCCGCGTNT%G%A%A%T%T%G%G%G%A%T%G%C%T%G%T%T%T%NNNNNNNNN 
               
               
                   
                 NNNNNNNNNNNNNNNT%T%T%A%T%T%T%T%A%C%T%G%T%C%T%T%T%A%GGTACCCC 
               
               
                   
                 A 
               
               
                   
               
               
                 #1 adapter-fwd**1 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGT 
               
               
                   
               
               
                 #1 adapter-rev**1 
                 ACAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**2 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGAA 
               
               
                   
               
               
                 #1 adapter-rev**2 
                 TTCAGAGATCGGAAGAGCGTCGTGTAGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**3 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCAA 
               
               
                   
               
               
                 #1 adapter-rev**3 
                 TTGCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**4 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACT 
               
               
                   
               
               
                 #1 adapter-rev**4 
                 AGTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**5 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATT 
               
               
                   
               
               
                 #1 adapter-rev**5 
                 AATGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**6 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCATGA 
               
               
                   
               
               
                 #1 adapter-rev**6 
                 TCATGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**7 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATGCT 
               
               
                   
               
               
                 #1 adapter-rev**7 
                 AGCATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**8 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTAGT 
               
               
                   
               
               
                 #1 adapter-rev**8 
                 ACTAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**9 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTAA 
               
               
                   
               
               
                 #1 adapter-rev**10 
                 TTAGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**10 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGTA 
               
               
                   
               
               
                 #1 adapter-rev**11 
                 TACTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**11 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACT 
               
               
                   
               
               
                 #1 adapter-rev**12 
                 AGTACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**12 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGT 
               
               
                   
               
               
                 #1 adapter-rev**13 
                 ACAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**13 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTAA 
               
               
                   
               
               
                 #1 adapter-rev**14 
                 TTAGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**14 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGTA 
               
               
                   
               
               
                 #1 adapter-rev**14 
                 TACTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**15 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACT 
               
               
                   
               
               
                 #1 adapter-rev**15 
                 AGTACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #1 adapter-fwd**16 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGT 
               
               
                   
               
               
                 #1 adapter-rev**16 
                 ACAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGG 
               
               
                   
               
               
                 #2A primer-fwd 
                 AATGATACGGCGACCAC 
               
               
                   
               
               
                 #2A primer- 
                 GTTCAGACGTGTGCTCTTCCGATCTNNNNAGTGGTGAGCGTGACG 
               
               
                 rev*CCR5A 
                   
               
               
                   
               
               
                 #2A primer-rev*ATM 
                 GTTCAGACGTGTGCTCTTCCGATCTNNNNACGCGGAGTGGGGTACC 
               
               
                   
               
               
                 #2A primer- 
                 CAGACGTGTGCTCTTCCGATCNNNNAGCGTGGAGTCCCGAGG 
               
               
                 rev*CCR5B 
                   
               
               
                   
               
               
                 #2B primer-fwd 
                 AATGATACGGCGACCAC 
               
               
                   
               
               
                 #2B primer-rev**1 
                 CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTC 
               
               
                   
               
               
                 #2B primer-rev**2 
                 CAAGCAGAAGACGGCATACGAGATACGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTC 
               
               
                   
               
               
                 #2B primer-rev **3 
                 CAAGCAGAAGACGGCATACGAGATTCTAACATGTGACTGGAGTTCAGACGTGTGCTCTTC 
               
               
                   
               
               
                 #2B primer-rev **4 
                 CAAGCAGAAGACGGCATACGAGATCGGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTC 
               
               
                   
                 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG 
               
               
                   
               
               
                 #1 Lib. adapter - 
                 GTACCCAGATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTT 
               
               
                 fwd*CCR5A 
                 CTGCTTG 
               
               
                   
               
               
                 #1 Lib. adapter - 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTG 
               
               
                 rev*CCR5A 
                   
               
               
                   
               
               
                 #1 Lib. adapter - 
                 GTACGATGCGATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTC 
               
               
                 fwd*ATM 
                 TTCTGCTTG 
               
               
                   
