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Multiplexed In Vivo DNA Assembly

Award Number: R01HG011676
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 10/24/2023
PERIOD OF PERFORMANCE END DATE: 08/31/2025

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Multiplexed In Vivo DNA Assembly - PROJECT SUMMARY/ABSTRACT High-throughput and low-cost synthesis of long DNA fragments could open up new frontiers in genomics and synthetic biology by facilitating the production of genes, genetic pathways, variant libraries, or entire synthetic genomes. Microarray printing technologies provide for high-throughput and low-cost synthesis of short oligonucleotides (currently up to 300 bases long), while new technologies, such as enzymatic synthesis, have the potential to synthesize longer sequences. Libraries of open reading frames or regulatory elements have also been constructed. However, assembly of the above or other DNA elements into longer fragments remains a challenge. Widely-used in vitro DNA assembly methods, such as Gibson assembly and polymerase chain assembly, require considerable hands-on time, are difficult to multiplex and scale, and have size constraints on the length of the assembled product. In addition, accurate long assemblies from de novo synthesized DNA are difficult to achieve without a high-throughput error correction step. To overcome these hurdles, we have developed a novel, multiplexed method to parse, sequence verify, and stitch together DNA of various sizes in vivo. Using this method, complex pools of array-synthesized oligonucleotides or other DNA elements can be turned into positionally-ordered cellular arrays, with each position containing a sequence-verified input DNA. Input DNA in these arrays can then be stitched together recursively by bacterial mating. A key feature of our technology is early error detection, whereby individual DNA inputs are sequence-verified prior to stitching. That is, errors that occur during short DNA synthesis are detected and removed, rather than propagated into longer assemblies. At maximal throughput, we estimate that our technology has the potential to produce thousands of DNA assemblies in parallel at a cost that is at least an order of magnitude cheaper than commercially available gene synthesis technologies. Because DNA stitching can occur within bacterial artificial chromosomes, in vivo DNA parsing and stitching can perform multiplexed assembly of DNA constructs exceeding 100kb, at least an order of magnitude longer than current in vitro assembly techniques. In vivo assembly uses a stepwise stitching process that can be branched, allowing the construction of a wide variety of variants at low incremental cost. Here, we propose to continue development of this technology i) by building many genes to learn the error modalities of the process, ii) by assembling a ~100kb product, and iii) by developing a multi-step assembly procedure to reduce assembly time and increase product accuracy. Together, this integrated set of aims will result in a new high-throughput DNA assembly platform capable of synthesizing long and complex DNA constructs and variant libraries at low cost.


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	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	002	4	3/13/2025	NON-COMPETING CONTINUATION	$0
2025	2024	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	000	4	1/10/2025	NON-COMPETING CONTINUATION	$0
2025	2023	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	001	3	1/10/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $794,324 )
2024	2024	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	006	4	3/19/2024	NON-COMPETING CONTINUATION	$808,339
2024	2024	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	007	4	7/26/2024	NON-COMPETING CONTINUATION	$71,852
2024	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	004	2	3/12/2024	NON-COMPETING CONTINUATION	-$4,546
2024	2023	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	005	3	3/12/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$4,546
2024	2023	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	003	3	1/10/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$0
2024	2023	BACSTITCH DNA INC.	1027 RAY AVE	LOS ALTOS	CA	94022	SANTA CLARA	USA	Human Genome Research	001	3	11/22/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$458,304
2024	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	002	2	12/13/2023	NON-COMPETING CONTINUATION	$65,961
2024	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	000	2	11/21/2023	NON-COMPETING CONTINUATION	-$610,132
														Subtotal = $794,324

Issue Date FY: 2023 ( Subtotal = $1,080,116 )
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	001	2	2/23/2023	NON-COMPETING CONTINUATION	$1,080,116
2023	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	000	1	12/14/2022	NEW	$0
														Subtotal = $1,080,116

Issue Date FY: 2022 ( Subtotal = $1,075,060 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Human Genome Research	000	1	5/10/2022	NEW	$1,075,060
														Subtotal = $1,075,060

Grand Total All Awards = $2,949,500

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Multiplexed In Vivo DNA Assembly

Award Number: R01HG011676

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 10/24/2023

PERIOD OF PERFORMANCE END DATE: 08/31/2025

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