CHEMOGENETIC & OPTOGENETIC TECHNOLOGIES FOR PROBING MOLECULAR & CELLULAR NETWORKS

The goal of our laboratory is to develop and disseminate transformative technologies for probing molecules and functional networks at both the sub-cellular and multi-cellular level, leveraging our laboratory’s unique strengths in protein engineering, directed evolution, proteomics, chemical synthesis, computational design, and microscopy. While we strive to develop technologies that are broadly applicable across biology, we also pursue applications of our methods to neuroscience and mitochondrial biology in our own laboratory and through collaborations. Our research program is broadly divided into three areas: (1) molecular recorders for scalable, single-cell recording of past cellular events; (2) molecular editors for the precise manipulation of cellular biomolecules, pathways, and organelles; and (3) proximity labeling for unbiased discovery of functional molecules.

MOLECULAR RECORDERS

Molecular recorders are scalable, single cell technologies that fulfill a longstanding need in biology by creating stable records of past cellular events, simultaneously applicable across thousands of cells. The “records” can be read out by RNA sequencing, FACS, imaging, or if desired, altered cellular properties and physiology.

FLiCRE, FLARE, and scFLARE are molecular calcium recorders designed as membrane-anchored transcription factors whose proteolytic release and activation are gated by calcium and blue light. Because activated neurons increase their cytosolic Ca+2, FLiCRE can provide a stable snapshot of transiently activated neural circuits if a brain-implanted optical fiber delivers a brief (60-second) light pulse during a behavior of interest. By coupling the transcriptional read-out of FLiCRE with high-throughput single-cell RNA sequencing and subsequently, with expression of a channellrhodopsin for direct manipulation of FLiCRE-tagged neurons, we discovered a new striatal cell type that drives aversion in mice.

We have also designed molecular recorders for GPCR activation, glutamate and dopamine release, intracellular protein-protein interactions, and protein trafficking. HiLITR is a molecular recorder that gives mCherry expression when its engineered protease and transcription factor are colocalized to the same organelle. We used HiLITR in combination with an sgRNA library in a CRISPRi human cell line to identify genes that regulate the targeting of mitochondrial and ER tail-anchored (TA) proteins. Our screen led to the unexpected discovery of SAE1 (SUMO activating enzyme) as a regulator of tail-anchored (TA) protein insertion into mitochondrial membranes and EMC10 as an antagonist in the insertion of TA proteins into ER membranes.

In ongoing work, we are developing molecular recorders that detect extracellular antigens, and we are exploring novel recorder designs that enable the recording of dynamic cell histories.

MOLECULAR EDITORS

In addition to molecular recorders that store cell history in a format readable by FACS and scRNA-seq, we are building molecular editors that can be used for the precise manipulation of cellular biomolecules, pathways, and organelles.

For example, in the area of neuroscience, we are creating optogenetic and chemogenetic tools that can be used for the temporally-precise inactivation of defined cell-cell contacts. Methods for manipulating the activity of genetically-defined cells (e.g., via channelrhodopsin and DREADDs) have revolutionized systems neuroscience by enabling scientists to probe the causal relationships between specific cell populations and behavior. However, no methods exist for the precise manipulation of defined cell-cell contacts, which are the fundamental unit of neural computation. We are developing two-part tools in which trans-synaptic recognition leads to specific and local synapse inactivation. These tools could be impactful for dissecting the causal basis of behavior at the level of neural connections.

For editing of biomolecules, we are focused on the engineering of enzymes that can manipulate DNA, RNA, and proteins precisely in living cells. For example, we used directed evolution to enhance the turnover and reprogram the specificity of the workhorse protease TEV.

PROXIMITY LABELING

Proximity labeling mediated by promiscuous enzymes such as APEX and TurboID have enabled proteomic and transcriptomic mapping and discovery in living cells with unprecedented spatial and temporal resolution. Though proximity labeling technology is fairly mature, we are pursuing several new directions which may expand the breadth and impact of this method. First, we are combining orthogonal proximity labeling enzymes targeted to different compartments, and performing tandem labeling in cells and tissue, in order to map the trafficking of proteomes both within cells and between cells (“TransitID”). Second, we are developing novel proximity labeling enzymes and labeling chemistries. Third,  we are developing “functional proximity labeling” in which proximity labeling is combined with other methods for enrichment of specific protein functional classes in order to simultaneously map proteome localization, timing, and function in a single experiment. Fourth, we are developing single-cell versions of our proximity labeling methods such as APEX-seq for high-resolution transcriptome mapping.