PUBLICATION
Understanding functional miRNA-target interactions in vivo by site-specific genome engineering
- Authors
- 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.
- ID
- ZDB-PUB-140820-8
- Date
- 2014
- Source
- Nature communications 5: 4640 (Journal)
- Registered Authors
- Sauka-Spengler, Tatjana
- Keywords
- none
- MeSH Terms
-
- Base Sequence
- Transcriptional Activation/genetics
- Transcriptional Activation/physiology
- Deoxyribonucleases/genetics*
- Deoxyribonucleases/physiology
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/physiology
- Drosophila
- Endonucleases/genetics*
- Endonucleases/physiology
- Sequence Analysis
- Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
- Clustered Regularly Interspaced Short Palindromic Repeats/physiology
- Animals
- Response Elements/genetics*
- Response Elements/physiology
- Genetic Engineering/methods*
- HEK293 Cells
- Zebrafish
- Transfection
- MicroRNAs/genetics*
- MicroRNAs/physiology
- Molecular Sequence Data
- Humans
- PubMed
- 25135198 Full text @ Nat. Commun.
Citation
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. (2014) Understanding functional miRNA-target interactions in vivo by site-specific genome engineering. Nature communications. 5:4640.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping