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Together we Drive the Future

CRISPR & Genome Engineering

2017-04-272017-09-182017-08-18
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Day 1 Day 2
   
Day 1 – Thursday, May 26, 2016
   
   
12:00 Registration & Continental Breakfast
   
   
1:25 Welcome & Opening Remarks
   
   
Update: Current CRISPR Technologies & Techniques
Moderator: Miguel A. Moreno-Mateos, Associate Research Scientist, Department of Genetics, Yale University School of Medicine
   
  FEATURED PRESENTATION
1:30 A New Hope in Functional Genomics: Genetic Screens with CRISPR
 

 

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

  Functional genomics attempts to understand the genome by disrupting the flow of information from DNA to RNA to protein and then observing how the cell or organism changes in response. Both RNAi and CRISPR technologies are simply hacks of systems that originally evolved to silence viruses, reprogrammed to target genes we’re interested in studying, as decoding the function of genes is a critical step towards understanding how gene dysfunction leads to disease. Here we will discuss the development and optimization of CRISPR technology for genome-wide genetic screens and its application to multiple biological problems.
   
   
2:15
Jonathan D. Chesnut
Sr. Director, Synthetic Biology R&D
Life Sciences Solutions Group
Thermo Fisher Scientific
   
   
2:40 Understanding and Tuning CRISPR with RNA Modification
  Keith T. Gagnon
Assistant Professor , Biochemistry & Molecular Biology , Chemistry & Biochemistry
Southern Illinois University
  Although CRISPR technology is revolutionizing genome editing and synthetic biology applications, predictable targeting in the laboratory and safety in the future clinic will require better mechanistic understanding and careful engineering. The RNA components of the CRISPR system present an opportunity to probe and optimize activity through chemical modification. The prototypical CRISPR-associated protein, Cas9 from S. pyogenes, naturally binds two RNAs, a CRISPR RNA (crRNA) guide and a trans-acting CRISPR RNA (tracrRNA), to assemble a tripartite CRISPR ribonucleoprotein (crRNP). Using simple base substitution and chemical modification of the crRNA, we have discovered rules that govern crRNP activity. We have also identified modification patterns that can alter crRNP activity, such as enhanced target cleavage. Our results help establish a rationale for how to use nucleic acid chemistry to tune the Cas9 RNP for current and future applications, such as therapeutics.
   
   
3:05 Functional Genomics Using CRISPR-Cas9 for the Identification & Validation of Novel Oncology Targets
  Dennis France

Vice President, Oncology R&D

Horizon Discovery
  RNA interference possesses disadvantages for precise and accurate functional genomics, including incomplete knockdown and off-target effects, especially when deployed in the highly-efficient pooled drop-out screen format. These issues are particularly acute in the search for synthetic lethal targets to exploit vulnerabilities exposed by cancer-driving mutations in tumour suppressors and undruggable oncogenes. A facile method enabling complete knockdown should allow the identification of targets previously unreachable to functional genomic screening and with the discovery of the RNA-guided Cas9 nuclease, such a technology is now within our grasp. Following Feng Zhang and others, we have deployed a pooled lentiviral CRISPR-Cas9 knock-out screening approach employing large-scale complex libraries and data deconvolution via next generation sequencing. Using a whole genome sgRNA library, we have performed small molecule resistance screens and identified both previously published and novel hits. The availability of the haploid cell lines eHap/Hap1 enables exceptionally efficient target validation. We have created a sgRNA library based on a subset of the druggable genome to perform unbiased large-scale functional screens across cell line panels to identify synthetic lethal interactions with common cancer genotypes. The phosphatidylinositol-3-kinase (PI3K) pathway, and in particular the catalytic subunit PIK3CA, is frequently mutated in various cancer types. Using isogenic cell lines sensitivity screens were conducted to identify targets that show selective lethality with the activating PIK3CA E545K mutation found in a colon carcinoma cell line. Using this approach we identified several novel hits, as well as PI3K-pathway associated genes that validate the application of CRISPR-Cas9 technology to identify cancer-specific vulnerabilities.
   
   
3:30 Afternoon Networking Break
   
   
4:00 CRISPRscan: Designing Highly Efficient SgRNAs for CRISPR-Cas9 Targeting in Vivo
  Miguel A. Moreno-Mateos
Associate Research Scientist, Department of Genetics
Yale University School of Medicine
  CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5’ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9-sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3’-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo.
   
