LIVE – The CRISPR/Cas9 Revolution and Gene Editing: 2016 WARREN ALPERT FOUNDATION PRIZE SYMPOSIUM
Reporter: Aviva Lev-Ari, PhD, RN
Each year the recipient(s) of the Warren Alpert Foundation Prize are recognized at a scientific symposium hosted by Harvard Medical School.
OCTOBER 6, 2016 –
2016 WARREN ALPERT FOUNDATION PRIZE SYMPOSIUM
The CRISPR/Cas9 Revolution and Gene Editing
In honor of Rodolphe Barrangou, Emmanuelle Charpentier, Jennifer Doudna, Philippe Horvath, Viginijus Siksnys for remarkable contributions to the understanding of the CRISPR bacterial defense system and the revolutionary discovery that it can be adapted for genome editing.
Harvard Medical School
The Joseph B.Martin Conference Center
77 Avenue Louis Pasteur
Boston, MA 02115
Seating available on a first-come first served basis
Barbara J McNeil, MD, PhD, Acting Dean, HMS
Warren Alpert a very prestiguos Prize for advancements in Medicine, Treatment of disease and alleviation of suffering
Clifford Tabin, MD, Prof. of Genetics at HMS
- CRISPR as regulatory system
Featured Speakers include:
Rodolphe Barrangou, PhD
Todd R. Klaenhammer Distinguished Scholar in Probiotics Research
North Carolina State University
CRISPR-mediated immunity in bacteria: discovery and applications
Philippe Horvath, PhD
CRISPR-mediated immunity in bacteria: discovery and applications
- CRISPR-Cas: basics, history, applications, future
- cas1 (larger number of spacers) and Cas 2 almost universal
- DNA repeate in preKariotes, 2002 Milk coagulation, dairy industry – lactic acid, bacteriaphaging – failure of fermentation
- 2005 outstanding spacer polymorphism
- CRISPR genotype – phase sensitivity & resistance correlation
- CRISPR – Mechanism of Action
- 8/2005 Patent of bacteriaphage – Eureka:comercialization in 1990 200 sequence resistance to phage – Anti Phaging Hypothesis
- certain spacers in genome cross immunity against the phage –
- Spacers: engineeringCRISPR-encoded immunity resistence against phaging: ad spacer gain resistence
- cas9 disruption >> loss of phage resistence in dairy bacteria
- csn2 disruption -.. no subsequence acquisition of spacers
- no phenotypic resistance loss
- RNSi – A putative RNA-interference-based immune system
- Science 2007
- Discovery of the CRISPR motif (PAM): resistence in Streptococcus Thermophilus
- Immunity is mediated by small CRISPR RNAs (crRNAs)
- CRISPR Immunity – DNA encoded in bacteria
- CRISPR/Cas bacterial immune system cleaves bacteriaphage and plasmid DNA, Nature 11/2010
- Interference with expression of immunity – with invading nucleus by viral DNA infection
Applications for CRISPR
- Bacterial strain typing
- natural vaccination against phages: CRISPerization (cultivation, plating)
- Natural genetc tagging
- signature in the genome – genetic tag
- strain identification
- Patent for phage genome editing in 2009
- Lethal self-targegting in bacteria programmable antimicrobial is death
- genotype of interest selected
- Agriculture applications: contamination in food, starters probiotics
Perspective on last Decade
- phage resistance phenotype
- In silico & predictions in vitro
- success in Crops, Food, Animals
- Matters: IP (file for Patent, convert, publish), PR, Reg
Emmanuelle Charpentier, PhD
Prof. Dr.; Scientific Member of the Max Planck Society, Max Planck Director
Professor, Umeå University
The transformative genome engineering CRISPR-Cas9 technology: lessons learned from bacteria
- Non-infectious Disease: Cancer, Heart Genetic, Brain
- Infectious Diseases: Transmiable & Comnunicative
- Enzyme Cas9 S. Pyogenes: Group A Strep
- spacer acquisition – crRNA expression and maturationng CRISPR-CAS evolved into 6 types
- Human Bacterial host
- An mRNA : Type II CRISPR -Cas locus: TracrRNA – pre-crRNA
- Cas9 requires tracrRNA:crRNA to cleave DNA
- Genome editing with sequence specific nucleatease
- RNA -programmable CRISPR -Cas9
- Applications of CRISPR-Cas9 in human medicine: sequencing of Human genome – gene therapy to an organ, genetic predisposition of diseases
- trcrRNA is associated to Type II CRISPR-Cas
- Interchangeability among dual-RNA-Cas9 orthologs
- Cpf1 – Type V-A
- Adaptive Immune system
- Mechnism of maturation of CRISPR-RNA
Jennifer Doudna, PhD
Li Ka Shing Chancellor’s Chair in Biomedical and Health Sciences/HHMI Investigator
University of California, Berkeley
The Future of Genome Engineering: Biology, Technology and Ethics
- Technology – Gene Editing
- Adaptation – acquire and maintain genetic memory Prokaryotic cells
- crRNA Biogenesis
- Supercoiled plasmid target helps for the integration reaction
- Integration preceeds via a 3′-OH nucleophilic attack, 3′ – PO4
- What directs Cas1-Cas2 to the leader side of CRISPR Loci
- Integration Host Factor (IHF): alpha and beta
- IHF is required for spacer acquisition in vivo
- mechanism of spacer integration for DNA repair and repeat replication
- Harness integrase for genomic tagging
- CAS9 is a dual-RNA guided DNA Endonuclease
- Cas9 programmed by single chimeric RNA
- Most CISPR systems target dsDNA
- PAM binding drives DNA target recognition: protospacer — PAM– dsDNA
- C2c2 is an RNA-activated RNase: cis Cleavage vs trans Cleavage – used to detect specific RNA
- Chromatin search
- DNA repair
- RNA targeting
- CRISPR based white mushrooms programmed to resist browning
- human gene modification
Virginijus Siksnys, PhD
Professor and Chief Scientist/Department Head, Institute of Biotechnology
From mechanisms of microbial immunity to novel genome editing tools
- bacteria can absorb interference
- superinfection survival
- defense islands in genomes
