In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering

Research output: Contribution to journalArticle

Authors

  • Qianxin Wu
  • Quentin R V Ferry
  • Toni A Baeumler
  • Yale S Michaels
  • Dimitrios M Vitsios
  • Omer Habib
  • Xiaowei Jiang
  • Stefano Maio
  • Bruno R Steinkraus
  • Marta Tapia
  • Paolo Piazza
  • Ni Xu
  • Georg A Holländer
  • Thomas A Milne
  • Jin-Soo Kim
  • Anton J Enright
  • Andrew R Bassett
  • Tudor A Fulga

Colleges, School and Institutes

External organisations

  • Mouse Pipelines, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
  • Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.
  • European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom, CB10 1SD.
  • Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, 08826, Republic of Korea.
  • Molecular and Population Genetics Laboratory, Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
  • Weatherall Institute of Molecular Medicine, Developmental Immunology, University of Oxford, Oxford, OX3 9DS, UK.
  • Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford, OX3 9DS, UK.
  • Bioinformatics, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
  • Department of Biomedicine, Laboratory of Paediatric Immunology, University of Basel, CH-4058, Basel, Switzerland.
  • Department of Chemistry, Seoul National University, Seoul, 151-747, Republic of Korea.
  • Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK. tudor.fulga@imm.ox.ac.uk.

Abstract

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.

Details

Original languageEnglish
Article number2109
JournalNature Communications
Volume8
Issue number1
Publication statusPublished - 13 Dec 2017