Leaders in Pharmaceutical Business Intelligence (LPBI) Group

UPDATED – Medical Interpretation of the Genomics Frontier – CRISPR – Cas9:  Gene Editing Technology for New Therapeutics

Authors and Curators: Larry H Bernstein, MD, FCAP and Stephen J Williams, PhD

and

Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 3/17/2015

Status “Interference — Initial memorandum” – CRISPR/Cas9 – The Biotech Patent Fight of the Century: UC, Berkeley and Broad Institute @MIT

Reporter: Aviva Lev-Ari, PhD, RN

2.1.3.1   UPDATED – Medical Interpretation of the Genomics Frontier – CRISPR – Cas9:  Gene Editing Technology for New Therapeutics, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

http://pharmaceuticalintelligence.com/2016/01/06/status-interference-initial-memorandum-crisprcas9-the-biotech-patent-fight-of-the-century/

67 articles in PharmaceuticalIntelligence.com

http://pharmaceuticalintelligence.com/category/crisprcas9-gene-editing/

Nine Parties had come forward: Opposition Procedure to the Broad Institute’s first European CRISPR–Cas9 Patent

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2016/03/17/nine-parties-had-come-forward-opposition-procedure-to-the-broad-institutes-first-european-crispr-cas9-patent/

CRISPR: A Podcast from Nature.com on Gene Editing

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2016/03/17/crispr-a-podcast-from-nature-com-on-gene-editing/

UPDATED on 10/4/2015

Nobel Prize Predictions

Curator: Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/10/04/nobel-prize-predictions/

UPDATED on 9/24/2015

Nobel Gazing

Thomson Reuters predicts that the developers of the CRISPR/Cas9 genome editing approach may win the Nobel Prize in Chemistry.

This article appears as Chapter 21 in 

Genomics Orientations for Individualized Medicine

Volume One

Larry H Bernstein, MD, FCAP, Senior Editor

Leaders in Pharmaceutical Business Intelligence, Boston

Larry.bernstein@gmail.com

Stephen J. Williams, PhD, Editor

Leaders in Pharmaceutical Business Intelligence, Philadelphia

sjwilliamspa@comcast.net

and

Aviva Lev-Ari, PhD, RN, Editor

Editor-in-Chief BioMed E-Book Series

Leaders in Pharmaceutical Business Intelligence, Boston

avivalev-ari@alum.berkeley.edu

It will appear with modifications as Part One in

Volume Two:

Genomics Methodologies: NGS, BioInformatics & Simulations and the Genome Ontology

Stephen J. Williams, PhD, Senior Editor

Co-Editor(s) – TBA

Abbreviated eTOCs

Recent Advances in Gene Editing Technology Adds New Therapeutic Potential for the Genomic Era by Stephen J Williams, PhD

Chapter Introduction by Larry H Bernstein, MD, FCAP

The Voice of Aviva Lev-Ari, PhD, RN — Big Pharma, CRISPR and Cancer

 
21.1 Introducing CRISPR/Cas9 Gene Editing Technology – Works by Jennifer A. Doudna

21.1.1 Ribozymes and RNA Machines – Work of Jennifer A. Doudna

21.1.2 Evaluate your Cas9 gene editing vectors: CRISPR/Cas Mediated Genome Engineering – Is your CRISPR gRNA optimized for your cell lines?

21.1.3 2:15 – 2:45, 6/13/2014, Jennifer Doudna “The biology of CRISPRs: from genome defense to genetic engineering”

21.1.4  Prediction of the Winner RNA Technology, the FRONTIER of SCIENCE on RNA Biology, Cancer and Therapeutics  & The Start Up Landscape in BostonGene Editing – New Technology The Missing link for Gene Therapy? 

21.2 CRISPR in Other Labs

21.2.1 CRISPR @MIT – Genome Surgery

21.2.2 The CRISPR-Cas9 System: A Powerful Tool for Genome Engineering and Regulation

21.2.3 New Frontiers in Gene Editing: Transitioning From the Lab to the Clinic, February 19-20, 2015 | The InterContinental San Francisco | San Francisco, CA

21.2.4 Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global

21.2.5 CRISPR & MAGE @ George Church’s Lab @ Harvard

21.3 Patents Awarded and Pending for CRISPR

21.3.1 Litigation on the Way: Broad Institute Gets Patent on Revolutionary Gene-Editing Method

21.3.2 The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

21.3.3 Framing the battle over credit for CRISPR as Berkeley v. MIT is wrong.
http://www.wired.com/2015/10/battle-genome-editing-gets-science-wrong/

2.4 CRISPR/Cas9 Applications

21.4.1  Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas

21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF

21.4.3 In vivo validated mRNAs

21.4.4 Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

21.4.5 Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

21.4.6 Level of Comfort with Making Changes to the DNA of an Organism

21.4.7 Who will be the the First to IPO: Novartis bought in to Intellia (UC, Berkeley) as well as Caribou (UC, Berkeley) vs Editas (MIT)??

