The manipulation of DNA is central to advancing our understanding of fundamental biological principles and to realizing the promise of synthetic biology.
1. Development of tools for mammalian ‘genome writing’. Despite the advent of a vast suite of genome editing tools, we have lacked the tools to make multiple changes over long genomic windows or introduce large amounts of novel genetic material into cells at once. The bottom-up synthesis and introduction of long DNA constructs into cells has the potential to address this gap. However, these ‘synthetic genomics’ efforts have largely been restricted to microbial systems. I played a key role in the development of tools for the synthesis of large sections of mammalian genomes (>100kb) in yeast via homologous recombination and their subsequent delivery to mammalian cells in a controlled, site-specific manner.
Publications: Pinglay et al., Science 2022; Mitchell et al., Genetics 2021; Brosh et al., PNAS 2021; German et al., BioRxiv 2022.
2. Synthetic reconstitution to study regulation at HoxA and beyond!
I developed a ‘synthetic regulatory reconstitution’ framework for the study of gene regulation inspired by the bottom-up approaches of synthetic biology and biochemical reconstitution. The HoxA cluster is a master regulator of development. Multiple regulatory modules have been implicated in controlling HoxA in response to developmental signals. I constructed variants encoding combinations of regulatory elements that had been previously implicated in HoxA control. Subsequent delivery to an ectopic genomic locus directly tested the ability of these variant clusters to independently reconstitute distinct aspects of HoxA regulation, revealing the relative contributions of various locus constituents. This is a generalizable strategy to understand the logic of gene regulation at any locus. As part of the NIH CEGS funded ‘Dark Matter Project’, I am currently extending this approach to understand mammalian X-inactivation, the emergence of phenotypic novelty in moles, and to construct regulatory landscapes from first-principles in Drosophila.
Publications: Pinglay et al., Science 2022
My appearance on The Genomics Lab podcast discussing synthetic regulatory reconstitution
3. Genome writing as a platform for fundamental discovery and cellular engineering. The ability to synthesize and integrate large DNA constructs into mammalian cells portends to have an impact analogous to genome editing in fundamental research as well as in industrial applications, especially when combined with the latest advances in single-cell sequencing and protein design. I am interested in using these tools to construct better disease models, understand our developmental blueprint and probe the limits of genome structure. Mammalian genome writing allows for the introduction of sophisticated genetic programs into mammalian cells to endow them with novel behaviors for cell therapy, biologics production and cultivated meat. Please get in touch to discuss any ideas you may have!
Publications: Trolle et al., BioRxiv 2022 and more to come!
Complete list of primary research articles (*indicates equal contribution, # indicates corresponding author):
1. Pinglay, S.* , M. Bulajic*, D. Rahe, E. Huang, R. Brosh, N. E. Mamrak, B. R. King, S. German, J. A. Cadley, L. Rieber, N. Easo, T. Lionnet, S. Mahony, M.T. Maurano, L.J. Holt, E. O. Mazzoni# and J.D. Boeke#. (2022). “Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation”. Science
2. German, S., S. Pinglay, B. Camellatto, D. Fenyo, J.D. Boeke#. (2022). “MenDEL: automated search of BAC sets covering long DNA regions of interest”. BioRxiv
3. Trolle, J.*, R.M. McBee*, A. Kaufman, S. Pinglay, H. Berger, S. German, L. Liu, M.J. Shen, X. Guo, J.A. Martin, M. Pacold, D.R. Jones, J.D. Boeke#, H.H Wang#. (2021). “Resurrecting essential amino acid biosynthesis in a mammalian cell”. BioRxiv
4. Mitchell, L. A., L. H. McCulloch*, S. Pinglay*, H. Berger, M. Bulajic, J. A. Martin, M. S. Hogan, E. O. Mazzoni, M. T. Maurano and J. D. Boeke# (2021). "De novo assembly, delivery and expression of a 101 kb human gene in mouse cells." Genetics
5. Brosh, R*., J. M. Laurent*, R. Ordonez, E. Huang, M. S. Hogan, A. M. Hitchcock, L. A. Mitchell, S. Pinglay, J. A. Cadley, R. D. Luther, D. M. Truong, J.D. Boeke#, M. T. Maurano. (2021) “A versatile platform for locus-scale genome rewriting and verification.” PNAS
6. Sang, D., S. Pinglay, R. P. Wiewiora, M. E. Selvan, H.J. Lou, J. D. Chodera, B. E. Turk, Z. H. Gümüş, and L. J. Holt#. (2019) "Ancestral Reconstruction Reveals Mechanisms of Erk Regulatory Evolution." eLife
7. Delarue, M*., G. P. Brittingham*, S. Pfeffer*, I. V. Surovtsev, S. Pinglay, K. J. Kennedy, M. Schaffer, J. I. Gutierrez, D. Sang, G. Poterewicz, J. K. Chung, J. M. Plitzko, J. T. Groves, C. Jacobs-Wagner, B. D. Engel# and L. J. Holt# (2018). "mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding." Cell
8. Kuang, Z., S. Pinglay, H. Ji# and J. D. Boeke# (2017). "Msn2/4 regulate expression of glycolytic enzymes and control transition from quiescence to growth." eLife
9. Gowda, R. N., R. Redfern, J. Frikeche, S. Pinglay, J. W. Foster, C. Lema, L. Cope and S. Chakravarti# (2015). "Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in Pglyrp-2, -3 and -4." PLOS ONE
Other publications:
1. Laurent, J., S. Pinglay, L. A. Mitchell and R. Brosh (2019) "Probing the dark matter of the human genome with big DNA." The Biochemist