Current Research and Scholarly Interests
Endocytic Pathogens as Tools and Targets
Endocytic pathogens such as protein aggregates, viruses, protein toxins, and bacteria have evolved remarkable ways to enter the cell, disrupt homeostasis, and cause cell death. We use these agents both as probes to understand normal cellular trafficking and signaling events, and to find key targets for therapy.
Stress Signaling to the Cell Death Machinery
Cells have elaborate mechanisms of sensing diverse stresses (oxidative damage, nutrient deprivation, DNA breaks, etc), and must either repair damage or induce cell death. Misregulation of these pathways results in diseases such as cancer and Alzheimer’s. We would like to understand how these signals connect to the death pathway in health and disease in order to improve therapies.
Technology Development and Genetic Interaction Maps
Customized genome-scale gene perturbation libraries: Much of the work we do utilizes genetic screens enabled by novel high-coverage CRISPR/Cas9 libraries (10 sgRNAs/gene) and shRNA libraries (25 shRNAs/gene) we have developed. The high coverage greatly reduces false positive and false negative results. Our platform allows for easy creation of new library designs, and we use a pooled format that can be used to rapidly screen genome-scale libraries in ~1-2 weeks. Libraries can then be analyzed by deep sequencing to quantify changes in sgRNA/shRNA abundance.
Systematic comparison of gene perturbation technologies: We have systematically compared the ability of genome-wide RNAi and CRISPR/Cas9 deletion screens to identify drug targets and essential genes, and have found important differences in their outputs. For example, while both screens perform well in detecting a gold standard set of essential genes, they can identify distinct essential biological processes. Moreover, each technology exhibits its own set of off-target effects and limitations in on-target efficacy.
Using what we have learned about these technologies, we recently created new genome-wide CRISPR libraries that incorporate a number of improved sgRNA features and controls. With a novel statistical framework (casTLE) we can model on- and off-target effects accurately, markedly improve hit detection, and even combine results from screens to improve performance and further limit false positives and false negatives. These studies have demonstrated the utility of parallel screening approaches using complementary technologies to reveal a more complete biological picture.
Systematic genetic interaction maps: We have also developed strategies to systematically knock down/knock out pairs of genes. This has facilitated some of the first systematic genetic interaction maps in mammalian cells. Using these maps, we can understand coordinated gene functions and predict new functions for uncharacterized genes. They also allow us to quickly identify synergistic interactions under stress conditions that we hope to exploit for combination therapies.
Directed evolution using dCas9-targeted somatic hypermutation (CRISPR-X): More recently, we developed a strategy to re-purpose the somatic hypermutation machinery used in antibody diversification to create targeted populations of point mutations. Using dCas9 to recruit a hyperactive variant of the deaminase AID, we can target diverse point mutations within an ~100bp window centered on the sgRNA PAM site. These mutant populations can then be subjected to selection to evolve proteins with improved function or to map the sites of drug-protein interactions. For example, by tiling mutations across PSMB5, we could map known and novel mutations that affect binding to the chemotherapeutic bortezomib.