title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins.
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.
authors.affiliation: 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.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Van Nostrand
authors.affiliation: 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.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Gelboin-Burkhart
authors.affiliation: 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.
authors.forename: Ruth
authors.initials: R
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Pratt
authors.affiliation: 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.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Blue
authors.affiliation: 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.
authors.forename: Gene W
authors.initials: GW
authors.lastname: Yeo
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D004720:Endonucleases / Q000737:chemistry; D015723:Gene Library; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D059765:Homologous Recombination; D006801:Humans; D020014:K562 Cells; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D059467:Transcriptome
chemical_list: D000906:Antibodies; D010455:Peptides; D016601:RNA-Binding Proteins; D012313:RNA; D004720:Endonucleases
keywords: CLIP-seq; CRISPR/Cas9; Protein tagging; RNA binding protein; eCLIP