A comprehensive platform for highly multiplexed mammalian functional genetic screens

Area of Science:

• Functional genomics
• High-throughput screening
• Molecular biology

Background:

• Genome-wide screening using RNA interference (RNAi) and open reading frame (ORF) over-expression is a growing research method.
• A lack of detailed protocols and reagents hinders the deconvolution of large-scale genetic screens.
• The Gene Modulation Array Platform (GMAP) was developed to address this gap.

Purpose of the Study:

• To present a comprehensive platform for highly multiplexed functional genetic screens.
• To provide a microarray-based solution for deconvoluting pooled loss- and gain-of-function screens.
• To validate GMAP for use with commercially available gene modulation libraries in human, mouse, and yeast cells.

Main Methods:

• Development of the Gene Modulation Array Platform (GMAP), a single microarray-based detection solution.
• Testing GMAP with lentiviral-based plasmid pools containing approximately 78,000 and 90,000 shRNAs for RNAi screens.
• Comparison of GMAP with next-generation sequencing for deconvolution of shRNA screens.
• Evaluation of GMAP for deconvolution of ORF over-expression screens.

Main Results:

• GMAP successfully deconvoluted genome-wide shRNA "dropout" screens using pooled lentiviral libraries.
• Equivalent results were obtained using plasmid pools and genomic DNA from infected cells.
• GMAP and next-generation sequencing provide complementary and valid approaches for screen deconvolution.
• GMAP demonstrated equivalence to other microarray products for ORF over-expression screen deconvolution.

Conclusions:

• GMAP supports four major applications, including deconvolution of pooled RNAi screens with up to 90,000 distinct shRNAs.
• Detailed methodologies for pooled shRNA screen readout using GMAP are provided.
• A comparison of next-generation sequencing and GMAP-based deconvolution methods is presented.