AAV-mediated genome editing is influenced by the formation of R-loops.

Francesco Puzzo; Magdalena P Crossley; Aranyak Goswami; Feijie Zhang; Katja Pekrun; Jada L Garzon; Karlene A Cimprich; Mark A Kay

doi:10.1101/2024.05.07.592855

PubMed · 38766176

AAV-mediated genome editing is influenced by the formation of R-loops.

Francesco Puzzo·Magdalena P Crossley·Aranyak Goswami·Feijie Zhang·Katja Pekrun·Jada L Garzon·Karlene A Cimprich·Mark A Kay

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Abstract

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

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Francesco Puzzo, Magdalena P Crossley, Aranyak Goswami, Feijie Zhang, Katja Pekrun, Jada L Garzon, Karlene A Cimprich, Mark A Kay. 2024-05-09. AAV-mediated genome editing is influenced by the formation of R-loops.. https://doi.org/10.1101/2024.05.07.592855

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