Drug Discovery in the Age of the Customizable Genome: Implications of the CRISPR Revolution

The CRISPR/Cas9 system allows for unprecedented ease and access to editing the genome, offering drug discovery researchers creative and precise control when designing and developing screening assays. Application of CRISPR/Cas9 has spurred collaborations, led to key advances in diverse fields from neglected and rare diseases to cancer and gene therapy, and has the potential to shift the landscape of drug discovery.


The Unpredictable Path of CRISPR Discovery

In the less than two years since its discovery, CRISPR/Cas9 has unleashed a frenzy of research in diverse fields of biomedicine with important implications for human health. Nonetheless, CRISPR/Cas9’s discovery as a system for precise targeted genome editing was a long and circuitous path. Beginning in the 1980s, Dr. Atsuo Nakata and colleagues at Osaka University in Japan observed repeats in the genome of E. coli but did not have the means to understand their biological function [1]. It was not until 2007 that Dr. Philip Horvath and his team at Danisco, now Dupont, in France serendipitously discovered the function of these genomic regions, while searching for factors protecting the bacteria in yogurt from viral infection. They found that bacteria with the repeated genomic segments (which surrounded sequences matching viral DNA) were better adapted to survive infection, serving as a primitive form of innate immunity [2]. The implications were large – this system, which allowed for the sequence-specific targeting of DNA, could be immensely valuable if it could be repurposed for programmable genome editing, a long-sought goal of biomedical research. The only thing lacking was the identification of the enzyme responsible for binding and cutting the DNA. This crucial piece of the puzzle came in August 2012 from the intercontinental collaboration of the groups of Dr. Jennifer Doudna at University of California Berkeley and Dr. Emmanuelle Charpentier at Umea University in Sweden, who revealed the Cas9 endonuclease family’s ability to cleave DNA at specific sites when targeted with a single RNA molecule [3]. With an RNA-directed system for inducing double-stranded DNA breaks in hand, scientists all over the world began genome editing with unprecedented ease, specificity, and efficiency, offering a taste of the important advances to come for drug discovery.

What is CRISPR?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to the loci of bacterial genome that incorporates the DNA to be targeted. Cas9 (CRISPR associated protein 9 nuclease) is an endonuclease that is programmed by small RNAs to cleave DNA. Since its identification, researchers have made modified forms of the Cas9 enzyme to enable several customized applications: 1) the creation of genetic knockouts (with non homologous end-joining), 2) the targeted replacement of specific base pairs (with homology-directed repair), 3) upregulation of genes (through fusion to activation domains), 4) genome modifications (through fusion to DNA- or chromatin-modifying enzymes) and 5) the ability to image specific genetic loci (through fusion to fluorescent proteins) [4].

How has CRISPR/Cas9 been applied for biomedical innovation so far?

One of the most exciting applications of the CRISPR/Cas9 system has been for the identification of novel targets for therapeutics through functional genomic screens. Two examples include screens that identified essential human genes for anthrax and diphtheria intoxification [5] and the genes required for the development of drug resistance in cancer [6, 7]. CRISPR has also been used to validate hypotheses generated by high-throughput-sequencing analysis of drug-resistant tumors [8]. The genome-editing options made available by CRISPR/Cas9 will open the door to complex chemical genomics studies, for example the simultaneous screening of knockout, point mutant, and overexpression of the same target in human disease models. This will lead to interesting informatics challenges to learn from the massive data-collection efforts. Target validation and mechanistic studies will become much easier with the ability to specifically inactivate a gene or study the effect of surgical mutations, an especially important goal for cancer research where this technology can be applied to a whole array of tissue-specific cancer cell lines [9].

CRISPR/Cas9 can be used to modify the genome for gene and cell replacement therapies. Recently, researchers injected CRISPR/Cas9 into the livers of adult Fah-mutant mice and were able to reverse the phenotype in this model of the human disease hereditary tyrosinemia [10], providing a tantalizing glimpse of the potential of CRISPR/Cas9 as a direct treatment through adult genome engineering.

Beyond human cells, CRISPR/Cas9 allows researchers genetic access to organisms that have previously been intractable. Recently, researchers have applied CRISPR/Cas9 to the human malaria parasite P.falciparum, revealing new targets and mechanisms of drug resistance [11].

Finally, the role of CRISPR/Cas9 in bacteria is still under intense investigation. As our understanding of the system’s natural function grows deeper, we may identify new possibilities to exploit. For example, the role of CRISPR in the adaptation of multiple drug-resistant bacteria and drug-resistant tuberculosis is currently under investigation [12, 13].

How will collaborations enhance CRISPR’s application to drug discovery?

The numerous advantages of CRISPR/Cas9 over rival genetic modulation technologies such as ZFNs, TALENs, and RNAi give it enormous potential for application in biomedical research. Yet, it is still early days and there remain questions about CRISPR/Cas9’s off-target effects and optimal delivery methods. Thus, it will be important for the scientific community to continue to work together to understand and address these potential problems. CRISPR/Cas9 is poised to accelerate the production of new disease models and the rapid identification of gene functions, creating many new opportunities for high-throughput screening, target validation, and drug development and demanding contributions from a broad diversity of geneticists, cell biologists, and chemists. In the brief period since its first introduction as a tool for biomedical research, CRISPR/Cas9 has already captured the imagination of scientists with its wealth of potential, making it hard not to wonder what else lies in store.



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