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Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells

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Publisher
Oxford University Press
Publication Date
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PMC
Keywords
  • Articles
Disciplines
  • Biology
  • Design
  • Medicine

Abstract

gkr218 1..11 Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting Tomas Cermak1, Erin L. Doyle2, Michelle Christian1, Li Wang2, Yong Zhang1,3, Clarice Schmidt2, Joshua A. Baller1,4, Nikunj V. Somia1, Adam J. Bogdanove2,* and Daniel F. Voytas1,* 1Department of Genetics, Cell Biology & Development and Center for Genome Engineering, 321 Church Street SE, University of Minnesota, Minneapolis, MN 55455 2Department of Plant Pathology, 351 Bessey Hall, Iowa State University, Ames, IA 50011, USA, 3Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chendu 610054, China and 4Biomedical Informatics and Computational Biology Program, University of Minnesota Rochester, 111 South Broadway, Rochester, MN 55904, USA Received March 8, 2011; Revised March 23, 2011; Accepted March 24, 2011 ABSTRACT TALENs are important new tools for genome engin- eering. 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 custom- izable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN con- structs 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 35bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our re- agents. 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 con- structed de n

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