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Archive for the ‘CRISPR/Cas9 & Gene Editing’ Category


Optimization of CRISPR Gene Editing with Gold Nanoparticles

Reporter: Irina Robu, PhD

The CRISPR-Cas9 gene editing system has been welcomed as a hopeful solution to a range of genetic diseases, but the expertise has proven hard to deliver into cells. One plan is to open the cell membrane using an electric shock, but that can accidentally kill the cell. Another is to use viruses as couriers. Problem is, viruses can cause off-target side effects.

CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of DNA sequence. It is faster, cheaper and more accurate than previous techniques of editing DNA and can have a wide range of potential applications.

The CRISPR-Cas9 system consists of two key molecules that introduce a change into the DNA. One is an enzyme called Cas9 which acts as a pair of molecular scissors that can cut the two strands of DNA at a specific location in the genome where bits of DNA can be added or removed. The other one, is a piece of RNA which consists of a small piece of pre-designed RNA sequence located within a longer RNA scaffold. The scaffold part binds to the DNA and pre-designed sequence which contains Cas9. The RNA sequence is designed to find and locate specific sequence in the DNA. The Cas9 trails the guide RNA to the same location in the DNA sequence and makes a cut across both strands of DNA. At this point the cell distinguishes that the DNA is damaged and tries to repair it.

Researchers at Fred Hutchinson Cancer Research Center published new findings in Nature Materials suggested an alternative delivery method such as gold nanoparticles. The gold nanoparticles are packed with all the CRISPR components necessary to make clean gene edits. When the gold nanoparticles were tested in lab models of inherited blood disorders and HIV, between 10% and 20% of the targeted cells were effectively edited, with no toxic side effects.

The researchers use gold nanoparticles to deliver CRISPR to blood stem cells. Each gold nanoparticle contains four CRISPR components, including the enzyme needed to make the DNA cuts. But Fred Hutchinson researchers chose Cas12a, which they believed would lead to more efficient edits. Plus, Cas12a only needs one molecular guide, while Cas9 requires two.
In one experiment, they sought to disturb the gene CCR5 to make cells resistant to HIV. In the second, they created a gene mutation that can protect against blood disorders, including sickle cell disease. They observed the cells encapsulated the nanoparticles within six hours and began the gene-editing process within 48 hours. In mice, gene editing peaked eight weeks after injection, and the edited cells were still in circulation 22 weeks after the treatment.
Researchers at Fred Hutchinson are now working on improving the efficiency of the gold-based CRISPR delivery system so that 50% or more of the targeted cells are edited and are also looking for a commercial partner to bring the technology to clinical phase in the next few years.

SOURCE:

https://www.fiercebiotech.com/research/fred-hutch-team-uses-gold-nanoparticles-to-improve-crispr-gene-editing

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Breakthrough in Gene Editing CRISPR–Cas systems: First example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.

 

Reporter: Aviva Lev-Ari, PhD, RN

 

CRISPR alternatives for editing genes without cutting: CRISPR 12, 12a, 13, 14 – Alternative Techniques to CRISPR/Cas9

 

  • Alternative to CRISPR/Cas9 – CAST (CRISPR-associated transposase) – A New Gene-editing Approach for Insertion of Large DNA Sequences into a Genome developed @BroadInstitute @MIT @Harvard

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2019/06/11/alternative-to-crispr-cas9-cast-crispr-associated-transposase-a-new-gene-editing-approach-for-insertion-of-large-dna-sequences-into-a-genome-developed-broadinstitute-mit-harvard/

 

  • Vertex Pharmaceuticals agreed to pay $420 million to acquire Exonics and to expand its partnership with CRISPR Therapeutics. The deal sets in motion a planto use CRISPR to treat Duchenne muscular dystrophy and myotonic dystrophy type 1.

 

  • In May, a team at the Fred Hutchinson Cancer Research Center described a method developed there to use gold nanoparticles to carry CRISPR components into cells and to use the Cas12a enzyme to make cleaner cuts than Cas9 typically does.

 

  • A UC Berkeley spinoff, GenEdit, is also developing a gold-based CRISPR system.