   
4:25 Rewriting the Genome 
  Adam Clore
Technical Director, Synthetic Biology
Integrated DNA Technologies
  The Synthetic Biology revolution has allowed unprecedented advances in the areas of disease treatment vaccine development, and small molecule discovery. Two advances have facilitated this growth, the inexpensive, fast and reliable synthesis of large fragments of DNA, and the ability to rapidly and accurately modify genomes using CRISPR technology. This presentation will discuss the development, methodology, and implementation of these technologies drawing from IDT’s in house research and recent publications.
   
   
4:50 A High-Throughput Workflow for CRISPR/Cas9 Mediated Targeted Mutagenesis to Model Human Disease Genes in Zebrafish
  Gaurav Varshney
Research Scientist, Translational & Functional Genomics
National Human Genome Research Institute
  Advances in sequencing technologies have enabled the rapid identification of human disease genes by GWAS or whole exome sequencing techniques. There is a large-gap between identification of human disease genes and their functional validation. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9 including a variety of tools and methods for guide RNA synthesis and mutant identification. While all the published techniques work, not all approaches are readily scalable to increase throughput. In addition, zebrafish have been shown to effectively recapitulate disease phenotypes, however models that fail to generate relevant phenotypes are rarely reported. We recently described a CRISPR/Cas9 based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here we present a complete workflow including target selection, cloning-free single guide RNA (sgRNA) synthesis, microinjection, validation of the target-specific activity of the sgRNAs, founder screening to identify germline transmitting mutations, determination of the exact lesion by PCR and next generation sequencing (including software for analysis), and genotyping in the F1 or subsequent generations. We used this high throughput pipeline to target all published human genes linked to non-syndromic deafness in zebrafish. We will present phenotyping data from homozygous mutants, and determine a rate of successful modeling in zebrafish.
   
   
5:15 Evening Reception & Poster Session
   
   
 
Day 1 Day 2
   
   
Day 2 – Friday, May 27, 2016
   
   
7:00 Continental Breakfast
   
   
Therapeutic Applications of CRISPR & Genome Engineering
Moderator: Guangbin Xia, Assistant Professor, Department of Neurology University of Florida
   
   
8:00 Engineering Human T Cell Circuitry 
  Alexander Marson

Principal Investigator

University of California, San Francisco
  Functional testing of human genome sequences in primary immune cells has been largely impossible until our advances in genome engineering methods that now permit direct DNA editing in human primary T cells. CRISPR/Cas9 has facilitated genome engineering in many cell types, but in human T cells Cas9 efficiency had been limited and Cas9 had not allowed targeted nucleotide replacements. We have now developed a CRISPR/Cas9-based platform that enables both knock-out and knock-in genome editing in primary human T cells by electroporation of Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs paired with homology-directed repair (HDR) template oligonucleotides can generate a high frequency of knock-in targeted genome modifications in primary T cells. The technology enables unprecedented explorations of genetic mechanisms that regulate T cell differentiation and function. We aim to understand how sequence variation throughout the human genome affects T cell circuits in health and disease. Cas9 RNP technology holds great potential for therapeutic genome engineering of human T cells for treatment of cancer, HIV, primary immune deficiencies, and autoimmune diseases.
     
   
8:25 TALEN®-based Targeted Genome Modifications for Improved CAR T-cell Adoptive Immunotherapy
 

 

Alexandre Juillerat
Project Leader Senior Scientist
Cellectis

 

 

After nearly two decades of gene editing, only a few nuclease platforms successfully transitioned from research laboratory to industrial and therapeutic products. Cellectis’ leading gene-editing platform TALEN® (transcription activator like effectors nuclease) already crossed the doors of research laboratories and possesses all the key features: precision, efficiency and specificity, to design molecular scissors for therapeutic gene editing applications.

Adoptive immunotherapy using engineered immune cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of immune cells through ex vivo genetic engineering and transfer of chimeric antigen receptors (CARs) or engineered TCRs. Cellectis proprietary nuclease-based gene editing technologies, combined with 16 years of gene editing experience, makes it possible to efficiently edit any gene in primary cells with very high precision and therefore offers unparalleled possibilities to design the next generation of immunotherapy products.

