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Posts Tagged ‘CRISPR’


CRISPR cuts turn gels into biological watchdogs

Reporter: Irina Robu, PhD

Genome editing if of significant interest in the prevention and treatment of human diseases including single-gene disorders such as cystic fibrosis, hemophilia and sickle cell disease. It also shows great promise for the prevention and treatment of diseases such as cancer, heart disease, mental illness and human immunodeficiency virus infection. However, ethical concerns arise when genome editing, using technologies such as CRISPR-Cas9 is used to alter human genomes.

James Collins, bioengineer at MIT and his team worked with water-filled polymers that are held together by strands of DNA, known as DNA hydrogels. To alter the properties of these materials, these scientists turned to a form of CRISPR that uses a DNA-snipping enzyme called Cas12a, which can be programed to recognize a specific DNA sequence. The enzyme then cuts its target DNA strand, then severs single strands of DNA nearby. This property lets scientists to build a series of CRISPR-controlled hydrogels encapsulating a target DNA sequence and single strands of DNA, which break up after Cas12a identifies the target sequence in a stimulus. The break-up of the single DNA strands activates the hydrogels to change shape or completely dissolve, releasing a payload.

According to Collins and his team, the programmed hydrogels will release enzymes, small molecules and human cells as part of a smart therapy in response to stimuli. However, in order to make it a smart therapeutic, the researchers in collaboration with Dan Luo, bioengineer at Cornell University placed the CRISPR- controlled hydrogels into electric circuits. The circuit is switched off in response to the detection of the genetic material of harmful pathogens such as Ebola virus and methicillin-resistant Staphylococcus aureus. The team used these hydrogels to develop a prototype diagnostic tool that sends a wireless signal to identify Ebola in lab samples.

Yet, it is evident that these CRISPR-controlled hydrogels show great potential for the prevention and treatment of diseases.

SOURCE

https://www.nature.com/articles/d41586-019-02542-3?utm_source=Nature+Briefing

 

 

 

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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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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Researchers have embraced CRISPR gene-editing as a method for altering genomes, but some have reported that unwanted DNA changes may slip by undetected. The tool can cause large DNA deletions and rearrangements near its target site on the genome. Such alterations can confuse the interpretation of experimental results and could complicate efforts to design therapies based on CRISPR. The finding is in line with previous results from not only CRISPR but also other gene-editing systems.

 

CRISPR -Cas9 gene editing relies on the Cas9 enzyme to cut DNA at a particular target site. The cell then attempts to reseal this break using its DNA repair mechanisms. These mechanisms do not always work perfectly, and sometimes segments of DNA will be deleted or rearranged, or unrelated bits of DNA will become incorporated into the chromosome.

 

Researchers often use CRISPR to generate small deletions in the hope of knocking out a gene’s function. But when examining CRISPR edits, researchers found large deletions (often several thousand nucleotides) and complicated rearrangements of DNA sequences in which previously distant DNA sequences were stitched together. Many researchers use a method for amplifying short snippets of DNA to test whether their edits have been made properly. But this approach might miss larger deletions and rearrangements.

 

These deletions and rearrangements occur only with gene-editing techniques that rely on DNA cutting and not with some other types of CRISPR modifications that avoid cutting DNA. Such as a modified CRISPR system to switch one nucleotide for another without cutting DNA and other systems use inactivated Cas9 fused to other enzymes to turn genes on or off, or to target RNA. Overall, these unwanted edits are a problem that deserves more attention, but this should not stop anyone from using CRISPR. Only when people use it, they need to do a more thorough analysis about the outcome.

 

References:

 

https://www.nature.com/articles/d41586-018-05736-3?utm_source=briefing-dy

 

https://www.ncbi.nlm.nih.gov/pubmed/28561021

 

https://www.ncbi.nlm.nih.gov/pubmed/30010673

 

https://www.ncbi.nlm.nih.gov/pubmed/24651067

 

https://www.ncbi.nlm.nih.gov/pubmed/25398350

 

https://www.ncbi.nlm.nih.gov/pubmed/24838573

 

https://www.ncbi.nlm.nih.gov/pubmed/25200087

 

https://www.ncbi.nlm.nih.gov/pubmed/25757625

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Scientists think excessive population growth is a cause of scarcity and environmental degradation. A male pill could reduce the number of unintended pregnancies, which accounts for 40 percent of all pregnancies worldwide.