 

  • Other recently proposed ideas for improving CRISPR include attaching a hairpin-like guide to RNA to improve the accuracy of DNA cuts and adding an on-off switch to Cas9 enzymes to ensure they can’t make edits anywhere other than the targeted sites.

 

  • The next step for Sternberg’s team at Columbia is to test the INTEGRATE technology in mammalian cells. They believe the technique could eventually be applied to a variety of products, such as gene therapies and engineered crops.

 

Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration

Abstract

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in E. coli requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. This work provides the first example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.

 SOURCE

A CRISPR alternative for editing genes without cutting

Scientists at Columbia University’s Vagelos College of Physicians and Surgeons are now proposing an alternative gene-editing system—one that sidesteps the need for DNA cutting altogether.

The researchers are using a “jumping gene,” or transposon, from a bacterium called Vibrio cholerae. The transposon is able to insert itself into different regions of the genome and can be programmed to carry any DNA sequence to any site. Therefore their technology, which they dubbed INTEGRATE, acts less like molecular scissors and more like molecular glue, they explained in the journal Nature.

“Rather than introduce DNA breaks and rely on the cell to repair the break, INTEGRATE directly inserts a user-defined DNA sequence at a precise location in the genome, a capability that molecular biologists have sought for decades,” said senior author Sam Sternberg, Ph.D., assistant professor of biochemistry and molecular biophysics at Columbia, in a statement. Sternberg recently joined Columbia after a stint working in the lab of CRISPR pioneer Jennifer Doudna at the University of California, Berkeley.

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Laboratory for Genomics Research (LGR) to be established by GSK ($67M investment in 5 years) at UC, Berkeley/UCSF to be lead by Prof. Jennifer Doudna focusing on immunology, oncology, and neuroscience disease-causing gene mutations and development of new technologies using CRISPR to accelerate new drug discovery

 

Reporter: Aviva Lev-Ari, PhD, RN

 

GlaxoSmithKline, UC’s Doudna Establish $67M Genomics, CRISPR Research Lab

Jun 13, 2019   |  staff reporter

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NEW YORK (GenomeWeb) – Drug company GlaxoSmithKline announced today that it will establish a laboratory for CRISPR technologies as part of a five-year collaboration with University of California researchers.

The new Laboratory for Genomics Research (LGR) will investigate disease-causing gene mutations and develop new technologies using CRISPR to accelerate the discovery of new drugs, with a focus on immunology, oncology, and neuroscience. The LGR will receive up to $67 million in funding over the five-year collaboration period, including facilities for 24 full-time university employees funded by GSK, plus up to 14 full-time GSK employees, the company said. GSK’s artificial intelligence and machine learning group will also aid in building any necessary bioinformatics pipelines. The laboratory will be based near the University of California, San Francisco’s Mission Bay campus.

The LGR aims to automate existing CRISPR approaches so that this work can be done at scale. Ultimately, the lab’s goal is to deepen researchers’ understanding of genetics and discover new drug targets. They’re also aiming to create next-generation technologies for the pharmaceutical industry, GSK added.

The LGR will also serve as a resource for investigators at both UCSF and the University of California, Berkeley who can access and use its technology to answer their own research questions and to develop new tools.

The LGR was developed by Berkeley professor and CRISPR expert Jennifer Doudna, UCSF Professor Jonathan Weissman, and GSK CSO and President of R&D Hal Barron.

“Over the last seven years, CRISPR has transformed academic research, but until the LGR, we haven’t had a focused effort to catalyze the kind of research we know will lead to new innovation using this CRISPR tool,” Doudna said in a statement. “LGR is about building that space where creative science is partnered with the development of robust technology that will help develop tomorrow’s drugs. I think we’re going to be able to do science that none of us can even imagine today.”

The collaboration will be governed by a Joint Steering Committee with equitable UC and GSK representation, with additional joint sub-committees covering patents, scientific, and project management, GSK noted. Doudna and Weissman will sit on the committee along with GSK’s new head of functional genomics, Chris Miller.

“One of our key goals is to advance the field overall and make these tools as broadly available as possible,” Weissman added in the statement. “The LGR screening center will enable labs at UCSF and Berkeley, and having access to it will give our scientists opportunities to advance their research in ways that would be very hard for them to do in their own labs.”

