Details

Autor(en) / Beteiligte

• Uhde-Stone, Claudia
• Sarkar, Nandita
• Antes, Travis
• Otoc, Nicole
• Kim, Young
• Jiang, Yan J
• Lu, Biao

Titel

A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells

Ist Teil von

• RNA (Cambridge), 2014-06, Vol.20 (6), p.948-955

Ort / Verlag

United States: Cold Spring Harbor Laboratory Press

Erscheinungsjahr

2014

Quelle

Electronic Journals Library

Beschreibungen/Notizen

• Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for bi-allelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.