The preclinical stage of drug discovery relies heavily on the ability to manipulate genomes. By altering the sequence or expression of genes, scientists can develop a variety of assays to identify disease targets and test therapeutic efficacy. However, the ability to perform genomic manipulations in a fast, accurate, and cost-effective manner has often been limited by technology.

The difficulties posed by first-generation gene-editing tools such as RNAi, ZFNs and TALENs have limited the adoption of genome editing in the drug discovery workflow. The picture is rapidly changing, however, with recent gene editing innovation: CRISPR (clustered regularly interspaced short palindromic repeats).

CRISPR/Cas9 consists of two components: a Cas9 protein with endonuclease activity, and a guide RNA (gRNA) that confers specificity to the system by sequence-dependent recognition. After binding of the gRNA to the targeted DNA, the endonuclease activity of Cas9 generates a double-stranded break at the targeted genomic DNA site. At this point, researchers can take advantage of the cell’s DNA repair mechanisms to fix the double-stranded break. If the objective is to simply knock-out expression of the target gene, the cell’s non-homologous end joining (NHEJ) pathway can be exploited.

This DNA repair pathway is error-prone by nature, as it often includes or deletes nucleotides at the site of the break during the end-joining process. The loss of nucleotides invariably interrupts the open reading frame – often resulting in the generation of a premature stop codon – which prevents the protein from being produced. This creates a knock-out (KO).

If a more precise edit of the gene is necessary, the homology-driven repair pathway (HDR) can be employed. By designing a repair template containing the desired sequence change, researchers can use homologous recombination to introduce (or knock-in) any alteration into the genomic DNA.

Target identification and validation in the drug discovery workflow

CRISPR-based high-throughput screens are commonly used to systematically knockout, inhibit or activate large numbers of candidate genes. Perturbations that exacerbate or hinder a disease can reveal potential drug targets.

For scientists in early-stage drug discovery, working with CRISPR “pools” is a high-throughput approach for initial target identification or for selecting a phenotype. These pools can be genome-wide or custom pools. The library is a pool in lentivirus which is transfected into cells, resulting in one guide RNA per cell on average. These changes are transient and there is no requirement for clonality.

Once a putative target is identified, further functional information is collected through in vitro and in vivo studies.

Screening for Clonality in target validation

For target validation and unravelling disease pathways and cellular processes, gene knock-outs (KO) are required for each gene and these need to be in stable cell lines. Clones are also needed for the multiple genes in the particular pathway being studied.

A high-throughput approach for doing this is to purchase arrayed synthetic guide RNA libraries (from vendors such as Dharmacon/Horizon, Synthego) and to reverse transfect these with Cas9-expressing cells. Single cells can then be seeded into wells of a 384 well plate, and then these wells are imaged using the dedicated Cell Metric whole well imager for colony outgrowth of surviving transfectants and confirmation of clonality for these wells.

Colony growth and clonality are fully documented in the Clonality Report (see below) within the Cell Metric software.

Clones will then be replica plated. Homozygous knockouts can be confirmed by NGS of one daughter plate, and the remaining plates will be cryopreserved.

To meet the high-throughput requirements of this workflow, customers can either use the Cell Metric CLD with incubated plate load and cassette for walkaway screening of 10 plates, or the Cell Metric can be linked through an API to a third-party robotic arm (e.g. Tecan, Hamilton, Thermo).

After a target has been validated, the next step is to use cell-based assays for screening compounds.

Hit Validation

CRISPR has profoundly impacted this stage of preclinical development by promoting the generation of models that accurately recapitulate diseases.

Instead of being limited to immortalised cell lines, scientists can produce primary cells, stem cells and even organoids with appropriate cellular and genetic backgrounds. For instance, human induced pluripotent stems cells (or iPSCs) can be used to produce nearly any cell type. From such stem cells, isogenic cell lines can easily be generated, and the genetic variation associated with the disease can be recreated through precise gene editing.

To initiate these disease models for all subsequent downstream work, the clonality and growth of the iPSCs is paramount, which can also be assessed by the Cell Metric.

The ability to then go on and produce such realistic disease models has greatly enhanced the efficacy of hit validation, enabling potential drugs to be assessed more accurately.

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