UC Davis

A commonly overlooked problem associated with current procedures in molecular biology that seek to gain information about the state of the genome, epigenome, transcriptome, proteome, or metabolome within a particular cell state is the necessary destruction of cell populations to extract genomic material. If a particular cell population is critical or valuable, this is a non-ideal reality of obtaining desired data. While highly sensitive single cell methodologies have evolved in recent years, many procedures still analyze bulk cell populations, often containing a high degree of complexity and heterogeneity. In addition, the information gathered from these types of multiple-omics studies—even at the single cell level—is not gathered in real-time, but after the fact as a snapshot of a particular cell state. One approach to solving these issues with current molecular biology multiple-omics techniques is to use biosensing technology. Biosensors are analytical devices that detect a specific target molecule within a heterogeneous molecular background. These tools operate in real-time or near real-time conditions to provide rapid detection capabilities for a wide range of molecular targets noninvasively (in situ) with high sensitivity and specificity.

Our approach to developing a new generation of live cell biosensors is based on a split reporter system, where a signal-producing protein is truncated into distinct segments which have very little to no activity individually but can recombine at a specific interface to produce a full-length, functional version of the protein. To this end, we have developed split luminescent reporter biosensors for both individual DNA sequences and chromatin loops anchored by cohesin and CTCF in living cells. Our platform for development of these biosensors has involved a combination of in silico rational design based on an independently optimized truncation of NanoLuc luciferase (Binary Technology of NanoLuc luciferase or NanoBiT system) and catalytically-inactive Cas9 (dCas9) enzymes from Streptococcus pyogenes and Staphylococcus aureus guided to bind target DNA and chromatin loop anchor regions via single guide RNAs (sgRNAs), functional testing in a variety of cell lines, and statistical analysis of signal-to-background data for comparison between on-target and background conditions and determination of sensitivity and specificity. This split reporter platform can be extended to other fluorescent, luminescent, and enzymatic reporters and energy transfer-based systems to yield a powerful modular design, construction, and validation methodology for live cell biosensors. In addition, facilitated by the use of additional biorecognition elements in biosensor design, we envisage application of our live cell biosensing platform to detection of additional molecular targets including various epigenetic marks, other nucleic acids such as RNA or R-loops, and small molecules or ligands. If applied in vitro, this platform could feasibly be used to detect pathogen genomic material and environmental contaminants, aiding in monitoring and screening efforts to improve public health across a wide range of fields.