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Improving prime editing and other RNA-guided technologies by engineering synthetic RNA Polymerase III promoters

Award Number: F31HG014983
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 03/22/2027
PERIOD OF PERFORMANCE END DATE: 03/21/2028

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Improving prime editing and other RNA-guided technologies by engineering synthetic RNA Polymerase III promoters - PROJECT SUMMARY/ABSTRACT RNA-programmed precision genome editors have revolutionized the life sciences and offer tremendous potential to cure genetic disease. Prime editing is unique among editing tools as an incredibly precise and versatile method of making a range of edits in living cells and organisms. Prime editors (PEs) do not introduce double-strand breaks nor rely on endogenous homologous recombination machinery like CRISPR-Cas9. Rather, by encoding a template in the prime editing guide RNA (pegRNA) that is written into the genome by the reverse transcriptase component of PEs, one can install any single nucleotide change and moderately sized indels with few to no off-target edits, making PEs applicable to the study and treatment of myriad genetic diseases and a powerful molecular tool for research. Despite this potential, PEs are limited by relatively low efficiency in many contexts and difficulties with in vivo delivery. The expression of pegRNAs is a significant limiting factor in PE efficiency and several studies have demonstrated that improving expression of pegRNAs can drastically improve editing. PegRNAs are transcribed by RNA Polymerase III (Pol III), typically by utilizing the U6 promoter. The U6 promoter has been the best-in-class option in use for over two decades without substantial improvement. The overall hypothesis of this proposal is that the use of promoters with increased Pol III activity to express pegRNAs will yield improvements to PE efficiency. To test this hypothesis, I will conduct an in-depth mutational screen of the U6 promoter and other Pol III regulatory elements in massively parallel reporter assay (MPRA) experiments. Leveraging the insights gained from this screen and other preliminary data, I will engineer potent, minimized synthetic Pol III promoters and test them in additional MPRAs to identify optimal configurations. Finally, I will implement the engineered promoters to express pegRNAs that encode corrections to pathogenic variants and test their improvements to PE. My preliminary data have already revealed promoter architectures with improved expression and editing compared to the U6 promoter. I anticipate that leveraging the insights gained from my proposed deep mutational screens and rational engineering will yield even more potent synthetic promoters. This work will provide insight into a relatively understudied fundamental biological process, regulation of transcription by Pol III, and directly improve the efficiency, and by enabling lower doses, the safety of prime editing and other RNA-guided technologies that are widely used throughout the life sciences and are emerging as powerful potential therapeutics to treat genetic disease. These aims, and the comprehensive research plan behind them, are aligned with the areas of emphasis articulated by the NHGRI including genomic technology development and high-throughput methods to study functional genomics.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $50,114 )
2026	2026	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Human Genome Research	000	1	9/23/2026	NEW	$50,114
														Subtotal = $50,114

Grand Total All Awards = $50,114

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Improving prime editing and other RNA-guided technologies by engineering synthetic RNA Polymerase III promoters

Award Number: F31HG014983

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 03/22/2027

PERIOD OF PERFORMANCE END DATE: 03/21/2028

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