               
               
                 #1 Lib. adapter - 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGCATC 
               
               
                 rev*ATM 
                   
               
               
                   
               
               
                 #1 Lib. adapter - 
                 TCGGGAACGTGATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTCTAATCTCGTATGCCGT 
               
               
                 fwd*CCR5B 
                 CTTCTGCTTG 
               
               
                   
               
               
                 #1 Lib. adapter - 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCACGTT 
               
               
                 rev*CCR5B 
                   
               
               
                   
               
               
                 #2A Lib. primer-rev 
                 CAAGCAGAAGACGGCATACGA 
               
               
                   
               
               
                 #2A Lib. primer- 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCG 
               
               
                 fwd*CCR5A 
                 TCACGCTCACCACT 
               
               
                   
               
               
                 #2A Lib. primer- 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 
               
               
                 fwd*ATM 
                 GTACCCCACTCCGCGT 
               
               
                   
               
               
                 #2A Lib. primer- 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCC 
               
               
                 fwd*CCR5B 
                 TCGGGACTCCACGCT 
               
               
                   
               
               
                 #2B Lib. primer-rev 
                 CAAGCAGAAGACGGCATACGA 
               
               
                   
               
               
                 #2B Lib. primer-fwd 
                 AATGATACGGCGACCAC 
               
               
                   
               
               
                 G adapter-fwd 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCT 
               
               
                   
               
               
                 G adapter-rev 
                 /5Phos/GATCGGAAGAGCACACGTCTGAACTCCA 
               
               
                   
               
               
                 G-B primer-fwd 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC 
               
               
                   
               
               
                 G-B primer-rev**1 
                 CAAGCAGAAGACGGCATACGAGATGTGCGGACGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**2 
                 CAAGCAGAAGACGGCATACGAGATCGTTTCACGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**3 
                 CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**4 
                 CAAGCAGAAGACGGCATACGAGATTCCGAAACGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**5 
                 CAAGCAGAAGACGGCATACGAGATTACGTACGGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**6 
                 CAAGCAGAAGACGGCATACGAGATATCCACTCGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**7 
                 CAAGCAGAAGACGGCATACGAGATAAAGGAATGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 G-B primer-rev**8 
                 CAAGCAGAAGACGGCATACGAGATATATCAGTGTGACTGGAGTTCAGACGTGTGCT 
               
               
                   
               
               
                 CCR5AonCfwd 
                 CGACGGTCTAGAGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT 
               
               
                   
               
               
                 CCR5Amut1fwd 
                 CGACGGTCTAGAGTCTTCATTACAtCTGCAcCTCTCATTTTCCATACAGT 
               
               
                   
               
               
                 CCR5Amut2fwd 
                 CGACGGTCTAGAGTCTTCAaTACACCTGtAGCTCTCATTTTCCATACAGT 
               
               
                   
               
               
                 CCR5Amut3fwd 
                 CGACGGTCTAGAGTCTTCgTTACACCTGCAtCTCTCATTTTCCATACAGT 
               
               
                   
               
               
                 CCR5Amut4fwd 
                 CGACGGTCTAGAGTCTTaATTgCACCTGCAGCTCTCATTTTCCATACAGT 
               
               
                   
               
               
                 CCR5AonCrev 
                 CCGACGAAGCTTTTCTTCCAGAATTGATACTGACTGTATGGAAAATGA 
               
               
                   
               
               
                 CCR5Amut1rev 
                 CCGACGAAGCTTTTCTTaCAGAATTcATACTGACTGTATGGAAAATGA 
               
               
                   
               
               
                 CCR5Amut2rev 
                 CCGACGAAGCTTTTCcTCCAGAgTTGATACTGACTGTATGGAAAATGA 
               
               
                   
               
               
                 CCR5Amut3rev 
                 CCGACGAAGCTTTTCTTCCtGAATTGATAaTGACTGTATGGAAAATGA 
               
               
                   
               
               
                 CCR5Amut4rev 
                 CCGACGAAGCTTTTCTTCCAGcATTGtTACTGACTGTATGGAAAATGA 
               
               
                   
