Sequencing of long-donor insertions after gene therapy in hemophilia mice

CRISPR-Cas9-mediated integration of large transgenes into target cells has revolutionized in vivo gene therapy for various diseases, including hemophilia A. Successful low-level targeted integration of B-domain-deleted F8 at the Alb locus in hepatocytes has cured this hemostasis disorder in mice. To assess the safety profile of this therapy, we aimed to comprehensively analyze the spectrum of DNA integrated at the Alb target site. We devised a strategy for characterizing complex inserted sequences at the on-target edited locus using barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, and long amplicon enrichment with magnetic beads, followed by nanopore sequencing.