De novo assembly and delivery to mouse cells of a 101 kb functional human gene
Open accessAbstract
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
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The two synthetic BAC constructs were sequence-verified using Illumina short-read sequencing.
“A variant of the eSwAP-In method24 was used to produce the two constructs, which were sequence-verified using illumina short-read sequencing (Figure S1A).”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [24]
The paper’s “Structural and sequence verification of assembled DNA” section says that pLM750 underwent PacBio sequence verification, not Illumina short-read sequencing. It describes several assembled plasmids but does not report Illumina verification of two constructs. (LLM, conf=0.84)
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Cites
Design and synthesis of a minimal bacterial genome
Hutchison, C. A., Chuang, R.-Y., Noskov, V. N., Assad-Garcia, N., Deerinck, T. J., Ellisman, M. H., Gill, J., Kannan, K., Karas, B. J., Ma, L., et al. (2016) · Science · doi:10.1126/science.aad6253 · Cited by 1,500 · see citing works
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Research works printed but not linked: 4
- Brosh R, Laurent JM, Ordoñez R, Huang E, Hogan MS, et al. 2021. A versa- tile platform for locus-scale genome rewriting and verification. Proc Natl Acad Sci USA. 118:e2023952118 10.1073/pnas.2023952118 · DOI
- Gibson DG. 2011. Gene and genome construction in yeast. Curr Protoc Mol Biol. Chapter 3: Unit3 22.
- Mitchell LA, Phillips NA, Lafont A, Martin JA, Cutting R, et al. 2015. qPCRTag analysis-a high throughput, real time PCR assay for Sc2.0 genotyping. J Vis Exp. (99):e52941. doi: 10.3791/52941. · DOI
- Townsend MH, Robison RA, O'Neill KL. 2018. A review of HPRT and its emerging role in cancer. Med Oncol. 35:89.
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Super-enhancers include classical enhancers and facilitators to fully activate gene expression
Blayney, J. W., Francis, H., Rampasekova, A., Camellato, B., Mitchell, L., Stolper, R., Cornell, L., Babbs, C., Boeke, J. D., Higgs, D. R., et al. (2023) · Cell · doi:10.1016/j.cell.2023.11.030 · Cited by 196 · see citing works
Self-citation
- Authors
- Mitchell, L. A., McCulloch, L. H., Pinglay, S., Berger, H., Bosco, N., Brosh, R., Bulajić, M., Huang, E., Hogan, M. S., Martin, J. A., Mazzoni, E. O., Davoli, T., Maurano, M. T., & Boeke, J. D.
- Affiliations
- NYU Langone Health (US)
- New York University (US)
- Corresponding author
- Mitchell, Leslie A
- Berger, Henri
- Martin, James A
- Boeke, Jef D
- ORCID
- Venue
- Genetics
- Publisher
- Oxford University Press (OUP)
- Published
- 2021-04-16
- Volume
- 218
- Issue
- 1
- ISSN
- 0016-6731 (ISSN-L)
- 1943-2631
- PMC
- PMC8128383
- OpenAlex
- W3137811709
- Cited by
- 55
- References
- 1
- Citations by year
- 2026: 10 · 2025: 14 · 2024: 11 · 2023: 6 · 2022: 10 · 2021: 2 · 2019: 2
- FWCI
- 2.28
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Genetics (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- NHGRI CEGS (RM1HG009491)
- DARPA (HR0011-16-2-0002)
- Cancer Research UK Grand Challenge
- Mark Foundation for Cancer Research (C5470/A27144)
- NCI (R00CA212621)
- NINDS (R01NS100897)
- NYSTEM Pre-doctoral Training (C322560GG)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- hypoxanthine-guanine phosphoribosyltransferase, mouse embryonic stem cells
- MeSH terms
- Animals, Cloning, Molecular, DNA, Genetic Engineering, Genetic Techniques, Humans, Hypoxanthine Phosphoribosyltransferase, Genome, Sequence Analysis, DNA, Gene Transfer Techniques, Genomics, Mice, Workflow
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