 

ADDITIONAL ARTICLES in GenomeWeb

SOURCE

https://www.genomeweb.com/business-news/glaxosmithkline-ucs-doudna-establish-67m-genomics-crispr-research-lab#.XQKQ8tNKgcg

 

Other related articles published in this Open Access Online Scientific Journal include the following 156 articles:

https://pharmaceuticalintelligence.com/?s=CRISPR

 

 

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Alternative to CRISPR/Cas9 – CAST (CRISPR-associated transposase) – A New Gene-editing Approach for Insertion of Large DNA Sequences into a Genome developed @BroadInstitute @MIT @Harvard

Reporter: Aviva Lev-Ari, PhD, RN

 

A new gene-editing CAST member

In Science, a team led by Jonathan Strecker, Alim Ladha, and core institute member Feng Zhang reports a new gene-editing approach that can precisely and efficiently insert large DNA sequences into a genome. The system, called CRISPR-associated transposase (CAST), is a completely new platform to integrate genetic sequences into cellular DNA, addressing a long-sought goal for precision gene editing. The team molecularly characterized and harnessed the natural CAST system from cyanobacteria, also unveiling a new way that some CRISPR-associated systems perform in nature: not to protect bacteria from viruses, but to facilitate the spread of transposon DNA. Check out more in coverage from STAT and New Scientist.

SOURCE

https://www.broadinstitute.org/news/research-roundup-june-7-2019

 

RNA-guided DNA insertion with CRISPR-associated transposases

Science  06 Jun 2019:
eaax9181
DOI: 10.1126/science.aax9181

Abstract

CRISPR-Cas nucleases are powerful tools to manipulate nucleic acids; however, targeted insertion of DNA remains a challenge as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase (CAST) from cyanobacteria Scytonema hofmanni which consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60-66 bp downstream of the protospacer. ShCAST integrates DNA into unique sites in the E. coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

 

SOURCE

https://science.sciencemag.org/content/early/2019/06/05/science.aax9181

 

Other related articel published in thies Open Access Online Scientific Journal, include:

Breakthrough in Gene Editing CRISPR–Cas systems: First example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2019/06/13/breakthrough-in-gene-editing-crispr-cas-systems-first-example-of-a-fully-programmable-rna-guided-integrase-and-lays-the-foundation-for-genomic-manipulations-that-obviate-the-requirements-for/

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eProceedings for BIO 2019 International Convention, June 3-6, 2019 Philadelphia Convention Center; Philadelphia PA, Real Time Coverage by Stephen J. Williams, PhD @StephenJWillia2

 

CONFERENCE OVERVIEW

Real Time Coverage of BIO 2019 International Convention, June 3-6, 2019 Philadelphia Convention Center; Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/05/31/real-time-coverage-of-bio-international-convention-june-3-6-2019-philadelphia-convention-center-philadelphia-pa/

 

LECTURES & PANELS

Real Time Coverage @BIOConvention #BIO2019: Machine Learning and Artificial Intelligence: Realizing Precision Medicine One Patient at a Time, 6/5/2019, Philadelphia PA

Reporter: Stephen J Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/05/real-time-coverage-bioconvention-bio2019-machine-learning-and-artificial-intelligence-realizing-precision-medicine-one-patient-at-a-time/

 

Real Time Coverage @BIOConvention #BIO2019: Genome Editing and Regulatory Harmonization: Progress and Challenges, 6/5/2019. Philadelphia PA

Reporter: Stephen J Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/05/real-time-coverage-bioconvention-bio2019-genome-editing-and-regulatory-harmonization-progress-and-challenges/

 

Real Time Coverage @BIOConvention #BIO2019: Precision Medicine Beyond Oncology June 5, 2019, Philadelphia PA

Reporter: Stephen J Williams PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/05/real-time-coverage-bioconvention-bio2019-precision-medicine-beyond-oncology-june-5-philadelphia-pa/

 

