A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells.
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.
Department of Biological Sciences, California State University, East Bay, Hayward, California 94542, USA.
Claudia
C
Uhde-Stone
System Biosciences (SBI), Mountain View, California 94043, USA.
Nandita
N
Sarkar
System Biosciences (SBI), Mountain View, California 94043, USA.
Travis
T
Antes
System Biosciences (SBI), Mountain View, California 94043, USA.
Nicole
N
Otoc
System Biosciences (SBI), Mountain View, California 94043, USA.
Young
Y
Kim
Endocrine Research Unit, Veterans Affairs Medical Center, Department of Medicine, University of California at San Francisco, San Francisco, California 94121, USA.
Yan J
YJ
Jiang
System Biosciences (SBI), Mountain View, California 94043, USA.
Biao
B
Lu
D000483:Alleles; D049109:Cell Proliferation; D004950:Esterases / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D015894:Genome, Human / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D010865:Pilot Projects; D015533:Transcriptional Activation / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics
C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D004950:Esterases
double knockout; gene-editing; homology-directed repair (HDR); microRNA; transcription activator-like effector nucleases (TALENs)