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Posts Tagged ‘CRISPR-Cas9’

2nd Annual Translational Gene Editing: Exploiting CRISPR/Cas9 for Building Tools for Drug Discovery & Development: June 16, 2016, Boston, MA

Posted in BioIT: BioInformatics, BioIT: BioInformatics, NGS, Clinical & Translational, Pharmaceutical R&D Informatics, Clinical Genomics, Cancer Informatics, Biological Networks, Computational Biology/Systems and Bioinformatics, CRISPR/Cas9 & Gene Editing, Pharmaceutical Drug Discovery, tagged advances in drug development, CRISPR-Cas9, CRISPR/Cas, Drug discovery, functional screens, gene editing, high throughput, RNAi on March 30, 2016| Leave a Comment »

2nd Annual Translational Gene Editing: Exploiting CRISPR/Cas9 for Building Tools for Drug Discovery & Development: June 16, 2016, Boston, MA

Reporter: Stephen J. Williams, PhD

Translational Gene Editing – June 16-17, 2016 in Boston, MA #CHIWPC16 Learn More | Sponsorship & Exhibit Details | Register by April 29 & SAVE up to $200! IMPROVING CRISPR FOR BETTER FUNCTIONAL SCREENING Optimized sgRNA Libraries for Genetic Screens with CRISPR-Cas9 John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT Optimizing CRISPR for Pooled Genome-Wide Functional Genetic Screens Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc. CRISPR-Cas9 Whole Genome Screening: Going Where No Screen Has Gone Before Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center Cross-Species Synthetic Lethal Screens and Applications to Drug Discovery Norbert Perrimon, Ph.D., Professor, Department of Genetics, Harvard Medical School and Investigator, Howard Hughes Medical Institute Interactive Breakout Discussion Groups with Continental Breakfast This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants. Topic: CRISPR/Cas9 System for In vivo Drug Discovery Moderator: Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research Impact of CRISPR/Cas9 system on in vivo mouse models Application of the CRISPR/Cas9 system in in vivo screens Technical limitations/safety issues Topic: Getting Past CRISPR Pain Points Moderators: John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MITStephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School Challenges and solutions for CRISPR gRNA design Methods for detecting engineered changes Topic: Cellular Delivery of CRISPR/Cas9 Moderator: Daniel E Bauer M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute GENE EDITING FOR SCREENING DISEASE PATHWAYS AND DRUG TARGETS Scouring the Non-Coding Genome by Saturating Edits Daniel E. Bauer, M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute 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 BUILDING THE CRISPR TOOLBOX Beyond Cas9: Discovering Single Effector CRISPR Tools Jonathan Gootenberg, Member, Laboratories of Dr. Aviv Regev and Dr. Feng Zhang, Department of Systems Biology, Harvard Medical School, and Broad Institute of Harvard and MIT CRISPR-Cas9 Genome Editing Improves Sub-Cellular Localization Studies Netanya Y. Spencer, M.D., Ph.D., Research Fellow in Medicine, Joslin Diabetes Center, Harvard Medical School TECHNOLOGY PANEL: Trends in CRISPR Technologies Panelists to be Announced This panel will bring together 2-3 technical experts from leading technology and service companies to discuss trends and improvements in CRISPR libraries, reagents and platforms that users can expect to see in the near future. (Opportunities Available for Sponsoring Panelists) APPLICATIONS OF CRISPR FOR DRUG DISCOVERY Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc. Application of Genome Editing Tools to Model Human Genetics Findings in Drug Discovery Myung Shin, Ph.D., Senior Principal Scientist, Genetics and Pharmacogenomics, Merck & Co. Inc. In vivo Application of the CRISPR/Cas9 Technology for Translational Research Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research DEVELOPING TOOLS FOR BETTER TRANSLATION Improving CRISPR-Cas9 Precision through Tethered DNA-Binding Domains Scot A. Wolfe, Ph.D., Associate Professor, Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing Daniel G. Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology Translating CRISPR/Cas9 into Novel Medicines Alexandra Glucksmann, Ph.D., COO, Editas Medicine

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Rice University researches develop new CRISPR-CAS9 strategy to reduce off-target gene editing effects

