CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

13CRISPR-Cas9 genome engineering has revolutionised high-throughput functional 14 genomic screens. However, recent work has raised concerns regarding the 15 performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a 16 p53-mediated DNA damage response (DDR) limiting the efficiency of generating 17 viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-18Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type 19 and TP53 knockout human retinal pigment epithelial cells using a focused dual guide 20 RNA library targeting 852 DDR-associated genes. Our work demonstrates that 21 although functional p53 status negatively affects identification of significantly depleted 22 genes, optimal screen design can nevertheless enable robust screen performance. 23Through analysis of our own and published screen data, we highlight key factors for 24 successful screens in both wild-type and p53-deficient cells. 25 26

The discovery of synthetic lethal interactions with genetic deficiencies in cancers has highlighted several candidate targets for drug development, with variable clinical success. Recent work has unveiled a promising synthetic lethal interaction between inactivation/inhibition of the WRN DNA helicase and tumours with microsatellite instability, a phenotype that arises from DNA mismatch repair deficiency. While these and further studies have highlighted the therapeutic potential of WRN inhibitors, compounds with properties suitable for clinical exploitation remain to be described. Furthermore, the complexities of MSI development and its relationship to cancer evolution pose challenges for clinical prospects. Here, we discuss possible paths of MSI tumour development, the viability of WRN inhibition as a strategy in different scenarios, and the necessary conditions to create a roadmap towards successful implementation of WRN inhibitors in the clinic.