Cambridge Healthtech Institute’s inaugural conference on Synergistic Use of Functional Genomics Technologies brings together experts from all areas of drug discovery and clinical research to talk about how and where functional genomics tools, particularly RNAi (RNA Interference) and CRISPR screens, can be best applied to answer the biological questions asked. Scientists and clinicians from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of functional genomics for target identification and validation, high-throughput screening, disease modeling and pathway analysis.

Final Agenda

Thursday, June 11

12:00 pm Registration

RNAi & CRISPR SCREENS FOR ONCOLOGY

2:00 Chairperson’s Opening Remarks

Charles Gersbach, Ph.D., Assistant Professor, Department of Biomedical Engineering, Center for Genomic and Computational Biology, Duke University

2:05 An RNAi Screen Utilizing a 3D Spheroid Model Suggests Destabilizing Tumor Architecture as a Potential Anti-Cancer Therapy

Geoffrey Bartholomeusz, Ph.D., Associate Professor and Director, siRNA Core Facility, Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas M.D. Anderson Cancer Center

The compact architecture of solid tumors results in hypoxia that contributes to chemotherapy/radiation resistance. We propose targeting non-neoplastic components within the tumor architecture as an effective alternative anti-cancer therapy. Using a high throughput RNAi screen and a multicellular tumor spheroid model we identified TLR4 as a potential target. Silencing TLR4 inhibited the expression of E-cadherin, altered the integrity of the spheroid architecture, reduced hypoxia, inhibited the hypoxic tolerance response and sensitized MCTS to radiation.

2:35 Modeling Cancer In vivo Using CRISPR/Cas9

Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT

Cancer genomics has revealed hundreds to thousands of mutations associated with human cancer. To test the roles of these mutations, we applied CRISPR/Cas9-mediated genome editing platform to engineer specific mutations in oncogenes and tumor suppressor genes. This results in tumorigenesis in several internal organs in mice. Our method expedites modeling of multigenic cancer with virtually any combination of mutations.

3:05 Combining RNAi and Genome Editing: New Avenues for Orthogonal Validation of Functional Genomic Profiles

Donald Apanovitch, Ph.D., Director, Functional Genomics, Oncology, Pfizer Research

RNAi-based functional genomics is a staple of gene pathway and drug target exploration. While great strides have been made in the reagents and workflows for shRNA and siRNA screening, there is a need for tools to provide rapid orthogonal validation of gene candidate that emerge from RNAi campaigns. CRISPR, CRISPRi, and CRISPRa are not only developing into primary screening platforms, they are a promising method to compliment RNAi and enhance the quality of functional genomic datasets.

3:35 Loss-of-Function Genetic Screens with Pooled CRISPR and sgRNA Libraries

Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.

Both CRISPR/Cas9 knockout and RNA interference (RNAi) have proven effective tools for targeted interruption of gene function. Large scale loss-of-function genetic screens with complex heterogeneous pools of lentiviral shRNA and sgRNA constructs directly identify genes regulating cellular responses that can then be targeted by novel therapeutics. This presentation will discuss the development of Cellecta's flexible platform to generate both RNAi and CRISPR libraries, and describe lethal interactions identified in recent screens of PDX-derived cell line pairs with our genome-wide sgRNA and shRNA libraries.

4:05 Refreshment Break in the Exhibit Hall with Poster Viewing

USING GENE EDITING FOR DRUG DISCOVERY

4:45 A Versatile Functional Genetics Platform for Malarial Parasites Enabled by Efficient CRISPR-Mediated Genome Editing

Jacquin C. Niles, M.D., Ph.D., Associate Professor of Biological Engineering, Massachusetts Institute of Technology

Functional genetics in the human malaria parasite, Plasmodium falciparum, has previously been frustratingly inefficient and time consuming. This bottleneck has limited the opportunities to validate and prioritize parasite targets to motivate the development of new therapeutics. We have now established strategies for robustly achieving controllable gene expression, and have integrated these into an experimental framework that facilitates efficient interrogation of virtually any target parasite gene using CRISPR/Cas9 editing. With these technologies, we are querying the essentiality of parasite proteins to validate them as potential drug targets and developing approaches to identify the targets of compounds having antimalarial activity.

