Cambridge Healthtech Institute’s Second Annual

New Frontiers in Gene Editing

Striving for Better Design, Precision, and Efficiency

March 10-11, 2016 | Hilton San Francisco Union Square | San Francisco, CA
Part of the 23rd International Molecular Medicine Tri-Conference


Gene editing is rapidly progressing from being a research/screening tool to one that promises important applications downstream in drug development, cell therapy and bioprocessing. Cambridge Healthtech Institute’s second annual symposium on New Frontiers in Gene Editing will bring together experts from all aspects of basic science and clinical research to talk about the progress being made in gene editing and how it’s being applied. Knowing the strengths and limitations of the different tools, how does one decide when to use the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system, as opposed to Transcription Activator-Like Effector Nucleases (TALENs), zinc finger nucleases (ZFNs) and other systems? What is being done to overcome some of the inherent challenges with design, delivery and off-target effects, associated with each of these techniques? Experts from pharma/biotech, academic and government labs will share their experiences leveraging the utility of gene editing for diverse applications.

Final Agenda

Thursday, March 10

7:30 am Registration and Morning Coffee


8:30 Chairperson’s Opening Remarks

Erik Sontheimer, Ph.D., Professor, RNA Therapeutics Institute and the Program in Molecular Medicine, University of Massachusetts Medical School

8:40 Activities and Applications of Neisseria meningitidis Cas9

Erik Sontheimer, Ph.D., Professor, RNA Therapeutics Institute and the Program in Molecular Medicine, University of Massachusetts Medical School

Diverse Cas9 orthologs have the potential to provide novel activities and targeting specificities to the genome engineering toolbox. The Sontheimer lab has established Neisseria meningitidis Cas9 (NmeCas9) as a compact genome-editing enzyme. This presentation will describe the NmeCas9 system’s features during native bacterial interference, as well as human gene targeting. These features include novel activities that are independent of the tracrRNA, which was previously considered an essential Cas9 co-factor.

9:10 Engineered Nucleases for Targeted Genome Integration

Pablo Perez-Pinera, M.D., Ph.D., Assistant Professor, Department of Bioengineering, University of Illinois at Urbana-Champaign

The CRISPR-Cas9 system can be used to inactivate genes by introducing double-strand breaks in genomic DNA that are preferentially repaired by non-homologous end joining, an error-prone DNA repair pathway that often causes mutations. However, tools for targeted gene insertion in genomes remain elusive. In this talk, I will summarize recent advances in methods for targeted integration of heterologous DNA within complex genomes.

9:40 In vivo Genome Engineering Using S. aureus Cas9: Development and Applications

Winston Yan, Graduate Student, M.D.-Ph.D. Program, Laboratory of Dr. Feng Zhang, Broad Institute of MIT and Harvard

The small Cas9 ortholog from Staphylococcus aureus (SaCas9) has proven to be a versatile and efficient RNA-guided endonuclease ideally suited for in vivo applications due to its ability to be packaged into the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we describe the characterization and structure of SaCas9, and its application in knocking down the cholesterol regulatory gene Pcsk9 in the adult liver as a prototype for efficient in vivo genome editing using CRISPR-Cas9.

Surrogen10:10 Precision Disease Modeling in Swine with TALENS

Scott Fahrenkrug, Ph.D. Founder & CEO Recombinetics, Parent company of Surrogen

Sole reliance on rodent preclinical models has resulted in inflated failure rates due to vast differences in size, anatomy and physiology compared to humans. Pigs are an excellent model of human anatomy and physiology, and we present a new frontier in preclinical models where pigs with precise human disease alleles are produced by gene editing.

10:40 Coffee Break with Exhibit and Poster Viewing


11:15 Engineering CRISPR for Visualizing Genome Organization

Wulan Deng, Ph.D., Helen Hay Whitney Fellow, Research Specialist, Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute

We have engineered the nuclease-deficient CRISPR/Cas9 for labeling genomic DNA in situ in fixed cells and tissues. Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and multi-color labeling of DNA elements and coding gene loci in mammalian cells. This rapid, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

11:45 Engineered Orthogonal Drug Switchable Precise Control for CRISPR Transcription Regulation

Xin (Cindy) Xiong, Ph.D., Research Scientist, Agenovir Corporation

We have engineered the CRISPRi/a system to precisely control transcription activity and dosage by drug. We identified several drug switchable protein dimerization modules that are highly efficient and specific when combined with CRISPR. By pairing these modules with orthogonal Cas9s, we developed orthogonal drug switches that enable independent transcriptional regulation (activation/repression) of distinct target genes according to the drug inputs.

