Normalized concentration values were then analyzed in GraphPad prism version 8 and differential levels of NADP and NADPH were expressed as the mean??standard error of the mean (SEM)

Normalized concentration values were then analyzed in GraphPad prism version 8 and differential levels of NADP and NADPH were expressed as the mean??standard error of the mean (SEM). olaparib as a surrogate. We identify and or in the HR pathway5C9. In addition, alterations in genes not directly related to the homologous recombination (HR) pathway, such as PTEN loss10 and translocation in TMPRSS2-ERG11 and EWSR1-FLI112, also result in the increased sensitivity to PARP inhibitors, suggesting that other molecular pathways unrelated to the HR pathway may also contribute to PARP inhibitor sensitivity. Hence, a better understanding of genetic determinants that contribute to PARP inhibitor sensitivity will extend the clinical utility of PARP inhibitors. One of the challenges in the further development of PARP inhibitors is to fully understand the molecular mechanisms contributing to PARP inhibitor sensitivity13. To address this challenge, several studies have reported candidate genes that cause synthetic lethality with PARP inhibitors from genome-wide RNAi profiling and functional studies5,14,15. These studies identified genes involved in DNA damage response (DDR) and repair pathways, such as BRCA1, NBN, FANCD, FANCC, RAD51, LIG3, RAD51C, RAD51D, RAD21, ESCO1, and SMC3, as well as genes involved in replication and cell cycle progression, such as MCM proteins, TOP3A, POLB, and CDK7 as modulators of sensitivity to Toxoflavin PARP inhibitors5. However, the clinical benefit of targeting these genes to enhance sensitivity to PARP inhibitors is questionable given that chemical inhibition of these genes may likely sensitize normal cells as well as cancer cells to PARP inhibitors. Therefore, there is an urgent need to explore additional synthetic lethal targets for PARP inhibitors. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed Thy1 Cas9-mediated endonuclease activity can disrupt specific genetic sequences in the genome and provide a means to perform loss-of-function genetic screens16C18. When combined with sub-lethal doses of PARP inhibitor, this approach may allow identification of genetic factors that, when disrupted, contribute to PARP inhibitor sensitivity or resistance. Unlike RNAi approaches, CRISPR/Cas9 system provides more thorough depletion of target gene expression with less off-target effects when the guide RNA is appropriately designed19C21. We therefore used CRISPR/Cas9 system to perform a genome-scale loss-of-function screen to identify modifiers of olaparib sensitivity in cancer cells. From this screen, we identified (Fig. 1d, e). All three gRNAs show consistent, dose-dependent increase in abundance following olaparib treatment (Supplementary Fig. 1A). These results are consistent with a previous report indicating that PARP1 depletion results in resistance to PARP inhibitors24,25. While enrichment of gRNAs following olaparib selection indicates that target genes contribute to olaparib sensitivity, the depletion of gRNAs following olaparib selection indicates that target genes contribute to olaparib resistance. We identified (were depleted following olaparib treatment, suggesting that disruption of gene sensitizes the cells to olaparib. We also identified and as other candidate genes that decrease sensitivity to olaparib (Fig. 1f, g). Next, we transiently knocked down individual gene with pooled siRNAs to validate the potential candidates. We selected those candidates that have at least two different gRNAs being consistently and dose-dependently enriched or depleted after olaparib treatment for further validation, including and (Supplementary Fig. 1ACB). Open in a separate window Fig. 1 Genome-scale CRISPR knockout screening and candidate identification. a Stable expression of Cas9 endonuclease in A2780 cells. FLAG-tagged Cas9 was detected by western blot with anti-FLAG antibody. -Actin was used as a loading control. b Olaparib sensitivity in parental A2780 and A2780-Cas9 cells. Dose-response curves were generated from SRB assays. IC50 was calculated in Prism 6 software. c The flowchart shows the time scheme of the CRISPR/Cas9 knockout screen. dCg Potential dropout and enriched candidates were Toxoflavin identified after olaparib selection. The top 10 candidates that were most significantly enriched after olaparib selection are ranked by a modified robust ranking aggregation (RRA Toxoflavin score) (d) or KD resulted in an increase in IC50 of olaparib in A2780 cells. The IC50 for olaparib was calculated from dose-response curves. b The real-time RT-PCR analysis demonstrates the KD of TIGAR and with corresponding pooled siRNAs. Three replicates are included. c Colony formation assay showed that TIGAR KD decreased the clonogenic survival of cells treated with olaparib and KD enhanced the clonogenic survival. Representative images of.