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

ABSTRACT:

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 t

SUBMITTER: Zhao JJ 

PROVIDER: S-EPMC10874558 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature