Fig 2: PARG loss sensitizes cells to PARGi.a, Ranking of co-essential genes with PARGi treatment on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screening with a DDR library in HEK293A cells. The NormZ score was used to determine a possible synthetic lethality gene under PARGi treatment. Drug-sensitive genes were marked in red; drug-resistant genes were marked in blue on the basis of the false discovery rate (FDR, 0.05 cut-off). b, HEK293A WT cells and HEK293A PARG KO cells were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay. c, HEK293A PARG KO cells, reconstituted with either full-length PARG or catalytic domain mutation of PARG, were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay.

Fig 3: PARG is essential for cell survival.a, Diagram of full-length PARG was presented with the indicated gRNAs which target different regions in the C-terminal catalytic domain. The boundary of the catalytic domain was depicted based on Uniprot annotation. b, Immunoblotting was conducted to confirm the loss of PARG in PARG complete/conditional knockout (cKO) cells derived from HEK293A and HeLa cells, which were cultured in the presence of 100 nM olaparib. c, Clonogenic assay results of WT and PARG cKO cells treated with or without PARPi (100nM) for 7 days. d, Left: The immunoblots to confirm reconstitution with WT PARG or catalytic inactivation PARG in HEK293A PARG cKO cells. Right: Results of clonogenic survival assay with HEK293A PARG cKO cells reconstituted with WT or catalytic inactivation mutant of PARG for 7 days. e, Representative clonogenic results conducted in HEK293A PARG cKO cells treated with NAM (100 μM) or NMN (1 mM) for 7 days. f, HEK293A PARG cKO cells were synchronized with double thymidine block (DTB). Cells remained with DTB or were released from DTB for 4 hours, and then fixed and stained with anti-pADPr antibody and FxCycle Violet dye. g, Immunoblots of soluble and chromatin-bound PARP1 and pADPr levels in HEK293A WT and PARG cKO cells treated with DMSO or olaparib (10 μM) for 2 hours.

Fig 4: CRISPR screening identifies regulators of pADPr and cell viability.a, Workflow of whole-genome CRISPR screens. For FACS-based CRISPR screening, 5 days after puromycin selection, cells were treated with PARGi (10uM) for 4 hours and then stained with anti-pADPr antibody and sorted with flow cytometry. Cells with strong signals (top 25%, TOP) and weak signals (bottom 25%, BOT) were selected. The sgRNAs from these cells were then sequenced and analyzed. For cell viability screening, cells were treated with or without PARGi for 21 days before collection. b, Scatter plot of DrugZ scores of PARG KO cells treated with or without PARGi treatment. The genes in the same pathway were marked with specific colors. A positive score indicates an enhanced pADPr signal, while a minus score indicates a decreased pADPr signal. c and d, Ranking of PARGi co-essential genes on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screens performed with Toronto Knock Out Library (version 3) in HEK293A PARG KO cells and HEK239A cells. e, Analysis of biological processes of PARGi co-essential genes identified in HEK293A PARG KO cells and HEK239A cells. f, HEK293A WT cells, PARG KO, POLB KO, LIG1 KO, XRCC1 KO, and LIG3 knockdown cells were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay. g, The cell Viability of HEK293A WT and PARG cells under POLB knockdown to PARGi. Cells were treated with different doses of PARGi for 72 hours.

Fig 5: Prolonged PARGi treatment induces pADPr throughout the cell cycle and DDR in PARG KO cells.a, Immunoblots of chromatin-bound PARP1 and PARylated proteins in HEK293A WT and PARG KO cells treated with PARGi (10 μM) for 4 hours. PARylated proteins were enriched by Af1521 beads. b, Sensitivity of HEK 293A PARG/PARP1/2 TKO and PARP1 reconstitution cells to PARGi. Cells were treated with different doses of PARGi for 72 hours, and cell viability was determined by the CellTiter-Glo assay. c, Prolonged PARGi treatment induces pADPr throughout the cell cycle in PARG KO cells. HEK293A WT and PARG KO cells were treated with PARGi (10 μM) for the indicated time and then fixed and stained with anti-pADPr antibody and violet. d, Immunoblotting of γH2A.X signals and other indicated proteins and modifications induced by prolonged PARGi+/‒MMS treatment.