 

But, big drug companies long ago dropped out of the search for a male contraceptive pill which is able to chemically intercept millions of sperm before they reach a woman’s egg. Right now the chemical burden for contraception relies solely on the female. There’s not much activity in the male contraception field because an effective solution is available on the female side.

 

Presently, male contraception means a condom or a vasectomy. But researchers from Center for Drug Discovery at Baylor College of Medicine, USA are renewing the search for a better option—an easy-to-take pill that’s safe, fast-acting, and reversible.

 

The scientists began with lists of genes active in the testes for sperm production and motility and then created knockout mice that lack those genes. Using the gene-editing technology called CRISPR, in collaboration with Japanese scientists, they have so far made more than 75 of these “knockout” mice.

 

They allowed these mice to mate with normal (wild type) female mice, and if their female partners don’t get pregnant after three to six months, it means the gene might be a target for a contraceptive. Out of 2300 genes that are particularly active in the testes of mice, the researchers have identified 30 genes whose deletion makes the male infertile. Next the scientists are planning a novel screening approach to test whether any of about two billion chemicals can disable these genes in a test tube. Promising chemicals could then be fed to male mice to see if they cause infertility.

 

Female birth control pills use hormones to inhibit a woman’s ovaries from releasing eggs. But hormones have side effects like weight gain, mood changes, and headaches. A trial of one male contraceptive hormone was stopped early in 2011 after one participant committed suicide and others reported depression. Moreover, some drug candidates have made animals permanently sterile which is not the goal of the research. The challenge is to prevent sperm being made without permanently sterilizing the individual.

 

As a better way to test drugs, Scientists at University of Georgia, USA are investigating yet another high-tech approach. They are turning human skin cells into stem cells that look and act like the spermatogonial cells in the testes. Testing drugs on such cells might provide more accurate leads than tests on mice.

 

The male pill would also have to start working quickly, a lot sooner than the female pill, which takes about a week to function. Scientists from University of Dundee, U.K. admitted that there are lots of challenges. Because, a women’s ovary usually release one mature egg each month, while a man makes millions of sperm every day. So, the male pill has to be made 100 percent effective and act instantaneously.

 

References:

 

https://www.technologyreview.com/s/603676/the-search-for-a-perfect-male-birth-control-pill/

 

https://futurism.com/videos/the-perfect-male-birth-control-pill-is-coming-soon/?utm_source=Digest&utm_campaign=c42fc7b9b6-EMAIL_CAMPAIGN_2017_03_20&utm_medium=email&utm_term=0_03cd0a26cd-c42fc7b9b6-246845533

 

http://www.telegraph.co.uk/women/sex/the-male-pill-is-coming—and-its-going-to-change-everything/

 

http://www.mensfitness.com/women/sex-tips/male-birth-control-pill-making

 

http://health.howstuffworks.com/sexual-health/contraception/male-bc-pill.htm

 

http://europe.newsweek.com/male-contraception-side-effects-study-pill-injection-518237?rm=eu

 

http://edition.cnn.com/2016/01/07/health/male-birth-control-pill/index.html

 

http://www.nhs.uk/Conditions/contraception-guide/Pages/male-pill.aspx

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LIVE 9/21 8AM to 10:55 AM Expoloring the Versatility of CRISPR/Cas9 at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Targettaking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

#BostonDOT16

@BostonDOT

 

COMMENTS BY Stephen J Williams, PhD

EXPLORING THE VERSATILITY OF CRISPR/Cas9

 

8:00 Chairperson’s Opening Remarks

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

@CRISPRTX

 

8:10 Functional Genomics Using CRISPR-Cas9: Technology and Applications

Neville Sanjana, Ph.D., Core Faculty Member, New York Genome Center and Assistant Professor, Department of Biology & Center for Genomics and Systems Biology, New York University

 

CRISPR Cas9 is easier to target to multiple genomic loci; RNA specifies DNA targeting; with zinc finger nucleases or TALEEN in the protein specifies DNA targeting

 