               
               
                 ATMonAfwd 
                 CGACGGTCTAGATTTGAATTGGGATGCTGTTTTTAGGTATTCTATTCAAATT 
               
               
                   
               
               
                 ATMmut1fwd 
                 CGACGGTCTAGATTTGAATTGGGtTGCTGTTTTTAGGTATTCTATTCAAATT 
               
               
                   
               
               
                 ATMmut2fwd 
                 CGACGGTCTAGATTTGAATTGcGATGCTGTTTTTAGGTATTCTATTCAAATT 
               
               
                   
               
               
                 ATMmut3fwd 
                 CGACGGTCTAGATTTGAgTTGGGATGCTGTTTTTAGGTATTCTATTCAAATT 
               
               
                   
               
               
                 ATMmut4fwd 
                 CGACGGTCTAGATTTGAATTGGGATGCTGaTTTTAGGTATTCTATTCAAATT 
               
               
                   
               
               
                 ATMonArev 
                 CCGACGAAGCTTAATAAAGACAGTAAAATAAATTTGAATAGAATACCTAAAA 
               
               
                   
               
               
                 ATMmut1rev 
                 CCGACGAAGCTTAATAAAGACAGTgAAATAAATTTGAATAGAATACCTAAAA 
               
               
                   
               
               
                 ATMmut2rev 
                 CCGACGAAGCTTAATAAAGAtAGTAAAATAAATTTGAATAGAATACCTAAAA 
               
               
                   
               
               
                 ATMmut3rev 
                 CCGACGAAGCTTAATAAAGACAGTAAgATAAATTTGAATAGAATACCTAAAA 
               
               
                   
               
               
                 ATMmut4rev 
                 CCGACGAAGCTTAATAAcGACAGTAAAATAAATTTGAATAGAATACCTAAAA 
               
               
                   
               
               
                 CCR5BonBfwd 
                 CGACGGTCTAGAAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCA 
               
               
                   
               
               
                 CCR5Bmut1fwd 
                 CGACGGTCTAGAGTCTTCATTACACCTGtAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5Bmut2fwd 
                 CGACGGTCTAGAGTCTTCATaACACCTGCAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5Bmut3fwd 
                 CGACGGTCTAGAGTCTTCATTACACCcGCAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5Bmut4fwd 
                 CGACGGTCTAGAGTCTTCATaACACCTGtAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5Bmut5fwd 
                 CGACGGTCTAGAGTCTTCATTAtACCTaCAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5Bmut6fwd 
                 CGACGGTCTAGAGTCTTCATTgCACCcGCAGCTCTCATTTTC 
               
               
                   
               
               
                 CCR5BonBreV 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTGACTGTATGGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut1rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTaACTGTATGGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut2rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTGACTGTATcGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut3rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTGACTGaATGGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut4rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACcGACTGTATGGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut5rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTaACTGTATcGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut6rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTGAaTGTgTGGAAAATGAGAGCT 
               
               
                   
               
               
                 CCR5Bmut7rev 
                 CCGACGAAGCTTTCTTCCAGAATTGATACTGAtaGTATGGAAAATGAGAGCT 
               
               
                   
               
               
                 pUC19Ofwd 
                 GCGACACGGAAATGTTGAATACTCAT 
               
               
                   
               
               
                 pUC19Orev 
                 CAGCGAGTCAGTGAGCGA 
               
               
                   
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
            
               
                 DNA substrate name 
                 Oligonucleotide Combination 
               
               
                   
               
               
                 A1 
                 ATMmut1fwd + ATMonArev 
               
               
                   
               
               
                 A2 
                 ATMmut2fwd + ATMonArev 
               
               
                   
               
               
                 A3 
                 ATMonAfwd + ATMmut1rev 
               
               
                   
               
               
                 A4 
                 ATMonAfwd + ATMmut2rev 
               
               
                   
               
               
                 A5 
                 ATMmut2fwd + ATMmut2rev 
               
               
                   
               
               
