Gene editing has very rapidly established its importance as a research/screening tool in drug discovery, and promises its utility downstream in drug development, cell therapy and bioprocessing, as well. Cambridge Healthtech Institute’s third annual symposium on New Frontiers in CRISPR-based Gene Editing will bring together experts from all aspects of basic science and clinical research to talk about the recent progress made in gene editing and it’s growing applications. Knowing the strengths and limitations, how does one decide when to use the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system, as opposed to alternate gene editing systems? What is being done to overcome some of the inherent challenges in design, delivery and off-target effects, associated with CRISPR and some of its alternatives that are being developed? Experts from pharma/biotech, academic and government labs will share their experiences leveraging the utility of CRISPR-based gene editing for diverse applications including, creating cell lines and knock-out models, for functional screening, cellular pathway visualization, and for therapeutics.
Thursday, February 23
7:00 am Registration and Morning Coffee
8:25 Chairperson’s Opening Remarks
Bruce R. Conklin, M.D., Investigator, Gladstone Institute of Cardiovascular Disease and Professor, Division of Genomic Medicine, University of California, San Francisco
8:30 FEATURED PRESENTATION: CRISPR-Based Screens in Human Cardiac Disease Models
Bruce R. Conklin, M.D., Investigator, Gladstone Institute of Cardiovascular Disease and Professor, Division of Genomic Medicine, University of California, San Francisco
We have developed efficient methods to edit one residue at a time in human iPS cells. These “isogenic” lines form models that are yielding phenotypes that help to explain the molecular basis of several human diseases. Recently, we developed CRISPR-inhibition (CRISPRi) cell lines for high-throughput gene inactivation of thousands of genes. CRISPRi screens could help us construct more mature human tissues and improved disease models.
9:00 Development and Optimization of CRISPR Gene Editing for Drug Discovery Applications
John Feder, Ph.D., Associate Director, Genome Biology and Emerging Technologies, Department of Genetically Defined Diseases and Genomics, Bristol-Myers Squibb
New CRISPR systems, modalities and methods are being discovered and published at an unprecedented pace such that unbiased and agnostic comparisons and protocol optimizations are warranted if the promise of genome engineering is to be realized in the pharmaceutical setting. We will present our results to date for generating highly optimized method for gene editing in induced pluripotent stem cells.
9:30 Tailored Pre-Clinical Models with CRISPR-Based Genome Editing
Lukas Edward Dow, Ph.D., Assistant Professor, Medicine, Weill Cornell Medicine
CRISPR/Cas9 genome editing has changed the way we design and execute in vivo experiments. We are using CRISPR-based genome editing in stem cells and in adult mice to generate tailored pre-clinical models. This allows both a deeper understanding of the genetic underpinnings of cancer progression and provides a platform to interrogate new therapeutic strategies in specific genetic contexts, which is key for realizing the potential of personalized medicine.
10:00 Use of Synthetic sgRNA to Improve CRISPR Editing Efficiency
Kevin Holden, Ph.D., Head, Synthetic Biology, Synthego
CRISPR has made genome editing accessible for a wide range of cell-types. However, obtaining consistent editing efficiencies remains a challenge. Synthego has developed novel RNA synthesis technology to produce 100-mer sgRNA for CRISPR at a practical scale and price. We demonstrate that Synthego sgRNA produces consistent and superior genome editing.
10:15 Moving Towards a Cost-Effective, SMRT® Sequencing-Based Assay for the Genome-Wide Detection of CRISPR/Cas9 Nuclease Activity
Paul Kotturi, Ph.D., PacBio
10:30 Coffee Break with Exhibit and Poster Viewing
11:15 Whole-Genome CRISPR Screening for Necroptosis Resistance and Tumor Suppressor Synthetic Lethality
Mike Costa, Ph.D., Senior Scientific Manager, Department of Discovery Oncology, Genentech Research & Early Development
We have developed efficient whole-genome pooled gRNA libraries and applied them to positive and negative selection screens. Screening for resistance to necroptotic cell death identifies known necrosome components and additional hits that reveal new regulators. Screens in cancer cell lines detect novel dependencies conferred by mutations in tumor suppressors. We will discuss successful strategies for increasing screen throughput, computational identification of hits, and hit validation.