21.4.8 CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

Summary 

Larry H Bernstein, MD, FCAP 

Advances in Gene Editing Technology: New Gene Therapy Options in Personalized Medicine

Curators: Stephen J Williams, PhD and Aviva Lev-Ari, PhD, RN

Recent Advances in Gene Editing Technology Adds New Therapeutic Potential for the Genomic Era

Author and Curator: Stephen J Williams, PhD

The fundamental shift presently occurring within the medical field as well as our understanding of underlying biology has been brought on by revolutionary advances in the disciplines referred to as ‘OMICS’ (genomics, metabolomics, transcriptomics, proteomics). This paradigm shift has brought a new, more “personalized” mindset in investigating, treating, detecting, and policy-decision making disease as well as the physician-patient relationship. This Volume One of Genomics explains this paradigm shift as our classical understanding of the gene has evolved with rapid development of molecular technologies and high-end computational methods to a vision beyond the classic model. This new model involves big data to focus of the “code of OMIC signature”, moving from our investigational focus of “one gene at a time” to analysis of the changes in the networks of protein and gene expression occurring during disease progression.

Moving toward this promise of genome-based therapeutics has required the concomitant development of methodologies unavailable to the researcher and drug developer for most of the 20^th century. These new technologies have allowed for the sequencing of the whole genome (advanced and inexpensive pyrosequencing), analyze the proteome for changes in post-translational modifications (new mass spectroscopy techniques combined with automated high-throughput gel electrophoresis on robotic platforms), ability to track all the changes happening to a patient’s metabolic profile (LC-MS in combination with an array of biocurated database functions), and develop new therapeutics based on discrete disease-specific changes in protein, enzyme, and DNA/RNA (mutational analysis, and advanced molecular techniques to allow for manipulation of DNA/RNA such as gene editing and therapeutic vectors) all advancements being dependent on the massive advancements in computing power and software development.

Although this final chapter on a specific technology (Cas9-mediated gene editing) might seem out of place to the reader for the subject of this Genomics volume, as discussed above, the development of these omics-related technologies have spurred the advent of personalized therapies. For example, in the 1990’s (as highlighted in the earlier chapters of this book) Dr. Craig Venter founded Celera Genomics with the goals of 1) sequencing the human genome in a cost effective manner (using new DNA sequencing technology and workflow he and colleagues had developed, and 2) use the information from whole genome sequencing to develop a new line of genomic-based therapeutics. Other companies such asHuman Genome Sciences, Myriad Genetics, Seattle Genetics and recently new ventures from 23andMe and Google Ventures were also founded based on the promise that high-end sequencing information could directly lead to this new era of genome-based therapeutics. And although many in the medical field have felt that the primary goal of these companies, in particular using genomic analysis to enhance drug development has been a bit disappointing, AS IN ALL SCIENTIFIC AND MEDICAL DISCOVERY, which involves both SERENDIPITY and INDIRECT HAPPENSTANCE, three important breakthroughs, directly related to the development of a post-genomics era personalized medicine approach, resulted from the aforementioned efforts. These were:

• The detection of disease-specific mutations in exomes resulting in “druggable” protein targets and ability to define the respective drug-responsive patient cohorts

Chronic myelogenous (or myeloid or myelocytic) leukemia (CML) was one of the first cancers attributed to a specific chromosomal aberration, namely the translocation event resulting in a fusion protein between part of the BCR(“breakpoint cluster region”) gene from chromosome 22 with the ABL gene on chromosome 9. Early drug development efforts were directed against the tyrosine kinase activity of the aberrant BCR/ABL protein. The first of this new class of drugs was imatinib mesylate (Gleevec™) showed early success but was later noticed that a subset of patients had significantly greater response rates. This led to more detailed investigation of Gleevec’s mechanism of action and was determined that Gleevec’s therapeutic action depended on the drug’s ability to bind to an ATP binding pocket within the BCR/ABL. Patients with a specific mutation in this ATP pocket (C944T and T1052C) were found resistant to Gleevec. This finding, that pateint’s DNA could be sequenced to stratify them in responder versus nonresponder groups became a cornerstone for tyrosine kinase inhibitor (TKI) development for various cancers. One example is the development of crizotanib, a TKI directed against a mutant version of the anaplastic lymphomakinase (ALK) enzyme, namely in patients carrying the ALK-EML4 fusion gene. As with Gleevec, certain mutations in the ATP binding pocket confer resistance to the inhibitory effects of crizotanib. Therefore, the Whole Exome Sequencing (WES) has shown its utility not only in drug development against cancer-specific mutant targets but stratifies patient cohorts into eligible versus non-eligible for a specific personalized therapy.