                 A6 
                 ATMmut3fwd + ATMmut3rev 
               
               
                   
               
               
                 A7 
                 ATMmut1fwd + ATMmut1rev 
               
               
                   
               
               
                 A8 
                 ATMmut4fwd + ATMmut4rev 
               
               
                   
               
               
                 C1 
                 CCR5Amut1fwd + CCR5AonCrev 
               
               
                   
               
               
                 C2 
                 CCR5Amut2fwd + CCR5AonCrev 
               
               
                   
               
               
                 C3 
                 CCR5Amut3fwd + CCR5AonCrev 
               
               
                   
               
               
                 C4 
                 CCR5Amut4fwd + CCR5AonCrev 
               
               
                   
               
               
                 C5 
                 CCR5AonAfwd + CCR5AmutArev 
               
               
                   
               
               
                 C6 
                 CCR5AonAfwd + CCR5Amut2rev 
               
               
                   
               
               
                 C7 
                 CCR5AonAfwd + CCR5Amut3rev 
               
               
                   
               
               
                 C8 
                 CCR5AonAfwd + CCR5Amut4rev 
               
               
                   
               
               
                 B1 
                 CCR5Bmut1fwd + CCR5BonBrev 
               
               
                   
               
               
                 B2 
                 CCR5Bmut2fwd + CCR5BonBrev 
               
               
                   
               
               
                 B3 
                 CCR5Bmut3fwd + CCR5BonBrev 
               
               
                   
               
               
                 B4 
                 CCR5BonBfwd + CCR5Bmut1rev 
               
               
                   
               
               
                 B5 
                 CCR5BonBfwd + CCR5Bmut2rev 
               
               
                   
               
               
                 B6 
                 CCR5BonBfwd + CCR5Bmut3rev 
               
               
                   
               
               
                 B7 
                 CCR5BonBfwd + CCR5Bmut4rev 
               
               
                   
               
               
                 B8 
                 CCR5Bmut4fwd + CCR5BonBrev 
               
               
                   
               
               
                 B9 
                 CCR5Bmut5fwd + CCR5BonBrev 
               
               
                   
               
               
                 B10 
                 CCR5Bmu6fwd + CCR5BonBrev 
               
               
                   
               
               
                 B11 
                 CCR5BonBfwd + CCR5Bmut5rev 
               
               
                   
               
               
                 B12 
                 CCR5BobBfwd + CCR5Bmut6rev 
               
               
                   
               
               
                 B13 
                 CCR5BonBfwd + CCR5Bmut7rev 
               
               
                   
               
               
                 B14 
                 CCR5Bmut1fwd + CCR5Bmut1rev  
               
               
                   
               
               
                 B15 
                 CCR5Bmut2fwd + CCR5Bmut2rev 
               
               
                   
               
               
                 B16 
                 CCR5Bmut1fwd + CCR5Bmut3rev 
               
               
                   
               
            
           
           
               
            
               
                 C 
               
            
           
           
               
               
               
               
            
               
                 Site 
                 Fwd primer 
                 Rev primer 
                 PCR 
               
               
                   
               
               
                 OnCCR5A 
                 TCACTTGGGTGGTGGCTGTG 
                 GACCATGACAAGCAGCGGCA 
                   
               
               
                   
               
               
                 OffC-1 
                 AGTCCAAGACCAGCCTGGGG 
                 AAGAACCTGTTGTCTAATCCAGCA 
                   
               
               
                   
               
               
                 OffC-2 
                 GAACCTGTTGTCTAATCCAGCGTC 
                 CTGCAAAGAAGGCCAGGCA 
                   
               
               
                   
               
               
                 OffC-3 
                 AGTCCAAGACCAGCCTGGGG 
                 AAGAACCTGTTGTCTAATCCAGCA 
                   
               
               
                   
               
               
                 OffC-4 
                 TGACCTGTTTGTTCAGGTCTTCC 
                 CCATATGGTCCCTGTCGCAA 
                   
               
               
                   
               
               
                 OffC-5 
                 TCCAGTTGCTGTCCCTTCAGA 
                 ACAGGGAGAGCCACCAATGC 
                   
               
               
