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

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

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

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 sequenceverified
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.
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