Here, we describe how the TALEN® gene-editing technology allows creating allogeneic CAR T-cells (derived from healthy donors rather than from patients themselves) but also empowering cells with additional safety and efficacy attributes. These new features include, among other possibilities, control properties designed to prevent engineered cells from attacking healthy tissues, to prevent auto-destruction, and to enable these cells to tolerate standard oncology treatments.

   
   
8:50 Delivery Technologies and Therapeutic Genome Editing
  Hao Yin
Massachusetts Institute of Technology
 

The CRISPR/Cas9 system has emerged as a transforming genome editing tool. CRISPR/Cas9 genome editing has been applied to correct disease-causing mutations in mouse zygotes and human cell lines, but delivery to adult mammalian organs to correct genetic disease genes has not been reported prior to our study. The liver disease hereditary tyrosinemia type I is a suitable model for gene repair-based therapy because the repaired hepatocytes will expand and repopulate the liver. Mutation of fumarylacetoacetate hydrolase (FAH), the last enzyme catalyzing the tyrosine catabolic pathway, leads to accumulation of toxic metabolites and severe liver damage. The Fahmut/mut mouse model harbors the same homozygous G to A point mutation of the last nucleotide of exon 8 as causes the human disease. This causes splicing skipping of exon 8 and truncated Fah mRNA.

In our first proof-of-concept study, we demonstrate CRISPR/Cas9-mediated correction of the Fah point mutation in hepatocytes in the Fahmut/mut mice. Delivery of the CRISPR/Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 (0.4%) liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that for the first time CRISPR/Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

Clinical implementation of CRISPR system requires safe and effective delivery to target tissue. In our second study, we combined lipid nanoparticle—mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce gene repair in the Fahmut/mut mice. The efficiency of correction was >6% of hepatocytes after a single application with minimal off-target effects, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

   
   
9:15 Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
  Daniel Anderson

Associate Professor, Department of Chemical Engineering, Institute for Medical Engineering and Science, Harvard-MIT Division of Health Sciences & Technology, and The David H. Koch Institute for Integrative Cancer Research

Massachusetts Institute of Technology
  High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our work on high throughput methods for developing and characterizing RNA delivery and gene editing systems. Libraries of degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to delivery RNA, both in vitro and in vivo. A number of delivery formulations have been developed with in vivo efficacy, and show potential therapeutic application for the treatment of genetic disease, viral infection, and cancer.
   
   
9:40 Efficient Correction of the Sickle Mutation in Human Hematopoietic Stem Cells Using a Cas9 Ribonucleoprotein Complex
  Mark DeWitt
Postdoctoral Researcher
University of California, Berkeley
  Sickle Cell Disease (SCD) is a serious recessive genetic disorder caused by a single nucleotide polymorphism (SNP) in the ß-globin gene (HBB). Sickle hemoglobin polymerizes within red blood cells (RBCs), causing them to adopt an elongated “sickle” shape. Sickle RBCs damage vasculature, leading to severe symptoms, ultimately diminishing patient quality of life and reducing lifespan. Here, we use co-delivery of a pre-formed Cas9 ribonucleoprotein complex (RNP) and a single-stranded DNA (ssDNA) oligonucleotide donor to drive sequence replacement at the SCD SNP in human CD34+ hematopoietic stem/progenitor cells (HSPCs), achieving up to 34% editing in vitro. Corrected HSPCs from SCD patients produce 2-fold less sickle hemoglobin protein, produce wild-type hemoglobin, and 8-fold more fetal hemoglobin, when differentiated into erythroblasts. When injected into immunocompromised mice, treated HSPCs maintain editing long-term at reduced levels (2%). These results demonstrate that the Cas9 RNP/ssDNA donor approach can achieve persistent editing HSPC gene editing and could form the basis for treatment of SCD by autologous hematopoietic cell transplantation.
   
   
10:05 Morning Networking Break
   
     
11:10 NextGEN™ CRISPR for Enhancing Therapeutics
 

 

Eric Ostertag

CEO

Poseida Therapeutics

 

Poseida is currently developing CAR-T and gene therapy products for cancer and orphan liver diseases. Poseida has best-in-class genome engineering technology for stable transgene integration or targeted gene editing. Proprietary platform technologies include the piggyBac™ DNA Modification System, capable of delivering 300kb+ of cargo for stable integration and long-term expression, the Footprint-Free™ Gene Editing System and NextGEN™ CRISPR and XTN™ TALEN site-specific nucleases.