  • This feature of crisper allows you to make a quick big and cheap array of a GENOME SCALE Crisper Knock out (GeCKO) screening library
  • How do you scale up the sgRNA for whole genome?; for all genes in RefSeq, identify consitutive exons using RNA-sequencing data from 16 primary human tissue (alot of genes end with ‘gg’) changing the bases on 3’ side negates crisper system but changing on 5’ then crisper works fine
  • Rank sequences to be specific for target
  • Cloned array into lentiviral and put in selectable markers
  • GeCKO displays high consistency betweens reagents for the same gene versus siRNA; GeCKO has high screening sensitivity
  • 98% of genome is noncoding so what about making a library for intronic regions (miRNA, promoter regions?)
  • So you design the sgRNA library by taking 100kb of gene-adjacent regions
  • They looked at CUL3; (data will soon be published in Science)
  • Do a transcription CHIP to verify the lack of binding of transcription factor of interest
  • Can also target histone marks on promoter and enhancer elements
  • NYU wants to explore this noncoding screens
  • sanjanalab.org

 

@nyuniversity

 

8:40 Therapeutic Gene Editing With CRISPR/Cas9

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

NEHJ is down and dirty repair of single nonhomologous end but when have two breaks the NEHJ repair can introduce the inversions or deletions

 

    • High-throughput screens are fine but can limit your view of genomic context; genome searches pick unique sites so use bioinformatic programs  to design specific guide Rna
    • Bioinformatic directed, genome wide, functional screens
    • Compared COSMID and CCTOP; 320 COSMID off-target sites, 333 CCtop off target
    • Young lab GUIDESeq program genome wide assay useful to design guides
    • If shorten guide may improve specificity; also sometime better sensitivity if lengthen guide

 

  • Manufacturing of autologous gene corrected product ex vivo gene correction (Vertex, Bayer, are partners in this)

 

 

They need to use a clones from multiple microarrays before using the GUidESeq but GUIDEseq is better for REMOVING the off targets than actually producing the sgRNA library you want (seems the methods for library development are not fully advanced to do this)

 

The score sometimes for the sgRNA design programs do not always give the best result because some sgRNAs are genome context dependent

9:10 Towards Combinatorial Drug Discovery: Mining Heterogeneous Phenotypes from Large Scale RNAi/Drug Perturbations

Arvind Rao, Ph.D., Assistant Professor, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center

 

Bioinformatics in CRISPR screens:  they looked at image analysis of light microscopy of breast cancer cells and looked for phenotypic changes

 

  • Then they modeled in a small pilot and then used the algorithm for 20,000 images (made morphometric measurements)
  • Can formulate training statistical algorithms to make a decision tree how you classify data points
  • Although their algorithms worked well there was also human input from scientists

Aggregate ranking of hits programs available on web like LINKS

 

@MDAndersonNews

 

10:25 CRISPR in Stem Cell Models of Eye Disease

Alexander Bassuk, M.D., Ph.D., Associate Professor of Pediatrics, Department of Molecular and Cellular Biology, University of Iowa

 

Blind athlete Michael Stone, biathlete, had eye disease since teenager helped fund and start the clinical trial for Starbardt disease; had one bad copy of ABCA4, heterozygous (inheritable in Ahkenazi Jewish) – a recessive inheritable mutation with juvenile macular degeneration

  • Also had another male in family with disease but he had another mutation in the RPGR gene
  • December 2015 paper Precision Medicine: Genetic Repair of retinitis pigmentosa in patient derived stem cells
  • They were able to correct the iPSCs in the RPGR gene derived from patient however low efficiency of repair, scarless repair, leaves changes in DNA, need clinical grade iPSCs, and need a humanized model of RPGR

@uiowa

10:55 CRISPR in Mouse Models of Eye Disease

Vinit Mahajan, M.D., Ph.D., Assistant Professor of Ophthalmology and Visual Sciences, University of Iowa College of Medicine

  • degeneration of the retina will see brown spots, the macula will often be preserved but retinal cells damaged but with RPGR have problems with peripheral vision, retinitis pigmentosa get tunnel vision with no peripheral vision (a mouse model of PDE6 Knockout recapitulates this phenotype)
  • the PDE6 is linked to the rhodopsin GTP pathway
  • rd1 -/- mouse has something that looks like retinal pigmentosa; has mutant PDE6; is actually a nonsense mutation in rd1 so they tried a crisper to fix in mice
  • with crisper fix of rd1 nonsense mutation the optic nerve looked comparible to normal and the retina structure restored
  • photoreceptors layers- some recovery but not complete
  • sequence results show the DNA is a mosaic so not correcting 100% but only 35% but stil leads to a phenotypic recovery; NHEJ was about 12% to 25% with large deletions
  • histology is restored in crspr repaired mice
  • CRSPR off target effects: WGS and analyze for variants SNV/indels, also looked at on target and off target regions; there were no off target SNVs indels while variants that did not pass quality control screening not a single SNV
  • Rhodopsin mutation accounts for a large % of patients (RhoD190N)
  • injection of gene therapy vectors: AAV vector carrying CRSPR and cas9 repair templates