• Ability to define at-risk populations based on genomic data and development of corresponding genetic risk assessment for disease

Tremendous advances have been made in the area of risk-assessment for a plethora of diseases, including various malignancies, heart disease, and metabolic diseases. These risk factors have been identified given our advances in whole genome sequencing and proteomic and metabolomics. And, although the aforementioned companies had not developed therapeutic agents using these technologies, their major contribution has been the development of the diagnostic tests which identify at-risk patients and susceptible populations for a given disease. For example, the development of tests for carriers of the BRCA1/BRAC2 breast/ovarian cancer susceptibility mutation or APC (for colon cancer) has led to the appearance of Family Risk Assessment Programs and radically changed the discourse between patient and physician. And although determining risk factors to a disease such as cardiac disease in a large population can be fraught with complexities, the advanced research tools together with gene-directed technologies discussed in this Volume and current chapter may give better clarity in this regard. In essence, the technology had been developed well before its use in the clinic had been identified.

• Supplying and verifying linkages of specific genetic alterations to heritable diseases and offering a framework for future advances in gene-replacement and mutation-correction therapy

Our abilities to phenotypically correct inheritable diseases thru a gene-therapy (either by gene replacement or correction of mutated genes) have been hampered by three main areas. First identifying the specific mutations for a given inheritable disease used to be an arduous time-consuming process (linkage analysis), especially in small affected populations. However as whole exome sequencing rapidly evolved this had no longer become a rate-limiting step toward the development of a gene-directed therapy. Second and more troubling was determining a process which could deliver therapeutic genes in a safe, reliable and persistent manner. The first attempts at gene-therapy, relying on DNA virus and retroviral based delivery met with disaster and set back the field of gene therapy for decades (this story is too long for an introduction but for reference see thelink.)   Recently there have been improvements in therapeutic gene-therapy delivery systems such as the use of conditionally replicative adenovirus (cRADs) and novel serotype AAV (Recombinant adeno-associated virus, a nonpathogenic single stranded DNA human parvovirus) which have greatly improved safety and therapeutic profiles). The third issue, directly related to this chapter on Cas9-mediatied DNA editing) is the ability to integrate therapeutic DNA into the genome in a safe manner or correct mutations in their proper place. It is well established that the random integration of pieces of DNA has spurious effects on gene expression or contribute to transformation by an insertional mutagenesis mechanism.

This chapter will discuss how CRISPR/Cas9-mediated gene editing is being used in ex vivo strategies, namely to insert T-cell specific genes, in definable and safe loci, for the development of the new CAR-T cancer immuno-based therapies.   In addition CRISPR/Cas9-mediated gene editing has much hope and promise for correcting specific mutations related to inheritable diseases, although investigations are at an infantile yet rapidly expanding area. As discussed above, new technologies have preceded their clinical use, mostly in a serendipitous and advantageous manner. Therefore it is a natural progression, using the concepts and curations in previous chapters, to investigate how a new technology, such as CRISPR/Cas9 medicated gene editing will fit into the ‘OMICS era of medicine.

Introduction to Chapter 21

Larry H Bernstein, MD, FCAP

The recent development of advanced methods for genome engineering has superceded methods already in used in recent years of the 21^st century.  Genome editing technologies enable the deletion, insertion or correction of DNA at specific targeted sites within an organism’s genome. The power of the technology lies in its ability to specifically target any site in the genome and to alter the DNA sequence at that site. Researchers working in the biomedical field use these techniques to address diseases that are known to have a genetic origin. Early genome-editing research focused on the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs), which laid important foundations in establishing genome engineering as a potential approach for treating human diseases. These were superceded by the recent discovery of CRISPR-Cas9, followed by work demonstrating its advantages over traditional approaches. CRISPR-Cas9 has truly democratized genome editing and transformed the potential for treatment of genetic disorders. Genetic disorders may or may not be heritable, i.e., passed down from the parents’ genes. In non-heritable genetic disorders, defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line.

