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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. Cell-based transcriptional reporters are an invaluable part of highthroughput screening, but many such reporters have weak or transient responses. Here, the authors describe a digitizer circuit for amplifying reporter activity, increasing sensitivity, and retaining memory of pathway activation.

SUBMITTER: Wong N

PROVIDER: S-EPMC8536748 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOne problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits.ResultsWe first measured the stability of two BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and the mutations that caused their loss-of-function. The first circuit, T9002, loses function in less than 20 generations and the mutation that repeatedly causes its loss-of-function is a deletion between two homologous transcriptional terminators. To measure the effect between transcriptional terminator homology levels and evolutionary stability, we re-engineered six versions of T9002 with a different transcriptional terminator at the end of the circuit. When there is no homology between terminators, the evolutionary half-life of this circuit is significantly improved over 2-fold and is independent of the expression level. Removing homology between terminators and decreasing expression level 4-fold increases the evolutionary half-life over 17-fold. The second circuit, I7101, loses function in less than 50 generations due to a deletion between repeated operator sequences in the promoter. This circuit was re-engineered with different promoters from a promoter library and using a kanamycin resistance gene (kanR) within the circuit to put a selective pressure on the promoter. The evolutionary stability dynamics and loss-of-function mutations in all these circuits are described. We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.ConclusionsA wide variety of loss-of-function mutations are observed in BioBrick-assembled genetic circuits including point mutations, small insertions and deletions, large deletions, and insertion sequence (IS) element insertions that often occur in the scar sequence between parts. Promoter mutations are selected for more than any other biological part. Genetic circuits can be re-engineered to be more evolutionary robust with a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

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Engineering digitizer circuits for chemical and genetic screens in human cells

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