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) |