Fig. 4
A high-density tiling CRISPR-Cas9 screen enables functional annotation of a PARP1 mutation observed in an olaparib-resistant patient. a Positions of sgRNA target sites in the dense PARP1 tiling library mapped onto the PARP1 coding DNA sequence shown in b. b Positions of mutations identified in the dense PARP1 tiling screen. Number of mutant reads for in-frame mutant alleles affecting each base is shown on the y-axis normalised to per-base coverage (mutant reads/total coverage × 1000). Only sites with coverage >500 reads are shown. The experiment was repeated in duplicate from two independently tagged SUM149 PARP1-GFP cell lines (clone 5 and 8, shown in red and blue). Arrows highlight the mutations observed at inter-domain contacts as shown in e. c Positions of protein domains mapped onto the PARP1 protein sequence. Red triangles indicate DNA–protein contacts based on crystal structures, blue circles show the location of the patient mutation identified in this study. Grey arcs represent key inter-domain contacts as shown in e and Fig. 2g. d Ribbon plot of ZnF1 and ZnF2 bound to a single-strand break (PDB: 2N8A). Ribbons are coloured red by residue based on the frequency of mutations observed in the tiling screen affecting that residue as shown in b. The thickness of the ribbon is proportional to the frequency of mutations observed affecting that residue. e Ribbon plot of the PARP1-DSB crystal structure (PDB: 4OQB) highlighting the clustering of mutations along the hydrogen-bonding axis postulated in Fig. 2g and the A925 residue that abuts Y848 (inset 2). Regions marked 1 and 2 are magnified. f Analysis of trapping for the R591C patient mutation by microirradiation with or without talazoparib as shown. Although the R591C mutant can bind DNA at sites of damage (blue line), it is not trapped in the presence of talazoparib (purple line)
