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Programmable Mitochondrial DNA Annealase for Scarless, Human-Endogenous Genome Editing and Functional Screening

Award Number: R01GM141627
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/10/2021
PERIOD OF PERFORMANCE END DATE: 04/30/2030

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Programmable Mitochondrial DNA Annealase for Scarless, Human-Endogenous Genome Editing and Functional Screening - Project Abstract Gene editing technologies have revolutionized biomedical research, yet significant challenges remain in achieving safe, efficient, and immunologically compatible integration of large DNA sequences. Current technologies, including CRISPR-Cas based transposase, base-editing, prime-editing, or phage-derived integrases and recombinases, are limited by their non-human origins, which can trigger toxic cellular responses and restrict editing capabilities. During our previous NIGMS funding period, we successfully developed a phage-derived recombineering system (REDIT) that achieves efficient kilobase-scale editing without DNA cutting, significantly reducing off-target effects compared to Cas9 editing. While successful, this system still faced challenges including variable efficiencies, large system size constraining delivery, and potential immunogenicity from non-human components when applied in primary cell or complex models like stem cell derived organoids. To overcome these limitations, we made a breakthrough discovery through structural homology mining: human mitochondrial Twinkle helicase possesses DNA unwinding-annealing capabilities with structural and functional similarity to phage SSAPs, despite low sequence homology. Our preliminary data show that engineered nuclear-targeted Twinkle facilitates homology-driven DNA insertion with double-digit efficiency for kilobase-sized sequences. Additionally, we confirm Twinkle editor could be guided by CRISPR system or function independently from CRISPR via homology annealing. This renewal application proposes to develop this human-derived mitochondrial enzyme into a versatile platform for proteome-wide functional screening. We will optimize Twinkle through protein and donor engineering, elucidate DNA repair mechanisms affecting editing efficiency, and validate the system through pooled knock-in screening in cancer and stem cell models. Our goal is to develop the first fully human gene-editing platform capable of efficient kilobase-scale insertion, enabling scalable genome engineering for both basic research and disease studies.


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 = $1,684,760 )
2026	2026	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	5	4/28/2026	COMPETING CONTINUATION	$1,684,760
														Subtotal = $1,684,760

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	4	8/5/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $417,908 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	002	4	7/15/2024	NON-COMPETING CONTINUATION	$41,790
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	4	4/15/2024	NON-COMPETING CONTINUATION	$376,118
2024	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	3	2/13/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $417,908

Issue Date FY: 2023 ( Subtotal = $417,908 )
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	3	4/27/2023	NON-COMPETING CONTINUATION	$417,908
2023	2022	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	2	2/9/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $417,908

Issue Date FY: 2022 ( Subtotal = $417,908 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	2	5/16/2022	NON-COMPETING CONTINUATION	$417,908
														Subtotal = $417,908

Issue Date FY: 2021 ( Subtotal = $391,444 )
2021	2021	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	1	8/10/2021	NEW	$391,444
														Subtotal = $391,444

Grand Total All Awards = $3,329,928

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Programmable Mitochondrial DNA Annealase for Scarless, Human-Endogenous Genome Editing and Functional Screening

Award Number: R01GM141627

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/10/2021

PERIOD OF PERFORMANCE END DATE: 04/30/2030

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