Lonza12:15 pm Genomic Editing, Nucleofection, and the Generation of Cell Lines for Cell-Based Assays

Gregory Alberts, Ph.D. Global Subject Matter Expert, Lonza Pharms Bioscience Solutions

Using primary cells in cell-based assays can improve the assays, which should translate more effectively into in vivo models. Lonza’s Nucleofector™ technology easily transfects primary cells, and with CRISPR, primary cells can be specifically modified at the genomic level, creating isogenic strains of specific cells that differ in only one specific aspect.

12:30 Session Break

12:45 Luncheon Presentation: CRISPR and RNAi: Gene editing and functional genomic screening approaches

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

While RNAi screens have proven effective genome-wide loss-of-function pooled screens, CRISPR/Cas9 provides a newer attractive alternative. We have developed pooled sgRNA libraries that complement our established shRNA ones, and then compared how each type performs in genetic screens on PDX-derived cell lines.

1:15 Session Break


1:50 Chairperson’s Remarks

James Inglese, Ph.D., Head, Assay Development & Screening Technologies, National Center for Advancing Translational Sciences, NIH

2:00 Genome-Edited Reporter Systems to Enable Cell-Based HTS Assays for Chemical Biology and Drug Discovery

James Inglese, Ph.D., Head, Assay Development & Screening Technologies, National Center for Advancing Translational Sciences, NIH

The targeting precision of genome editing was used in combination with advances in reporter gene design to modify the genetic loci of neurologic target genes to create HTS assays for compound library interrogation. Our goal was to identify transcriptionally active pharmacological agents acting by a variety of mechanisms, including through chromatin co-regulators accessible by our assay design. Specific case studies will serve to illustrate progress and findings to date.

2:30 Optimizing CRISPR-Cas9 System to Improve Genome-Wide Knockout Screening Performance

Haoquan Wu, Ph.D., Associate Professor, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center

CRISPR-Cas9 system enables genome-wide knockout screening in human cells. One of the limitations is that the knockout efficiency of sgRNAs targeting the same gene can vary significantly, or even dramatically. Here we present data to improve knockout efficiency generally to improve the screening performance of CRISPR-Cas9-mediated knockout screening.

3:00 Refreshment Break with Exhibit and Poster Viewing

3:30 Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets

Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University

We have developed high-complexity shRNA libraries (25 shRNAs/gene) that greatly reduce false negatives/false positives, and have adapted these libraries to knock down gene pairs to perform systematic genetic interaction maps in mammalian cells. Using this strategy in parallel with the CRISPR/Cas9 system, we have uncovered new insights into the biology of stress signaling and identified novel drug targets.

4:00 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.

4:30 Recent Progress towards Efficient Targeted Gene Modification in Primary Human Hematopoietic Cells

David Rawlings, M.D., Director, Center for Immunity and Immunotherapies Seattle Children’s Research Institute; Professor of Pediatrics and Immunology, University of Washington School of Medicine

We have utilized RNA-based nuclease and AAV-mediated donor co-delivery to drive targeted gene modification in primary hematopoietic cells. Using this approach, we achieve ~60% gene targeting in T-cells and we have generated “targeted CAR” T-cells with potent functional activity. We have also applied this method to edit CD34+ stem cells. Overall, primary cells with myriad novel properties can be generated with high-efficiency using this clinically feasible gene editing approach.