- CCGG-family: specificity of restriction enzymes that recognize different nucleotides
- meganucleases: ZFN, TALEN
- CRISPR-Cas are transportable: CRISPR3 was transferred to e-Coli and plasmid
- isolate Cas9 protein – RNA-guided endonuclease – adaptive immunity in bacteria
- generate Cas9 variants
- Cas9 – restriction enzyme – targeting 2 sites on a pUC18 plasmid
- Cas9 specificityis encoded by crRNA: REases
- Cas9- versatilegenome editing tool: induce DNA breaks, gene editing of Human cells, animals plants
- Cas target is composite – >1000 Cas9 orthologues are known: 20 nt protospacer PAM sequence PAM Assay: PAM depends on Cas9 concentration
- RNP assembly Cas9
- Type II-CCRISPR-Cas for B. laterosporus
- PAM preference for Blat Cas9
- Maze genome
- Off-target cleavage: role of PAM, PAM contribute to cleavage at off-target site: Stringent PAM restriction on Cas9
Luhan Yang, PhD
Chief Scientific Officer
Rewriting the pig genome to transform Xenotransplantation
- Organ transplantation unmet needs
- natural bioreactor for organ transplants manufacturing
Obstacles for Xenotransplantation
- immunological Incompatibility
- New tools: CRISPR-Cas9 multiplexible genome engineering
- Infectivity of virus – Infectivity is real: gRNA to destroy catalytic PERVs
- Eridicate of PERVs activates in PK15 cells
- generate viable PERV free embryo
- Genotyping of Clone 40
- viral transmission
- a disruptive technology across tissue and organ types
- therapeutic applications
- write the Genome
- Next Generation of Gene Editing Tools
Austin Burt, PhD
Professor of Evolutionary Genetics
Imperial College London
Developing CRISPR-based gene drive for malaria control
- Genetically MODIFICATION of the mosquito strains that brings Malaria to Humans
- Driving Y chromosom – convert all population of mosquitos to MALE: don’t bite, don’t transmit and do not contribute to next generation
- Homing: natural process endonuclease genes in many microbes
- Find Gene needed for female fertility: Ovary expression : sterile non-sterile
- gene needed for vector competence
- target gene validation: number of Larvae
- CRISPR-based homing at target gene – Frequency
- Issues arising form this approach: Resistance, ecological and biodiversity, Governance and acceptance, step by step development pathway
Warren Alpert Foundation Prize Recipients
For remarkable contributions to the understanding of the CRISPR bacterial defense system and the revolutionary discovery that it can be adapted for genome editing.
For their pioneering discoveries in chemistry and parasitology, and personal commitments to translate these into effective chemotherapeutic and vaccine-based approaches to control malaria – their collective work will impact millions of lives globally particularly in the developing countries.
For seminal contributions to our understanding of neurotransmission and neurodegeneration.
For their seminal contributions to concepts and methods of creating a genetic map in the human, and of positional cloning, leading to the identification of thousands of human disease genes and ushering in the era of human genetics.
For the discovery, preclinical and clinical development of bortezomib to FDA approval and front line therapy for the treatment of patients with multiple myeloma.
In recognition of their extraordinary contributions to medicine and innovations in bioengineering.
For the expansion and differentiation of human keratinocyte stem cells for permanent skin restoration in victims of extensive burns.
For the discovery, characterization and implementation of laser panretinal photo-coagulation, which is used to treat proliferative diabetic retinopathy.
For work leading to the development of a vaccine against human papillomavirus.
For their contribution to the development of the breast cancer therapy Herceptin, the first target-directed cancer treatment for solid tumors.
For discovering angiogenesis and its relationship to disease, and for championing the concept of anti-angiogenic therapies.
For her seminal contributions to the understanding of how the antitumor agent Taxol kills cancer cells.
For their pioneering work on the purification, characterization, and cloning of human interferon-alpha.
For his pioneering work in understanding the role of vitamin A supplementation in preventing blindness and life-threatening infections in children in the developing world.
For their pioneering work in cardiovascular research which has dramatically reduced the mortality rate for heart attacks.
For their research that contributed to the development of a drug that effectively treats chronic megelogenous leukemia and other forms of cancer.
For their research in the development of statins which lower the level of cholesterol in the heart.
For elucidating the pathway forming the leukotrienes and their role in bronchial asthma.
For their discoveries of molecules that regulate the growth and differentiation of bone marrow cells in health and disease.
For the development of the lung surfactant used for treating pulmonary hyaline membrane disease.
For identifying Helicobacter pylori as the organism that causes gastric and duodenal ulcers.
For developing a complete description of thalassemia at the molecular level.
For discovering the enzymatic basis of Gaucher’s disease leading to its effective treatment.
For designing a powerful new approach to the treatment of high blood pressure and congestive heart failure.
For pioneering the use of DNA in the diagnosis of congenital anemias.
For defining the genetic basis of muscular dystrophy.
For elaborating the genetics of Hepatitis B as the basis for its vaccine.