RNAi screening comes of age: improved techniques and complementary approaches

Abstract

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

Figure 1. Gene silencing by RNAi

RNAi reagents can be introduced into cells through different routes: siRNAs or endoribonuclease-prepared siRNAs (esiRNAs) can be transfected into mammalian (and other) cells; short hairpin RNAs (shRNAs) can be virally transduced into mammalian (and other) cells; double-stranded RNAs (dsRNAs) in solution can be applied to Drosophila melanogaster cells resulting in their uptake; dsRNAs (D. melanogaster or C. elegans) or shRNAs (D. melanogaster or mice) can be expressed from transgenic constructs; dsRNAs can be microinjected (C. elegans, D. melanogaster and some non-model insects); and Escherichia coli expressing dsRNAs can be fed to living animals (C. elegans or Planaria). Once in the cells, reagents such as dsRNAs are recognized by DICER (not shown), which processes them into siRNAs of 21–23 nucleotides in length. The synthetic or endogenously processed siRNA molecules are then incorporated into the RNA-induced silencing complex (RISC) and mediate gene silencing through target mRNA cleavage (if perfect sequence complementarity exists between the target mRNA and the siRNA) or translational interference (if the complementarity is partial; not shown).

Figure 2. Strategies for validating RNAi screen results

a | RNA-induced silencing complex (RISC)-incorporated siRNAs mediate target mRNA cleavage upon perfect sequence complementarity in either the coding region or the 3′ untranslated region (UTR) of the mRNA (depending on the siRNA design). For the siRNA shown, the 5′ seed region is in green, the middle region is in yellow and the 3′ end is in orange. b | Testing for potential off-target effects of a given siRNA can be carried out using the C911 method. siRNA bases 9–11 are mutated while the seed region (bases 2–8) remains intact. This maintains off-target interactions mediated by seed region matches but perturbs on-target silencing. c | On-target specificity by phenotypic rescue can be demonstrated by the co-expression of RNAi-resistant versions of the target mRNA. Synonymous mutations in the siRNA-targeted region of the mRNA can be introduced to prevent RISC-mediated silencing while preserving function. Alternatively, a homologous gene from a related species that has sufficient sequence divergence in the siRNA targeting region to be RNAi resistant, but also sufficient similarity to elicit function, can be used to test on-target specificity of the RNAi construct.

Figure 3. Genome-engineering approaches offer new opportunities for assay development, screening and validation

A number of points of intersection exist between RNAi screening (or other types of large-scale screening, such as overexpression of open reading frame (ORF) clones, or microRNA (miRNA) mimics or inhibitors (BOX 2)) and genome-engineering technologies (step 1) such as TAL effector nucleases (TALENs) and the CRISPR (clustered regularly interspaced short palindromic repeats)– Cas9 systems (highlighted in blue). Genome engineering can be used to create robust, well-controlled assays (step 2) in cell lines and model organisms by introducing various mutations such as gene knockouts, diseaseassociated mutations and knock-in of selectable markers or in-frame fusions or reporter genes. CRISPR–Cas9-mediated knockouts (step 3) in mouse or human cells have been reported as an effective method for pooled-format screens. These can be performed in parallel to RNAi screens, followed by comparison of results from the two types of screens. Independently of the screening approach, genome engineering can be used to modify cells or organisms for follow-up studies of specific gene candidates (step 4). In this case, the CRISPR–Cas9 system or TALENs can be used to knock out genes identified in RNAi screens, with a concordance of the knockdown and knockout phenotypes providing a high degree of confidence in the results. They can also be used to create other types of modifications useful for follow-up studies, for example, transcriptional upregulation or downregulation, using modified forms of Cas9, or introducing fluorescence tags or reporters using a knock-in approach.