Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable...

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genomics

Background:

• Transcription activator-like effector (TALE) repeats are crucial for engineering DNA-binding domains.
• TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) are powerful tools for genome editing and gene regulation, respectively.
• Efficient construction of TALE repeat arrays is essential for their application.

Purpose of the Study:

• To describe the fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method for constructing engineered TALE repeats.
• To present improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server for designing TALE arrays.
• To enable high-throughput generation of TALEN and TALE-TF expression plasmids.

Main Methods:

• Utilized the FLASH assembly method for automated or manual construction of TALE repeat arrays.
• Employed an automated liquid handling robot for high-throughput DNA fragment construction.
• Integrated FLASH with the ZiFiT Targeter web server for streamlined TALE array design.

Main Results:

• Automated FLASH enables construction of up to 96 DNA fragments encoding TALE repeat arrays per day.
• Sequence-verified TALEN or TALE-TF expression plasmids can be generated in one week or less.
• ZiFiT Targeter improvements facilitate high-throughput design and construction of FLASH TALE repeat arrays.

Conclusions:

• The FLASH method significantly accelerates the generation of engineered TALE repeats for research applications.
• This high-throughput approach facilitates the rapid development of custom TALENs and TALE-TFs.
• The described methods and tools lower the barrier for utilizing TALE-based technologies in various organisms and cell types.