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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.

SUBMITTER: Van Nostrand EL

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

REPOSITORIES: biostudies-literature

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

Van Nostrand Eric L EL Gelboin-Burkhart Chelsea C Wang Ruth R Pratt Gabriel A GA Blue Steven M SM Yeo Gene W GW

Methods (San Diego, Calif.) 20161218

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 desc ...[more]

PMID: 28003131

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Project description:BackgroundPichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.ResultsA non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris.ConclusionsA CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Dataset Information

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

Publications

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

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