# Multiplexing CRISPR/Cas9-based Continuous Evolution for Improved Epigenome Editing > **NIH NIH F30** · UNIVERSITY OF PENNSYLVANIA · 2020 · $50,520 ## Abstract Cas9-guided fusion proteins target specific DNA sequences for custom chemical modification. Cas9-bound error- prone DNA polymerases (EvolvRs) diversify pre-specified DNA segments to facilitate user-defined mutagenesis and accelerate the pace of directed evolution. However, EvolvRs are limited by short target length and have yet to be applied to non-contiguous sites within a protein coding sequence, a common feature of enzyme active sites. Cas9-guided ten-eleven translocation (TET) enzymes promote targeted demethylation of modified cytosine bases to precisely alter gene expression. These Cas9-TET epigenome editors have the potential to elucidate the biological effects of specific epigenetic marks and provide therapies for numerous diseases. However, Cas9- TET epigenome editors often achieve incomplete demethylation, limiting their effect on gene expression as well as their technological and clinical promise. In Cas9-TET epigenome editing, dose-response relationships have been shown between effective TET concentration, demethylation, and gene expression. These correlations suggest that TET’s catalytic activity may limit the ultimate efficiency of Cas9-TET epigenome editing. Prior work has demonstrated that TET’s catalytic activity can be increased through mutation of active site residues and that simultaneous active site mutations can be synergistic. This study aims to multiplex EvolvR-based diversification to evolve non-contiguous protein regions comprising the TET active site and increase TET’s catalytic activity. The project will leverage nature’s array-based generation of Cas-targeting RNA molecules to parallelize EvolvR-based evolution of many DNA sequences. Multiplexed EvolvR’s function will be validated by rescue of fluorescence in GFP reporters and analysis with flow cytometry and deep sequencing. Multiplexed EvolvR will next be applied to increase TET activity through parallel mutagenesis of non-contiguous TET active site regions and enrichment of hyperactive TET variants through immunoprecipitation. Catalytic activity of hyperactive TET variants will be characterized in vitro. Subsequently, this study will apply known and novel TET variants to improve the efficiency of Cas9-TET epigenome editing. Hyperactive TET variants will be fused to catalytically inactive Cas9 and targeted to methylated promoters in reporter and endogenous systems. By way of bisulfite and RNA sequencing, changes in demethylation and gene expression will be assessed among Cas9-TET fusions with variable activity to determine whether TET activity limits efficiency of current epigenome editing. These experiments may yield engineered TET variants with improved activity and push epigenome editing technologies towards clinical utility. Combined with bioethics coursework, this research will train an MD/PhD student to become an independent physician-scientist who can clinically translate genome and epigenome editing technologies and guide policy makers in their responsible us... ## Key facts - **NIH application ID:** 10140674 - **Project number:** 1F30HG011578-01 - **Recipient organization:** UNIVERSITY OF PENNSYLVANIA - **Principal Investigator:** Niklaus Hoyt Evitt - **Activity code:** F30 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $50,520 - **Award type:** 1 - **Project period:** 2020-09-15 → 2023-08-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10140674 ## Citation > US National Institutes of Health, RePORTER application 10140674, Multiplexing CRISPR/Cas9-based Continuous Evolution for Improved Epigenome Editing (1F30HG011578-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10140674. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*