A One-Step System for Convenient and Flexible Assembly of Transcription Activator-Like Effector Nucleases (TALENs).
Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.
From Innovative Cellular Therapeutics Co., Ltd, (formerly SiDanSai Biotechnology Co., Ltd), Shanghai 201203, China.
Jinlong
J
Zhao
From Innovative Cellular Therapeutics Co., Ltd, (formerly SiDanSai Biotechnology Co., Ltd), Shanghai 201203, China.
Wenye
W
Sun
From Innovative Cellular Therapeutics Co., Ltd, (formerly SiDanSai Biotechnology Co., Ltd), Shanghai 201203, China.
Jing
J
Liang
From Innovative Cellular Therapeutics Co., Ltd, (formerly SiDanSai Biotechnology Co., Ltd), Shanghai 201203, China.
Jing
J
Jiang
From Innovative Cellular Therapeutics Co., Ltd, (formerly SiDanSai Biotechnology Co., Ltd), Shanghai 201203, China.
Zhao
Z
Wu
D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D000069896:Transcription Activator-Like Effector Nucleases / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism
D000069896:Transcription Activator-Like Effector Nucleases; C483995:BsaI endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific
genome editing; molecular genetics; transcription activator-like effector nuclease (TALEN)