CAPN mouse models

  • family in Iowa have dominant mutation in CAPN5; retinal degenerates
  • used CRSPR to generate mouse model with mutation in CAPN5 similar to family mutation
  • compared to other transgenic methods CRSPR is faster to produce a mouse model

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Overall good meeting #s:

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CRISPR’s Unwanted off-target effects: Need for safety study designs with Gene-Editing

Reporter: Stephen J. Williams, Ph.D.

From CafePharma at https://www.statnews.com/2016/07/18/crispr-off-target-effects/

Do CRISPR enthusiasts have their head in the sand about the safety of gene editing?

WASHINGTON — At scientific meetings on genome-editing, you’d expect researchers to show pretty slides of the ribbony 3-D structure of the CRISPR-Cas9 molecules neatly snipping out disease-causing genes in order to, everyone hopes, cure illnesses from cancer to muscular dystrophy. Less expected: slides of someone kneeling on a beach with his head in the sand.

Yet that is what Dr. J. Keith Joung of Massachusetts General Hospital showed at the American Society of Hematology’s workshop on genome-editing last week in Washington. While the 150 experts from industry, academia, the National Institutes of Health, and the Food and Drug Administration were upbeat about the possibility of using genome-editing to treat and even cure sickle cell disease, leukemia, HIV/AIDS, and other blood disorders, there was a skunk at the picnic: an emerging concern that some enthusiastic CRISPR-ers are ignoring growing evidence that CRISPR might inadvertently alter regions of the genome other than the intended ones.

“In the early days of this field, algorithms were generated to predict off-target effects and [made] available on the web,” Joung said. Further research has shown, however, that such algorithms, including one from MIT and one calledE-CRISP, “miss a fair number” of off-target effects. “These tools are used in a lot of papers, but they really aren’t very good at predicting where there will be off-target effects,” he said. “We think we can get off-target effects to less than 1 percent, but we need to do better,” especially if genome-editing is to be safely used to treat patients.

Off-target effects occur because of how CRISPR works. It has two parts. RNA makes a beeline for the site in a genome specified by the RNA’s string of nucleotides, and an enzyme cuts the genome there. Trouble is, more than one site in a genome can have the same string of nucleotides. Scientists might address CRISPR to the genome version of 123 Main Street, aiming for 123 Main on chromosome 9, only to find CRISPR has instead gone to 123 Main on chromosome 14.

In one example Joung showed, CRISPR is supposed to edit a gene called VEGFA (which stimulates production of blood vessels, including those used by cancerous tumors) on chromosome 6. But, studies show, this CRISPR can also hit genes on virtually every one of the other 22 human chromosomes. The same is true for CRISPRs aimed at other genes. Although each CRISPR has zero to a dozen or so “known” off-target sites (where “known” means predicted by those web-based algorithms), Joung said, there can be as many as 150 “novel” off-target sites, meaning scientists had no idea those errors were possible.

One reason for concern about off-target effects is that genome-editing might disable a tumor-suppressor gene or activate a cancer-causing one. It might also allow pieces of two different chromosomes to get together, a phenomenon called translocation, which is the cause of chronic myeloid leukemia, among other problems.

Many researchers, including those planning clinical trials, are using web-based algorithms to predict which regions of the genome might get accidentally CRISPR’d. They include the scientists whose proposal to use CRISPR in patients was the first to be approved by an NIH committee. When scientists assure regulators that they looked for off-target effects in CRISPR’d cells growing in lab dishes, what they usually mean is that they looked for CRISPR’ing of genes that the algorithms flagged.

As a result, off-target effects might be occurring but, because scientists are doing the equivalent of the drunk searching for their lost keys only under the lamppost, they’re not being found.

