ABSTRACT
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
Subject(s)
BRCA1 Protein/genetics , Bone Neoplasms/drug therapy , Breast Neoplasms/drug therapy , DNA End-Joining Repair , Drug Resistance, Neoplasm , Osteosarcoma/drug therapy , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proteins/metabolism , Recombinational DNA Repair , Animals , BRCA1 Protein/deficiency , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle Proteins , Cell Line, Tumor , Cisplatin/pharmacology , DNA Breaks, Double-Stranded , DNA-Binding Proteins , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Female , HEK293 Cells , Humans , Mad2 Proteins/genetics , Mad2 Proteins/metabolism , Mice , Multiprotein Complexes , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Proteins/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
