Next-generation libraries for robust RNA interference-based genome-wide screens.

摘要: Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches mammalian cell culture has enormous potential, RNA interference (RNAi)-based remains method choice several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin (shRNA) libraries can largely overcome problem RNAi off-target effects genome-wide screens. Here, we systematically optimize aspects our shRNA library, including promoter and microRNA context for expression, selection guide strands, features relevant postscreen sample preparation deep sequencing. present next-generation high-complexity targeting human mouse protein-coding genes, which grouped into 12 sublibraries function. A pilot screen suggests library performs comparably to current CRISPR (CRISPRi)-based yield complementary results with high sensitivity specificity.