5:15 Use of CRISPR/Cas9 Technology to Study Retinal Development and Disease

Donald Zack, M.D., Ph.D., Associate Professor of Ophthalmology and Neuroscience, Johns Hopkins University School of Medicine

Advances in human stem cell technology have made possible the differentiation of retinal eyecups in vitro. We have been using CRISPR/Cas9 technology to generate retinal cell type-specific reporter ES and iPS lines and to introduce retinal degeneration-associated mutations. These reporter lines can be used to follow retinal neuronal specification during differentiation, they allow the purification of specific cell types by sorting and immunopanning, and they also are useful for the development of drug screening assays.

5:45 Manipulating Cell Phenotype with CRISPR/Cas9-Based Epigenome Editing

Charles Gersbach, Ph.D., Assistant Professor, Department of Biomedical Engineering, Center for Genomic and Computational Biology, Duke University

New methods for programming cell phenotype have broadly enabled drug screening, disease modeling, and regenerative medicine. However many of the current protocols are slow, inefficient, and lead to heterogeneous cell populations. We are exploring genome engineering tools, such as CRISPR/Cas9-based gene regulation and epigenome editing, to more precisely reprogram gene networks and control cellular decision making. We have successfully used these tools to generate cell sources useful for many areas of biotechnology.

6:15 Close of Day

Friday, June 12

EXPLORING NEW ASSAYS & PLATFORMS

8:35 Chairperson’s Remarks

James Inglese, Ph.D., Head Assay Development & Screening Technologies, National Center for Advancing Translational Sciences (NCATS) and Adjunct Investigator, National Human Genome Institute (NHGRI)

9:30 CRISPR-based Genome Editing Tools: New Applications and Streamlined Workflows

Xiquan Liang, Ph.D., Staff Scientist, Synthetic Biology R&D, Thermo Fisher Scientific

CRISPR-Cas9 is rapidly evolving as the tool of choice for genome editing in mammalian cells. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain two steps that limit overall efficiency and general ease of use. Here we describe novel methods for rapid synthesis of gRNA and delivery of Cas9 protein/gRNA complexes into a variety of cells. This workflow enables highly efficient genome editing and biallelic knockout of multiple genes in hard-to-transfect cells in as little as three days. The reagent preparation and delivery to cells requires no plasmid manipulation so is amenable for high throughput, multiplexed genome-wide cell engineering. This CRISPR-based gene-editing platform represents the latest in the rapid evolution of editing tools for mammalian genomes by simplifying and increasing the cell engineering workflow and providing a pre-designed, ready to use platform for efficient compound screening in mammalian cell lines.

9:45 High Content RNAi Screening with Persomics: Reduction of Scale and Cost with Turnkey Printed Libraries

Neil Emans, Ph.D., CEO, Persomics USA, Inc.

RNA interference is routinely used in High Content and Phenotypic screening. However, set-up and operational costs of conventional methods remain beyond the scope of individual labs or limit screens in facilities. Persomics technology miniaturizes, accelerates and de-industrializes RNAi screening. Turnkey, preprinted libraries enable off-the-shelf focused screens and integrate with High Content platforms, and existing image analysis strategies. This lowers cost, labor, waste and overall time for any scale of screening; enabling individual labs or facilities to do more.

10:00 Evolution of Target Discovery and Validation – From Large Scale RNAi to Focused CRISPR/Cas9

Joseph Murphy, Ph.D., Director, Science, R&D, Charles River Laboratories

David F. Fischer, Ph.D., Senior Director, Biology, & Head, Leiden Site, Charles River Nederland BV

RNAi screening has been the method of choice to discover and validate novel drug targets and recently moved to human primary/patient-derived and stem cell models, for instance with our adenoviral (SilenceSelect) library. Target validation in mouse using CRISPR is also an integrated part of the discovery of new drug targets.

