CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

Eric L Van Nostrand; Chelsea Gelboin-Burkhart; Ruth Wang; Gabriel A Pratt; Steven M Blue; Gene W Yeo

doi:10.1016/j.ymeth.2016.12.007

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

Methods. 2017 Apr 15:118-119:50-59. doi: 10.1016/j.ymeth.2016.12.007. Epub 2016 Dec 18.

Authors

Eric L Van Nostrand¹, Chelsea Gelboin-Burkhart¹, Ruth Wang¹, Gabriel A Pratt², Steven M Blue¹, Gene W Yeo³

Affiliations

¹ Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA.
² Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA; Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA.
³ Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA; Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Molecular Engineering Laboratory, A*STAR, Singapore. Electronic address: geneyeo@ucsd.edu.

Abstract

Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA interaction landscape.

Keywords: CLIP-seq; CRISPR/Cas9; Protein tagging; RNA binding protein; eCLIP.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Antibodies / chemistry
Base Sequence
Binding Sites
CRISPR-Cas Systems*
Cloning, Molecular
Endonucleases / chemistry
Gene Library*
HEK293 Cells
High-Throughput Nucleotide Sequencing / methods*
Homologous Recombination
Humans
K562 Cells
Peptides / chemistry
Polymerase Chain Reaction
Protein Binding
RNA / chemistry*
RNA / genetics
RNA / metabolism
RNA-Binding Proteins / genetics*
RNA-Binding Proteins / metabolism
Sequence Analysis, RNA / methods
Staining and Labeling / methods*
Transcriptome

Substances

Antibodies
Peptides
RNA-Binding Proteins
RNA
Endonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding