Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Leveraging CRISPR-Cas9 for Accurate Detection of AAV-Neutralizing Antibodies: The AAV-HDR Method.

The effectiveness of adeno-associated virus (AAV)-based gene therapy is frequently constrained by the presence of AAV-neutralizing antibodies (NAbs). Existing detection techniques have shown inconsistencies across laboratories and cellular dependencies, challenging their universal applicability. Here, we redefine the NAb titer concept to represent the capability to neutralize a specific number of AAV virions per milliliter of serum. We present the AAV-homology-directed repair (HDR) assay, which harnesses the CRISPR-Cas9 system, offering a precise and sensitive means of detecting AAV NAbs. This assay employs a promoterless AAV HDR vector for integration into electroporated cells, facilitating the stable expression of a quantifiable fluorescent reporter and subsequent NAb titer assessment. Comparative evaluations indicated that the AAV-HDR method outperforms the traditional AAV overexpression (AAV-OE) assay regarding sensitivity and consistency. Crucially, it produced consistent outcomes across various cell lines, suggesting its potential as a universal standard for NAb titer measurement. We further confirmed the validity of the AAV-HDR titration approach by juxtaposing it with the established NT50 assay. Notably, the AAV-HDR method correlated robustly with both the AAV-OE assay and NT50 NAb titer values, and it exhibited heightened efficacy in identifying low-titer antibodies compared with the NT50 method. Given its ability to address AAV NAb detection challenges, the AAV-HDR assay holds promise for refining therapeutic strategies in gene therapy, particularly in tailoring AAV doses to neutralize preexisting NAbs.