Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles

Dmitrii Degtev (), Jack Bravo, Aikaterini Emmanouilidi, Aleksandar Zdravković, Oi Kuan Choong, Julia Liz Touza, Niklas Selfjord, Isabel Weisheit, Margherita Francescatto, Pinar Akcakaya, Michelle Porritt, Marcello Maresca (), David Taylor () and Grzegorz Sienski ()
Additional contact information
Dmitrii Degtev: AstraZeneca
Jack Bravo: University of Texas at Austin
Aikaterini Emmanouilidi: AstraZeneca
Aleksandar Zdravković: AstraZeneca
Oi Kuan Choong: AstraZeneca
Julia Liz Touza: AstraZeneca
Niklas Selfjord: AstraZeneca
Isabel Weisheit: AstraZeneca
Margherita Francescatto: AstraZeneca
Pinar Akcakaya: AstraZeneca
Michelle Porritt: AstraZeneca
Marcello Maresca: AstraZeneca
David Taylor: University of Texas at Austin
Grzegorz Sienski: AstraZeneca

Nature Communications, 2024, vol. 15, issue 1, 1-15

Abstract: Abstract Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

Date: 2024
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DOI: 10.1038/s41467-024-53418-8

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