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G Gupta and D RamsdenCongrats on the new publication: G.P.Gupta and Ramsden labs! “Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks” Nucleic Acids Research. This was a collaborative effort with UNC Biochemistry and Biophysics, UNC Lineberger Comprehensive Cancer Center, UNC Department of Radiation Oncology, The Curriculum in Genetics and Molecular Biology, and UNC Pharmacy.

Interested in DSB repair pathway choice? This paper is on NGS and dPCR methods to quantify DSB repair pathway use, without needing fluorescent reporters, in essentially any cell type.

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.

Wanjuan Feng, Dennis A Simpson, Jang-Eun Cho, Juan Carvajal-Garcia, Chelsea M Smith, Kathryn M Headley, Nate Hathaway, Dale A Ramsden, Gaorav P Gupta, Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks, Nucleic Acids Research, 2021;, gkab299, https://doi.org/10.1093/nar/gkab299

Congrats to all authors in UNC Biochemistry and Biophysics, UNC Lineberger Comprehensive Cancer Center, UNC Department of Radiation Oncology, The Curriculum in Genetics and Molecular Biology, and UNC Pharmacy.