11:45 Genome Editing on iPSCs for Drug Discovery
Nazish Sayed, M.D., Ph.D., Instructor, Cardiovascular Institute, Stanford University School of Medicine
This presentation will describe the use of genome editing technology for assessing the pathogenicity related to variant of uncertain significance (VUS). In addition, we will describe examples of how control vs. genome-edited iPSC-derived cardiomyocytes are being used for drug screening and drug discovery applications.
12:15 pm Nucleofection, Genome Editing, and the Transfection of Clinically-Relevant Cells
Greg Alberts, Ph.D., Lonza Walkersville, Inc
The LV Nucleofector is the latest addition to the Nucleofector platform, and can transfect up to 2 billion cells, with the same performance of other Nucleofector devices. Nucleofection is a proven choice for genome modification applications like CRISPR, and is poised to play a comprehensive role in new innovative therapies.
12:30 Session Break
12:40 Luncheon Presentation: Functional Genome-Wide Analysis Using Optimized CRISPR Pooled Screens
Paul Diehl, Ph.D., COO, Cellecta, Inc.
1:15 Session Break
1:50 Chairperson’s Remarks
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University
2:00 Fixing the Broken Heart by Myoediting
Chengzu Long, Ph.D., Assistant Professor, Division of Cardiology, New York University School of Medicine
Using CRISPR-mediated genomic editing, we successfully prevented muscular dystrophy in the germline and postnatal muscles of mdx mouse model (Long et al. Science 2014; Long et al. Science 2016). Recently, we have advanced it to novel strains of humanized mice and patients’ cardiomyocytes. This has enabled us to optimize the correction of DMD mutations, providing a path toward a potential cure of the disease in patients.
2:30 Precise Gene Editing in Human Pluripotent Stem Cells
Krishanu Saha, Ph.D., Assistant Professor, Department of Biomedical Engineering, & Wisconsin Institute for Discovery, University of Wisconsin, Madison
Human pluripotent stem cells are important resources for drug discovery, toxicology, disease modeling, tissue engineering and regenerative medicine. Recently, we developed new CRISPR-Cas9 strategies to correct pathogenic point mutations and introduce transgenes precisely using homology-directed DNA repair. These strategies reduce and, in some cases, eliminate undesired allelic modifications associated with non-homologous end joining.
3:00 High Throughput Screening: Best Technology and Practices
Caroline Beckett, Global CRISPR Product Manager, MilliporeSigma
CRISPR revolutionized gene editing, but multi-target screening remains a complex goal. MilliporeSigma shares best approaches learned over years of genome editing. We also explore the best CRISPR tools from small gene panels to whole genome pooled and arrayed screening libraries.
3:30 Refreshment Break and Poster Competition Winner Announced in the Exhibit Hall
4:15 Development of New CRISPR/Cas9-Based Tools to Study Drug Interactions through Knockout and Directed Evolution
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University
We have used parallel shRNA and CRISPR screening to explore the biology of essential and non-essential genes, and have identified the target and mechanism of action of a novel host-targeting antiviral drug. More recently, we have used pairwise expression of sgRNAs to identify synergistic combinations of drug targets, and adapted our screening systems for new applications in mutagenesis and directed evolution.
4:45 Applying Inducible and Multiplexed CRISPR/Cas System in Functional Cancer Genetic Studies
Jian Cao, Ph.D., Associate Research Scientist, Department of Pathology, Yale University
We have developed a highly efficient doxycycline-inducible Cas9 system for uniform temporal control and efficient gene disruption even in a polyclonal setting. We also established a simple one-step cloning approach for multiple-sgRNA expression in an improved vector. By combining our inducible and multiplex genome editing approaches, we performed functional studies to identify cancer driver genes.
