Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dosedependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

Proliferation history and transcription factor levels drive direct conversion.

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies.

Synthetic gene circuits as tools for drug discovery.

Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays.

Engineering cell fate: Applying synthetic biology to cellular reprogramming.

Cellular reprogramming drives cells from one stable identity to a new cell fate. By generating a diversity of previously inaccessible cell types from diverse genetic backgrounds, cellular reprogramming is rapidly transforming how we study disease. However, low efficiency and limited maturity have limited the adoption of in vitro-derived cellular models. To overcome these limitations and improve mechanistic understanding of cellular reprogramming, a host of synthetic biology tools have been deployed. Recent synthetic biology approaches have advanced reprogramming by tackling three significant challenges to reprogramming: delivery of reprogramming factors, epigenetic roadblocks, and latent donor identity. In addition, emerging insight from the molecular systems biology of reprogramming reveal how systems-level drivers of reprogramming can be harnessed to further advance reprogramming technologies. Furthermore, recently developed synthetic biology tools offer new modes for engineering cell fate.