Posted in CRISPR/Cas9 & Gene Editing, tagged CRISPR-Cas9, gene editing technology platform on March 22, 2016| Leave a Comment »

Rice University researches develop new CRISPR-CAS9 strategy to reduce off-target gene editing effects

Reporter: Stephen J. Williams, PhD

Series B, Volume 2:

Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS & BioInformatics, Simulations and the Genome Ontology

https://www.amazon.com/dp/B08385KF87

New strategies, tools offered for genome editing

 Reported from Science Daily at  https://www.sciencedaily.com/

Bioengineer Gang Bao and team explore CRISPR-Cas9 alternatives

Date:

February 8, 2016

Source:

Rice University

Summary:

Bioengineers have studied alternative CRISPR-Cas9 systems for precision genome editing, with a focus on improving its accuracy and limiting ‘off-target’ errors.

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FULL STORY

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A Cas9 protein (light blue) with guide RNA (purple) and DNA (red) shows a DNA bulge, marking a sequence that would be considered off-target for CRISPR-Cas9 editing. The Rice University lab of bioengineer Gang Bao has developed Web-based tools to search for such off-targets.

Credit: Bao Lab/Rice University

 

 

Rice University bioengineers have found new techniques for precision genome editing that are more accurate and have fewer off-target errors.

The new strategies are shared in three papers in an upcoming special issue of the Nature journal Molecular Therapy on improving the revolutionary genome-editing technique called CRISPR-Cas9.

Bioengineering Professor Gang Bao and his colleagues present ideas for maximizing on-target gene editing with biological catalysts capable of cutting DNA called “engineered nucleases.” Several such systems have been studied for years, but for the past three, the promise of cut-and-paste editing via CRISPR-Cas9 has captured the attention of scientists worldwide.

CRISPR-Cas9, a naturally occurring defense system in bacteria, allows researchers to design a short sequence of RNA called “guide RNA” that targets a specific section of genetic code (DNA) in a cell. An associated Cas9 protein then cuts the section, disrupts it or replaces it with the desired code.

That’s how bacteria use CRISPR-Cas9 to immunize themselves from disease. Exposure to an invader causes the bacteria to adapt by adding the invader’s genetic signature to a CRISPR database. The bacteria then recognize future enemies and destroy them with an appropriate Cas9 protein.

About three years ago researchers discovered that bacterial CRISPR-Cas9 could be modified to edit DNA in human cells by, for instance, replacing mutant sequences with normal, or “wild-type,” sequences in much the same way a bacterium banks an invader’s DNA signature. The technique is seen as having great potential for disease modeling and treatment, synthetic biology and molecular pathway dissection.

But CRISPR-Cas9 is still vulnerable to snipping the wrong sequences — called “off-targets” — in addition to the right ones. In therapeutic applications, Bao said, off-target cutting by CRISPR-Cas9 could cause many detrimental effects, including cancer.

Bao, who moved to Rice’s BioScience Research Collaborative (BRC) in 2015 with a grant from the Cancer Prevention and Research Institute of Texas, is studying ways to refine CRISPR-Cas9, which he described as “nanoscissors for editing genes.”

One of his goals is to treat the hereditary disease sickle cell anemia, which he hopes CRISPR-Cas9 will eventually cure. But first the therapy must become much better at avoiding off-targets that can cause unwanted side effects.

In two of the papers, the researchers study different orthologs: Cas9 proteins from species with the same ancestors as the Streptococcus pyogenes (Spy)bacterium commonly used in CRISPR/Cas9.

“Our approach in these papers is to explore the possibility of using different Cas9 orthologs,” Bao said. “There are many possibilities.”

In the first paper, Bao and his group used experiments on mammalian cells to characterize a CRISPR-Cas9 system from the Neisseria meningitides (Nme) bacterium. It differs from Spy in a way that bioengineers can use to reduce the risk of off-target edits, he said.

That difference lies primarily in a sequence of code that is not part of the target, but close by. Known as a protospacer-adjacent motif (PAM), it’s a marker for target DNA sequences and necessary for Cas9 protein binding. InSpyCas9 editing, the PAM sequence is generally three nucleotides long. For Nme, the required PAM sequence is significantly longer — eight nucleotides. While Nme may find fewer targets, those targets are more likely to be the correct ones, according to the researchers. That, they argue, may make it a safer alternative for gene editing.