                   
               
               
                 OffC-6 
                 GCCCGGCCTGTCCTGTATTT 
                 CACCCACACATGCACTTCCC 
                   
               
               
                   
               
               
                 OffC-7 
                 TGGCTATTCTAGTTCTTTTGCAT 
                 CCATGCCCTAGGGATTTGTGGA 
                   
               
               
                   
               
               
                 OffC-8 
                 CGCTGAAGGCTGTCACCCTAA 
                 TGGACCTAAGAGTCCTGCCCAT 
                   
               
               
                   
               
               
                 OffC-9 
                 CCACCACCACACAACTTCACA 
                 CAGCTGGCGAGAACTGCAAA 
                 ND 
               
               
                   
               
               
                 OffC-10 
                 TTCCAGGTCCTTTGCACAAATA 
                 GCAAGGTCGTTGGATAGAAGTTGA 
                   
               
               
                   
               
               
                 OffC-11 
                 CACCGAAAGCAACCCATTCC 
                 TGATCTGCCCACCCCAGACT 
                   
               
               
                   
               
               
                 OffC-12 
                 TTCATTCTCACCATCTGGAATTGG 
                 TCTGGCTGGACTGCTCTGGTT 
                   
               
               
                   
               
               
                 OffC-13 
                 TGGCATGTGGATCAGTACCCA 
                 TAGAACATGCCCGCGAACAG 
                   
               
               
                   
               
               
                 OffC-14 
                 CTGACGTCCATGTCAACGGG 
                 TTTGAATTCCCCCTCCCCAT 
                   
               
               
                   
               
               
                 OffC-15 
                 GCTCCTTTCTGAGAAGCACCCAT 
                 GGCAGATGGTGGCAGGTCTT 
                   
               
               
                   
               
               
                 OffC-16 
                 ATGAGGGCTTGGATTGGCTG 
                 CCACCTCCCCCACTGCAATA 
                   
               
               
                   
               
               
                 OffC-17 
                 GGAGGCCTTCATTGTGTCACG 
                 AACTCCACCTGGGTGCCCTA 
                   
               
               
                   
               
               
                 OffC-18 
                 CGTGGTCCCCCAGAAATCAC 
                 GGAGCAGGAGTTGGTGGCAT 
                   
               
               
                   
               
               
                 OffC-19 
                 GATTGCATAGGTTAGCATTGCC 
                 GCCCCTGTTGGTTGACTCCC 
                   
               
               
                   
               
               
                 OffC-20 
                 TTCCAGCGAATGGAAAGTGCT 
                 AAGCCCAGGAATAAGGGCCA 
                   
               
               
                   
               
               
                 OffC-21 
                 AAGCATGCTCACACTGTGGTGTA 
                 TTGCTTGAGGCGGAAGTTGC 
                   
               
               
                   
               
               
                 OffC-22 
                 TGACCCTCCAGCAAAGGTGA 
                 CCCCAGGGACTGAGCATGAG 
                   
               
               
                   
               
               
                 OffC-23 
                 GCTTTGCTTGCACTGTGCCTT 
                 GGGGACAGACTGTGAGGGCT 
                   
               
               
                   
               
               
                 OffC-24 
                 TCAAAAGGATGTGATCTGCCACA 
                 GGCCTCTTTGAGGGCCAGTT 
                   
               
               
                   
               
               
                 OffC-25 
                 CCAGGGCTCAATTCTTAGACCG 
                 AAAAGAGCAGGGCTGCCATC 
                   
               
               
                   
               
               
                 OffC-26 
                 TGTTCATGCCTGCACAGTGG 
                 TGGATGTGCCCTCTACCACA 
                   
               
               
                   
               
               
                 OffC-27 
                 TTTGGCAAGGAATTCACAGTTC 
                 TCATGCCTGCACAGTGGTTG 
                   
               
               
                   
               
               
                 OffC-28 
                 GGAGGATGTCTTTGTGGTAGGGG 
                 CGCTGCCAAGCAAACTCAAA 
                   
               
               
