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Directed evolution of polymerases that can read and write extremely long sequences

Award Number: R01EB027202
ORGANIZATION: NATIONAL INSTITUTE Of BIOMEDICAL IMAGING & BIOENGINEERING
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 03/15/2020
PERIOD OF PERFORMANCE END DATE: 11/30/2025

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Directed evolution of polymerases that can read and write extremely long sequences - Project Summary Advances in synthetic biology have accelerated to the point where the synthesis of entire genomes is now possible. However, the technologies for these feats are painstaking, and the production of a new chromosome or genome requires multiple years of effort, working from small fragments to ever larger assemblies. The cumbersome assembly process is due in large measure to the need to carry out an ordered series of hierarchical homologous recombination steps that proceed through transformations into organisms, primarily yeast. The speed (and ultimately scale) of large fragment assembly would be greatly improved if it were possible to routinely amplify very long stretches of DNA (> 100 kb) in vitro. To that end, this proposal is focused on the further development of a novel directed evolution method known as Compartmentalized Self-Replication (CSR), in which polymerases expressed in cells in emulsions undergo thermal cycling to amplify their own genes, to generate long read DNA polymerases that should prove capable of generating PCR amplicons > 100 kb in length, with few errors. To achieve this goal, we propose to develop a novel library construction method that most efficiently brings together sequence and structural domains from a variety of DNA polymerase variants to form diverse chimeras (Aim 1.1), and to sieve these libraries using improvements to CSR that will allow us to select for extreme processivity in yeast (Aim 1.2) and efficient error-correction (Aim 1.3). The variants that result will be characterized for their ability to synthesize long amplicons in vitro (Aim 2.1), for their fidelity (Aim 2.2), and for their detailed kinetic properties (Aim 2.3). Finally, to better ensure the processivity of the resultant polymerase chimeras, we will append either DNA-binding domains (Aim 3.1) or clamps (Aim 3.2) that should lead to much better ability to grip DNA. In addition to accelerating the ongoing revolution in genome synthesis, such long-read polymerases should also pave the way to new sequencing technologies, including for single molecule sequencing and for single cell sequencing.


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: 2025 ( Subtotal = $0 )
2025	2024	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	4	11/19/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $343,817 )
2024	2024	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	4	11/2/2023	NON-COMPETING CONTINUATION	$315,750
2024	2024	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	4	4/29/2024	NON-COMPETING CONTINUATION	$28,067
														Subtotal = $343,817

Issue Date FY: 2023 ( Subtotal = $344,576 )
2023	2023	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	3	11/29/2022	NON-COMPETING CONTINUATION	$315,750
2023	2023	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	3	1/30/2023	NON-COMPETING CONTINUATION	$35,084
2023	2020	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	002	1	7/5/2023	SUPPLEMENT FOR EXPANSION	-$6,258
														Subtotal = $344,576

Issue Date FY: 2022 ( Subtotal = $350,834 )
2022	2022	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	2	6/27/2022	NON-COMPETING CONTINUATION	$35,084
2022	2022	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR MN 13	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	2	1/6/2022	NON-COMPETING CONTINUATION	$315,750
														Subtotal = $350,834

Issue Date FY: 2021 ( Subtotal = $0 )
2021	2020	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR MN 13	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	1	9/10/2021	SUPPLEMENT FOR EXPANSION	$0
2021	2020	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR MN 13	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	1	2/3/2021	NEW	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $512,711 )
2020	2020	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR MN 13	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	1	8/31/2020	SUPPLEMENT FOR EXPANSION	$182,969
2020	2020	UNIVERSITY OF TEXAS AT AUSTIN	110 INNER CAMPUS DR MN 13	AUSTIN	TX	78712	TRAVIS	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	1	3/13/2020	NEW	$329,742
														Subtotal = $512,711

Grand Total All Awards = $1,551,938

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Directed evolution of polymerases that can read and write extremely long sequences

Award Number: R01EB027202

ORGANIZATION: NATIONAL INSTITUTE Of BIOMEDICAL IMAGING & BIOENGINEERING

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 03/15/2020

PERIOD OF PERFORMANCE END DATE: 11/30/2025

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