Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Martin Peterka (), Nina Akrap, Songyuan Li, Sandra Wimberger, Pei-Pei Hsieh, Dmitrii Degtev, Burcu Bestas, Jack Barr, Stijn Plassche, Patricia Mendoza-Garcia, Saša Šviković, Grzegorz Sienski, Mike Firth and Marcello Maresca ()
Additional contact information
Martin Peterka: BioPharmaceuticals R&D Unit, AstraZeneca
Nina Akrap: BioPharmaceuticals R&D Unit, AstraZeneca
Songyuan Li: BioPharmaceuticals R&D Unit, AstraZeneca
Sandra Wimberger: BioPharmaceuticals R&D Unit, AstraZeneca
Pei-Pei Hsieh: BioPharmaceuticals R&D Unit, AstraZeneca
Dmitrii Degtev: BioPharmaceuticals R&D Unit, AstraZeneca
Burcu Bestas: BioPharmaceuticals R&D Unit, AstraZeneca
Jack Barr: BioPharmaceuticals R&D Unit, AstraZeneca
Stijn Plassche: BioPharmaceuticals R&D Unit, AstraZeneca
Patricia Mendoza-Garcia: BioPharmaceuticals R&D Unit, AstraZeneca
Saša Šviković: BioPharmaceuticals R&D Unit, AstraZeneca
Grzegorz Sienski: BioPharmaceuticals R&D Unit, AstraZeneca
Mike Firth: Discovery Sciences, AstraZeneca
Marcello Maresca: BioPharmaceuticals R&D Unit, AstraZeneca

Nature Communications, 2022, vol. 13, issue 1, 1-9

Abstract: Abstract Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

Date: 2022
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DOI: 10.1038/s41467-022-28771-1

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