Gene editing, particularly using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system, has gained importance both as a research tool in drug discovery and for drug therapy. Cambridge Healthtech Institute’s fourth annual symposium on New Frontiers in CRISPR-Based Gene Editing will bring together experts from research and clinical laboratories to talk about the recent progress in gene editing and its growing applications. However, the technology is not without limitations. What is being done to overcome some of the inherent challenges in guide RNA design, delivery and off-target effects associated with CRISPR/Cas and what are some of the alternatives being developed? Experts from pharma/biotech, academic and government labs, and technology companies will share their experiences leveraging the utility of CRISPR-based gene editing for creating cell lines and disease models, for functional in vitro, in vivo and ex vivo screening, for target and cellular pathway identification, and for therapeutic use.

Thursday, February 15

7:00 am Registration Open and Morning Coffee

UNDERSTANDING THE NUANCES OF CRISPR-BASED EDITING

8:25 Chairperson’s Opening Remarks

Kevin Davies, Ph.D., Executive Editor, The CRISPR Journal; Author, The $1,000 Genome

8:30 The CRISPR Revolution: Highlights and Challenges in Gene Editing

Kevin Davies, Ph.D., Executive Editor, The CRISPR Journal; Author, The $1,000 Genome

Rarely has a technology transformed the world of life sciences and biotechnology as we are seeing with CRISPR-Cas9. Developed barely 5 years ago, CRISPR shows immense power to revolutionize medicine, agriculture, animal sciences, public health, and much more. Naturally there are opportunities – CRISPR has already given rise to a multi-billion-dollar biotech boom – and challenges – scientific, medical, and ethical – to be overcome. In this general introduction, I will offer a breezy introduction to the field and comment on some recent milestones and controversies.

 9:00 KEYNOTE PRESENTATION: Our Editable, Hence Druggable Genome

Fyodor Urnov, Ph.D., Associate Director, Altius Institute for Biomedical Sciences and Adjunct Professor, Department of Molecular & Cell Biology, University of California, Berkeley

Current clinical trials of genome editing have relied on advanced in genomics, delivery, nuclease design, and disease animal models to establish the fundamental feasibility of engaging a genomic target in the clinic by editing it. A survey of open INDs and ongoing late-stage preclinical efforts reveals a set of challenges that the field must address to fully realize the promise of the “if it is in the genome, it is a druggable target” concept ushered in by the development of genome editing.

9:30 Cellular Factors that Modify Cas9 Editing Outcomes

Stephen Floor, Ph.D., Assistant Professor, Department of Cell and Tissue Biology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco

CRISPR–Cas9 and its guide RNA must travel intact through a eukaryotic cell’s cytoplasm into its nucleus in order to edit its genome. I will present data on how factors native to human cells affect editing efficiency and outcomes.

10:00 Wholesale and Retail CRISPR Editing in Human Pluripotent Stem Cell and Their Derivatives

Ajamete Kaykas, Ph.D., Senior Investigator, Neuroscience Department, Novartis Institutes for Biomedical Research

We extensively optimized the CRISPR-Cas9 system in human pluripotent stem cells (hPSCs) and conducted a genome-scale pooled based screen. This screen identified multiple new genes involved in hPSC survival and pluripotency.

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

11:15 Translational and Basic Research-Based CRISPR-Cas9 Genome Editing of Human Hematopoietic Stem Cells

Daniel P. Dever, Ph.D., Instructor, Laboratory of Dr. Matthew Porteus, Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University Medical Center

Genome editing by homologous recombination (gene targeting) in hematopotieic stem and progenitor cells (HSPCs) is crucial for understanding human hematopoiesis and for developing novel cell and gene therapies for diseases of the blood and immune system with unmet medical needs. Here, I present our data that summarizes the biotechnological advancements of CRISPR-Cas9 and rAAV6-mediated gene targeting in HSPCs for basic and translational research purposes.

11:45 Novel Nanoparticles with CRISPR Ribonucleoproteins for Precise Gene Editing of Human Cells

Krishanu Saha, Ph.D., Assistant Professor, Department of Biomedical Engineering & Wisconsin Institute for Discovery, University of Wisconsin-Madison

Writing specific DNA sequences into the human genome with CRISPR/Cas9 is challenging, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular nanoparticle strategy to complex Cas9 ribonucleoproteins with a nucleic acid donor template and other molecular cargoes. In stem cells, this strategy increased the ratio of precisely-edited to imprecisely-edited alleles. These versatile, pre-assembled reagents could advance precision medicine, drug discovery and in vivo gene editing.

