Systematic identification of G-quadruplex sensitive lethality by genome-wide genetic screening

A375 and HT1080 cells are treated with G-quadruplex ligands PDS or PhenDC3 following genome-wide shRNA knockdown. This enables the identification of genes that when silenced, specifically compromises cell growth in the presence of the ligand. First, a pilot screen was performed to determine a ligand concentration and experimental duration that caused ligand-specific, significant changes in shRNA levels. Second, a genome-wide screen was performed to globally evaluate G4-ligand synthetic lethal interactions. Third, to corroborate the G4-sensitisers uncovered in the genome-wide screen, a focussed screen was performed with a custom shRNA pool.

本研究先对A375与HT1080细胞开展全基因组小发夹RNA（shRNA）敲低实验，随后使用G-四链体（G-quadruplex）配体PDS或PhenDC3进行处理，借此可鉴定出当某基因被沉默后，会在该配体存在的条件下特异性损害细胞增殖能力的基因。整个实验分为三个阶段：
首先开展预筛选实验，以确定可使shRNA水平出现配体特异性显著变化的配体浓度与实验时长；
其次开展全基因组筛选，以全局性评估G4配体的合成致死相互作用；
最后，为验证全基因组筛选中发现的G4增敏基因，研究人员使用定制化shRNA文库开展了聚焦筛选实验。