title: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.
abstract: TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
authors.affiliation: Department of Genetics, Cell Biology & Development and Center for Genome Engineering, 321 Church Street SE, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Tomas
authors.initials: T
authors.lastname: Cermak
authors.forename: Erin L
authors.initials: EL
authors.lastname: Doyle
authors.forename: Michelle
authors.initials: M
authors.lastname: Christian
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Clarice
authors.initials: C
authors.lastname: Schmidt
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Baller
authors.forename: Nikunj V
authors.initials: NV
authors.lastname: Somia
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Bogdanove
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012984:Software; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014974:Xanthomonas / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific