Using CRISPR for Target Identification

Drug development is a long, complex, and expensive process, typically costing over $1 billion and taking up to 15 years to bring a drug to market and into clinical use. In the past, drugs have been discovered either through the identification of the active ingredient from a traditional medicine, or by simple serendipity. Now, modern drug discovery begins with target identification—finding gene targets associated with the disease of interest that are safe, efficacious, and ‘druggable’—meaning that the target should be accessible to a drug molecule resulting in a measured biological effect.

Target identification relies on elucidating and understanding cellular pathways that play a role in disease progression and then identifying potential molecular candidates, including genes, RNA, or proteins that when inhibited or activated elicit a therapeutic outcome. Targets can be identified from data mining, genetic association, studying mRNA expression profiles, or performing in vitro cell-based mechanistic studies. The recent development of CRISPR-Cas9 gene-editing technology provides a powerful tool to perform genome-wide, phenotypic analysis, allowing whole genomes to be interrogated to investigate the functional consequence of gene expression. This article discusses how CRISPR technology can be used to elucidate biological pathways for target identification.

Unbiased CRISPR pooled library screening

The aim of functional genomics is to link genotype with biological phenotype—simply put, change the expression of a gene, and observe what happens. CRISPR-Cas9 allows genome-wide loss of function studies to be performed at high specificity and within the technical capabilities of most laboratories. CRISPR screening begins with the creation of a sgRNA library that targets every gene in the genome—the role of the sgRNA is to then target the Cas9 endonuclease to the site of interest where it induces a double-strand break resulting in a frameshift mutation and subsequent gene knockout. Delivery of the sgRNA library by lentiviral vector allows screens to be performed in a pooled format—as each transgene stably integrates, this effectively provides each cell with a barcode so the sgRNA that have been enriched or depleted following the screen can be identified by massively parallel sequencing. Early work using genome-wide CRISPR loss of function screens showed their promise for target identification, recapitulating known mechanisms of action to several drugs, including 6-thioguanine, etoposide, and vemurafenib.

Exploiting cancer vulnerabilities as potential therapeutic targets

Dysregulation of cellular signaling pathways is often a driver of cancer initiation, leading to aberrant cell proliferation and/or survival. CRISPR-based knockout screening provides researchers with a robust and unbiased means to interrogate genomes and elucidate biological mechanisms, and therefore identify potential molecular features of a tumor to help find cellular targets for drug discovery. For example, Steinhert et al conducted a genome-wide screen in pancreatic ductal adenocarcinoma (PDAC) cells with an RNF43 mutation and discovered that proliferation of these cells was dependent on the Wnt signaling pathway involving a FZD5 receptor—a druggable vulnerability that could be exploited for therapeutic effect.¹ Screens can also be performed in vivo—Wu et al employed an in vivo CRISPR screen to identify five tumor suppressor genes that significantly promote lung tumorigenesis, including the histone demethylase Utx, which is particularly sensitive to EzH2 inhibition.²

Leveraging synthetic lethal interactions

One approach to target identification for cancer therapeutics is to exploit synthetic lethal interactions, which is where disruption of two genes independently has no effect on cell survival but in combination results in cell death—a good example of this phenomenon is of tumors carrying the BRCA-1 mutation, which are selectively sensitive to PARP enzyme inhibition. Previous screens looking for synthetic lethal interactions utilized isogenic cell pairs, but recent developments to CRISPR technology now mean screens can be completed using paired sgRNAs—termed combinatorial or multiplex screening. A recent study by Thompson et al looked at 1191 gene pairs and identified 105 combinations of genes that when disrupted simultaneously resulted in cell death. The study identified 27 gene pairs that affected cellular fitness across several different cancer cell lines, which included FAM50A and FAM50B. Interestingly, many of these gene pairs were paralogues, which suggests that paralogous genes could be a potential therapeutic target.³

Beyond loss of function: expanding the CRISPR toolkit

Precursor technologies to CRISPR-Cas9, such as shRNA screens and Genetrap-based approaches, while powerful, were very much limited to loss of function genome-wide screens—and initially this was the case with CRISPR too. However, many cancers are affected by gain of function mutations—where mutations that activate genes drive resistance and/or sensitivity to drugs. To explore these pathways on a genome-wide scale, scientists adapted the CRISPR-Cas9 technology—tethering a catalytically inactive Cas9 (dCas9) to modulators of DNA, including transcriptional repressors (CRISPRi) and transcriptional activators (CRISPRa). In a recent paper, CRISPRa was used in an in vivo gain of function screen to identify genes involved in resistance to BRAF inhibitors in cutaneous melanoma—the authors identified SMAD3 as a potential target to reduce resistance to BRAFi treatment.⁴ In addition to CRISPRi and CRISPRa, single nucleotide changes are now possible with base editing, which has been successfully used in a high-throughput screen. Keown et al used CRISPR base editing to perform a functional assessment of BRCA1 variants, identifying several previously unknown pathogenic variants. Such screens may provide insights and potential targets for drug resistance and thus improve therapeutic outcomes.⁵

Drug discovery with CRISPR

Pooled library screening with CRISPR offers an unbiased and robust means of interrogating entire genomes to elucidate biological mechanisms and identify potential targets for drug discovery. The inherent flexibility of the system and expanded toolkit mean that a range of high-throughput screens are available both in vitro and in vivo—allowing researchers to study different effects on gene expression on phenotype in an orthologous manner to improve target identification for downstream validation.

Key Takeaways

• The aim of target identification is to find gene targets associated with the disease of interest that are safe, efficacious, and druggable
• Target identification relies on characterizing and understanding cellular pathways that play a role in disease progression
• CRISPR-Cas9 screening provides a robust and unbiased means of performing genome-wide phenotypic analysis of the functional consequences of gene expression with high specificity
• CRISPR-Cas9 screens can be utilized to identify molecular candidates that can be targeted for clinical therapeutics

References

1. Steinhart, Z., Pavlovic, Z., Chandrashekhar, M. et al. Genome-wide CRISPR screens reveal a Wnt–FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors. Nat Med 23, 60–68 (2017). 

2. Wu, Qibiao et al. In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis. Proceedings of the National Academy of Sciences of the United States of America 115,17 (2018) 

3. Thompson, N.A., Ranzani, M., van der Weyden, L. et al. Combinatorial CRISPR screen identifies fitness effects of gene paralogues. Nat Commun 12, 1302 (2021). 

4. Gautron, A., CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance. EMBO Mol Med 13 (2021) 

5. Kweon, J., Jang, AH., Shin, H.R. et al. A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants. Oncogene 39, 30–35 (2020).