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

Qianxin Wu; Quentin R V Ferry; Toni A Baeumler; Yale S Michaels; Dimitrios M Vitsios; Omer Habib; Roland Arnold; 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

doi:10.1038/s41467-017-00686-2

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

Nat Commun. 2017 Dec 13;8(1):2109. doi: 10.1038/s41467-017-00686-2.

Authors

Qianxin Wu^{1

2}, Quentin R V Ferry¹, Toni A Baeumler¹, Yale S Michaels¹, Dimitrios M Vitsios³, Omer Habib⁴, Roland Arnold⁵, Xiaowei Jiang⁵, Stefano Maio⁶, Bruno R Steinkraus¹, Marta Tapia⁷, Paolo Piazza⁸, Ni Xu¹, Georg A Holländer^{6

9}, Thomas A Milne⁷, Jin-Soo Kim^{4

10}, Anton J Enright³, Andrew R Bassett^{11

2}, Tudor A Fulga¹²

Affiliations

¹ Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.
² Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
³ European Molecular Biology Laboratory-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.
⁴ Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, 08826, Republic of Korea.
⁵ Molecular and Population Genetics Laboratory, Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
⁶ Weatherall Institute of Molecular Medicine, Developmental Immunology, University of Oxford, Oxford, OX3 9DS, UK.
⁷ Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford, OX3 9DS, UK.
⁸ Wellcome Trust Centre for Human Genetics, Oxford, OX3 7BN, UK.
⁹ Department of Biomedicine, Laboratory of Paediatric Immunology, University of Basel, CH-4058, Basel, Switzerland.
¹⁰ Department of Chemistry, Seoul National University, Seoul, 151-747, Republic of Korea.
¹¹ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RF, UK.
¹² 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

3' Untranslated Regions / genetics
Animals
CRISPR-Cas Systems / genetics*
Clustered Regularly Interspaced Short Palindromic Repeats / genetics
Gene Editing / methods*
Genome / genetics
Humans
MicroRNAs / genetics
RNA / genetics*
Regulatory Sequences, Nucleic Acid / genetics*
Response Elements / genetics

Substances

3' Untranslated Regions
MicroRNAs
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding