CRISPR in Drug Screening

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) allows scientists to create breaks in DNA at precise locations using an enzyme, Cas, which acts like molecular scissors. Once a gene has been sliced, the region of interest may be removed with no further modification (e.g., gene knockout), or replaced by a new gene (knockin).

Although CRISPR has been adapted to do a great many things, the method is not without its limitations. CRISPR editing of large numbers of mature cells is difficult, which has impeded clinical applications. There have also been issues with efficiency and accuracy. Not every cell treated with CRISPR undergoes editing, and off-target effects, although uncommon, can have serious consequences in clinical settings.

When CRISPR was first introduced as a general method, in 2013, it competed with two other gene-editing protocols, zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), both of which are engineered versions of endogenous nucleases. CRISPR is derived from a naturally occurring bacterial defense mechanism. Today, CRISPR dominates gene editing but many groups continue to pursue applications in ZFN and TALENs.

CRISPR applications

CRISPR’s low cost, adaptability, speed, accuracy, and general accessibility, combined with rapidly expanding knowledge of animal genomes, have fueled interest in gene editing for a variety of applications, even in difficult contexts such as the genetic reprogramming of primary mammalian cells. One obvious avenue, therapeutic gene editing, is showing great promise in its discovery/development phase, most notably in cell-based therapies; more is surely to come in treating heritable and somatic gene-based disorders.

CRISPR’s role in drug discovery arises from its ability to create, through editing of one or multiple genes, cell- and animal-based models of human disease to facilitate drug discovery and validation. The good news on this front is that CRISPR editing is applicable to all species currently used in preclinical drug testing.

CRISPR enables wide-ranging gene editing, including knockouts, knockins, specific insertions or mutations, and control over transcription. These capabilities, combined with the physiologic relevance of cell- or animal-based models, create nearly limitless possibilities for tailor-made drug discovery platforms. The most common embodiments are deployed at the cellular and organism level, through target discovery/validation (most practical in cells) and direct screening in gene-edited animals.

Researchers have traditionally relied on RNA interference (RNAi) or inhibition by small-molecule agents to silence genes for loss-of-function studies for validation and screening purposes. While RNAi was useful, it suffers from poor specificity and efficiency. While CRISPR does not completely eliminate these issues it greatly improves on them, with the added capability of gene insertion.

CRISPR in cell-based assays

Cell-based assays (CBAs), particularly cells cultured in three dimensions (3D), are rapidly replacing animal testing and experiments based on immortalized cells. Many advanced 3D cultures (e.g., organoids, organ-chips) employ induced pluripotent stem cells (iPSCs) that are subsequently differentiated into the desired cell type, plus scaffolding and partner cells to confer physiologic relevance. When applied at the iPSC stage, CRISPR editing has the potential for:

• Gene knockout, to establish the connection between phenotype and upstream molecular events. CRISPR achieves this without construction of either a selection marker or targeting vector, as illustrated by the SUCCESS method developed at Osaka University.
• Gene knockin, to introduce exogenous nucleotide sequences and disease-related phenotypes in subsequent progeny
• Transcription activation or repression
• Genome-wide screening. While RNAi can silence genes at the messenger RNA level, CRISPR targets gene knockout or transcription inhibition

Similar approaches may be used to modify primary cells or even immortalized cell lines used in suspension, 2D, or 3D drug screens.

CRISPR-based screening is a specialized technique that begins with a list of genetic targets believed to act within biological pathways critical to the disease under investigation. CRISPR screening knocks out each of these genes, but only one gene per cell. This results in a library of cells that are identical except for the single knocked-out gene. Some cells do not survive this manipulation and others appear to thrive post-knockout.

After expanding these modified cells, investigators apply next-generation screening to determine which sequences have been affected. This exercise identifies not only sequences required for cells to survive, but which ones survive under specific conditions, e.g., drug administration. Various tagging methods are used to identify which cells are reporting the selected response. In “full discovery” mode, that is, when a set of suspected genetic players is unavailable, knockouts could include a much larger set of genes, up to the organism’s entire genome.

CRISPR-edited animals

CRISPR’s extension from cells to organisms promises a range of benefits, including the production of stably modified animals suitable for drug screening.

CRISPR holds numerous advantages over more conventional genetic engineering methods, including speed and reduced cost (compared with breeding strategies, which often don’t work), scalability (compared with transient gene expression, RNAi, or most other knockdown methods), and the ability to target or insert any gene compatible with the organism’s survival. Genetic modifications possible include knockout, knockin, knockdown, and transgenic animals. Companies offering CRISPR-engineered mice and rats, for example, advertise their products based in part on time and cost savings.

A corollary benefit of organisms over cells is the ability to confer multiple mutations, including those whose function is poorly understood (and which may involve more than one tissue), in a single organism. CRISPR also provides the opportunity for more complete humanization of animal models.

For example, zebrafish models have become popular organisms in drug development for their ease of genetic manipulation. When applied to these animals, CRISPR enables, through knockout and knockins, access to studies on transcriptional modulation, epigenome editing, live genome imaging, and lineage tracing.

CRISPR-edited mouse and rat models are of course widely available. Unlike specially bred rodents, which may exhibit just one or two phenotypic or genomic characteristics, these models are capable of recapitulating many of the complexities of disease states, for example in non-alcoholic fatty liver disease, familial hypercholesterolemia, cardiovascular disease, Alzheimer’s disease, and of course cancer.

Conclusion

CRISPR-based genetic engineering, though lagging on the therapeutic front, is already paying big dividends to companies exploiting this technology in disease modeling. At the cellular level, researchers can create, within very reasonable timeframes and at relatively low cost, animal cells exhibiting complex genotypes and phenotypes. The application of CRISPR to stem cells, in particular, addresses scaleup and industrialization of cell-based assays, particularly of the 3D variety.

By no means is CRISPR’s ability to recapitulate multifactorial disease genetics limited to rodents, as the technique is applicable to canine and primate models as well.

If drug screening and disease research is only as good as the test platform or model used, the model is only as good as the premises underlying it. Many diseases are multifactorial, and by no means are all relevant associations between gene and illness known, or when known appreciated. Epigenetics introduces an entirely new level of complexity, for example. As more of these causal relationships are discovered, CRISPR editing may one day yield the equivalent of “fully humanized” (at least in terms of the disease under investigation) cell- and animal-based screening platforms.