Real Time @BIOConvention #BIO2019:#Bitcoin Your Data! From Trusted Pharma Silos to Trustless Community-Owned Blockchain-Based Precision Medicine Data Trials, 6/5/2019, Philadelphia PA

Reporter: Stephen J Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/05/real-time-bioconvention-bio2019bitcoin-your-data-from-trusted-pharma-silos-to-trustless-community-owned-blockchain-based-precision-medicine-data-trials/

 

Real Time Coverage @BIOConvention #BIO2019: Keynote Address Jamie Dimon CEO @jpmorgan June 5, 2019, Philadelphia, PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/05/real-time-coverage-bioconvention-bio2019-keynote-address-jamie-dimon-ceo-jpmorgan-june-5-philadelphia/

 

Real Time Coverage @BIOConvention #BIO2019: Chat with @FDA Commissioner, & Challenges in Biotech & Gene Therapy June 4, 2019, Philadelphia, PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/04/real-time-coverage-bioconvention-bio2019-chat-with-fda-commissioner-challenges-in-biotech-gene-therapy-june-4-philadelphia/

 

Falling in Love with Science: Championing Science for Everyone, Everywhere June 4 2019, Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/04/real-time-coverage-bioconvention-bio2019-falling-in-love-with-science-championing-science-for-everyone-everywhere/

 

Real Time Coverage @BIOConvention #BIO2019: June 4 Morning Sessions; Global Biotech Investment & Public-Private Partnerships, 6/4/2019, Philadelphia PA

Reporter: Stephen J Williams PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/04/real-time-coverage-bioconvention-bio2019-june-4-morning-sessions-global-biotech-investment-public-private-partnerships/

 

Real Time Coverage @BIOConvention #BIO2019: Understanding the Voices of Patients: Unique Perspectives on Healthcare; June 4, 2019, 11:00 AM, Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/04/real-time-coverage-bioconvention-bio2019-understanding-the-voices-of-patients-unique-perspectives-on-healthcare-june-4/

 

Real Time Coverage @BIOConvention #BIO2019: Keynote: Siddhartha Mukherjee, Oncologist and Pulitzer Author; June 4 2019, 9AM, Philadelphia PA

Reporter: Stephen J. Williams, PhD. @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/04/real-time-coverage-bioconvention-bio2019-keynote-siddhartha-mukherjee-oncologist-and-pulitzer-author-june-4-9am-philadelphia-pa/

 

Real Time Coverage @BIOConvention #BIO2019:  Issues of Risk and Reproduceability in Translational and Academic Collaboration; 2:30-4:00 June 3, 2019, Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/03/real-time-coverage-bioconvention-bio2019-issues-of-risk-and-reproduceability-in-translational-and-academic-collaboration-230-400-june-3-philadelphia-pareal-time-coverage-bioconvention-bi/

 

Real Time Coverage @BIOConvention #BIO2019: What’s Next: The Landscape of Innovation in 2019 and Beyond. 3-4 PM June 3, 2019, Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/03/real-time-coverage-bioconvention-bio2019-whats-next-the-landscape-of-innovation-in-2019-and-beyond-3-4-pm-june-3-philadelphia-pa/

 

Real Time Coverage @BIOConvention #BIO2019: After Trump’s Drug Pricing Blueprint: What Happens Next? A View from Washington; June 3, 2019 1:00 PM, Philadelphia PA

Reporter: Stephen J. Williams, PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/03/real-time-coverage-bioconvention-bio2019-after-trumps-drug-pricing-blueprint-what-happens-next-a-view-from-washington-june-3-2019-100-pm-philadelphia-pa/

 

Real Time Coverage @BIOConvention #BIO2019: International Cancer Clusters Showcase June 3, 2019, Philadelphia PA

Reporter: Stephen J. Williams PhD @StephenJWillia2

https://pharmaceuticalintelligence.com/2019/06/03/real-time-coverage-bioconvention-bio2019-international-cancer-clusters-showcase-june-3-philadelphia-pa/

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Real Time Coverage @BIOConvention #BIO2019: Genome Editing and Regulatory Harmonization: Progress and Challenges

Reporter: Stephen J Williams, PhD @StephenJWillia2

 

Genome editing offers the potential of new and effective treatments for genetic diseases. As companies work to develop these treatments, regulators are focused on ensuring that any such products meet applicable safety and efficacy requirements. This panel will discuss how European Union and United States regulators are approaching therapeutic use of genome editing, issues in harmonization between these two – and other – jurisdictions, challenges faced by industry as regulatory positions evolve, and steps that organizations and companies can take to facilitate approval and continued efforts at harmonization.

