FLASH assembly of TALENs for high-throughput genome editing.

Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK.
Source: Nat Biotechnol
Publication Date: (2012)
Issue: 30(5): 460-465
Research Area:
Cancer Research/Cell Biology
Basic Research
Molecular Biology
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector® X-Unit
Nucleofection of U2OS derived cells with 500 ng of each TALEN plasmid DNA and 50 ng ptdTomato-N1 plasmid DNA using a Lonza 4D-Nucleofector System, Solution SE and program DN-100. As a negative control, 1 µg of ptdTomato-N1 plasmid was transfected alone. Cells were assayed for EGFP and tdTomato expression at 2 and 5 days post-transfection using flow cytometry. Summary (Lonza): The authors developed a system for large scale construction of transcription activator–like effector nucleases (TALENs) genome editing tools. The method, named fast ligation-based automatable solid-phase high-throughput (FLASH), is publically available, rapid and cost-effective. 1µg of TALEN plasmid pair (500ng each) were transfected into U2OS derived cells, using the 4D-Nucleofector™ System. Using this method, the authors were able to efficiently introduce targeted alterations in 84 out of 96 endogenous human genes implicated in cancer and epigenetic regulation.
Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.