ZFIN ID: ZDB-PUB-140820-8
Understanding functional miRNA-target interactions in vivo by site-specific genome engineering
Bassett, A.R., Azzam, G., Wheatley, L., Tibbit, C., Rajakumar, T., McGowan, S., Stanger, N., Ewels, P.A., Taylor, S., Ponting, C.P., Liu, J.L., Sauka-Spengler, T., Fulga, T.A.
Date: 2014
Source: Nature communications   5: 4640 (Journal)
Registered Authors: Sauka-Spengler, Tatjana
Keywords: none
MeSH Terms:
  • Animals
  • Base Sequence
  • Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats/physiology
  • DNA-Binding Proteins/genetics
  • DNA-Binding Proteins/physiology
  • Deoxyribonucleases/genetics*
  • Deoxyribonucleases/physiology
  • Drosophila
  • Endonucleases/genetics*
  • Endonucleases/physiology
  • Genetic Engineering/methods*
  • HEK293 Cells
  • Humans
  • MicroRNAs/genetics*
  • MicroRNAs/physiology
  • Molecular Sequence Data
  • Response Elements/genetics*
  • Response Elements/physiology
  • Sequence Analysis
  • Transcriptional Activation/genetics
  • Transcriptional Activation/physiology
  • Transfection
  • Zebrafish
PubMed: 25135198 Full text @ Nat. Commun.
MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.