New publication by the Roukos lab on multilevel characterization of genome editor nuclease activity with BreakTag
Longo GMC, Sayols S, Roukos V (2025) Multilevel characterization of genome editor nuclease activity with BreakTag. Nat Protoc, doi:10.1038/s41596-025-01271-4 Link
Abstract:
BreakTag is a scalable next-generation sequencing-based method for the unbiased characterization of programmable nucleases and guide RNAs at multiple levels. BreakTag allows off-target nomination, nuclease activity assessment and the characterization of scission profile, that, in Cas9-based gene editing, is mechanistically linked with the indel repair outcome. The method relies on digestion of genomic DNA by Cas9 and guide RNAs in ribonucleoprotein format, followed by enrichment of blunt and staggered DNA double-strand breaks generated by CRISPR nucleases at on- and off-target sequences. Next-generation sequencing and data analysis with BreakInspectoR allows high-throughput characterization of Cas nuclease activity, specificity, protospacer adjacent motif frequency and scission profile. Here we first describe a detailed BreakTag protocol for the nomination of CRISPR off-targets and multilevel characterization of engineered Cas variants and second, we describe a step-by-step protocol for data analysis using BreakInspectoR. Third, we provide a web interface for XGScission, a machine learning model amenable to training with scission-aware BreakTag data to predict the relative frequency of blunt and staggered double-strand breaks at new sequences unseen by the model. XGScission allows a preselection of target sequences predicted to be cut in staggered configuration that are preferably repaired as single-nucleotide templated insertions. Furthermore, XGScisson can be used to assess sequence determinants of blunt and staggered cleavage by SpCas9 and engineered nuclease variants. As a companion strategy, we describe HiPlex for the generation of hundreds to thousands of single guide RNAs in pooled format for the production of robust BreakTag datasets. The BreakTag library preparation takes ~6 h, and the entire protocol can be completed in ~3 d, including sequencing, data analysis with BreakInspectoR and XGScission model training.
Read the full paper here: https://www.nature.com/articles/s41596-025-01271-4
