Year: 2025
Type: preprint
Abstract: DNA base editing is increasingly used for human genetic modification, but methods for monitoring off-target editing are nascent. Here we present a simple model-independent workflow for identifying sites of off-target base-editing in relevant cell types on a genome-wide level. We report that sites of off-target editing by the ABE8e editor could be identified using an ABE8e derivative with restored DSB cleavage activity. This allows marking of enzyme-generated double-stranded (ds) DNA breaks by incorporation of dsDNA oligonucleotides that are transfected into primary target cells. DNA sequencing at sites of oligonucleotide incorporation reported both the genomic location of off-target cleavage and the extent of base-editing nearby. We present a platform combining this cellular (BEiGUIDE-Seq) and computational workflow (CRISPRito) to generate optimized amplicon panels for convenient monitoring of off-target base editing. This work introduces a generalizable strategy to evaluate off-target edits in patient-derived cells, addressing a critical safety gap for clinical base editing.
Institution: University of Pennsylvania
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1852790566, Engineering T cells to Provide Durable Control of HIV-1 Replication, A Phase I Clinical Trial Testing Feasibility of Hematopoietic Stem Cell Gene Therapy Using Platelet Factor VIII to Safely Improve Hemostasis for Severe Hemophilia A with Inhibitory Antibodies, G4739483508, G9000000688, G9000033535, G9000127251, G9000138654, G9000266210, Gene therapy for SCID-X1 with low dose busulfan and a SIN-lentiviral vector
OpenAlex ID: W4413766317