Unit

UNIT 12.16 Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-Based Automatable Solid-Phase High-Throughput (FLASH) Assembly

  1. Deepak Reyon1,2,
  2. Morgan L. Maeder1,3,
  3. Cyd Khayter1,
  4. Shengdar Q. Tsai1,2,
  5. Jonathan E. Foley1,
  6. Jeffry D. Sander1,2,
  7. J. Keith Joung1,2,3

Published Online: 1 JUL 2013

DOI: 10.1002/0471142727.mb1216s103

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Reyon, D., Maeder, M. L., Khayter, C., Tsai, S. Q., Foley, J. E., Sander, J. D. and Joung, J. K. 2013. Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-Based Automatable Solid-Phase High-Throughput (FLASH) Assembly. Current Protocols in Molecular Biology. 103:12.16:12.16.1–12.16.18.

Author Information

  1. 1

    Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts

  2. 2

    Department of Pathology, Harvard Medical School, Boston, Massachusetts

  3. 3

    Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 JUL 2013
  2. Published Print: JUL 2013

Abstract

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. Curr. Protoc. Mol. Biol. 103:12.16.1–12.16.18. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • FLASH;
  • TALEN;
  • TALE;
  • TAL effector;
  • TALE activator