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

SUBMITTER: Van Nostrand EL 

PROVIDER: S-EPMC5411315 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature