P14

ePsCas9 (eSpOT-ON): An Engineered High-Fidelity Genome Editor for Lipid Nanoparticle Delivery

D Degtev(1) J Bravo(2) A Emmanouilidi(1) O K Choong(1) J L Touza(1) N Selfjord(1) I Weisheit(1) P Akcakaya(1) M Porritt(1) K Holden(3) A Gittos(3) C Jowdy(3) M Maresca(1) D Taylor(2) G Siensky(1)

1:Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden; 2:Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA; 3:Synthego, Redwood City, CA, USA

CRISPR-Cas technologies hold great promise for therapeutic genome editing, but many Cas enzymes lack sufficient in vivo activity for clinical use. Our previous work introduced PsCas9 (SpOT-ON), a high-fidelity Type II-B Cas9 from Parasutterella secunda, achieving comparable editing levels to SpCas9 with fewer off-target effects and reduced chromosomal translocations. This study aimed to address reduced PsCas9 efficacy with non-adenoviral delivery methods. Cellular studies revealed PsCas9 activity was limited under low intracellular concentrations due to lower DNA-binding affinity compared to SpCas9. To enhance activity, we used structural insights to engineer the enzyme. While sgRNA optimization modestly improved activity, targeted mutagenesis increased editing efficiency up to 20-fold across multiple targets, enhancing DNA-binding affinity while maintaining high fidelity. 

The optimized enzyme, ePsCas9 (eSpOT-ON), was tested for therapeutic genome editing in vivo. Delivered via lipid nanoparticles, ePsCas9 effectively edited the Pcsk9 gene in mouse liver, reducing plasma Pcsk9 protein levels more efficiently than SpCas9. 

ePsCas9’s precision, efficiency, and fidelity make it well-suited for therapeutic applications. eSpOT-ON tools, including synthetic guide RNAs and purified nuclease, are now available through a Synthego-AstraZeneca partnership to advance CRISPR-based medicine.