(a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA-binding module consists of 34 amino acids, where the RVDs in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA-binding modules are flanked by nonrepetitive N and C termini, which carry the translocation, nuclear localization (NLS) and transcription activation (AD) domains. A cryptic signal within the N terminus specifies a thymine as the first base of the target site.

(b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: four monomer plasmids to be used as templates for PCR amplification, four TALE-TF and four TALEN cloning backbones corresponding to four different bases targeted by the 0.5 repeat. CMV, cytomegalovirus promoter; N term, nonrepetitive N terminus from the Hax3 TALE; C term, nonrepetitive C terminus from the Hax3 TALE; BsaI, type IIs restriction sites used for the insertion of custom TALE DNA-binding domains; ccdB + CmR, negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS, nuclear localization signal; VP64, synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A, 2A self-cleavage linker; EGFP, enhanced green fluorescent protein; polyA signal, polyadenylation signal; FokI, catalytic domain from the FokI endonuclease.

(c) TALEs can be used to generate custom TALE-TFs and modulate the transcription of endogenous genes from the genome. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription.

(d) TALENs can be used to generate site-specific double-strand breaks to facilitate genome editing through nonhomologous repair or homology directed repair. Two TALENs target a pair of binding sites flanking a 16-bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites, respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN-binding sites.

0001 TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA 
0061 CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT 
0121 AGGATGGCAA GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT 
0181 ATTAATTTCC CCTCGTCAAA AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT 
0241 GAATCCGGTG AGAATGGCAA AAGTTTATGC ATTTCTTTCC AGACTTGTTC AACAGGCCAG 
0301 CCATTACGCT CGTCATCAAA ATCACTCGCA TCAACCAAAC CGTTATTCAT TCGTGATTGC 
0361 GCCTGAGCGA GGCGAAATAC GCGATCGCTG TTAAAAGGAC AATTACAAAC AGGAATCGAG 
0421 TGCAACCGGC GCAGGAACAC TGCCAGCGCA TCAACAATAT TTTCACCTGA ATCAGGATAT 
0481 TCTTCTAATA CCTGGAACGC TGTTTTTCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA 
0541 TCAGGAGTAC GGATAAAATG CTTGATGGTC GGAAGTGGCA TAAATTCCGT CAGCCAGTTT 
0601 AGTCTGACCA TCTCATCTGT AACATCATTG GCAACGCTAC CTTTGCCATG TTTCAGAAAC 
0661 AACTCTGGCG CATCGGGCTT CCCATACAAG CGATAGATTG TCGCACCTGA TTGCCCGACA 
0721 TTATCGCGAG CCCATTTATA CCCATATAAA TCAGCATCCA TGTTGGAATT TAATCGCGGC 
0781 CTCGACGTTT CCCGTTGAAT ATGGCTCATA TTCTTCCTTT TTCAATATTA TTGAAGCATT 
0841 TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA 
0901 ATAGGGGTCA GTGTTACAAC CAATTAACCA ATTCTGAACA TTATCGCGAG CCCATTTATA 
0961 CCTGAATATG GCTCATAACA CCCCTTGTTT GCCTGGCGGC AGTAGCGCGG TGGTCCCACC 
1021 TGACCCCATG CCGAACTCAG AAGTGAAACG CCGTAGCGCC GATGGTAGTG TGGGGACTCC 
1081 CCATGCGAGA GTAGGGAACT GCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT 
1141 GGGCCTTTCG CCCGGGCTAA TTAGGGGGTG TCGCCCTTCG CTGAACTCAC CCCAGAGCAG 
1201 GTCGTGGCAA TTGCGAGCCA TGACGGGGGA AAGCAGGCAC TCGAAACCGT CCAGAGGTTG 
1261 CTGCCTGTGC TGTGCCAAGC GCACGGACGT CAAAAGGGCG ACACAAAATT TATTCTAAAT 
1321 GCATAATAAA TACTGATAAC ATCTTATAGT TTGTATTATA TTTTGTATTA TCGTTGACAT 
1381 GTATAATTTT GATATCAAAA ACTGATTTTC CCTTTATTAT TTTCGAGATT TATTTTCTTA 
1441 ATTCTCTTTA ACAAACTAGA AATATTGTAT ATACAAAAAA TCATAAATAA TAGATGAATA 
1501 GTTTAATTAT AGGTGTTCAT CAATCGAAAA AGCAACGTAT CTTATTTAAA GTGCGTTGCT 
1561 TTTTTCTCAT TTATAAGGTT AAATAATTCT CATATATCAA GCAAAGTGAC AGGCGCCCTT 
1621 AAATATTCTG ACAAATGCTC TTTCCCTAAA CTCCCCCCAT AAAAAAACCC GCCGAAGCGG 
1681 GTTTTTACGT TATTTGCGGA TTAACGATTA CTCGTTATCA GAACCGCCCA GGGGGCCCGA 
1741 GCTTAAGACT GGCCGTCGTT TTACAACACA GAAAGAGTTT GTAGAAACGC AAAAAGGCCA 
1801 TCCGTCAGGG GCCTTCTGCT TAGTTTGATG CCTGGCAGTT CCCTACTCTC GCCTTCCGCT 
1861 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 
1921 TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA 
1981 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT 
2041 AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC 
2101 CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 
2161 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 
2221 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 
2281 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 
2341 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 
2401 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GGCTAACTAC 
2461 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 
2521 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 
2581 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 
2641 TCTACGGGGT CTGACGCTCA GTGGAACGAC GCGCGCGTAA CTCACGTTAA GGGATTTTGG 
2701 TCATGAGCTT GCGCCGTCCC GTCAAGTCAG CGTAATGCTC TGCTTT