12:15 pm Synthetic sgRNA for CRISPR: Enabling Automation and Therapeutics

Kevin Holden, Ph.D., Head, Synthetic Biology, Synthego

Achieving consistent and high editing efficiencies with CRISPR is critical for automation and therapeutic applications with primary cells, and remains a significant challenge. Through a collaborative effort, we demonstrate that use of synthetic sgRNA for CRISPR yields improved and consistent editing efficiencies that are required for such applications.

12:30 Session Break

12:40 Luncheon Presentation: Optimization of CRISPR Technology for Large-Scale Genetic Screens

Paul Diehl, Ph.D., COO, Cellecta, Inc.

Complex lentiviral-based pooled sgRNA libraries provide one of the most effective tools currently available to identify genes that play a role in biological responses, disease pathologies, drug efficacy. Pooled CRISPR screens uncover the functional genetic elements required for a particular phenotype. Selection of the sgRNA targets and the design influence the outcome of a genetic screen for CRISPR-KO, CRISPRa or CRISPRi applications. A set of application-specific tools will be presented.

1:15 Session Break

NEXT-GENERATION GENE EDITING TOOLS

1:55 Chairperson’s Remarks

Martin Kampmann, Ph.D., Assistant Professor, Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases, University of California, San Francisco

2:00 Next-Generation Functional Genomics with CRISPRi and CRISPRa

Martin Kampmann, Ph.D., Assistant Professor, Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases, University of California, San Francisco

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) enable high-precision genome-wide loss-of-function and gain-of-function screens. We have adapted this strategy for use in human iPSC-derived cells, in order to characterize disease mechanisms in patient-derived cells and isogenic control cells, as well as relevant differentiated cell types, such as neurons.

2:30 Next-Generation CRISPR Technologies for Efficient Genome Engineering

C. B. Gurumurthy (Guru), Ph.D., Associate Professor, Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and Director, Mouse Genome Engineering Core Facility, University of Nebraska Medical Center

During this talk, I will discuss the latest CRISPR strategies and tools available for complex animal genome editing projects, with particular emphasis on strategies for increasing homology directed repair mechanism to enable insertion of longer sequences. A few examples of designing knock-in animal models and the workflow of generating the models will be presented.

3:00 Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans

Carsten Charlesworth, Doctoral Student, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University Medical Center

The CRISPR-Cas9 system is a powerful tool for genome editing however the most commonly used homologs of Cas9 are derived from the bacteria Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), which frequently inhabit humans. We screened for the presence of pre-existing adaptive immune responses to these proteins in humans and found that we could detect antibodies against both SaCas9 and SpCas9 as well as T-cell responses to SaCas9.

3:30 Refreshment Break and Poster Competition Winner Announced in the Exhibit Hall

4:15 i-GONAD: A Method for Genome Editing Rodents without ex vivo Handling of Embryos

Masato Ohtsuka, Ph.D., Associate Professor, Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University

We developed a novel technique called GONAD (Genome-editing via Oviductal Nucleic Acids Delivery) which enables genome editing in mice through intraoviductal instillation of CRISPR components followed by an in vivo electroporation. GONAD can be applicable to generate gene disruption, large deletion, or knock-in in the mouse genome without ex vivo manipulation of zygotes.

4:45 TECHNOLOGY PANEL: Latest in CRISPR Tools and Strategies

Investigators and faculty come together with service providers to discuss current gaps in know-how and technology for CRISPR research. They will discuss current challenges, share best practices and their experiences using various CRISPR reagents and tools for gene editing.

Panelists: 

Paul Diehl, Ph.D., COO, Cellecta, Inc.