Other articles on CRISPR and Gene Editing on this Open Access Journal Include:

FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials

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

CRISPR, the Genome Editing Technology is Nearing Human Trials: Human T cells will soon be modified using the CRISPR technique in a clinical trial to attack cancer cells

Use of CRISPR & RNAi for Drug Discovery, CHI’s World PreClinical Congress – Europe, November 14-15, 2016, Lisbon, Portugal

CRISPR: A Podcast from Nature.com on Gene Editing

AND Please See Our Following ebooks available on Amazon containing interviews with Dr. Jennifer Duodna

Volume One: Genomics Orientations for Personalized Medicine

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

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Best in Precision Medicine: RNA May Surpass DNA in Precision Medicine

Curator: Larry H. Bernstein, MD, FCAP

 

  • “A more complicated application of CRISPR technology is to use it for gene activation,” adds Dr. Tedesco. “Cellecta plans to optimize this application to bring forth highly efficient, inexpensive, high-throughput genetic screens based on their pooled libraries.

 

RNA May Surpass DNA in Precision Medicine

http://www.genengnews.com/gen-news-highlights/rna-may-surpass-dna-in-precision-medicine/81252507/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Mar22_2016_Rgocs_RNASeqAlignment1872484040.jpg

Scientists based at the Translational Genomics Research Institute have published a review heralding the promise of RNA sequencing (RNA-seq) for precision medicine. The scientists also note that progress will be needed on analytical, bioinformatics, and regulatory fronts, particularly in light of the transcriptome’s variety, dynamism, and wealth of detail. In this image, one aspect of RNA-seq is shown, the alignment with intron-split short reads. It reflects the alignment of mRNA sequence obtained via high-throughput sequencing and the expected behavior of the alignment to the reference genome when the read falls in an exon–exon junction. [Rgocs, Wikipedia]

It’s not an either/or situation. Both DNA sequencing and RNA sequencing hold clinical promise—diagnostically, prognostically, and therapeutically. It must be said, however, that RNA sequencing reflects the dynamic nature of gene expression, shifting with the vagaries of health and disease. Also, RNA sequencing captures more biochemical complexity, in the sense that it allows for the detection of a wide variety of RNA species, including mRNA, noncoding RNA, pathogen RNA, chimeric gene fusions, transcript isoforms, and splice variants, and provides the capability to quantify known, predefined RNA species and rare RNA transcript variants within a sample.

All these potential advantages were cited in a paper that appeared March 21 in Nature Reviews Genetics, in an article entitled, “Translating RNA Sequencing into Clinical Diagnostics: Opportunities and Challenges.” The paper, contributed by scientists based at the Translational Genomics Research Institute (TGen), was definitely optimistic about the clinical utility of RNA sequencing, but it also highlighted the advances that would have to occur if RNA sequencing is to achieve its promise.

In general, the very things that make RNA sequencing so interesting are the same things that make it so challenging. RNA sequencing would take the measure of a world—the transcriptome—that is incredibly rich. To capture all the relevant subtleties of the transcriptome, scientists will have to develop sensitive, precise, and trustworthy analytical techniques. What’s more, scientists will need to find efficient and reliable means of processing and interpreting all of the transcriptome data they will collect. Finally, they will need to continue integrating RNA-based knowledge with DNA-based knowledge. That is, RNA sequencing results can be used to guide the interpretation of DNA sequencing results.

In their Nature Reviews Genetics paper, the TGen scientists review the state of RNA sequencing and offer specific recommendations to enhance its clinical utility. The TGen scientists make a special point about the promise held by extracellular RNA (exRNA). Because exRNA can be monitored by simply taking a blood sample, as opposed to taking a tumor biopsy, it could serve as a noninvasive diagnostic indicator of disease.

“Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities,” wrote the authors. “However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease.”

The first test measuring exRNA was released earlier this year, the paper said, for use measuring specific exRNAs in lung cancer patients. And, the potential for using RNA-seq in cancer is expanding rapidly. Commercial RNA-seq tests are now available, and they provide the opportunity for clinicians to profile cancer more comprehensively and use this information to guide treatment selection for their patients.

In addition, the authors reported on several recent applications for RNA-seq in the diagnosis and management of infectious diseases, such as monitoring for drug-resistant populations during therapy and tracking the origin and spread of the Ebola virus.

