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Mitigating Indel Impact: Precision Genome Editing through Homology Independent Targeted Integration (HITI)

G Turnbull(1) T Roberts(1) A Bray(2) S C Hyde(1) D R Gill(1)

1:University of Oxford; 2:Oxford BioMedica

The efficacy of homology-independent targeted integration (HITI) for disrupting dominant gain-of-function mutations was investigated. This was exemplified by targeting mutations in the clinically relevant SERPINA1 gene encoding Alpha-1-antitripsin (AAT), which cause misfolded  aggregates  in the liver and the loss of AAT protective function in the lungs  leading to Alpha-1-antitripsin deficiency (AATD).

HEK293T HITI reporter cells were transfected using mNeonGreen donor plasmids targeting an mCherry reporter cassette and SERPINA1. Editing was assessed by fluorescence imaging and droplet digital PCR. The integrated sequence was analysed using sanger sequencing of TOPO clones and next generation sequencing (NGS) of pooled genomic DNA. High resolution melting (HRM) analysis confirmed the status of indels throughout the edited sequence.

HITI successfully achieved targeted gene disruption and replacement (6.3% editing) in the HITI reporter cell line at the SERPINA1 locus. Notably, indels were restricted to donor-genome junctions, preserving donor sequence integrity and minimizing unintended mutations. Interestingly, NGS erroneously reported additional indels within the integrated donor sequence, but further analysis by HRM suggested these sequences were indistinguishable from known control sequences (p>0.9999).


This study demonstrates the use of HITI for precise gene editing of SERPINA1, offering the potential to address dominant gain-of-function genetic disorders such as AATD, while mitigating the risk of unintended mutations.

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