RRC ID 82147
Author Degtev D, Bravo J, Emmanouilidi A, Zdravković A, Choong OK, Liz Touza J, Selfjord N, Weisheit I, Francescatto M, Akcakaya P, Porritt M, Maresca M, Taylor D, Sienski G.
Title Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles.
Journal Nat Commun
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.
Volume 15(1)
Pages 9173
Published 2024-11-7
DOI 10.1038/s41467-024-53418-8
PII 10.1038/s41467-024-53418-8
PMID 39511150
PMC PMC11544209
MeSH Animals CRISPR-Associated Protein 9* / genetics CRISPR-Associated Protein 9* / metabolism CRISPR-Cas Systems* Cryoelectron Microscopy Gene Editing* / methods Genetic Therapy / methods HEK293 Cells Humans Lipids / chemistry Liposomes Liver* / metabolism Mice Mice, Inbred C57BL Nanoparticles* / chemistry Proprotein Convertase 9* / genetics Proprotein Convertase 9* / metabolism RNA, Guide, CRISPR-Cas Systems* / genetics
Resource
Human and Animal Cells HuH-7(RCB1366)