Ng, A. H. | bioRxiv Science

bioRxiv2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

synthetic biology↗

bioRxiv2016

The Effect of Compositional Context on Synthetic Gene Net-works

It is well known that synthetic gene expression is highly sensitive to how comprising genetic elements (promoter structure, spacing regions between promoter and coding sequences, ribosome binding sites, etc.) are spatially configured. An important topic that has received far less attention is how the physical layout of entire genes within a synthetic gene network affects their individual expression levels. In this paper we show, both quantitatively and qualitatively, that compositional context can significantly alter expression levels in synthetic gene networks. We also show that these compositional context effects are pervasive both at the transcriptional and translational level. Further, we demonstrate that key characteristics of gene induction, such as ultra-sensitivity and dynamic range, are heavily dependent on compositional context. We postulate that supercoiling can be used to explain these interference effects and validate this hypothesis through modeling and a series of in vitro supercoiling relaxation experiments. On the whole, these results suggest that compositional context introduces feedback in synthetic gene networks. As an illustrative example, we show that a design strategy incorporating compositional context effects can improve threshold detection and memory properties of the toggle switch.

synthetic biology↗