{"doi":"10.1093/nar/gkab299","title":"Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks","abstract":"Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.","journal":"Nucleic Acids Research","year":2021,"id":167281,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":32,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9578,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":436438,"name":"Dennis A. Simpson","orcid":"0000-0001-5332-8243","position":1,"is_corresponding":false},{"id":304363,"name":"Jang-Eun Cho","orcid":"0000-0003-1005-6393","position":2,"is_corresponding":false},{"id":605590,"name":"Juan Carvajal-Garcia","orcid":"0000-0001-7257-3674","position":3,"is_corresponding":false},{"id":694132,"name":"Chelsea M. Smith","orcid":"0000-0001-8263-9817","position":4,"is_corresponding":false},{"id":694651,"name":"Kathryn M. Headley","orcid":null,"position":5,"is_corresponding":false},{"id":694652,"name":"Nate Hathaway","orcid":null,"position":6,"is_corresponding":false},{"id":448788,"name":"Dale A. Ramsden","orcid":"0000-0003-1575-4748","position":7,"is_corresponding":false},{"id":457309,"name":"Gaorav P. Gupta","orcid":"0000-0001-9177-552X","position":8,"is_corresponding":false},{"id":476097,"name":"Wanjuan Feng","orcid":null,"position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-18T23:45:54.042727Z","pmid":"33963863","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}