Fig. 1
A genome-wide CRISPR screen for PARP inhibitor resistance identifies in-frame Parp1 mutants. a Experimental scheme. b Locations of Parp1 guide RNA target sites in exon two of the mouse Parp1 gene. c Parp1 western blot of lysates from talazoparib-resistant clones identified in the CRISPR screen. Individual clones are colour-coded according to sgRNA present (see key). Clones 1, 2, 6, 7, 9, 12 and 13 with Parp1 sgRNAs have lost Parp1 protein expression, whilst Parp1 sgRNA clone 8 (BR8, *) has retained Parp1 expression. d Clone BR8 has an in-frame Parp1 deletion and a Parp1 substitution mutation. Sanger sequencing trace of the Parp1 sgRNA target site is shown, illustrating a 3 bp deletion on both alleles and a heterozygous c.130T>A substitution mutation (p.44F>I) close to the CRISPR PAM site. e Parp1 is not trapped in the chromatin fraction by PARP inhibitor in the BR8 clone. Western blots illustrating Parp1 in the chromatin and nuclear soluble fractions of wild-type ES cells and Parp1 mutant BR8 cells exposed to talazoparib. Data shown are representative of two experiments. f PARP1 protein with a p.[43delM;44F>I] mutation has impaired recruitment to damaged DNA and does not initiate PAR synthesis at damaged DNA. Localisation of PARP1-GFP to damaged DNA was estimated by visualising GFP signal at the microirradiated spot, as was the generation of PAR at the damaged site by use of a PAR-binding PBZ-mRuby2 probe (see schematic). The time course of PARP1-GFP and PBZ-mRuby2 signals from CAL51 PARP1–/– cells transfected with PARP1-GFP (top) and PARP1-p.[43delM;44F>I]-GFP (bottom) are shown. “Damage” denotes time at which microirradiation was carried out. g, h Dose response curves illustrating that clone BR8 is resistant to both talazoparib (g) and olaparib (h). BR13 is an ES cell mutant with no Parp1 protein expression (see panel c); a transposon-mutagenised, Parp1 null mutant7 is also shown (red). Clone BR8 vs. BR13 and Parp1–/– transposon, p = ns, ANOVA. Parp1 mutant clones vs. wild-type cells, p < 0.0001, ANOVA. Mean of five replicates plotted, error bars show SD. Surviving fractions were calculated relative to DMSO-exposed cells for each mutant