Genes causing rare heritable childhood diseases are being discovered at an accelerating pace driven by the decreasing cost and increasing accessibility of next-generation DNA sequencing combined with the maturation of strategies for successful gene identification. The findings are shedding light on the biological mechanisms of childhood disease and broadening the phenotypic spectrum of many clinical syndromes. Most are due to a defect in an enzyme or transport protein, which results in a block in a metabolic pathway. Nearly every metabolic disease has several forms that vary in age of onset, clinical severity, and, often, mode of inheritance. Newborn screening tests can identify some of these disorders.

This chapter introduces some giants in the 20th century study of genetic medicine, such as, Victor McKusick, widely known as the “father of medical genetics” and Elizabeth F. Neufeld, among others.

RNA has a role in suppressing translation, as do proteins by allosteric effects. In addition, the most common diseases involved in age related change are strongly responsive to extracellular matrix effects, ionic fluxes, effects on the cellular matrix, and involve multicentric genome expression. This mode of expression leads one to think hard about the therapeutic target, or targets. The effect of RNA or of protein interacting with the genome is not an element of the classic construct. The phenotypic presentations may have genomic associations, and there may also be population variants.

The use of genomic profiling has rapidly emerged in the laboratory armamentarium. We consider Pompe’s disease, polymorphisms in the long non-coding RNA, and the role of Lipoprotein Lipase in Atherosclerosis, altered Stress Hormones that affect the ability to bounce back from trauma, and genome engineering with CRISPR-Cas9 (Jennifer A. Doudna and Emmanuelle Charpentier) in this review as examples of important work in recent years.

This document is a review and of the brilliant accomplishment of the Doudna Laboratory at University of California, Berkeley. It also traces the developments leading up to this groundbreaking work. The principle investigator is a young woman of significant accomplishments with the astounding publication of 4 papers at this time in 2015 and 20 in 2014. She is a member of the National Academy of Sciences, and recipient of the Breakthrough Prize and the Lurie Prize in Biomedical Sciences, R. B. Woodward Visiting Professor, Harvard University (2000-2001). She achieved the Henry Ford II Professor of Molecular Biophysics and Biochemistry, Center for Structural Biology, Department of Molecular Biophysics and Biochemistry, Yale University (1994-2002) nine years after completion of her B.A. at Pomona College, and her Ph.D. under Jack Stozak at Harvard in 1989, became a Searle Scholar in 1996, and a Howard Hughes Investigator in 1997.

Her work has encompassed the editing of genes using the CRISPR-Cas9 system, and her team replaced a gene in a human cell which was convincing replicated in the Broad Laboratory at Harvard. The laboratory is currently working on the just reported immunological implications for CRISPR-Cas9 with respect to editing prokaryotic CRISPR-Cas genomic loci that encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. This is because acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome.

Of special note are the following applications:

21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF

Reporter: Aviva Lev-Ari, PhD, RN

21.4.3 In vivo validated mRNAs

Doudna’s Interview from the National Academy of Science in 2004

Doudna discusses her current work with signal recognition particles, a type of RNA that is found in virtually all cell types and is responsible for directing specific proteins to specific membranes. She also discusses how advances in genomic sequencing may help catalog the complete range of functional RNA molecules. (9 minutes)

SOURCE

http://www.nasonline.org/news-and-multimedia/podcasts/interviews/jennifer-doudna.html

The Doudna lab pursues mechanistic understanding of fundamental biological processes involving RNA molecules. Research in the lab is currently focused on three major areas:

1. bacterial immunity via the CRISPR system,
2. RNA interference in eukaryotes, and
3. translational control logic.

• Bacterial immunity

http://rna.berkeley.edu/crispr.html

• RNA interference

http://rna.berkeley.edu/rnai.html

Different subunits are colored with invader RNAs in the background. Art by Gerard W.M. Staals

• Translational Control

http://rna.berkeley.edu/translation.html

Alu-element regulated miRNA interactions

The Voice of Aviva Lev-Ari, PhD, RN

Big Pharma, CRISPR and Cancer

In January, the pharmaceutical giant Novartis announced that it would be using Doudna’s CRISPR technology for its research into cancer treatments. It plans to edit the genes of immune cells so that they will attack tumors.

https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaJdJh5t

• In 1987, Yoshizumi Ishino and colleagues at Osaka University in Japan published the sequence of a gene called iap belonging to the gut microbe E. coli.
  https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaL6md3C
  

http://www.businessinsider.com/the-biggest-biotech-discovery-of-the-century-is-about-to-change-medicine-forever-2015-2?IR=T

• In 2007, Blake Wiedenheft joined Doudna’s lab as a postdoctoral researcher, eager to study the structure of Cas enzymes to understand how they worked.
  https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaNXI2R4
• In a new paper in Nature Reviews Genetics, Koonin and Mart Krupovic of the Pasteur Institute in Paris argue that the CRISPR-Cas system got its start when mutations transformed casposons from enemies into friends.
  https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaN6Ucnk

• In January 2013, the scientists went one step further: They cut out a particular piece of DNA in human cells and replaced it with another one.

