Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent 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.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Scalable biological signal recording in mammalian cells using Cas12a base editors.

Biological signal recording enables the study of molecular inputs experienced throughout cellular history. However, current methods are limited in their ability to scale up beyond a single signal in mammalian contexts. Here, we develop an approach using a hyper-efficient dCas12a base editor for multi-signal parallel recording in human cells. We link signals of interest to expression of guide RNAs to catalyze specific nucleotide conversions as a permanent record, enabled by Cas12's guide-processing abilities. We show this approach is plug-and-play with diverse biologically relevant inputs and extend it for more sophisticated applications, including recording of time-delimited events and history of chimeric antigen receptor T cells' antigen exposure. We also demonstrate efficient recording of up to four signals in parallel on an endogenous safe-harbor locus. This work provides a versatile platform for scalable recording of signals of interest for a variety of biological applications.

Multiple Input Sensing and Signal Integration Using a Split Cas12a System.

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.