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Pooled CRISPR screening

Pooled CRISPR screening

Genetic screens are powerful tools used to identify genes involved in diseases and identify new drug targets (1). CRISPR screens enable unbiased discovery of drug targets or study underlying causes of disease. OXGENE’s screening platform offers CRISPR-knockout (CRISPRko) and activation screens (CRISPRa) using sgRNA libraries against either the whole genome or specific gene families. In addition, we can also quickly and efficiently build custom-designed pooled sgRNA libraries and provide complete workflows for CRISPR pooled screens; from cell line selection and optimisation, through to bioinformatic analysis of the screening results.

Activating mutations of Epidermal Growth Factor Receptor (EGFR) have been identified in 10–30% non-small cell lung cancers (NSCLC). Over the last decade, multiple EGFR-tyrosine kinase inhibitors (TKIs) have been developed to target NSCLC carrying these mutations. However, treatment of NSCLC with these targeted TKIs eventually leads to the emergence of resistance (3). By identifying essential kinases in lung cancer, we may be able to reveal additional targets and inform new treatment strategies.

Here we developed and validated a focused sub-pool sgRNA CRISPR library against the human kinome in a NSCLC adenocarcinoma cell line, PC-9, which has an EGFR activating mutation. This mutation is the oncogenic driver for this cell line and is essential for its survival. In addition, we used CRISPRa to perform an arrayed screen using the Synergistic Activation Mediator (SAM) and VP64-p65-Rta (VPR) system, with sgRNAs against several genes that may play a role in resistance to TKIs.

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