CRISPR Adams

Utilising a bespoke paired guide CRISPR library to identify novel synthetically lethal gene targets in multiple cancer types. The project involves large-scale CRISPR screens in multiple cell lines for four cancer types: pancreas, non-small cell lung cancer (NSCLC), melanoma, and uveal melanoma.

This project aims to uncover novel synthetically lethal gene targets in multiple cancer types using a bespoke paired guideRNA CRISPR library and is a collaboration between the Dave Adams group, CGaP, and DNA Pipelines. This library contains guides for genes predicted to be lethal from a variety of sources. The aim is to identify two genes that may have redundancy in the genome and that can compensate for each other; if one of these genes is lost or mutated in cancer cells and the other gene is targeted, cancer cells would die. However, “normal” cells should be able to rely on the second gene for survival. 42 cell lines will be screened from four different cancer types: pancreas, non-small cell lung cancer (NSCLC), melanoma, and uveal melanoma with the paired guide library. Using a paired guide library, two guides in the same virus vector are expressed simultaneously targeting different genes. This provides practical advantages over the use of single-guide libraries as it significantly reduces the scale of the screens without sacrificing performance.

In CGaP, there are two main stages of the CGaP pipeline. The first stage includes creating a bank of each cell line, carrying out antibiotic titrations and the paired gRNA library titration. The second stage includes the paired gRNA library transduction; puromycin selection, 80 million passages as required, and pelleting the cells 28 days post transduction. Each screen is carried out in triplicate, making it a very large-scale project with 25x T150 flasks used at transduction and 18x T875 flasks used at each 80 million passage. Pelleting is the endpoint process in CGaP and pellets are transferred to the Dave Adams group for DNA extraction. They are then sent to DNA pipelines for sequencing and the Dave Adams group carry out the data analysis.