• In the same month, separate teams of scientists at Harvard University and the Broad Institute reported similar success with the gene-editing tool.

https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaOF9xi4

• Breakthrough DNA Editor Borne of Bacteria

• At Editas, a company based in Cambridge, Massachusetts, scientists have been investigating the Cas9 enzyme made by another species of bacteria, Staphylococcus aureus and Streptococcus pyogenes

https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaMPy9Yw

• In Hopes Of Fixing Faulty Genes, One Scientist Starts With The Basics

• Writing last year in the journal Reproductive Biology and Endocrinology, Motoko Araki and Tetsuya Ishii of Hokkaido University in Japan predicted that doctors will be able to use CRISPR to alter the genes of human embryos “in the immediate future.”
  https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaOg9jyD

International regulatory landscape and integration of corrective genome editing into in vitro fertilization, Motoko Araki and Tetsuya Ishii*

http://www.rbej.com/content/12/1/108

The Human Germ Line

Don’t edit the human germ line

Industry Body Calls for Gene-Editing Moratorium

By Antonio Regalado on March 12, 2015

Doudna’s Interview from the National Academy of Science in 2004

Track 6: Future Directions (requires RealPlayer)
Doudna discusses her current work with signal recognition particles, a type of RNA that is found in virtually all cell types and is responsible for directing specific proteins to specific membranes. She also discusses how advances in genomic sequencing may help catalog the complete range of functional RNA molecules. (9 minutes)

SOURCE

http://www.nasonline.org/news-and-multimedia/podcasts/interviews/jennifer-doudna.html

VIEW VIDEOS on Gene Editing

https://www.physicsforums.com/threads/breakthrough-prize-genome-editing-with-crispr-cas9.798959/

https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/

The Structural Biology of CRISPR-Cas Systems

Curr Opin Struct Biol. 2015 Feb 24;30C:100-111

Authors: Jiang F, Doudna JA

Abstract
Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding.

PMID: 25723899 [PubMed – as supplied by publisher]

SOURCE

feed://eutils.ncbi.nlm.nih.gov/entrez/eutils/erss.cgi?rss_guid=1T1hTO9Bp3LXOpYicP1VJ2xtLRuwKXiCBHyYsbxh719dOd_gXi

About the Significance of the CRISPR Discovery

“This technology will revolutionize biology in the same way PCR did,” Rudolf Jaenisch introducing Jennifer Doudna, 6/13/2014 @KI Symposium @MIT.

Koch Institute for Integrative Cancer Research @MIT – Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 8:30AM – 4:30PM, Kresge Auditorium @MIT

http://pharmaceuticalintelligence.com/2014/06/16/lecture-contents-delivered-at-koch-institute-for-integrative-cancer-research-summer-symposium-2014-rna-biology-cancer-and-therapeutic-implications-june-13-2014-mit/

Top CRISPR Related Publications

http://blog.appliedstemcell.com/top-crispr-related-publications/

What is CRISPR? Why are Cas9-CRISPR services so popular?

http://blog.appliedstemcell.com/what-is-crispr-why-are-cas9-crispr-services-so-popular/ 

Custom Rat Model Generation Service Using CRISPR/Cas9

http://www.appliedstemcell.com/services/animal-models/

Jennifer A. Doudna

Dr. Jennifer Doudna is a member of the departments of Molecular and Cell Biology and Chemistry atUC Berkeley, the Howard Hughes Medical Institute, and Lawrence Berkeley National Lab, along with the National Academy of Sciences, and the American Academy of Arts and Sciences.

http://rna.berkeley.edu/people.html

AWARDS for the Discovery

http://www.google.com/search?hl=en&q=Jennifer+Doudna+Awards&gbv=2&sa=X&oi=image_result_group&ei=VDIHVdrFOe7dsATYtoCoCQ&ved=0CD8QsAQ&tbm=isch

Jennifer Doudna, cosmology teams named 2015 Breakthrough Prize winners

Jennifer Doudna, The winner of the 2014 Lurie Prize in the Biomedical Sciences

CRISPR-Cas9 Discovery and Development of Programmable Genome Engineering – Gabbay Award Lectures in Biotechnology and Medicine – Hosted by Rosenstiel Basic Medical Sciences Research Center, 10/27/14 3:30PM Brandeis University, Gerstenzang 121

Doudna was a Searle Scholar and received a 1996 Beckman Young Investigators Award, the 1999 NAS Award for Initiatives in Research and the 2000 Alan T. Waterman Award. She was elected to the National Academy of Sciences in 2002 and to the Institute of Medicine in 2010. In 2014, Doudna was awarded the Lurie Prize in Biomedical Sciences from the Foundation for the National Institutes of Health as well as the Dr. Paul Janssen Award for Biomedical Researchand Breakthrough Prize in Life Sciences, both shared with Emanuelle Charpentier.

