Postdoctoral Fellow, University of California, San Francisco, Cellular and Molecular Pharmacology (2013)
Ph.D., Harvard University, Biological and Biomedical Sciences (2005)
B.S., University of Wisconsin, Madison, Biochemistry and Molecular Biology (1996)
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.
As an example of this work, we have used high-coverage shRNA libraries and genetic interaction maps to explore the biology of the retrograde-trafficking toxin ricin. We have identified new proteins involved in retrograde trafficking, new components and functions for the vesicle tethering TRAPP complex, and a new role for the small ribosomal subunit RPS25, all of which are under current study.
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
Much of the work we do utilizes genetic screens enabled by novel high-coverage shRNA libraries (~25 shRNAs/gene) we have developed. The high coverage greatly reduces false positive and false negative results that have plagued traditional RNAi strategies. We use a pooled format that can be rapidly screened in large bioreactors, and analyze screens by deep sequencing to quantify changes in shRNA abundance.
Importantly, our library design allows us to knock down pairs of genes, and has facilitated the first systematic genetic interaction maps in mammalian cells. Using these maps, we can understand coordinated gene functions, predict new functions for uncharacterized genes, and identify drug targets. They also allow us to quickly identify synergistic interactions under stress conditions that we hope to exploit for combination therapies. Together with a broad network of collaborators, we are continuing to use this platform to develop new technologies for disrupting and interrogating gene function on a genomic scale.
- Advanced Genetics
GENE 205 (Win)
- Biology and Applications of CRISPR/Cas9: Genome Editing and Epigenome Modifications
BIOS 268 (Spr)
Independent Studies (11)
- Biomedical Informatics Teaching Methods
BIOMEDIN 290 (Aut, Win, Spr)
- Directed Reading and Research
BIOMEDIN 299 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Genetics
GENE 299 (Win, Spr)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
GENE 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOMEDIN 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
GENE 370 (Win, Spr)
- Supervised Study
GENE 260 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
GENE 199 (Win, Spr, Sum)
- Biomedical Informatics Teaching Methods
- Prior Year Courses
Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens
CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.
View details for DOI 10.1038/ncomms15178
View details for Web of Science ID 000400851300001
View details for PubMedID 28474669
Population- and individual- specific regulatory variation in Sardinia
2017; 49 (5): 700-?
Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.
View details for DOI 10.1038/ng.3840
View details for Web of Science ID 000400051400010
View details for PubMedID 28394350
Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions
2017; 35 (5): 463-?
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.
View details for DOI 10.1038/nbt.3834
View details for Web of Science ID 000400809800021
View details for PubMedID 28319085
Human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy.
Current opinion in biotechnology
2017; 48: 127-134
The development of broad-spectrum, host-acting antiviral therapies remains an important but elusive goal in anti-infective drug discovery. To replicate efficiently, viruses not only depend on their hosts for an adequate supply of pyrimidine nucleotides, but also up-regulate pyrimidine nucleotide biosynthesis in infected cells. In this review, we outline our understanding of mammalian de novo and salvage metabolic pathways for pyrimidine nucleotide biosynthesis. The available spectrum of experimental and FDA-approved drugs that modulate individual steps in these metabolic pathways is also summarized. The logic of a host-acting combination antiviral therapy comprised of inhibitors of dihydroorotate dehydrogenase and uridine/cytidine kinase is discussed.
View details for DOI 10.1016/j.copbio.2017.03.010
View details for PubMedID 28458037
The mTOR Complex Controls HIV Latency
CELL HOST & MICROBE
2016; 20 (6): 785-797
A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.
View details for DOI 10.1016/j.chom.2016.11.001
View details for Web of Science ID 000392843500014
View details for PubMedID 27978436
View details for PubMedCentralID PMC5354304
Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.
Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.
View details for DOI 10.1038/nmeth.4038
View details for PubMedID 27798611
E2A-PBX1 remodels oncogenic signaling networks in B-cell precursor acute lymphoid leukemia.
There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR(+)) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR(+)/E2A-PBX1(+) leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.
View details for PubMedID 27758892
Bithionol blocks pathogenicity of bacterial toxins, ricin, and Zika virus
Diverse pathogenic agents often utilize overlapping host networks, and hub proteins within these networks represent attractive targets for broad-spectrum drugs. Using bacterial toxins, we describe a new approach for discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pathways. This approach can be widely used, as it combines genetic-based target identification with cell survival-based and protein function-based multiplex drug screens, and concurrently discovers therapeutic compounds and their protein targets. Using B-lymphoblastoid cells derived from the HapMap Project cohort of persons of African, European, and Asian ancestry we identified host caspases as hub proteins that mediate the lethality of multiple pathogenic agents. We discovered that an approved drug, Bithionol, inhibits host caspases and also reduces the detrimental effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, Botulinum neurotoxin, ricin, and Zika virus. Our study reveals the practicality of identifying host proteins that mediate multiple disease pathways and discovering broad-spectrum therapies that target these hub proteins.
View details for DOI 10.1038/srep34475
View details for Web of Science ID 000384291100001
View details for PubMedID 27686742
View details for PubMedCentralID PMC5043268
Translation readthrough mitigation
2016; 534 (7609): 719-?
A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.
View details for DOI 10.1038/nature18308
View details for Web of Science ID 000378676000044
View details for PubMedID 27281202
View details for PubMedCentralID PMC5054982
Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes
2016; 34 (6): 634-636
We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.
View details for DOI 10.1038/nbt.3567
View details for Web of Science ID 000377846400030
View details for PubMedID 27159373
View details for PubMedCentralID PMC4900911
Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
NATURE CHEMICAL BIOLOGY
2016; 12 (5): 361-?
Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.
View details for DOI 10.1038/NCHEMBIO.2050
View details for Web of Science ID 000374322800011
View details for PubMedID 27018887
View details for PubMedCentralID PMC4836973
Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.
Nucleic acids research
2014; 42 (19): 12169-12176
The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3' regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency. These studies provide new rules that should significantly improve on siRNA/shRNA design.
View details for DOI 10.1093/nar/gku854
View details for PubMedID 25270879
Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps
2014; 9 (8): 1825-1847
Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and for defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of 'hit' genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each round of screening can be implemented in ∼2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and we present complete experimental procedures, as well as a full computational analysis suite for the identification of hits in pooled screens and generation of genetic interaction maps. Although the protocol outlined here was developed for our original shRNA-based approach, it can be applied more generally, including to CRISPR-based approaches.
View details for DOI 10.1038/nprot.2014.103
View details for Web of Science ID 000340039700004
View details for PubMedID 24992097
Next-Generation NAMPT Inhibitors Identified by Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens.
Chemistry & biology
2013; 20 (11): 1352-1363
Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.
View details for DOI 10.1016/j.chembiol.2013.09.014
View details for PubMedID 24183972
A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility.
2013; 152 (4): 909–22
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
View details for DOI 10.1016/j.cell.2013.01.030
View details for PubMedID 23394947
Rapid creation and quantitative monitoring of high coverage shRNA libraries.
2009; 6 (6): 443–45
Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.
View details for DOI 10.1038/nmeth.1330
View details for PubMedID 19448642