The second paper, a collaboration with colleagues at the University of Freiburg, Germany, addresses highly specific human-gene editing using yet another bacteria’s immune system. For this study, Cas9 proteins from Spy were replaced with Streptococcus thermophiles (Sth) proteins that also recognize longer PAMs. Tests carried out in human cells found Sth proteins with more stringent PAM requirements were significantly better than SpyCas9 proteins at avoiding off-targets.

Bao and company also looked at the effect of bulges in DNA and RNA that can influence targeting. Bulges appear when a sequence is one nucleotide longer or one nucleotide shorter than the expected DNA sequence targeted by guide RNA.

“We found that even with DNA or RNA bulges, the Cas9 protein can still cut,” he said. “That’s a unique contribution. Nobody saw that would be the case, but we demonstrated it. Consequently, we’ve developed a Web-based tool to search for three cases of potential off-target sites that contain base mismatches, RNA bulges and DNA bulges.”

Bao noted the Nme and Sth Cas9 proteins, unlike Spy, are small enough to be packaged within an adeno-associated virus for delivery to and treatment of specific cells in an animal. “That’s another advantage, and why we want to go on to explore these two systems,” he said.

The third paper is a review of current CRISPR-Cas9 techniques that focuses on genome-editing tools available for target selection, experimental methods and validation. Bao and his team also lay out a list of challenges yet to be solved to eliminate off-target effects.

He said there is a path forward, represented in part by his investigation of two new bacterial systems as well as the fact that CRISPR-Cas9 is a much easier technique to implement in the lab than other genome-editing systems such as TALEN and zinc finger nuclease.

Bao said that unlike those older genome-editing techniques, CRISPR-Cas9 is straightforward enough for students to learn and use in a short time.

Bao hopes to establish his lab as a focal point for genome editing in the Texas Medical Center. To that end, he brought the TMC genome-editing community together for a well-attended workshop at the BRC last December.

“We had a lot of good discussions,” he said. “One thing I would like to stimulate is the formation of a consortium among the many labs in TMC using CRISPR. They have needs to design CRISPR systems for different applications, but there are a lot of common issues. If we work together, it will be easier to address them.”

 

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Story Source:

The above post is reprinted from materials provided by Rice University.Note: Materials may be edited for content and length.

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Journal References:

1. Ciaran M. Lee, Thomas J. Cradick, Gang Bao. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells. Molecular Therapy, 2016; DOI:10.1038/mt.2016.8
2. Maximilian Müller, Ciaran M Lee, Giedrius Gasiunas, Timothy H Davis, Thomas J Cradick, Virginijus Siksnys, Gang Bao, Toni Cathomen, Claudio Mussolino. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. Molecular Therapy, 2015; DOI: 10.1038/mt.2015.218
3. Ciaran M. Lee, Thomas J. Cradick, Eli J Fine, Gang Bao. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Molecular Therapy, 2016; DOI: 10.1038/mt.2016.1

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Rice University. “New strategies, tools offered for genome editing: Bioengineer Gang Bao and team explore CRISPR-Cas9 alternatives.” ScienceDaily. ScienceDaily, 8 February 2016. <www.sciencedaily.com/releases/2016/02/160208135449.htm>.

 

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RNA-Based Drugs Turn CRISPR/Cas9 On and Off

Posted in 3D Printing for Medical Application, Cell Level, CRISPR/Cas9 & Gene Editing, tagged CRISPR-Cas9, CRISPR/Cas and crispr, gene editing, RNA on February 17, 2016| Leave a Comment »

From GEN News Highlights

Reposted from GEN News

Nov 18, 2015

2.2.8   RNA-Based Drugs Turn CRISPR/Cas9 On and Off, 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

RNA-Based Drugs Turn CRISPR/Cas9 On and Off

• This image depicts a conventional CRISPR-Cas9 system. The Cas9 enzyme acts like a wrench, and specific RNA guides act as different socket heads. Conventional CRISPR-Cas9 systems act continuously, raising the risk of off-target effects. But CRISPR-Cas9 systems that incorporate specially engineered RNAs could act transiently, potentially reducing unwanted changes. [Ernesto del Aguila III, NHGRI]

  By removing parts of the CRISPR/Cas9 gene-editing system, and replacing them with specially engineered molecules, researchers at the University of California, San Diego (UCSD) and Isis Pharmaceutical hope to limit the CRISPR/Cas9 system’s propensity for off-target effects. The researchers say that CRISPR/Cas9 needn’t remain continuously active. Instead, it could be transiently activated and deactivated. Such on/off control could prevent residual gene-editing activity that might go awry. Also, such control could be exploited for therapeutic purposes.