SOURCE

http://en.wikipedia.org/wiki/Jennifer_Doudna

 

21.1 Introducing CRISPR/Cas9 Gene Editing Technology – Works by Jennifer A. Doudna

21.1.1 Ribozymes and RNA Machines – Work of Jennifer A. Doudna

Reporter: Aviva Lev-Ari Ph.D. RN

21.1.2 Evaluate your Cas9 gene editing vectors: CRISPR/Cas Mediated Genome Engineering – Is your CRISPR gRNA optimized for your cell lines?

Reporter: Aviva Lev-Ari Ph.D. RN

21.1.3 2:15 – 2:45, 6/13/2014, Jennifer Doudna “The biology of CRISPRs: from genome defense to genetic engineering”

Reporter: Aviva Lev-Ari Ph.D. RN

21.1.4  Prediction of the Winner RNA Technology, the FRONTIER of SCIENCE on RNA Biology, Cancer and Therapeutics  & The Start Up Landscape in BostonGene Editing – New Technology The Missing link for Gene Therapy?

Curator: Aviva Lev-Ari, PhD, RN

21.2 CRISPR in Other Labs

21.2.1 CRISPR @MIT – Genome Surgery

21.2.2 The CRISPR-Cas9 System: A Powerful Tool for Genome Engineering and Regulation

Yongmin Yan and Department of Gastroenterology, Hepatology & Nutrition, University of Texas M.D. Anderson Cancer, Houston, USADaoyan Wei^*

21.2.3 New Frontiers in Gene Editing: Transitioning From the Lab to the Clinic, February 19-20, 2015 | The InterContinental San Francisco | San Francisco, CA

Reporter: Aviva Lev-Ari Ph.D. RN

21.2.4 Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global

Reporter: Aviva Lev-Ari Ph.D. RN

21.2.5 CRISPR & MAGE @ George Church’s Lab @ Harvard

Genome Engineering: CRISPR & MAGE 
Multiplex Automated Genome Engineering (MAGE), is an intentionally broad term. In practice, it has come to be associated with a very efficient oligonucleotide allele-replacment (lambda red beta), so far restricted mainly to E.coli. CRISPR, in contrast, works in nearly every organism tested.

Relevant companies: EnEvolv, Egenesis, Editas.

News:
Editas (NextBigFuture, 28-Nov-2013, Brian Wang)
A Call to Fight Malaria One Mosquito at a Time by Altering DNA (NY Times, 17-Jul-2014, Carl Zimmer)

Resources:
* Vectors: Addgene
* Computational: Center for Causal Consequences of Variation (CCV)

Relevant Lab Publications:
2013 Probing the limits of genetic recoding in essential genes. Science.
2013 Genomically Recoded Organisms Impart New Biological Functions. Science.
2013 CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotech.
2009 Programming cells by multiplex genome engineering and accelerated evolution. Nature.

SOURCE

http://arep.med.harvard.edu/gmc/B2.html

21.3 Patents Awarded and Pending for CRISPR

21.3.1 Litigation on the Way: Broad Institute Gets Patent on Revolutionary Gene-Editing Method

Reporter: Aviva Lev-Ari, PhD, RN

21.3.2 The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

Reporter: Aviva Lev-Ari, PhD, RN

2.4 CRISPR/Cas9 Applications

21.4.1  Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas 

Kennedy EM¹, Kornepati AV¹, Goldstein M², Bogerd HP¹, Poling BC¹, Whisnant AW¹, Kastan MB², Cullen BR³.

21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF

Reporter: Aviva Lev-Ari, PhD, RN

21.4.3 In vivo validated mRNAs

Author & Curator: Larry H. Bernstein, MD, FCAP

21.4.5 Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

Author: Larry H. Bernstein, MD, FCAP

21.4.6 Level of Comfort with Making Changes to the DNA of an Organism

Curator: Aviva Lev-Ari, PhD, RN

21.4.7 Who will be the the First to IPO: Novartis bought in to Intellia (UC, Berkeley) as well as Caribou (UC, Berkeley) vs Editas (MIT)??

