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FLASH assembly of TALENs for high-throughput genome editing.

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.

Pubmed ID: 22484455

Authors

  • Reyon D
  • Tsai SQ
  • Khayter C
  • Foden JA
  • Sander JD
  • Joung JK

Journal

Nature biotechnology

Publication Data

May 18, 2012

Associated Grants

  • Agency: NIGMS NIH HHS, Id: DP1 GM105378
  • Agency: NIH HHS, Id: DP1 OD006862
  • Agency: NIH HHS, Id: DP1 OD006862
  • Agency: NIH HHS, Id: DP1 OD006862-01
  • Agency: NIH HHS, Id: DP1 OD006862-02
  • Agency: NHGRI NIH HHS, Id: P50 HG005550
  • Agency: NHGRI NIH HHS, Id: P50 HG005550-01
  • Agency: NHGRI NIH HHS, Id: P50 HG005550-02
  • Agency: NHGRI NIH HHS, Id: P50HG005550
  • Agency: NCI NIH HHS, Id: T32 CA009216
  • Agency: NCI NIH HHS, Id: T32 CA009216
  • Agency: NCI NIH HHS, Id: T32 CA009216-30
  • Agency: NCI NIH HHS, Id: T32 CA009216-31

Mesh Terms

  • Automation
  • Base Sequence
  • Biotinylation
  • Computational Biology
  • DNA Restriction Enzymes
  • Endonucleases
  • Epigenesis, Genetic
  • Gene Targeting
  • Genetic Techniques
  • Genome, Human
  • Green Fluorescent Proteins
  • Humans
  • Molecular Sequence Data
  • Mutation
  • Neoplasms
  • Sequence Analysis, DNA
  • Sequence Homology, Nucleic Acid
  • Transcription, Genetic