 

CBER:  because of the nature of these gene therapies, which are mainly orphan, there is expedited review.  Since they started this division in 2015, they have received over 1500 applications.

Spark: Most of the issues were issues with the primary disease not the gene therapy so they had to make new endpoint tests so had talks with FDA before they entered phase III.   There has been great collaboration with FDA,  now they partnered with Novartis to get approval outside US.  You should be willing to partner with EU pharmas to expedite the regulatory process outside US.  In China the process is new and Brazil is behind on their gene therapy guidance.  However there is the new issue of repeat testing of your manufacturing process, as manufacturing of gene therapies had been small scale before. However he notes that problems with expedited review is tough because you don’t have alot of time to get data together.  They were lucky that they had already done a randomized trial.

Sidley Austin:  EU regulatory you make application with advance therapy you don’t have a national option, the regulation body assesses a committee to see if has applicability. Then it goes to a safety committee.  EU has been quicker to approve these advance therapies. Twenty five percent of their applications are gene therapies.  Companies having issues with manufacturing.  There can be issues when the final application is formalized after discussions as problems may arise between discussions, preliminary applications, and final applications.

Sarepta: They have a robust gene therapy program.  Their lead is a therapy for DMD (Duchenne’s Muscular Dystrophy) where affected males die by 25. Japan and EU have different regulatory applications and although they are similar and data can be transferred there is more paperwork required by EU.  The US uses an IND for application. Global feedback is very challenging, they have had multiple meetings around the world and takes a long time preparing a briefing package….. putting a strain on the small biotechs.  No company wants to be either just EU centric or US centric they just want to get out to market as fast as possible.

 

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People with two copies of the Δ32 mutation died at rates 21 percent higher than those with one or no copies – application of CRISPR @Berkeley

Reporter: Aviva Lev-Ari, PhD, RN

 

CCR5-∆32 is deleterious in the homozygous state in humans

CRISPR baby mutation significantly increases mortality

“Here is a functional protein that we know has an effect in the organism, and it is well-conserved among many different species, so it is likely that a mutation that destroys the protein is, on average, not good for you,” he said. “Otherwise, evolutionary mechanisms would have destroyed that protein a long time ago.”

SOURCE

https://news.berkeley.edu/2019/06/03/crispr-baby-mutation-significantly-increases-mortality/

In the UK Biobank data they found two lines of evidence to suggest that these days, CCR5 actually is a net negative. In the first analysis they tracked how long people survived after enrolling in the Biobank study. They found that between the ages of 41 and 78, people with two copies of the Δ32 mutation had significantly higher death rates. They also observed that far fewer people with two copies enrolled in the study than expected, which they interpreted to mean that these individuals were less likely to survive into middle age than the general population. “Something has removed people with two copies of the mutation, and the likely explanation is increased mortality,” says Nielsen.

A child was born missing a large chunk of DNA in its CCR5 gene. This gene coded for a receptor on the surface of immune cells useful for coordinating responses to invading pathogens. And this spontaneous deletion torpedoed CCR5 production—one copy shrunk the number of receptors on cells, two copies erased the receptor altogether.

Today, the Δ32 mutation occurs in about 10 percent of the population of Europe, in a decreasing gradient from north to south. Natural selection pushed it through the population about 100 times faster than if it were a neutral change to the genome. But with the invention of vaccines, and the eradication of diseases like smallpox over the last century, the mutation has become less useful. According to Nielsen and Wei’s analysis, it’s now downright detrimental.

SOURCE

https://www.wired.com/story/a-study-exposes-the-health-risks-of-gene-editing-human-embryos/?mbid=social_linkedin&utm_brand=wired&utm_campaign=wired&utm_medium=social&utm_social-type=owned&utm_source=linkedin

 

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