Engineering digitizer circuits for chemical and genetic screens in human cells

Abstract

Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.

Programmable Mixed-Signal Biocomputers in Mammalian Cells

“…A significant challenge this work seeks to address is the inability to couple robust logic computation with tunable levels of circuit output. Many circuits benefit from the digitization of an analog input signal [21] [22] [23] [24], as it allows for the propagation of signals through multiple components with high signal and low noise. However, natural biological signals operate in the analog space, where different levels or combinations of input signals yield variable levels of the output response.…”

“…However, the function of this circuit is quantitatively accurate ( = 2.93°), and would likely perform as intended if there was no need to reuse fluorescent reporters in different states. Both distributed and combined versions of the CREATE platform should be advantageous for many applications, such as drug screening platforms where SSRs are driven from inducible promoters to digitize and amplify effects on biological signals related to the tested compounds [24] [23].…”

“…Synthetic biological signaling in the analog space often suffers from a loss of signal as it is transferred between circuit elements. Previous work has shown that digitizing these signals can lead to amplification of weak input signals [23] [24] [22] [20] [21] and allows for high fidelity information transfer despite passing multiple circuit modules. By coupling digital processing circuits with an analog output signal in this work, we expand the use of digitizer modules to settings where analog input signals are received, and analog output signals are required.…”

“…Previous attempts to combine the analog programmability of TFs and regulatory RNAs with SSR-mediated digital logic and memory have focused on placing SSRs under the control of a TF [18, 20] [21–24] ( Figure 1B , top ). While this design allows for potent signal amplification and information transfer with a high signal-to-noise ratio, it is fundamentally limiting in scope for the types of computation that may be performed.…”

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts

“…Conversely, recombination sites with enhanced activity are needed for the development of specific efficient gene editing tools. For example, highly active site-specific recombination could boost the efficiency of tools for optogenetics 36 , 76 , 94 , 95 , cell lineage tracing 96 , gene regulation 82 , genetic circuit design 4 , 97 and gene expression cascades 3 . Moreover, the 16 orthogonal recombination sites that we identified in this study might help to further expand these existing technologies, as they can be used simultaneously without cross-reactivity.…”