Understanding functional miRNA-target interactions in vivo by site-specific genome engineering

被引：63

作者：

Bassett, Andrew R. ^{[1
]}

Azzam, Ghows ^{[2
]}

Wheatley, Lucy ^{[2
]}

Tibbit, Charlotte ^{[1
]}

Rajakumar, Timothy ^{[2
]}

McGowan, Simon ^{[3
]}

Stanger, Nathan ^{[2
]}

Ewels, Philip Andrew ^{[4
]}

Taylor, Stephen ^{[3
]}

Ponting, Chris P.

Liu, Ji-Long ^{[1
]}

Sauka-Spengler, Tatjana ^{[2
]}

Fulga, Tudor A. ^{[2
]}

机构：

[1] Univ Oxford, MRC, Funct Genom Unit, Dept Physiol Anat & Genet, Oxford OX1 3PT, England

[2] Univ Oxford, Weatherall Inst Mol Med, Radcliffe Dept Med, Oxford OX3 9DS, England

[3] Univ Oxford, Weatherall Inst Mol Med, Radcliffe Dept Med, Computat Biol Res Grp, Oxford OX3 9DS, England

[4] Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-10691 Stockholm, Sweden

来源：

NATURE COMMUNICATIONS | 2014年 / 5卷

基金：

欧洲研究理事会; 英国医学研究理事会;

关键词：

CRISPR-CAS9; SYSTEM; HUMAN-CELLS; EFFICIENT; DROSOPHILA; IDENTIFICATION; EXPRESSION; REPRESSION; BINDING; PROTEIN; DESIGN;

D O I：

10.1038/ncomms5640

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：11

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