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OR11

A four-color AAV6 system for efficient biallelic homology-directed genome editing (HDR) without AAV vector integration into CRISPR-Cas9 induced double-strand breaks

M J Kleppa¹   M Rothe¹   L Unsöld¹   J Pötzsch¹   T C Ha¹   L Wielmann¹   A Schambach¹ ³   H Büning¹   A Schwarzer¹

1:Hannover Medical School;   2:Harvard Medical School; 3:Boston Children's Hospital

Genome editing by Cas9-ribonucleoproteins (RNPs) in combination with AAV6-DNA repair templates has become a promising tool for complex genome editing tasks and is rapidly moving towards clinical application. However, AAV6 vectors tend to integrate into Cas9-induced DNA double-strand breaks (DSB). We find that this remains undetected by commonly used sequencing techniques to assess HDR due to the presence of intact AAV ITR structures that prevent sequencing. To map the integration profile of AAV in the context of CRISPR-Cas9 RNP and AAV6 donor-mediated HDR, we developed a sequencing pipeline that uses ligation-mediated PCR with AAV-ITR specific primers. We find high frequency of AAV6 genome integrations into the HDR target site as well as into predicted sgRNA-off-target sites. Integration of AAV6 disrupts the gene to be targeted by HDR, leading to a loss of function of the affected allele. We developed a modular four color AAV6 vector system to detect and accurately quantify these events and to prospectively isolate cells carrying the desired HDR-editing outcome on both alleles. Using this system, we quantify the effect of different inhibitors of NHEJ and MMEJ and show that inhibition of NHEJ by M3814 leads to a significant increase in HDR efficacy from 36.1% to 54.3% . Our results show that unwanted integration of AAV genomes is a frequent adverse event and must be excluded when developing cell-based therapies using CRISPR-Cas9 and AAV6. Our vector system enhances the applicability of CRISPR-RNP-AAV6 genome editing by enabling the isolation of truly HDR-corrected cells in experimental and pre-clinical applications.

Sekretariat der DG-GT e.V.
Institut für Experimentelle Hämatologie
Hildegard Büning
Carl-Neuberg-Str. 1
30625 Hannover

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© 2021 Die Deutsche Gesellschaft für Gentherapie e.V.

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