High-resolution functional mapping of RAD51C by saturation genome editing

Rebeca Olvera-León; Fang Zhang; Victoria Offord; Yajie Zhao; Hong Kee Tan; Prashant Gupta; Tuya Pal; Carla Daniela Robles-Espinoza; Fernanda G Arriaga-González; Larissa Satiko Alcantara Sekimoto Matsuyama; Erwan Delage; Ed Dicks; Suzana Ezquina; Charlie F Rowlands; Clare Turnbull; Paul Pharoah; John R B Perry; Maria Jasin; Andrew J Waters; David J Adams

doi:10.1016/j.cell.2024.08.039

High-resolution functional mapping of RAD51C by saturation genome editing

Cell. 2024 Sep 16:S0092-8674(24)00968-1. doi: 10.1016/j.cell.2024.08.039. Online ahead of print.

Authors

Rebeca Olvera-León¹, Fang Zhang², Victoria Offord³, Yajie Zhao⁴, Hong Kee Tan³, Prashant Gupta³, Tuya Pal⁵, Carla Daniela Robles-Espinoza¹, Fernanda G Arriaga-González¹, Larissa Satiko Alcantara Sekimoto Matsuyama³, Erwan Delage³, Ed Dicks⁶, Suzana Ezquina⁶, Charlie F Rowlands⁷, Clare Turnbull⁸, Paul Pharoah⁹, John R B Perry⁴, Maria Jasin¹⁰, Andrew J Waters¹¹, David J Adams¹²

Affiliations

¹ Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK; Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, Querétaro, Mexico.
² Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
³ Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
⁴ MRC Epidemiology Unit, University of Cambridge School of Clinical Medicine, Institute of Metabolic Science, Cambridge, UK.
⁵ Department of Medicine, Division of Genetic Medicine, Vanderbilt University Medical Center (VUMC)/Vanderbilt-Ingram Cancer Center (VICC), Nashville, TN, USA.
⁶ Department of Public Health and Primary Care, University of Cambridge, Robinson Way, Cambridge, UK.
⁷ Division of Genetics and Epidemiology, The Institute of Cancer Research, London, UK.
⁸ Division of Genetics and Epidemiology, The Institute of Cancer Research, London, UK; National Cancer Registration and Analysis Service, National Health Service (NHS) England, London, UK; Cancer Genetics Unit, The Royal Marsden NHS Foundation Trust, London, UK.
⁹ Department of Computational Biomedicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
¹⁰ Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹¹ Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. Electronic address: aw28@sanger.ac.uk.
¹² Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. Electronic address: da1@sanger.ac.uk.

PMID: 39299233
DOI: 10.1016/j.cell.2024.08.039

Abstract

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.

Keywords: RAD51C; cancer predisposition; homologous recombination; saturation genome editing.