C. B. Gurumurthy (Guru), Ph.D., Associate Professor, Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and Director, Mouse Genome Engineering Core Facility, University of Nebraska Medical Center

Kevin Holden, Ph.D., Head, Synthetic Biology, Synthego

Martin Kampmann, Ph.D., Assistant Professor, Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases, University of California, San Francisco

Ajamete Kaykas, Ph.D., Senior Investigator, Neuroscience Department, Novartis Institutes for Biomedical Research

5:45 Reception in the Exhibit Hall with Poster Viewing

6:45 Close of Day

Friday, February 16

8:00 am Registration Open and Morning Coffee

BUILDING PRECISION & EFFICIENCY IN GENE EDITING

8:25 Chairperson’s Remarks

Paul W. Burridge, Ph.D., Assistant Professor, Department of Pharmacology, Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine

8:30 Validating Genomic Predictors of Chemotherapy-Induced Cardiotoxicity Using CRISPR-Mediated Genome Editing of Human iPSC

Paul W. Burridge, Ph.D., Assistant Professor, Department of Pharmacology, Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine

The combination of CRISPR/Cas9 and hiPSC modeling represents a powerful tool for the validation of SNPs discovered by GWAS as causative in specific drug responses. We have developed a human model of chemotherapy-induced cardiotoxicity, showing that hiPSC-cardiomyocytes can accurately recapitulate a patient’s phenotype. We show that that specific drug responses can be traced back to a single base pair and that by validating this hit we discover new drugs to reverse or enhance this predisposition.

9:00 AAV-saCas9 and Multiplex sgRNA Gene Therapy to Eradicate HIV/AIDS

Wenhui Hu, M.D., Ph.D., Associate Professor, Center for Metabolic Disease Research, Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University

AAV-mediated gene therapy is promising in clinical trials. The smaller size of saCas9 allows duplex or even quadruplex sgRNA-expressing cassettes to be delivered in all-in-one AAV vector for high titer packaging and robust gene transduction both in vitro and in vivo. Here, we demonstrate the feasibility and efficiency of quadruplex sgRNAs/saCas9 AAV-DJ/8 deliveries to excise HIV-1 provirus in various tissues/organs of three different mouse models.

9:30 Therapeutic Strategies via Genome Engineering: Some New Approaches

Prashant Mali, Ph.D., Assistant Professor, Department of Bioengineering, University of California San Diego

The recent advent of RNA-guided effectors derived from CRISPR–Cas systems have dramatically transformed our ability to engineer the genomes of diverse organisms. As unique factors capable of co-localizing RNA, DNA, and protein, tools and techniques based on these are paving the way for unprecedented control over cellular organization, regulation, and behavior. Here I will describe some of our ongoing efforts towards engineering this and related systems for enabling therapeutic applications.

10:00 Engineering Cellular and Animal Models of Rare Disease Using CRISPR-Cas9 Genome Editing

Jeffrey Y. Huang, Ph.D., Lead Scientist, Multidisciplinary Lysosomal Storage Disorders Program, Children’s Hospital of Orange County

At CHOC Children’s, we are seeking to develop personalized therapeutics for rare pediatric disorders such as Pompe disease (PD). PD is a serious, progressive disorder caused by loss-of-function mutations on acid alpha glucosidase (GAA) – an enzyme that recycles muscle cell glycogen. Using CRISPR-Cas9 genome editing, we aim to generate and characterize new models of PD with single nucleotide GAA mutations commonly found in human patients.

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

CRISPR FOR ONCOLOGY DRUG DISCOVERY

11:15 The Story of a Screen: How We Used CRISPR/Cas9 to Back-Up the ‘Precision’ in Precision Medicine

Arthur L. Shaffer III, Ph.D., Staff Scientist, Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health

CRISPR/Cas9 is the most powerful molecular tool developed to precisely alter the human genome. Our lab spent 3 years to set up a whole genome screen for gene essential in aggressive lymphoma. This talk will cover the technical details of using CRISPR/Cas9 from cell line engineering to data processing to therapeutic discovery.

11:45 Identification of Synthetic Lethal Targets Using Genome-Wide CRISPR/Cas9 Knockout Screens

Ning Sun, Ph.D., Senior Scientist II, Functional Genomics, AbbVie, Inc.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer that is characterized by the deficiency of Von Hippel-Lindau (VHL) tumor suppressor. To identify VHL synthetic lethal genes, we created two isogenic pairs of VHL-deficient and -reconstituted cell lines and performed genome-wide CRISPR/Cas9 screens and RNAseq analysis. Our work uncovers novel genetic vulnerabilities in VHL-deficient ccRCC and suggests new therapeutic targets.

12:15 Q&A with session speakers

12:45 Close of Symposium