Scalable prime editor evolution for correction of inherited retinal diseases

The National Eye Institute awarded the University of California Irvine $158,960 on September 30, 2026, under the Vision Research program (CFDA 93.867) to develop scalable prime editor variants for correction of inherited retinal diseases.

The recipient will conduct a large-scale evolution campaign on prime editors directed toward clinically relevant targets using a continuous biomolecular evolution system in yeast based on orthogonal DNA replication (ORTHOREP). The work will leverage a recently developed approach to overcome limitations in scalable application of the ORTHOREP system to genome editors, facilitating transfer of hypermutating linear plasmids from edited to unedited cells with high efficiency. Through high numbers of replicates per unique target, the research will gain insights into molecular mechanisms behind prime-editor improvements, sequence and edit-type specialization, and the translatability of prime editors evolved in yeast to mammalian-cell editing. Prime editing technology addresses limitations of early gene-editing approaches such as CRISPR-Cas9 by avoiding double-stranded breaks and enabling more precise editing at clinically relevant loci.

Work will be performed in Irvine, California, with a period of performance from September 30, 2026, through September 29, 2029. The assistance type is a Project Grant.

Abstract

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.