Decoding the Complexity of On-Target Integration: Characterizing DNA Insertions at the CRISPR-Cas9 Targeted Locus Using Nanopore Sequencing

Abstract

Background CRISPR-Cas9-facilitated integration of sizable transgenes into target cells has revolutionized in vivo gene therapy for various disorders, including hemophilia A. The effective targeted incorporation of F8 at the Alb locus in hepatocytes has cured this hemostasis disorder in mice. However, assessing the safety and specificity of this therapy is crucial. We developed a strategy to characterize intricate inserted sequences at the on-target edited locus using barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, long amplicon enrichment with magnetic beads, and nanopore sequencing. Results Our findings unveiled not only the expected F8 insertion but also diverse fragment combinations stemming from in vivo linearization of the double-cut plasmid donor. Impressively, our study is the first to report insertions exceeding 10 kbp. Furthermore, we discovered that a minor fraction of these insertions originated from sources other than donor plasmids, such as Cas9-sgRNA plasmids, genomic DNA fragments, or LINE-1 elements. Conclusions We established a robust method for assessing on-target editing complexity, especially in vivo long insertions where donor template integration is often inefficient. Our report presents a novel tool for quality control in gene editing outcomes, highlighting the need for comprehensive characterization of edited genomic sequences. This research can help improve the safety and efficacy of CRISPR-Cas9-facilitated gene therapy for treating various disorders, including hemophilia A.