2016 Archived Content
Cambridge Healthtech Institute’s 13th annual two-part conference on Advances in Gene Editing and Gene Silencing will cover the latest in the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-based gene editing and RNA interference (RNAi) for use in drug discovery and for developing novel drug therapies.
Part 2 will cover the latest in the use of CRISPR/Cas9 and RNAi for functional screening. It will cover everything from assay design to data analysis when conducting low and high throughput screens and generating cellular models, both in vitro and in vivo, using CRISPR/Cas9, siRNA (small interfering RNA), and shRNA (short hairpin RNA), in a way that will evoke thought-provoking discussions and sharing of best practices. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of these diverse screening platforms for a wide range of applications.
Final Agenda
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Wednesday, September 21
11:20 am Conference Registration Open
11:25 Enjoy Lunch on Your Own
2:40 Refreshment Break in the Exhibit Hall with Poster Viewing
3:20 Chairperson’s Opening Remarks
Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
3:35 Differences between CRISPR/RNAi Necessitate Re-Thinking Analysis Algorithms
Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
Recent CRISPR screens illustrate their low rate of false positives. Although initial CRISPR screens published have been analyzed using methods developed first for RNAi screening, the data is distinctly different. We will quantify the differences in false positives between the two screening modalities and suggest how analysis methods can be better tailored to CRISPR screens.
4:20 CARD: An Interactive Web-Based Application for Comprehensive Analysis of RNAi-Screen Data
Bhaskar Dutta, Ph.D., Principal Biomedical Informatics Scientist, AstraZeneca
Screen data can be susceptible to myriad of experimental biases, many of which can be corrected by computational analysis. We have developed a web-based platform for integrated analysis and visualization of RNAi screen data, named CARD. CARD allows the user to seamlessly carry out sequential steps, including normalization, off-target analysis, integration of gene expression data, optimal thresholds for hit selection and network/pathway analysis.
5:05 Refreshment Break in the Exhibit Hall with Poster Viewing
5:40 Extracting Novel Insight from Genome-Scale Genetic Screens through Integrated Iterative Analysis
Iain Fraser, Ph.D., Investigator, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
The interpretation of genome-wide screens through annotated databases of known molecular processes has been shown to increase the validation rates and the interpretability of those screens, yet these approaches come at a cost of missing novel, non-annotated hits. We will present an approach that integrates the advantages of annotated databases with statistical and network corrections allowing novel hits to still emerge.
6:10 PANEL DISCUSSION: Strategies to Interpret and Prioritize Data from CRISPR and RNAi Screens
Moderator: Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
Panelists: Session Speakers
Each speaker will spend a few minutes sharing their ideas and experiences on tools and techniques for handling data analysis. Attendees will have an opportunity to ask questions and share their opinions.
6:40 End of Day
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Thursday, September 22
7:30 am Registration Open and Morning Coffee
8:30 Chairperson’s Remarks
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT
8:45 Strategies and Applications Using shRNA and CRISPR Technology for Identification of New Druggable Targets
Donald Apanovitch, Ph.D., Director, Functional Genomics (Oncology), Pfizer Research
Application of RNAi loss of function negative selection screens is a well-documented platform for identification of essential gene function regulating oncogenic pathways and tumorigenesis. In collaboration with the Cold Spring Harbor and the IBB group of Pfizer Oncology we have designed and validated druggable and target specific lentiviral shRNA libraries. Overview of our mir-based libraries and screening strategy will be presented along with CRISPR applications as an orthogonal tool to characterize differences in shRNA rescue experiments.
9:15 High Throughput Phenotypic Screening in Drug Discovery Using the CRISPR-Cas9 System
Greg Hoffman, Ph.D., Investigator III, Developmental & Molecular Pathways Department, Novartis Institutes for Biomedical Research
The CRISPR-Cas9 system has revolutionized high throughput forward genetics in mammalian cells. I will present our work applying CRISPR screening for the discovery of novel targets in complex models of human disease using FACS based assays. I will describe results of a systematic comparison of pooled CRISPR and shRNA libraries, insights on improving gRNA activity, and methods for validation of hits from CRISPR screens.
9:45 CRISPR Libraries for Functional Genomics: Optimizing On-Target Activity and Avoiding Off-Target Effects
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT
The use of more active and specific CRISPR libraries is critical for maximizing the productivity of genetic screens. Here I will discuss considerations for genome-wide pooled screens using conventional CRISPR knockout libraries using Cas9 from S. pyogenes. I will also discuss the use of other Cas9 proteins for this purpose, as well as orthogonal approaches, such as CRISPRa and CRISPRi technologies.
10:15 Coffee Break in the Exhibit Hall with Poster Viewing and Poster Competition Winner Announced
11:10 A High Throughput Functional Genomics Screening Approach to Identify Modulators of Nonsense-Mediated mRNA Decay to Treat Mendelian Disorders
Madhu Lal-Nag, Ph.D., Group Leader, Trans-NIH RNAi Facility, National Center for Advancing Translational Sciences, National Institutes of Health
Many genetic disorders are attributed to a premature termination codon (PTC), and cells have evolved a surveillance pathway called nonsense-mediated mRNA decay (NMD) to eliminate PTC-containing transcripts. Identification of regulatory effectors could help manipulate the NMD machinery, allowing targeted interventions for PTC-associated diseases. A genome-wide RNAi screen identified known and novel players of the NMD pathway, and the top hits were validated with CRISPR to determine the extent of pathological phenotypes for NMD.