Reporter: Aviva Lev-Ari, PhD, RN

21.4.8 CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

Author: Stephen J. Williams, Ph.D.

Summary 

Larry H Bernstein, MD, FCAP 

The field of biology is now experiencing a transformative phase with the advent of facile genome engineering in animals and plants using RNA-programmable CRISPR-Cas9. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems, which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR associated protein Cas9 is an endonuclease uses a guide sequence within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA. This Review illustrates the power of the technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy.

Targeted gene knockdown by RNA interference (RNAi) has provided researchers with a rapid, inexpensive and high-throughput alternative to homologous recombination. However, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects, and provides only temporary inhibition of gene function. These restrictions impede researchers’ ability to directly link phenotype to genotype and limit the practical application of RNAi technology.

CRISPR/Cas9 allows the targeted genome editing for efficient and reliable ways to make precise, targeted changes to the genome of living cells. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference (RNAi).

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence.

There are several CRISPR biopharmaceutical companies using CRISPR-Cas9 technologies to develop transformative medicines for serious human diseases. CRISPR Therapeutics is focused on translating CRISPR-Cas9 gene-editing technology into transformative medicines for serious human diseases. There are also Cambridge-based Editas Medicine and Caribou Biosciences, among the biotech startups working to advance a much-watched new technology for precise gene editing. Doudna and her collaborator, Emmanuelle Charpentier of the Helmholtz Center for Infection Research in Braunschweig, Germany, and Umeå University in Sweden, figured out how to transform a bacterial defense against viral infection into a tool to edit out abnormal sections of genes, such as those that cause hereditary diseases.

In addition, researchers from North Carolina State University (NC State) and the University of North Carolina at Chapel Hill (UNC-CH) have created and utilized a nanoscale vehicle composed of DNA to deliver the CRISPR-Cas9 gene editing complex into cells both in vitro and in vivo.

Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:

1. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
2. CRISPR/CAS9 Use in Developing Models of Disease

• Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other ‘omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.

The current methods of producing the CRISPR–Cas9 components provide great flexibility in terms of expression and delivery, and biologists can exploit these options to control when and where DSBs are generated in an organism. To introduce DSBs and generate modifications early in development, the CRISPR–Cas9 components can be injected as DNA, RNA, or protein into most developing organisms. This approach, which has been widely used, generates mosaic organisms for analysis. To gain control over which tissues are affected, a plasmid expressing Cas9 under the control of tissue-specific enhancers can be used. Since each cell has a choice of whether to repair a breakthrough NHEJ or HDR, a variety of different repair events will be present in the injected organism (and in individual cells).

The relative ease of generating mutant animals will yield many additional animal models of disease and supply a means of testing whether specific polymorphisms are the proximal cause of disease in vivo. Additionally, the CRISPR–Cas9 system is amenable to application in organisms not widely used for genetic studies. Organisms that may be better suited to mimic human disease can now be more easily used to generate disease models. For example, mouse models of the bleeding disorder von Willebrand disease fail to fully recapitulate the human disease.

Apart from point mutations and gene deletions, large chromosomal rearrangements can drive specific cancers. By simultaneously introducing gRNAs targeting two different chromosomes or two widely separated regions of the same chromosome, RGNs have been used to introduce targeted inversions and translocations into otherwise wild-type human cells. These engineered cells will ultimately allow for studies of the causative role of these gene fusions in cancer progression. The first RGN based genetic screens were recently carried out in cultured mammalian cells. The screens identified targets affecting the DNA mismatch repair pathway, resistance to bacterial and chemical toxins, and cell survival and proliferation. The Zheng group also compared the results of their screen for genes involved in resistance to a drug that inhibits B-Raf with a prior RNAi screen that used the same cell line and drug, which revealed that gRNAs identified targets that could be validated more consistently and efficiently than shRNAs, pointing to the potential advantages of using gRNAs to knock out, rather than knock down, gene function in genetic screens.

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice.

A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations

The conclusions from a recent meeting held in Napa have been summarized as follows.

The summary statement is as follows: “A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed.”

They make 4 more specific recommendations.

1. Strongly discourage clinical application of this technology at this time.
2. Create forums for education and discussion
3. Encourage open research to evaluate the utility of CRISPR-Cas9 technology for both human and nonhuman model systems.
4. Hold an international meeting to consider these issues and possibly make policy recommendation.

