Editing a Zinc Finger Inducible Promoter Via Recombinases to Change Expression

Engineering mammalian cells provides key insight into translatable research, but it requires precise control of gene expression to recapitulate and manipulate biology. Furthermore, natural systems often exhibit expression changes over time which can be difficult to precisely control with current synthetic biology tools.

Toolkits like Composable Mammalian Elements of Transcription (COMET) provide design rules to vary the strength of a zinc finger (ZF) dependent promoter, but their features are often statically encoded. Furthermore, current methods of gene expression control are binary or transient, limiting the extent to which we can engineer and recast biological systems.

Therefore, we explore a new method that can introduce a stable change in gene expression between two non-zero levels upon a stimulus. To this end, we apply the COMET toolkit and the Cre-Lox recombinase system to edit the DNA length between the last ZF binding site and the TATA box to increase gene expression.

In transient transfection, excising the spacer causes various fold increases in the downstream reporter gene expression, which corresponds with the previously established COMET rules. We showed that longer spacer lengths start at lower levels of gene expression, but all reporters converge at the same expression level after excision.

Due to toxicity and challenges associated with delivering multiple plasmids, we sought to consolidate the ZF expression and the reporter gene into one cassette. Despite maintaining the same promoter architecture, we found that differences in DNA syntax affect expression profiles of both ZF and reporter gene. Tandem expression yielded the best expression pattern, with overall highest expression levels and no bimodal distribution seen with other orientations.

In addition to varying the spacer length, ZF expression can modify the output gene expression level. This modularity of our design allows multiple ways to alter the reporter gene expression, which can enable multiplexing a variety of inputs to confer downstream changes. Using multiple parameters can be useful in constructing gene circuits with editable setpoint levels. To demonstrate the utility of both ZF-expression mediated expression with editing spacer length, we hope to use these promoters to change the setpoint of a previously published antisense-based integral controller.

This work using recombinases to edit the DNA composition of a ZF-dependent promoter allows heritable changes to tune gene expression. Altogether, these designs can add to the synthetic biology toolkit by facilitating complex biological behaviors in mammalian cell engineering.