11:40 Fas-Mediated Apoptosis Overcomes Resistance to Kras-Silencing in Lung Cancer Cells
Haiwei Mou, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Wen Xue, RNA Therapeutics Institute and Program in Molecular Medicine, University of Massachusetts Medical School
When oncogenic KRAS is suppressed by RNA interference, tumors initially regress but eventually relapse in a KRAS-independent manner. To investigate the mechanisms of Kras-independence, we established Kras knockout cells using CRISPR and found these cells are viable and exhibit increased invasive ability. Based on RNAseq, we found that Fas activation in Kras knockout cells is an “Achilles heel” that could be exploited to prevent relapse.
12:10 pm Arrayed CRISPR Screening with Synthetic crRNA Libraries for High-Throughput Loss-of-Function Studies
Louise Baskin, Senior Product Manager, Dharmacon, GE Healthcare
While pooled lentiviral sgRNAs screens have demonstrated utility for loss-of-function studies, there are limitations in the assay types that can be utilized. Arrayed screens offer the advantage of more sophisticated assays, such as high-content microscopy. High-throughput synthesis allows rapid generation of large collections of CRISPR RNAs (crRNAs) in arrayed formats. We will demonstrate the application of arrayed screening with synthetic crRNA libraries across multiple assay types, and present considerations for experimental success.
12:40 Session Break
12:50 Luncheon Presentation: Optimizing CRISPR for in vitro and in vivo Pooled Functional Genetic Screens
Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.
Well-designed pooled lentiviral-based CRISPR/Cas9 libraries provide an efficient tool for genome-wide loss-of-function genetic screening to identify genes required for biological responses or disease pathologies. Generating robust screening results with complex heterogeneous pools of sgRNA, however, requires effective and efficient targeted gene interruption. We will review improvements we have discovered that produce more consistent results with stronger signals, and show how these enhancements provide better results in both in vitro and in vivo pooled CRISPR screens.
1:30 Refreshment Break in the Exhibit Hall with Poster Viewing
2:15 Chairperson’s Remarks
Roderick Beijersbergen, Ph.D., Group Leader, Netherlands Cancer Institute and Head, NKI Robotics and Screening Center
2:20 Large Scale CRISPR Screens for Discovery of Genotype Specific Combination Therapies
Roderick Beijersbergen, Ph.D., Group Leader, Netherlands Cancer Institute and Head, NKI Robotics and Screening Center
The complexity and heterogeneity of cancer, the extensive crosstalk between pathways and unanticipated feedback control are underlying the limited long-term success of targeted therapeutics in the clinic. We use large-scale functional genomic screening technologies including shRNA/CRISPR-based gene editing in combination with (clinically) relevant screening models for the identification of dependencies in the context of specific genetic alterations. Using this platform we have identified novel effective drug combinations that are currently in the clinic.
2:50 GPCR-Mediated cAMP as an Immune Checkpoint in Cancer Identified by RNAi Screening
Tillmann Michels, Head of Research Group, Immune Checkpoint Inhibitors, Department of Interventional Immunology, Regensburg Center for Interventional Immunology; Member, Department of Translational Immunology, German Cancer Research Center
Immune checkpoint blockade has revolutionized cancer therapy. Our group screened several tumor entities in conjunction with tumor-infiltrating lymphocytes (TILs) for novel immune modulators. We found that many immune checkpoints are tumor-restricted but the underlying mechanisms are shared between tumor entities. These mechanism range from inhibition of TIL-mediated apoptosis to cAMP-mediated inhibition of TILs.
3:20 Session Break
3:30 CRISPR-Based Mutagenesis Approach for Cancer Drug Target Identification
Junwei Shi, Ph.D., Assistant Professor, Department of Cancer Biology, University of Pennsylvania School of Medicine
CRISPR-Cas9 genome editing technology provides high-throughput genetic knockout screening strategies for cancer therapeutic target identification. We recently reported a domain-focused CRISPR screening approach to nominate protein domains that would sustain cancer cell growth and are suitable for drug targeting. Here, I will present an optimized CRISPR system, which could achieve robust genome editing efficiency in a broad variety of human cancer cell lines.
4:00 Applying Functional Genomics in Mouse Models of Human Cancer
Yejing Ge, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Elaine Fuchs, Department of Mammalian Cell Biology and Development, Rockefeller University
Using skin as paradigm, we describe the first panoramic view of microRNAs during the development, homeostasis and malignant transformation of skin epithelium. We devised lentiviral microRNAs expression platform and conducted pooled in vivo functional screens for oncomiRs in mice. Empowered by mouse genetics and high throughput approaches, we unveiled a rich set of putative microRNAs that drive skin malignancy, and their oncogenic targets.
4:30 A CRISPR/Cas9 System to Increase Homologous Recombination Repair
Ciro Bonetti, Ph.D., Postdoctoral Scientist, Laboratory of Dr. Andrea Ventura, Memorial Sloan-Kettering Cancer Center
CRISPR/Cas9 genome editing technology has been shown to be very effective to perform inactivation or activation of specific genes. However, engineering precise genomic modifications remains a challenge due to the low efficiency of repair of the double strand breaks through the homologous recombination pathway. Here, I will discuss a novel method to skew the repair away from non-homologous end-joining towards homologous repair pathway.
5:00 Close of Conference
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