Assay Development and Screening

NGS-based Genome-wide Genetic Screens for RNA Processing Regulators

• 

  Hai-Ri Li, CA

  Scientist
  University of California, San Diego

High throughput genetic and chemical screens have been powerful tools to comprehensively identify regulators in specific cellular pathways or drug leads in both industry and academia. However, most screens rely on one or a few functional readouts, even with so-called high content screens. We have developed a robust and fully automatable screen platform that couples with NGS (next-generation sequencing) to monitor the expression of hundred-to-thousand endogenous genes associated with a phenotype without need to purify RNA. The platform is based on annealing a cohort of specific oligonucleotides to specific transcripts and/or their isoforms followed by solid phase selection, RNA-templated oligonucleotide ligation, and PCR amplification using bar-coded primers. We refer this assay strategy as RASL-seq (RNA Annealing, Selection, Ligation coupled with NGS). Pooled samples from up to 1500 reactions in 384 wells can then be sequenced in a single lane of an Illumina sequencing flowcell to obtain quantitative information on targeted transcripts.

We have previously identified a gene signature associated with activated androgen receptor (AR) on prostate cancer cells and applied RASL-seq to identify chemicals that can specific inactivate the AR pathway, thus establishing the proof-of-concept for gene signature-based chemical screens, which are equally applicable to both druggable and non-druggable targets. We have also utilized the RASL-seq platform to screen for regulators involved in the regulation of pre-mRNA splicing and alternative polyadenylation in mammalian cells. Coupled with our efforts in technology development, we have developed a bioinformatics pipeline to process the data for quantitative and network analyses. The RASL-seq platform thus offers a general solution to pathway dissection in both genetic and chemical screens.