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NIH grant 1F32EY037536-01A1: Scalable Prime Editor Evolution for Correction of Inherited Retinal Diseases
Institute: {'code': 'EY', 'abbreviation': 'NEI', 'name': 'National Eye Institute', 'admin_org_id': '12500073', 'admin_funding_url': ''}. PI: Derek Aspacio. Award: $79480.
Start: 2026-09-30T00:00:00.
PROJECT SUMMARY
The gene-editing revolution is underway. However, early gene-editing technologies based on
programmable nucleases, such as CRISPR-Cas9, are limited in editing scope and cause double-stranded
breaks, leading to undesirable outcomes. Precision genome-editing technologies, such as prime editing,
address these issues but are often insufficiently effective at editing clinically relevant loci.
It is well established that variability in prime editor efficiency is influenced by target sequence and edit
type. We hypothesize that prime-editor variants can be generated that edit different clinically relevant targets
more efficiently through protein alterations. Doing so would require a scalable prime-editor evolution approach
with sequence flexibility that would enable the targeting of numerous therapeutically relevant gene sequences.
No report has presented this achievement on a large scale across multiple user-defined sequence targets.
In this proposal, I describe a continuous biomolecular evolution system in yeast based on orthogonal DNA
replication (OrthoRep), developed by my Sponsor, Prof. Chang Liu. I will leverage my recently developed
approach, supported by preliminary results, that overcomes the limitation to scalable application of the
OrthoRep system to genome editors, thereby facilitating the transfer of hypermutating linear plasmids from
edited to unedited cells with high efficiency.
I propose to conduct a large-scale evolution campaign on prime editors directed toward clinically relevant
targets. With the statistical power derived from a high number of replicates per unique target, we will gain
insights into the molecular mechanisms behind prime-editor improvements, sequence and edit-type
specialization, as well as the general translatability of prime editors evolved in yeast to mammalian-cell editing.
Enabled by my unique training environment that bridges the University of California, Irvine School of
Engineering and the School of Medicine, our large-scale evolution campaign includes evolution of prime
editors to exactly target key DNA sites, including a frameshift mutation in the MERTK gene and a missense
mutation in the C1QTNF5 gene, known to cause inherited retinal disorders in clinical settings and model mice.
This training plan and project encompass two primary phases. Training Phase 1 (TP1) includes modern
biomedical engineering and evolution in the most genetically tractable eukaryotic model organism, the yeast
Saccharomyces cerevisiae, and directed evolution in mammalian tissue culture in Prof. Liu’s primary laboratory
space. In Training Phase 2 (TP2), translation will extend to mouse models of retinal degeneration by hands-on
collaboration with expert clinician scientists at Prof. Palczewski’s vivarium space. The successful
implementation of this training plan will provide experience throughout the full vertical pipeline of modern gene
editing research. Successful implementation of this project will resolve questions about the translation of prime
editors from yeast to editing in mammalian-cell tissue culture to editing in the murine retina in vivo.document fetched 2026-10-24 02:47