CRISPR-Cas9-based gene dropout screens: a powerful platform for drug discovery

Uncovering the links between genes and their biological functions helps researchers to understand disease biology and develop new drugs or improve existing drugs to stop/reverse the effects of diseases. Many drugs are discovered and tested every year to find potential beneficial effects against numerous diseases. At the early stage of drug discovery, thousands of compounds are considered as potential candidates for development as medical treatments; however, after early testing and functional validation, only a small fraction of compounds are suitable for further study. As a result, the process of researching and developing new drugs with advantageous effects to treat diseases in human subjects is continuously expanding in difficulty and time.¹

On average, it takes at least 10-12 years for a new drug to complete the development journey and reach the marketplace, with clinical trials alone taking six to seven years on average. The mean cost of developing a drug has been the subject of debate, with recent estimates ranging from $314 million to $2.8 billion. This figure includes the cost of failures – although thousands of drugs may be screened and validated early in the R&D process, only a few of which go on to receive approval. The overall probability of a new drug entering clinical trials and eventually being approved is estimated to be less than 12 percent. Thus, the drug discovery process is a highly complex, lengthy and costly one, associated with a high degree of uncertainty that a drug will succeed.²

At the same time, the emergence of drug resistance has accelerated, giving rise to life-threatening diseases that will not respond to existing drug treatments, meaning patients will continue to need novel drugs and therapies.³ To overcome these obstacles, new powerful technologies and guidelines for the drug discovery process should be generated, which would provide scientists in industry and academia with a foundation to support the discovery of drugs or improve existing drugs over a sustained period.

The long-anticipated recognition of the ground-breaking discovery of gene-editing tool CRISPR, which was awarded the Nobel Prize in Chemistry in 2020, has elated the scientific community. Since its breakthrough development in 2012, CRISPR has revolutionised many aspects of our daily lives and transformed our ability to precisely manipulate genetic material and generate testable hypotheses.⁴ CRISPR-Cas9 targeted genome editing techniques offer powerful tools for studying disease biology and have greatly contributed to advances in biomedical science.⁴ Many researchers worldwide have adopted this game-changing technique to study the functions of different genes and treat genetic illness, ranging from sickle cell disease to blood cancers and rare genetic disorders of the eyes.⁵ CRISPR has raised hope that direct manipulation of the genome could potentially transform the process of drug and therapeutic discovery. In particular, CRISPR-Cas9 technology is a key to unlock novel drug targets and serves as a promising tool for discovering potential approaches to overcome drug resistance.

CRISPR has gained popularity within the scientific community and become an indispensable tool for biological research. The technique is far cheaper, faster and more efficient than other gene editing methods. CRISPR technology enables a huge range of alterations to be made in genomes, including gene knockouts, knock-ins, generation of specific insertion or mutations and the ability to activate or repress gene functions. These changes, coupled with physiological relevance, make this technology more likely to have a tremendous impact across the entire breadth of the drug discovery process.⁴

CRISPR has also now become a simple and efficient tool for generating targeted loss of function (LOF) mutations.⁶ Previously, genome-wide screens were conducted using RNA interference (RNAi) technology, which is in the form of short interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries.⁷ Such screens have made significant contributions to the field of genomics; however, their success has been limited by the varying efficiencies of siRNAs/shRNAs to silence target genes required for genome-wide studies. In general, RNAi technology produces high off-target effects which cause issues with false-positive results. Furthermore, RNAi technology works by reducing gene expression at the post-transcriptional level by targeting RNA transcripts, meaning RNAi-based screens only result in the partial and short-term suppression of genes. CRISPR-Cas9-based approaches are expected to overcome such limitations and are now becoming a popular choice for administering large-scale pooled screens.⁸

In recent years, CRISPR-Cas9-based gene dropout screens have been used to probe the mechanics of model systems. In a genome-wide CRISPR screening platform, the endonuclease Cas9 and a pooled guide RNA library were used in combination, which allows for high-throughput systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Performing such genome-wide CRISPR-Cas9 LOF screens in relevant cell types under pharmacological pressure with different drugs would help to identify targets whose inhibition will enhance biological activity (eg, antitumour activity). The genes identified by CRISPR screens whose ‘dropout’ associates with increased sensitivity to specific drug treatment serve as a basis for selecting potential candidate targets for combination treatment with that drug. For example, Gilad et al, have performed a genome-scale CRISPR-Cas9 screen in MCF-7 breast cancer cells under conditions of pharmacological pressure with the steroid receptor coactivator 3 (SRC-3) small molecule inhibitor SI-12.

From this screen, they identified eight candidates for which small molecular inhibitors are commercially available and subsequently validated all these targets for their co-operative anticancer activity in the presence of SI-12 by targeted functional genomics and drug combination experiments. By extending the drug-gene vulnerabilities evaluation beyond the MCF-7 cell line, these researchers also discovered highly potent cancer-killing combinations of SI-12 in pancreatic, prostate and triple-negative breast cancer (TNBC) cells with DNMT and RhoA inhibitors.⁹ In another study, Tzelepis et al, used a CRISPR dropout screen and identified genetic vulnerabilities and therapeutic targets (DOT1L, BCL2, MEN1and many other genes) in acute myeloid leukaemia (AML).¹⁰

Over recent years, genome-wide CRISPR screens have been used by many researchers and are considered highly useful for studying the intricate networks of cellular signalling¹¹ and identifying synthetic lethal partners.¹² It is also used as a screening strategy for host dependency factors (HDFs) in viral infections.¹³ Other applications of genome-wide CRISPR screens include the study of mitochondrial metabolism,¹⁴ novel genetic drivers of metastatic progression¹⁵ and drug resistance in cancer,¹⁶ West Nile virus-induced cell death¹⁷ and networks of gene expression in immune cells.¹⁸ In recent years, researchers have aimed to combine genome-wide CRISPR screening with single-cell RNA-sequencing (RNA-seq). Studies utilising CRISP-seq,¹⁹ CROP-seq²⁰ and PERTURB-seq²¹ have successfully identified gene expression signatures for individual gene knockouts in a complex pool of cells. These methods have the added benefit of producing sgRNA-induced transcriptional profiles in target cell populations.

In summary, the recent emergence of CRISPR-Cas9 technology has tremendously increased our ability to perform large-scale LOF screens. The high versatility and programmability of Cas9 endonuclease, coupled with high knockout efficiency and low off-target effects, have made CRISPR a promising platform for many researchers engaging in gene targeting and editing. Furthermore, the establishment of CRISPR systems as a feasible high-throughput gene-editing technology has opened new horizons in drug discovery, dramatically increasing our ability to explore gene-drug interactions and reveal novel drugs or drug targets for the treatment of human diseases.

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