This presentation will focus on NextGEN™ CRISPR , which has solved the off-target cutting problem inherent in the first generation CRISPR systems. We present data showing that NextGEN™ CRISPR has undetectable levels of off-site mutation frequency at several predicted sites when compared to first generation CRISPR directed to the same site in the genome. There are many therapeutic applications where NextGEN™ CRISPR can be used as a tool to improve a therapeutic without being used as a therapeutic itself. NextGEN™ CRISPR can be paired with piggyBac™ technology to perform Footprint-Free™ Gene Editing, a method to create surgically-precise edits of as little as a single nucleotide in nearly any genome with the ability to select for rare events. In another application, we show data that NextGEN™ CRISPR is an effective tool for disrupting genes in T cells when creating allogeneic Chimeric Antigen Receptor (CAR)-T cell products.

     
   
11:10 Genome Therapy for Myotonic Dystrophy Type 1
  Guangbin Xia
Assistant Professor, Department of Neurology
University of Florida
 

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3′-untranslated region (3’ UTR) of the DMPK gene. Mutant transcripts (expanded CUG) leads to disease by RNA gain-of-function. Currently, there is no cure. The advancement of induced pluripotent stem (iPS) cell technology has introduced new possibilities for developing cell-based therapies and correcting the mutation in DM1 iPS cells would be an important step towards autologous stem cell therapy. We have developed genome editing strategies to prevent production of mutant transcripts in iPS cells. I will talk about the results of the approaches and I will also talk about the prospective of in vivo genome therapy.

Benefits:
1. Will provide introduction of nucleotide repeat expansion-mediated neurodegenerative disorders.
2. Will discuss our successful genome editing strategies in nucleotide repeat expansion-mediated neurodegenerative disorders.
3. We will report our experience of using TALEN, SpCas9 and SaCas9 in nucleotide repeat expansion-mediated neurodegenerative disorders.
4. We will report how we characterized genome-edited iPS clones.

   
   
11:35 Restoration of Hearing from Genetic Deafness by Protein Delivery and CRISPR/Cas9-medated Genome Editing
  Zheng-Yi Chen
Associate Professor, Eaton-Peabody Laboratory, Department of Otolaryngology
Massachusetts Eye & Ear Infirmary, Harvard Medical School
 

1 in 500 newborns suffer from genetic hearing loss. Over 100 loci have been linked to and hundreds more genes are likely to be responsible for genetic hearing loss when mutated. Approaches including AAV-mediated gene therapy, anti-sense oligos and shRNA have been developed as potential treatment. Due to the limitations of each method, new approach is needed for the treatment of different forms of genetic hearing loss. CRSIPR/Cas9-mediated genome editing has been increasingly explored as new type of potential treatment due to the permanent editing results. However most CRISPR/Cas9 has been performed in germline or in vitro by viral vectors or DNA, which raises long-term safety concerns. Further the in vivo efficiency has been generally low.

We have demonstrated that Cas9 with guide RNAs can be effectively delivered into mammalian inner ear in vivo by cationic lipid formulation, resulting in efficient genome editing in the sensory hair cells. To develop potential treatment based on CRISPR/Cas9-mediated genome editing for genetic hearing loss, we studied the delivery of Cas9 with gRNAs against mutant Pmca2, a plasma-membrane calcium pump, which is associated with human and mouse progressive deafness. Direct in vivo local injection of Cas9 protein with Pmca2 guide RNA targeting the mutation led to restoration of hearing in the Pmca2 Oblivion mutant mouse demonstrated by auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE). Long-term hearing restoration was achieved. Cas9:gRNA mediated genome editing recovers the function of outer hair cells, promotes hair cell survival and preserves neurites of auditory ganglion neurons. Thus transient in vivo inner ear delivery of Cas9 and gRNA complex is sufficient to induce genome editing and restore hearing in a genetic deaf mouse model. Similar approach can be applied to treat other genetic hearing loss in the animal models with the implication in the treatment of human genetic hearing loss.

   
     
12:00 T-Cell Receptor Gene Editing for Tumor Therapy
  Mark Osborn

Assistant Professor

University of Minnesota
  Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach are the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor anti-host reactivity. To address these limitations, we assessed the ability of three different TCR-alpha targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent, and assessed off target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off target cleavage and we used them for a translatable manufacturing process to produce safe cellular substrates for next generation immunotherapies.
     