5:15 HOT TOPIC DISCUSSION: The Role of FDA in Regulating Gene Editing
Fyodor Urnov, Ph.D., Associate Director,
Altius Institute for Biomedical Sciences and Adjunct Professor, Department of
Molecular & Cell Biology, University of California Berkeley
5:45 Reception with Exhibit and Poster Viewing
6:45 Close of Day
Friday, February 24
7:00 am Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee
8:00 Registration Open
8:25 Chairperson’s Remarks
Mark A. Kay, M.D., Ph.D., Dennis Farrey Family Professor, Departments of Pediatrics and Genetics, Vice Chair for Basic Research (Pediatrics), Stanford University
8:30 FEATURED PRESENTATION: Novel AAV Vectors for Classical and Genome Editing-Based Gene Therapy
Mark A. Kay, M.D., Ph.D., Dennis Farrey Family Professor, Departments of Pediatrics and Genetics, Vice Chair for Basic Research (Pediatrics), Stanford University
Recombinant AAV vectors show promise in gene therapy. However, vector selection based on animal studies is not necessarily predictive of human outcomes. I will discuss approaches to improve these predictions, and novel methods to create/select rAAV vectors with enhanced properties. We have also developed an AAV promoterless site-specific gene targeting approach without the use of nucleases and show preclinical efficacy in animal models of human disease.
9:00 In vivo Genome Editing via CRISPR-Cas9 Mediated Homology-independent Targeted Integration
Keiichiro Suzuki, Ph.D., Research Associate, Laboratory of Dr. Juan Carlos Izpisua Belmonte, Gene Expression Laboratory, The Salk Institute for Biological Studies
Non-dividing cells, the major constituents of adult tissues, are inaccessible for targeted knock-in with current technologies. We have developed a robust homology-independent targeted integration (HITI) strategy that allows for efficient targeted knock-in in both dividing and non-dividing cells in vitro and in vivo. Using this method, we achieved the therapeutic efficacy of a rat model of blindness retinitis pigmentosa in vivo.
9:30 Development of a CRISPR/Cas9 and Stem Cell Platform for Duchenne Muscular Dystrophy
April Pyle, Ph.D., Associate Professor, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles
We have developed a platform to restore the reading frame using CRISPR/Cas9 in patients with Duchenne Muscular Dystrophy (DMD). CRISPR/Cas9 mediated deletion of dystrophin up to 725kb in hiPSCs represents a therapeutic strategy applicable to up to 60% of DMD patients. Current efforts are aimed at translating this platform using stem cell mediated delivery of corrected skeletal muscle progenitor cells in animal models of DMD.
10:00 An Arrayed CRISPR Library for Individual, Combinatorial and Multiplexed Gene Knockout
Simon R.V. Knott, Ph.D., Assistant
Professor and Associate Director, Center for
Bioinformatics and Functional Genomics, Cedars-Sinai
Medical Institute
We have combined a machine-learning approach with other strategies to optimize the efficiency of sgRNAs for CRISPR screens and have constructed a genome-wide, sequence-verified, arrayed CRISPR library. This incorporates expression strategies to facilitate multiplexed or combinatorial screening. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.
10:30 Coffee Break with Exhibit and Poster Viewing
11:15 In vivo Genome Engineering via CRISPR-Cas Systems
Prashant Mali, Ph.D., Assistant Professor, Department of Bioengineering, University of California San Diego
The CRISPR-Cas systems have emerged as a powerful toolset for targeted genome engineering. Development of safe and efficient gene transfer platforms for these will transform our ability to study biological processes in their native in vivo settings and also target various human diseases. In this talk I will describe some of our ongoing efforts to improve ex vivo and in vivo genome engineering.
11:45 Mechanism and Therapeutic Application of RNA-Guided Immune Systems
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, University of California, Berkeley
12:15 pm Pooled CRISPR Screens in the Noncoding Genome
Neville Sanjana, Ph.D., Core Faculty Member, New York Genome Center and Assistant Professor, Department of Biology & Center for Genomics and Systems Biology, New York University
We have recently adapted CRISPR forward genetic screens into noncoding regions of the genome, where it can be challenging to identify functional elements. We find that mutations at specific noncoding elements lead to changes in transcription factor occupancy and that these changes coincide with modulation of gene expression. These results expand the potential of CRISPR screens for fundamental genomic discovery, gene regulation, and therapeutic development.
12:45 Close of Symposium