CRISPR-Cas-Mediated Tethering Recruits the Yeast Mating-Type Locus to the Nuclear Periphery but Fails to Silence Gene Expression.

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the and loci, both of which are well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the locus but does not detectably silence reporter gene expression.

Multi-input chemical control of protein dimerization for programming graded cellular responses.

Chemical and optogenetic methods for post-translationally controlling protein function have enabled modulation and engineering of cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed, we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics.

A Chemically Disrupted Proximity System for Controlling Dynamic Cellular Processes.

Chemical methods that allow the spatial proximity of proteins to be temporally modulated are powerful tools for studying biology and engineering synthetic cellular behaviors. Here, we describe a new chemically controlled method for rapidly disrupting the interaction between two basally colocalized protein binding partners. Our chemically disrupted proximity (CDP) system is based on the interaction between the hepatitis C virus protease (HCVp) NS3a and a genetically encoded peptide inhibitor.

CRISPR-Cas-Mediated Chemical Control of Transcriptional Dynamics in Yeast.

Synthetic CRISPR-Cas transcription factors enable the construction of complex gene-expression programs, and chemically inducible systems allow precise control over the expression dynamics. To provide additional modes of regulatory control, we have constructed a chemically inducible CRISPR activation (CRISPRa) system in yeast that is mediated by recruitment to MS2-functionalized guide RNAs. We use reporter gene assays to systematically map the dose dependence, time dependence, and reversibility of the system.

"Examining RAS pathway rewiring with a chemically inducible activator of RAS".

RAS signaling pathways govern diverse cellular processes, are dynamic, and exhibit marked plasticity. Yet, these features also present a considerable obstacle to their study. Here, we report the use of a recently described RAS rheostat, Chemically Inducible Activator of RAS (CIAR), to study two poorly understood phenomena in RAS biology.

A computationally engineered RAS rheostat reveals RAS-ERK signaling dynamics.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function.