Despite these advances, the authors also sounded a few cautionary notes. “There are currently few agreed upon methods for isolation or quantitative measurements and a current lack of quality controls that can be used to test platform accuracy and sample preparation quality,” they wrote. “Analytical, bioinformatics, and regulatory challenges exist, and ongoing efforts toward the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.”

Overall, the authors remain hopeful that precision medicine will embrace RNA sequencing. For example, lead author Sara Byron, research assistant professor in TGen’s Center for Translational Innovation, said, “RNA is a dynamic and diverse biomolecule with an essential role in numerous biological processes. From a molecular diagnostic standpoint, RNA-based measurements have the potential for broad application across diverse areas of human health, including disease diagnosis, prognosis, and therapeutic selection.”

 

Gene Editing Casts a Wide Net   

With CRISPR, Gene Editing Can Trawl the Murk, Catching Elusive Phenotypes amidst the Epigenetic Ebb and Flow

http://www.genengnews.com/gen-articles/gene-editing-casts-a-wide-net/5713/

http://www.genengnews.com/Media/images/Article/thumb_Marusina_iStock_52289586_GeneticCodeDNA1830871003.jpg

Gene-editing advances will not only open new avenues toward curing genetic diseases but will also rapidly increase the pace of new scientific discoveries about human and other types of genomes. [iStock/adventtr]

 

  • Genome editing, a much-desired means of accomplishing gene knockout, gene activation, and other tasks, once seemed just beyond the reach of most research scientists and drug developers. But that was before the advent of CRISPR technology, an easy, versatile, and dependable means of implementing genetic modifications. It is in the process of democratizing genome editing.

    CRISPR stands for “clustered, regularly interspaced, short palindromic repeats,” segments of DNA that occur naturally in many types of bacteria. These segments function as part of an ancient immune system. Each segment precedes “spacer DNA,” a short base sequence that is derived from a fragment of foreign DNA. Spacers serve as reminders of past encounters with phages or plasmids.

    The CRISPR-based immune system encompasses several mechanisms, including one in which CRISPR loci are transcribed into small RNAs that may complex with a nuclease called CRISPR-associated protein (Cas). Then the RNA guides Cas, which cleaves invading DNA on the basis of sequence complementarity.

    In the laboratory, CRISPR sequences are combined with a short RNA complementary to a target gene site. The result is a complex in which the RNA guides Cas to a preselected target.

    Cas produces precise site-specific DNA breaks, which, with imperfect repair, cause gene mutagenesis. In more recent applications, Cas can serve as an anchor for other proteins, such as transcriptional factors and epigenetic enzymes. This system, it seems, has almost limitless versatility.

  • Edited Stem Cells

    The Sanger Institute Mouse Genetic Program, along with other academic institutions around the world, provides access to thousands of genetically modified mouse strains. “Genetic engineering of mouse embryonic stem (ES) cells by homologous recombination is a powerful technique that has been around since the 1980s,” says William Skarnes, Ph.D., senior group leader at the Wellcome Trust Sanger Institute.

    “A significant drawback of the ES technology is the time required to achieve a germline transmission of the modified genetic locus,” he continues. “While we have an exhaustive collection of modified ES cells, only about 5,000 knockout mice, or a quarter of mouse genome, were derived on the basis of this methodology.”

    The dominant position of the mouse ES cell engineering is now effectively challenged by the CRISPR technology. Compared with very low rates of homologous recombination in fertilized eggs, CRISPR generates high levels of mutations, and off-target effects may be so few as to be undetectable.

    “We used the whole-genome sequencing to thoroughly assess off-target mutations in the offspring of CRISPR-engineered founder animals,” informs Dr. Skarnes. “A mutated Cas9 nuclease was deployed to increase specificity, resulting in nearly perfect targeting.”

    Dr. Skarnes explains that the major mouse genome centers are now switching to CRISPR to complete the creation of the world-wide repository of mouse knockouts. His own research is now focused on genetically engineered induced pluripotent stem cells (iPSCs). These cells are adult cells that have been reprogrammed to an embryonic stem cell–like state, and are thus devoid of ethical issues associated with research on human embryonic stem cells. The ultimate goal is to establish a world-wide panel of reference iPSCs created by high-throughput genetic editing of every single human gene.

