Assay Development and Screening
Applications of CRISPR Technology in Drug Discovery
Functional genomics approaches to identify novel therapeutic targets are rapidly gaining traction. Arrayed screening for the phenotypes resulting from gene-knockouts using CRISPR-Cas9 technology can yield results rapidly, with very little need for target deconvolution. Data can be further enhanced by the selection of disease relevant primary cells.
We have developed a high-efficiency, arrayed genome-editing screen in primary CD4+ T cells using CRISPR–Cas9 for the identification of genes associated with cytokine release. T-cells are isolated, purified and expanded before genome editing occurs via nucleofection. A 384-well nucleofector is used to deliver RNP complexes consisting of guide RNA (gRNA), transactivating CRISPR RNA (tracrRNA) and Cas9 enzyme. Edited cells are rested and activated before being utilised in downstream assays capturing multi-cytokine release and cell viability.
The development of miniaturised, robust nucleofection protocols and assays for T-cell screening allows integration of this challenging cell-type onto well-established liquid handling platforms and demonstrates the potential of genome-wide arrayed CRISPR-Cas9 screening of primary cells in a screening environment.