10:15 Coffee Break in the Exhibit Hall with Poster Viewing

11:00 Massively Parallel Combinatorial Genetics for Developing Combinatorial Therapeutics

Timothy Lu, M.D., Ph.D., Associate Professor, Synthetic Biology Group, Department of Electrical Engineering and Computer Science and Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology

We have developed technologies for the scalable and barcoded assembly of high-order combinatorial genetic libraries. These strategies enable multiplexed tracking of individual genetic combinations with next-generation sequencing in pooled screens. We have used these technologies to perform massively parallel high-order combinatorial genetics in bacteria and human cells and to modulate phenotypes relevant to important human diseases. Insights derived from massively parallel combinatorial genetics can inform the design of effective and novel combinatorial therapeutics.

11:30 Building, Using and Maintaining a Functional Genomics Arsenal

Robert Damoiseaux, Ph.D., Scientific Director, Molecular Shared Screening Resource, California NanoSystems Institute, University of California, Los Angeles

Functional Genomics is one of the most useful modalities for pre- and post-screen target identification and validation. Functional genomics platforms include cDNA, siRNA, lentiviral shRNA and CRISPR and each platform has unique advantages and constraints. Here, we will discuss these properties, talk about how to organize and build a functional genomics arsenal that contains such a comprehensive set of tools and cover some of the most common issues encountered during maintenance and use of these tools.

12:00 pm Multiplexed Gene Editing of Human Pluripotent Stem Cells

Krishanu Saha, Ph.D., Assistant Professor, Biomedical Engineering and Bioethics, University of Wisconsin, Madison

Human pluripotent stem cells possess unique ability to mature into any cell type of the body, and therefore are attractive platforms for disease modeling, toxicology and regenerative medicine research. We developed multiplexed CRISPR/Cas9 gene editing tools to insert reporters, knockout or correct candidate genes in patient-specific pluripotent stem cells. Our strategies exploit patterned biomaterial substrates with live-imaging to increase throughput and screening for desired phenotypes in edited cell populations.

12:30 Luncheon Presentation (Sponsorship Opportunity Available)

1:30 Session Break

REFINING THE USE OF CRISPR TECHNOLOGY

2:00 Chairperson’s Remarks (Sponsorship Opportunity Available)

2:05 CRISPR Tools for Gene Regulation Applications

Lei Stanley Qi, Ph.D., Assistant Professor, Department of Bioengineering, and Department of Chemical and Systems Biology, Stanford University

Precise regulation of genes for activation or repression is an important approach for cell engineering and disease modeling. We develop the bacterial CRISPR system as a toolset for sequence-specific gene regulation. The CRISPR tools enable multiplexable, inducible and high-throughput activation or repression of mammalian genes, allowing genome wide perturbation for probing gene networks. The CRISPR technology thus provides a powerful screening approach to studying gene function and chemical genomics in addition to the RNAi method.

2:35 Small Molecules Modulating CRISPR Editing

Sheng Ding, Ph.D., William K. Bowes, Jr. Distinguished Investigator, Gladstone Institute of Cardiovascular Disease; Professor, Department of Pharmaceutical Chemistry, University of California, San Francisco

CRISPR-Cas9 system has emerged as an effective tool for genome editing, but challenges remain. To enhance CRISPR-mediated gene editing, we screened chemical libraries and had identified distinct small molecules that can enhance either HDR-based gene knock-in or NHEJ-based knock-out. The use of small molecules provides a simple and effective strategy that enhances precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.

3:05 Application of Genome Editing to Generate Animal Models for Drug Discovery

Myung Shin, Ph.D., Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies in ES cells. Moreover, genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFNs and CRISPRs to generate various animal models for drug discovery programs.

3:35 Close of Conference