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

Abstract

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 screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.functional genomics | shRNA | genetic screen | pooled screen | microRNA F unctional genomics approaches in mammalian cells have the potential to dissect gene functions and to complement observational genomics approaches for the identification of disease mechanisms and therapeutic strategies. For many years, RNA interference (RNAi) was the technology of choice for loss-offunction screens in mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches recently developed by us and others (1-4) provide a highly promising orthogonal strategy. In particular, CRISPR interference (CRISPRi) has reduced off-target effects and can reach high levels (90-100%) of knockdown (1), and CRISPR cutting can generate null alleles.Despite the advantages of CRISPR-based strategies, there are still important uses for RNAi technology. RNAi is a singlecomponent system that works in otherwise unengineered cells, and can thus be used in challenging biological contexts. Because CRISPRi blocks transcription at the transcription start site of endogenous genes, it does not allow selective targeting of functionally distinct coding and noncoding RNAs derived from the same primary transcript, such as splice isoforms or noncoding RNAs embedded in the introns of coding transcripts, whereas RNAi reagents can be designed for specific targeting of mature RNAs (mRNAs).Hence, there is a continued need to improve RNAi-based screening platforms. We have previously established a quantitative framework to derive robust results from pooled screens of ultracomplex short-hairpin RNA (shRNA) libraries that target each gene with ∼25 independent shRNAs and contain thousands of negative-control shRNAs (5). Such complex libraries can be constructed using massively parallel oligonucleotide synthesis, and phenotypes in pooled populations can be determined using n...

The New State of the Art: Cas9 for Gene Activation and Repression

“…To find the ideal shRNA or sgRNA coverage and design, multiple studies have performed small-scale pooled screens with very high coverage of shRNAs or sgRNAs per gene (29,69,80,81). These studies each identified high-confidence hits and then computationally subdivided their libraries to (i) discover the number of sgRNAs required to distinguish true hits from the background and (ii) define rules that make more effective shRNAs and sgRNAs.…”

“…These studies each identified high-confidence hits and then computationally subdivided their libraries to (i) discover the number of sgRNAs required to distinguish true hits from the background and (ii) define rules that make more effective shRNAs and sgRNAs. Thus, bioinformatic modeling and iterative analysis and testing large pools of sgRNAs have resulted in significant improvements in both RNAi and CRISPRi (29,69). It is now possible to use a library with 10 shRNAs or 10 sgRNAs per gene to produce robust results in a screen, although both RNAi and CRISPRi will no doubt benefit from further refinement in the future.…”

Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

“…For example, CRISPRi modulates transcription at the TSSs of endogenous genes; therefore, it is difficult to target specific splice isoforms. By contrast, RNAi can be targeted to specific mature transcripts 74 . Therefore, the use of CRISPRi and RNAi in conjunction may hold the potential for more complete analysis of gene function.…”

Optimizing Crispr Cas9 Genome Editing System:A Review

“…CRISPR-mediated repression (CRISPRi) and activation (CRISPRa) has been demonstrated as robust tools for functional genome screening in gene expression modulation. CRISPRi has been efficiently exploited to inhibit the transcription of target genes in E. coli and mammalian cells when dCas9 is recruited to transcriptional inhibitory domain 22 , whereas to activate the expression of target endogenous genes when dCas9 is tethered to a transcriptional activator domain 31,32 . Genome-scale CRISPRi/a libraries have been successfully employed in identification of mediators for cellular sensitivity to a cholera-diptheria fusion toxin, as well as essential genes for proliferation, differentiation and tumor suppression.…”

“…Additionally, CRISPRa helps in identification of a novel gene with its gainof-function properties. dCas9 when complexes with sgRNA has shown to successful in simultaneous activation of multiple genes, up regulation of long noncoding RNA transcripts and identifying genes conferring resistance to a BRAF inhibitor in melanoma 32 signifying the versatility of this system in discovering crucial genes in various biological processes.…”