Nature communicationsNov 7, 2024

Engineered PsCas9 allows gene editing treatment in mouse liver using fat-based nanoparticles

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Updated Mar 29, 2026

Abstract

Engineered PsCas9 (ePsCas9) demonstrates up to 20-fold increased editing activity compared to its predecessor.

PsCas9 is a Type II-B enzyme capable of editing the mouse liver genome with adenoviral delivery, showing no detectable off-target effects.
Non-viral delivery of PsCas9 remains a challenge due to insufficient efficacy.
Ribonucleoprotein structure of PsCas9 was solved using cryo-electron microscopy, aiding its rational engineering.
Multiple guide RNA and protein variants were screened to develop ePsCas9, which retains safety advantages while enhancing activity.
A single administration of mRNA encoding ePsCas9 with lipid nanoparticles achieved high editing levels in the Pcsk9 gene, relevant for hypercholesterolemia treatment.

Simplified

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.

Key numbers

20×

Increase in Editing Activity

ePsCas9 activity compared to wild-type PsCas9 across various targets.

60%

Editing Efficiency in Mouse Liver

Average editing efficiency in the Pcsk9 gene after ePsCas9 delivery.

10×

Off-Target Activity Reduction

ePsCas9 off-target activity compared to SpCas9.

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Funding

Competing interests

D.D., A.E., A.Z., O.K.C., J.L.T., N.S., I.W., M.F., P.A., M.P., M.M. and G.S. are employees and shareholders of AstraZeneca. G.S. and M.M. are listed as co-inventors in a patent application filed by AstraZeneca Ab (application number: WO2022248645A1; status: published) related to this work covering aspects of protein engineering. J.B. and D.T. declare no competing interests. This work was supported by the National Institutes of Health grant R35GM138348 to D.T. and Welch Foundation Research Grant F-1938 to D.T; by the AstraZeneca Postdoctoral Fellowship to I.W.

PubMed

Engineered PsCas9 allows gene editing treatment in mouse liver using fat-based nanoparticles

Abstract

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