5:00 Reception with Exhibit and Poster Viewing

6:00 Close of Day

Friday, March 11

7:30 am Morning Coffee


7:55 Chairperson’s Remarks

Bruce R. Conklin, M.D., Investigator, Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes and Professor, Division of Genomic Medicine University of California, San Francisco

8:00 Precise Genome Engineering in Human iPS Cells to Model and Treat Disease

Bruce R. Conklin, M.D., Investigator, Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes; Professor, Division of Genomic Medicine University of California, San Francisco

We have combined droplet digital PCR (ddPCR) technology, TaqMan PCR system, and optimized iPSC culture system to develop Rare Allele Induction and Detection (RAID). This method allows for precise base-by-base genome editing in human iPSCs followed by efficient detection, sub-selection, and isolation of mutant clones. We have made a series of >20 isogenic iPSC-derived cardiomyocytes and observed cardiomyopathy phenotypes with several heterozygous and homozygous single base mutations.

8:30 Engineering Human Stem Cells by CRISPR

Su-Chun Zhang, M.D., Ph.D., Steenbock Professor in Behavioral and Neural Sciences and Professor of Neuroscience and Neurology, Waisman Center, University of Wisconsin

We have adapted the current genome editing technology for human cells. Using the optimized technology, we have engineered human stem cell lines with reporters, inducible gene expression and knockout, as well as functional switches. These genetically modified human cells substantially enable fundamental research, drug discovery, and potentially clinical applications.

9:00 Therapeutic Genome Editing for Blood Diseases

Matthew Porteus, M.D., Ph.D., Associate Professor, Pediatrics, Stanford University School of Medicine

The genome editing toolbox now offers powerful options in designing engineered nucleases and there are multiple different ways to utilize the engineered nucleases to create precise genomic modifications using both non-homologous end-joining and homologous recombination. Harnessing this toolbox so that it can be applied beyond just manipulating cancer cell lines, and instead, utilized to engineer therapeutically relevant cell types is now proceeding. Progress on modifying T-cells and hematopoietic stem and progenitor cells will be presented.

Sigma Life Science9:30 Practical Considerations for Genome Engineering of Model Cell Lines

Daniel C. Teasley, Ph.D., Genome Engineering Specialist, Cell Design Studio, MilliporeSigma

The widespread adoption of CRISPR-based genome editing technology has made cell line engineering more accessible than ever before. Despite these recent advances, engineering the genome of a model cell line remains a challenging task. Common decision points - such as choosing a parental cell line and nuclease - and potential stumbling blocks in the workflow will be discussed. Several case study engineering projects will be reviewed to demonstrate best practices to manage risk and maximize success in model cell line genome engineering.

GE Healthcare Logo10:00 Designing Functional and Specific Guide RNAs for Gene Knockout or Homology-Directed Repair

John A. Schiel, Ph.D., Research Scientist, R&D, Dharmacon part of GE Healthcare

We describe a CRISPR-Cas9 algorithm that incorporates parameters to predict functional gene knockout of gRNAs and the ability to detect potential off-target sites typically missed using existing tools. We also provide guidelines for design of donor templates for optimal HDR and knockins.

10:30 Coffee Break with Exhibit and Poster Viewing


11:00 CRISPR Libraries for Functional Genomics: Optimizing On-Target Activity, Avoiding Off-Target Effects

John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of
Harvard and MIT

Pooled screens with CRISPR technology have proven to be a powerful means of understanding gene function. Here I will discuss experiments and computational modeling approaches to optimize sgRNA sequence to both increase on-target activity and decrease off-target effects. The resulting libraries generate deeper, more meaningful hit lists.

11:30 CRISPR-EATING: A Method for Inexpensively Generating Large sgRNA Libraries

Andrew Lane, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Rebecca Heald, Department of Molecular and Cell Biology, University of California, Berkeley

CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for genome screening and labeling is lacking. Using a new method, CRISPR-EATING, to construct libraries from any source of DNA, we have labeled a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency.

12:00 pm Application of Genome Editing Tools to Model Human Genetics Findings in Preclinical Animals

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

Genome editing tools have allowed for rapid generation of genetically engineered models in various preclinical species. We will present how ZFN and CRISPR have been applied to efficiently generate various animal models to recapitulate findings based on human genetics and pathobiology to aid drug discovery process.

12:30 Close of Symposium


Thursday, March 10, 6:30-9:00 pm

 SC26:  A Primer to Gene Editing: Tools and Applications

Course Instructors:
John Doench, Ph.D., Research Scientist, Broad Institute of Harvard and MIT

Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University