High-throughput functional genomics using CRISPR-Cas9

Abstract

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

Figure 1. Molecular mechanisms underlying gene perturbation via lentiviral delivery of RNA interference reagents, Cas9 nuclease and dCas9 transcriptional effectors

a | Lentiviral transduction begins with the fusion of virus particles with the cell membrane and the insertion of the single-stranded RNA (ssRNA) viral genome into the cell cytoplasm. A reverse transcriptase then converts the ssRNA genome into double-stranded DNA (dsDNA) that is imported into the nucleus and integrates into the host cell genome. Short hairpin RNA (shRNA) or single guide RNA (sgRNA) transgenes are then expressed from an RNA polymerase III (Pol III) or Pol II promoter. b | For shRNA transgenes, maturation involves a series of nucleolytic processing steps that result in cytoplasmic small interfering RNA (siRNA) with sequence complementarity to the target mRNA. Drosha processing is required for reagents consisting of shRNAs embedded in microRNA precursors (shRNAmirs) but is usually bypassed for simple stem–loop shRNA reagents. Gene silencing is achieved by siRNA recruitment to the RNA-induced silencing complex (RISC) for mRNA degradation and translational inhibition. c,d | By contrast, both the Cas9 nuclease and catalytically inactive Cas9 (dCas9)-mediated transcriptional modulation act in the nucleus. The transgene-encoded Cas9–sgRNA complex targets a genomic locus through sequence complementarity to the 20-bp sgRNA spacer sequence (part c). For Cas9 nuclease-mediated knockout, double-strand break (DSB) formation is followed by non-homologous end-joining (NHEJ) DNA repair that can introduce an indel mutation and a coding frameshift. For dCas9-mediated transcriptional modulation, the modification of expression (white arrows) depends on the exact type of fusion of either dCas9 or sgRNA (part d) (FIG. 2). These induced nuclear events, together with endogenous transcript degradation and dilution through cell division, will result in a new steady-state expression level in the cytoplasm.

Figure 2. dCas9-mediated transcriptional modulation

The different ways in which catalytically inactive Cas9 (dCas9) fusions have been used to synthetically repress (CRISPRi) or activate (CRISPRa) expression are shown. All approaches use a single guide RNA (sgRNA) to direct dCas9 to a chosen genomic location. A | To achieve transcriptional repression, dCas9 can be used by itself (whereby it represses transcription through steric hindrance)^– (part Aa) or can be used as part of a dCas9–KRAB transcriptional repressor fusion protein^, (part Ab). B | For transcriptional activation, various approaches have been implemented that involve the VP64 transcriptional activator. One approach is a dCas9–VP64 fusion protein^,– (part Ba). In an alternative method aimed at signal amplification, dCas9 is fused to a repeating array of peptide epitopes, which modularly recruit multiple copies of single-chain variable fragment (ScFv) antibodies fused to transcriptional activation domain^, (part Bb). Another approach is a dCas9–VP64 fusion protein together with a modified sgRNA scaffold with an MS2 RNA motif loop. This MS2 RNA loop recruits MS2 coat protein (MCP) fused to additional activators such as p65 and heat shock factor 1 (HSF1) (part Bc). C | Multiplexed activation and repression was implemented using an array of modified sgRNAs with different RNA recognition motifs (MS2, PP7 or com) and corresponding RNA-binding domains (MCP, PCP or Com) fused to different transcriptional effector domains (KRAB or VP64). TSS, transcriptional start site. Parts Bb and C adapted from REF. and REF. , respectively, Cell Press; part Bc adapted from REF. , Nature Publishing Group.

Figure 3. Screening strategies in either arrayed or pooled formats

Genetic screens follow two general formats that differ in the way in which the targeting reagents are constructed and how cell targeting and readout is carried out. a | In arrayed screens, reagents are separately synthesized and targeting constructs are arranged in multiwell plates. Cell targeting is also conducted in multiwell plates using either transfection or viral transduction. Screen readout is based on cell population measurements in individual wells. b | In pooled screens, reagents are usually synthesized and constructed as a pool. Viral transduction limits transgene copy number (ideally, one perturbation per cell), and viral integration enables readout through PCR and next-generation sequencing. Readout is based on the comparison of the abundance of the different genomically integrated transgene reagents between samples. MOI, multiplicity of infection; sgRNA, single guide RNA; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Figure 4. Distinct expression distributions for knockdown and knockout of a gene

a | Theoretical target gene expression distribution following knockout mediated by lentiviral-delivered Cas9 nuclease is shown. This assumes an 80% level of allelic mutations that abolish gene function, combining out-of-frame and large deletions, close to complete allele modification rate and diploid cells. Although most cells will have a complete knockout in both alleles, some cells will retain at least one copy of a functional allele. b | Theoretical target gene expression distribution following catalytically inactive Cas9 (dCas9)-mediated transcriptional repression or RNA interference (RNAi)-mediated knockdown is shown. All transduced cells experience a similar perturbation that results in a shift in the target gene expression distribution.