   
12:25 Lunch Provided by GTCbio
   
   
Therapeutic Applications of CRISPR & Genome Engineering
Moderator: Ron Weiss, Professor of Biological Engineering, Massachusetts Institute of Technology
   
   
1:30 CRISPR/Cas9 Gene Editing in HPSC-based Neuron Disease Modeling
  William Hendriks

Instructor in Neurology

Massachusetts General Hospital
Harvard Medical School
  The isolation of human embryonic stem cells (hESCs) and the discovery of human induced pluripotent stem cell (hiPSC) reprogramming have sparked a renaissance in stem cell biology, in vitro disease modeling, and drug discovery. In general, hPSC-based disease models are well-suited to study genetic variation. Studies commonly compare patient-derived hiPSCs, e.g., with a disease-causing genetic mutation, and (age-matched) control subject-derived hiPSCs, typically differentiated to the disease-affected cell type, e.g., neurons. A major caveat of this disease-modeling strategy is the variability of differentiation propensities and phenotypic characteristics, even in hPSCs derived from the same donor. Still, even if the cellular phenotype of a given mutation is strong and highly penetrant, it may be lost due to confounding effects of differences in genetic background of unrelated hPSC lines. A very powerful approach to overcome this hurdle is to use custom-engineered endonucleases, such as CRISPR/Cas9 that enable precise and programmable modification of endogenous hPSC genomic sequences. In our lab we use this genome-engineering strategy to study the neurological movement disorder dystonia, in particular X-linked Dystonia Parkinsonism (XDP). In this talk I will show how we use CRISPR/Cas9 to elucidate the underlying molecular pathogenesis of XDP in hPSC-based neuron disease modeling. I will also discuss some of the potential problems or pitfalls one might face using gene editing in hPSC-based disease modeling.
   
   
CRISPR Regulation of Gene Expression
Moderator: Ron Weiss, Professor of Biological Engineering, Massachusetts Institute of Technology
   
1:55 Mammalian Synthetic Biology: From Parts to Modules to Therapeutic Systems
 

 

Ron Weiss
Professor of Biological Engineering
Massachusetts Institute of Technology

  Synthetic biology is revolutionizing how we conceptualize and approach the engineering of biological systems. Recent advances in the field are allowing us to expand beyond the construction and analysis of small gene networks towards the implementation of complex multicellular systems with a variety of applications. In this talk I will describe our integrated computational / experimental approach to engineering complex behavior in a variety of cells, with a focus on mammalian cells. In our research, we appropriate design principles from electrical engineering and other established fields. These principles include abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic biology different from all other existing engineering disciplines and discovering new design and construction rules that are effective for this unique discipline. We will briefly describe the implementation of genetic circuits and modules with finely-tuned digital and analog behavior and the use of artificial cell-cell communication to coordinate the behavior of cell populations. The first system to be presented is a multi-input genetic circuit that can detect and destroy specific cancer cells based on the presence or absence of specific biomarkers in the cell. We will also discuss preliminary experimental results for obtaining precise spatiotemporal control over stem cell differentiation for tissue engineering applications. We present a novel approach for generating and then co-differentiating hiPSC-derived progenitors with a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression. We initiate rapid emergence of all three germ layers as a combined function of GATA6 expression levels and tissue context. We ultimately obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype that includes haematopoietic and stromal cells, as well as a neuronal niche. We will conclude by discussing the design and preliminary results for creating an artificial tissue homeostasis system where genetically engineered stem cells maintain indefinitely a desired level of pancreatic beta cells despite attacks by the autoimmune response, relevant for diabetes.
   
Discovery Mechanisms of CRISPR Systems
Moderator: Ron Weiss, Professor of Biological Engineering, Massachusetts Institute of Technology
   
2:20 Targeted Genome Integration Using Engineered Nucleases
  Pablo Perez-Pinera
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 as well as some of their potential applications in genome engineering.
   
   
2:45 Channabasavaiah Gurumurthy
Assistant Professor-Genetics, Director- Mouse Genome Engineering Core Facility
University of Nebraska Medical Center
   
3:10 Conference Concludes
   
   
 
Day 1 Day 2