                   
               
               
                 OffC-29 
                 TCCCCCAACTTCACTGTTTTT 
                 GCAATGAGCATGTGGACACCA 
                   
               
               
                   
               
               
                 OffC-30 
                 TTCTCTGTTTCCAGTGATTTCAGA 
                 GTCGCAAAACAGCCAGTTGC 
                 +DMSO 
               
               
                   
               
               
                 OffC-31 
                 TGGCTTGGTTAATGGACAATGG 
                 CCTGCAAGGAGCAAGGCTTC 
                 +DMSO 
               
               
                   
               
               
                 OffC-32 
                 TGGGCTTCGTTGACTTAAAGAG 
                 GGACAAGAGGGCCAGGGTTT 
                   
               
               
                   
               
               
                 OffC-33 
                 TCTTAAACATGTGGAACCCAGTCAT 
                 TGAAAACCCACAGAGTGGGAGA 
                   
               
               
                   
               
               
                 OffC-34 
                 GCAGATTCATTAGCGTTTGTGGC 
                 TGCATGGGTGTAAATGTAGCAGAAA 
                   
               
               
                   
               
               
                 OffC-35 
                 CCAAGGATCAATACCTTTGGAGGA 
                 GCCCTCCCTTGAATCAGGCT 
                   
               
               
                   
               
               
                 OffC-36 
                 TTCCCCTAACCAGGGGCAGT 
                 GTGGTGAGTGGGTGTGGCAG 
                 +DMSO 
               
               
                   
               
               
                 OnATM 
                 AGCGCCTGATTCGAGATCCT 
                 AGCGCCTGATTCGAGATCCT 
                   
               
               
                   
               
               
                 OffA-1 
                 CCTGCCATTGAATTCCAGCCT 
                 TGTCTGCCTTTCCTGTCCCC 
                   
               
               
                   
               
               
                 OffA-2 
                 GACTGCCACTGCACTCCCAC 
                 GGATACCCTTGCCTCCCCAC 
                   
               
               
                   
               
               
                 OffA-3 
                 CCTCCCATTTTCCTTCCTCCA 
                 CTGGGAGACACAGGTGGCAG 
                   
               
               
                   
               
               
                 OffA-4 
                 TCCTCCAATTTTCCTTCCTCCA 
                 CTGGGAGACACAGGTGGCAG 
                   
               
               
                   
               
               
                 OffA-5 
                 CTGGGAGACACAGGTGGCAG 
                 AGGACCAATGGGGCCAATCT 
                   
               
               
                   
               
               
                 OffA-6 
                 CTGGGAGACACAGGTGGCAG 
                 AGGACCAATGGGGCCAATCT 
                   
               
               
                   
               
               
                 OffA-7 
                 CTGGGAGACACAGGTGGCAG 
                 AGGACCAATGGGGCCAATCT 
                   
               
               
                   
               
               
                 OffA-8 
                 GCATGCCAAAGAAATTGTAGGC 
                 TTCCCCCTGTCATGGTCTTCA 
                   
               
               
                   
               
               
                 OffA-9 
                 GCATCTCTGCATTCCTCAGAAGTGG 
                 AGAAACTGAGCAAGCCTCAGTCAA 
                   
               
               
                   
               
               
                 OffA-10 
                 GGGATACCAAAGAGCTTTTGTTTTGTT 
                 CAGAGGCTGCATGATGCCTAATA 
                   
               
               
                   
               
               
                 OffA-11 
                 TGCAGCTACGGATGAAAACCAT 
                 TCAGAATACCTCCCCGCCAG 
                   
               
               
                   
               
               
                 OffA-12 
                 GCATAAAGCACAGGATGGGAGA 
                 TCCCTCTTTAACGGTTATGTTGGC 
                   
               
               
                   
               
               
                 OffA-13 
                 TGGGTTAAGTAATTTCGAAAGGAGAA 
                 ATGTGCCCCACACATTGCC 
                 +DMSO 
               
               
                   
               
               