  The key, report the scientists, is the introduction of RNA-based drugs that can replace the guide RNA that usually serves to guide the Cas9 enzyme to a particular DNA sequence. When Cas9 is guided by a synthetic RNA-based drug, its cutting action can be suspended whenever the RNA-based drug is cleared. The Cas9’s cutting action can be stopped even more quickly if a second, chemically modified RNA drug is added, provided that it is engineered to direct inactivation of the gene encoding the Cas9 enzyme.

  Details about temporarily activated CRISPR/Cas9 systems appeared November 16 in the Proceedings of the National Academy of Sciences, in a paper entitled, “Synthetic CRISPR RNA-Cas9–guided genome editing in human cells.” The paper’s senior author, the USCD’s Don Cleveland, Ph.D., noted that the RNA-based drugs described in the study “provide many advantages over the current CRISPR/Cas9 system,” such as increased editing efficiency and potential selectivity.

  “Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells,” wrote the authors of the PNAS paper. “Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA.”

  Not only did the synthetic RNA functionally replace the natural crRNA, it produced enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. These findings, Dr. Cleveland explained, could provide a platform for multiple therapeutic applications, especially for nervous system diseases, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 activity. “In addition,” he said, “[these designer RNAs] can be synthesized efficiently, on an industrial scale and in a commercially feasible manner today.”

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Use of CRISPR/CAS9 to Edit Genome of Pigs: Recominetics announces $10M Funding Round

Posted in CRISPR/Cas9 & Gene Editing, Disease Biology, Gene Regulation, Uncategorized, tagged animal models of disease, CRISPR-Cas9, gene editing, minipig, porcine models, transgenics on February 12, 2016| Leave a Comment »

Gene-editing startup raising $10M to expand staff
Nov 25, 2015

Reporter: Stephen J. Williams,Ph.D.

From the Mineapolis/St. Paul Journal

source from: http://www.bizjournals.com/twincities/news/2015/11/25/gene-editing-startup-raising-10m-to-expand-staff.html 

Katharine Grayson
Staff reporter
Minneapolis / St. Paul Business Journal

Recombinetics Inc. is seeking $10 million in funding as it ramps up sales of its genetically tweaked animals.
The St. Paul-based biotech company’s recent round has already brought in about about $2.8 million from friends and family, said Chief Operating Officer Kyle Dawley. Company officials hope to close out the round within the next two months and add about 10 employees to its staff of 25.

 

 

Recombinetics edits pigs’ genes for biomedical research purposes. Photo source: Simone Van Den Berg

Recombinetics uses gene-editing technology to tweak animals for the agribusiness and biomedical markets. It’s biomedical business centers around pigs, which the company modifies for research purposes. That side of the company’s business already generates revenue, Dawley said, though he declined to reveal sales figures.

The company focuses on pigs, touting them as better research subjects than mice when it comes to testing medical devices and drugs for use in humans.

“Pigs are — size-wise and genetically — a lot more like humans than rats and mice,” Dawley said.

One of Recombinetics’ long-term goals is grow human organs inside pigs.

The company aims to modify livestock for food consumption as well. One of its projects calls for creating hornless cattle by taking a gene from one breed and putting into another.

Recombinetics expects food ventures may get a boost from the Food and Drug Administration’s recent approval of a genetically engineered salmon called “AquAdvantage.” The fish grows faster than traditional salmon thanks to the introduction of trout genes.

Recombinetics has raised $15 million since its founding.

Katharine Grayson covers med tech, clean tech, technology, health care and venture capital.

 

See also Surrogen, Inc., which produces transgenic pigs for purpose of large animal models of disease.

Other posts on this Open Access Journal where I have discussed the utility of the minipig as a large animal model of disease include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

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