CRISPR: A Screener's Guide

Abstract

The discovery of CRISPR-Cas9 systems has fueled a rapid expansion of gene editing adoption and has impacted pharmaceutical and biotechnology research substantially. Here, gene editing is used at an industrial scale to identify and validate new biological targets for precision medicines, with functional genomic screening having an increasingly important role. Functional genomic strategies provide a crucial link between observed biological phenomena and the genes that influence and drive those phenomena. Although such studies are not new, the use of CRISPR-Cas9 systems in this arena is providing more robust datasets for target identification and validation. CRISPR-based screening approaches are also useful later in the drug development pipeline for understanding drug resistance and sensitivity ahead of entering clinical trials. This review examines the developing landscape for CRISPR screening technologies within the pharmaceutical industry and explores the next steps for this constantly evolving screening platform.

Keywords: CRISPR; RNAi; cancer and cancer drugs; cell-based assays; gene editing; high-content screening; in vivo screening; shRNA.

Figure 1.

CRISPR screening’s main modalities are for either complete gene ablation via a fully active Cas9 (CRISPRko), a transcriptional suppression approach (CRISPRi) that uses catalytically inactive Cas9 fused to a KRAB domain, or CRISPRa that can drive site-specific transcriptional activation, and has multiple tools published.

Figure 2.

Pooled CRISPR screening workflows begin with the selection of targets and the design of guides associated with those genes. Once synthesized, these guides are then cloned into a suitable library vector and introduced into cells by a lentiviral cassette. Screening assays often involve the addition of a drug (drug–gene interaction screening) or the comparative analysis of variable genotype cells (e.g., genetic interaction screening), but in all cases samples are analyzed by deep sequencing the sgRNA span to quantitatively determine barcode/genotype abundance.

Figure 3.

Fundamental properties of pooled CRISPR screening. Depicted are sgRNAs (barcodes) with differential impact on cellular physiology. The blue sgRNAs target gene ablation, which has no positive or negative influence on cell proliferation. The yellow and purple sgRNAs target genes important for cellular fitness and viability, respectively, the loss of which will negatively impact cell survival within the cell population. The green sgRNAs eliminate genes that are normally inhibitory to cell proliferation. The overall impact of sgRNAs on cell physiology is translated into sgRNA abundance, which can be directly measured by NGS.

Figure 4.

Schematic of a pooled phenotypic CRISPR screen. Detection of changes in the expression of a biomarker, that is, through antibody-based detection of a target molecule, or an endogenous reporter system can be visualized and specifically selected through FACS methods. The sorted populations are then analyzed for enrichment or loss of CRISPR genotypes.

Figure 5.

Overview of droplet-based single-cell sorting and subsequent RNA-seq of individual cells originating from a pooled CRISPR screen population. For each genotype introduced into the screen by CRISPR perturbation, a full transcriptomic profile is developed to determine cellular signatures in response to perturbation and treatment.

Figure 6.

Arrayed CRISPR screening workflow. CRISPR-ready cells can be generated by a two-step process where cells are engineered to express Cas9, or by a one-shot approach where the Cas9 is introduced concomitantly with the sgRNA or crRNA:tracrRNA components. Either way, CRISPR-Cas9 reagents are added to cells in individual wells to allow perturbations to be constrained within individual wells. This has the disadvantage of lower throughput than pooled screening, but the distinct advantage of allowing multiplexed readouts or the monitoring of non-cell-autonomous effects on a well-by-well basis, directly linking phenotype(s) to genotype.