The need for the meeting resulted from the following work in Japan:

• Concerns surface on Chinese paper on genetic modification of human embryos
• http://www.ipscell.com/2015/04/doudna/

CRISPR-Cas is a prokaryotic defense system against invading genetic elements. In a collaboration with John van der Oost’s laboratory, we are studying the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). Recently, we showed that multiple Cas proteins and a crRNA guide assemble to recognize and cleave invader RNAs at multiple sites . Our negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes, suggesting a model for cleavage of the target RNA at periodic intervals (in collaboration with Eva Nogales, UC Berkeley, HHMI).

Double-stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms through a pathway known as RNA interference (RNAi). RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. We previously determined the crystal structure of an intact Dicer enzyme, revealing how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. Current work focuses on the mechanism of a complex of proteins known as the RISC loading complex (RLC) which load miRNA into the endonuclease Argonaute. The RLC contains the enzyme Dicer as well as TRBP, an RNA-binding protein hypothesized to interact with miRNA and Dicer during RISC loading. We seek to determine the molecular underpinnings of these interactions, along with the role of TRBP in RISC loading.

MicroRNAs (miRNAs) regulate endogenous eukaryotic genes by repressing gene expression through direct base-pairing interactions with their target messenger RNAs (mRNAs). To date, the rules used to predict miRNA-mRNA interactions have been based on one-dimensional sequence analysis. A more complete picture of miRNA-mRNA interactions should take into account the ability of RNA to form two- and three-dimensional structures. We are investigating the role of mRNA structure in the efficiency and specificity of targeting by miRNAs. Specifically, we are investigating the structure of Alu elements found within the 3′ untranslated regions (UTRs) of many human mRNAs and whether these structured domains serve as targets of a subset of human miRNAs. We are using in vitro biochemical methods and cell-based assays to probe the relationship between miRNA binding and mRNA structure.

The 5’ UTR of mRNA is also the site of multiple regulatory mechanisms, including upstream open reading frames (uORFs), internal ribosome entry sites (IRESs), protein binding sites, and stable secondary structures. Genes that profoundly influence cellular state often are controlled by multiple of these regulatory mechanisms. We are attempting to further understand regulatory elements in the 5′ UTR of mammalian mRNA using a combination of in vitro, cell-based and high-throughput techniques.

What does this mean for the development of therapeutics in the near future?

New methods for programming cell phenotype have broadly enabled drug screening, disease modeling, and regenerative medicine. Current research explores genome engineering tools, such as CRISPR/Cas9-based gene regulation and epigenome editing, to more precisely reprogram gene networks and control cellular decision making.

Donald Zack, M.D., Ph.D., Associate Professor of Ophthalmology and Neuroscience, Johns Hopkins University School of Medicine, is using CRISPR/Cas9 technology to generate retinal cell type-specific reporter ES and iPS lines and to introduce retinal degeneration-associated mutations. These reporter lines can be used to follow retinal neuronal specification during differentiation, they allow the purification of specific cell types by sorting and immunopanning, and they also are useful for the development of drug screening assays.

Jacquin C. Niles, M.D., Ph.D., Associate Professor of Biological Engineering, Massachusetts Institute of Technology is using CRISPR-Cas9 technology to study functional genetics in the human malaria parasite, Plasmodium falciparum. The team has established strategies for achieving controllable gene expression, and has integrated these into an experimental framework that facilitates efficient interrogation of virtually any target parasite gene using CRISPR/Cas9 editing.

Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and the Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology is observing that cancer genomics has revealed hundreds to thousands of mutations associated with human cancer. To test the roles of these mutations, we applied CRISPR/Cas9-mediated genome editing platform to engineer specific mutations in oncogenes and tumor suppressor genes. This results in tumorigenesis in several internal organs in mice. Our method expedites modeling of multigenic cancer with virtually any combination of mutations.

Samuel Hasson, Ph.D., Principal Investigator, Neuroscience, Pfizer, Inc., observes that while RNAi-based functional genomics is a staple of gene pathway and drug target exploration, there is a need for tools to provide rapid orthogonal validation of gene candidates that emerge from RNAi campaigns. CRISPR, CRISPRi, and CRISPRa are not only developing into primary screening platforms, they are a promising method to compliment RNAi and enhance the quality of functional genomic datasets.

These are some of the developments that will be discussed in detail at an upcoming meeting in Boston, MA in June titled ‘Gene Editing for Drug Discovery’.

 SOURCE

http://rna.berkeley.edu/crispr.html

http://rna.berkeley.edu/rnai.html

http://rna.berkeley.edu/translation.html

 

Doudna Lab Publications

http://rna.berkeley.edu/publications.html

The structural biology of CRISPR-Cas systems.