                 OffA-14 
                 GAGTGAGCCACTGCACCCAG 
                 CGTGTGGTGGTGGCACAAG 
                 ND 
               
               
                   
               
               
                 OffA-15 
                 CCTCCCTCTGGCTCCCTCCC 
                 ACCAGGGCCTGTTGGGGGTT 
                   
               
               
                   
               
               
                 OffA-16 
                 TGCTCCCTGACCTTCCTGAGA 
                 CCATTGGAATGAGAACCTTCTGG 
                   
               
               
                   
               
               
                 OffA-17 
                 GGTGGAACAATCCACCTGTATTAGC 
                 GAATGTGACACCACCACCGC 
                   
               
               
                   
               
               
                 OffA-18 
                 GGCTTTGCAAACATAAACACTCA 
                 CCTTCTGAGCAGCTGGGACAA 
                   
               
               
                   
               
               
                 OffA-19 
                 CACTGGAACCCAGGAGGTGG 
                 CCTCCCATTGGAGCCTTGGT 
                   
               
               
                   
               
               
                 OffA-20 
                 CAGCCTGCCTGGGTGACAG 
                 CATCTGAGCTCAAAACTGCTGC 
                 +DMSO 
               
               
                   
               
               
                 OffA-21 
                 GCCACTGCATTGCATTTTCC 
                 TGAGGGCAGGTCTGTTTCCTG 
                 ND 
               
               
                   
               
               
                 OffA-22 
                 GGGAGGATCTCTCGAGTCCAGG 
                 CCTTGCCTGACTTGCCCTGT 
                   
               
               
                   
               
               
                 OffA-23 
                 TGTTTAGTAATTAAGACCCTGGCTTTC 
                 GCGACAGGTACAAAGCAGTCCAT 
                   
               
               
                   
               
               
                 OffA-24 
                 GCCCTTTGATTTCATCTGTTTCCC 
                 CATTGCTGCCATTGCACTCC 
                   
               
               
                   
               
               
                 OffA-25 
                 AAACTGGCACATGTACTCCT 
                 ACATGATTTGATTTTTCATGTGTTT 
                   
               
               
                   
               
               
                 OffA-26 
                 GGGTGGAAGGTGAGAGGAGATT 
                 CGCAGATGGGCATGTTATTG 
                 ND 
               
               
                   
               
               
                 OffA-27 
                 CCTCCCATTTTCCTTCCTCCA 
                 GACTGCCACTGCACTCCCAC 
                   
               
               
                   
               
               
                 OffA-28 
                 AGCCAAGATTGCACCATTGC 
                 GTCCCTGACGGAGGCTGAGA 
                 ND 
               
               
                   
               
               
                 OffA-29 
                 TGGTTGGATTTTGGCTCTGTCAC 
                 TGTCAATATCAATACCCTGCTTTCCTC 
                   
               
               
                   
               
               
                 OffA-30 
                 TGGTTACTTTTAAAGGGTCATGATGGA 
                 AAAAATGGATGCAAAGCCAAA 
                 +DMSO 
               
               
                   
               
               
                 OffA-31 
                 GGGACACAGAGCCAAACCGT 
                 TGTGCACATGTACCCTAAAACT 
                 ND 
               
               
                   
               
               
                 OffA-32 
                 CAGTCATTGTTTCTAGGTAGGGGA 
                 TTGGCAATTTGGGTGCAACA 
                   
               
               
                   
               
               
                 OffA-33 
                 TGGATAACCTGCAGATTTGTTTCTG 
                 TGAGCCCAGGAGTTTCAGGC 
                   
               
               
                   
               
               
                 OffA-34 
                 TCGTGTGTGTGTGTTTGCTTCCA 
                 CAGTGGTTCGGGAAACAGCA 
                   
               
               
                   
               
               
                 OffA-35 
                 TGGGAATGTAAATCTGACTGGCTG 
                 CTGGAACTCTGGGCATGGCT 
                   
               
               
                   
               
               
                 OffA-36 
                 GCTGCAATTGCTTTTTGGCA 
                 TGGACCCCTCCCTTACACC