    “We are poised to begin a large-scale phenotypic analysis of human genes,” declares Dr. Skarnes. His lab is releasing the first set of functional data on 100 DNA repair genes. “By knocking out individual proteins involved in DNA repair and sequencing the genomes of mutant cells,” declares Dr. Skarnes, “we hope to better understand the mutational signatures that occur in cancer.”

  • Pooled CRISPR Libraries

    Researchers hope to gain a better understanding of the mutational signatures found in cancers by using CRISPR techniques to knock out individual proteins involved in DNA repair and then sequencing the genomes of mutant cells. [iStock/zmeel]

    Connecting a phenotype to the underlying genomics requires an unbiased screening of multiple genes at once. “Pooled CRISPR libraries provide an opportunity to cast a wide net at a reasonably low cost,” says Donato Tedesco, Ph.D., lead research scientist at Cellecta. “Screening one gene at a time on genome scale is a significant investment of time and money that not everyone can afford, especially when looking for common genetic drivers across many cell models.”

    Building on years of experience with shRNA libraries, Cellecta is uniquely positioned to prepare pooled CRISPR libraries for genome-wide or targeted screens of gene families. While shRNA interferes with gene translation, CRISPR disrupts a gene and the genomic level due to imperfections in the DNA repair mechanism.

    To determine if these different mechanisms for inactivating genes affect the results of genetic screens, the team conducted a side-by-side comparison of Cellecta’s Human Genome-Wide Module 1 shRNA Library, which expresses 50,000 shRNA targeting 6,300 human genes, with the library of 50,000 gRNA targeting the same gene set. The concordance between approaches was very high, suggesting that these technologies may be complementary and used for cross-confirmation of results.

    Also, a recently completed Phase I NIH SBIR Grant was used to create and test guiding strand RNA (sgRNA) structures to drastically improve efficiency of gene targeting. For this work, Cellecta used a pool library strategy to simultaneously test multiple sgRNA structures for their efficiency and specificity. An early customized Cellecta pooled gRNA library was successfully utilized for screening for epigenetic genes. This particular screen is highly dependent on a complete loss of function, and could not have been accomplished by shRNA inhibition.

    Scientists from Epizyme interrogated 600 genes in a panel of 100 cell lines and, in addition to finding many epigenetic genes required for proliferation in nearly all cell lines, were able to identify validate several essential epigenetic genes required only in subsets of cells with specific genetic lesions. In other words, pooled cell line screening was able to distinguish targets that are likely to produce toxic side effects in certain types of cancer cells from gene targets that are essential in most cells.

    “A more complicated application of CRISPR technology is to use it for gene activation,” adds Dr. Tedesco. “Cellecta plans to optimize this application to bring forth highly efficient, inexpensive, high-throughput genetic screens based on their pooled libraries.

  • Chemically Modified sgRNA

    Scientists based at Agilent Research Laboratories and Stanford University worked together to demonstrate that chemically modified single guide RNA can be used to enhance the genome editing of primary hepatopoietic stem cells and T cells. This image, which is from the Stanford laboratory of Matthew Porteus, M.D., Ph.D., shows CD34+ human hematopoietic stem cells that were edited to turn green. Editing involved inserting a construct for green fluorescent protein. About 1,000 cells are pictured here.

    Researchers at Agilent Technologies applied their considerable experience in DNA and RNA synthesis to develop a novel chemical synthesis method that can generate long RNAs of 100 nucleotides or more, such as single guide RNAs (sgRNAs) for CRISPR genome editing. “We have used this capability to design and test numerous chemical modifications at different positions of the RNA molecule,” said Laurakay Bruhn, Ph.D., section manager, biological chemistry, Agilent.

    Agilent Research Laboratories worked closely with the laboratory of Matthew Porteus, M.D., Ph.D., an associate professor of pediatrics and stem cell transplantation at Stanford University. The Agilent and Stanford researchers collaborated to further explore the benefits of chemically modified sgRNAs in genome editing of primary hematopoetic stem cells and T cells.

    Dr. Porteus’ lab chose three key target genes implicated in the development of severe combined immunodeficiency (SCID), sickle cell anemia, and HIV transmission. Editing these genes in the patient-derived cells offers an opportunity for novel precision therapies, as the edited cells can renew, expand, and colonize the donor’s bone marrow.

    Dr. Bruhn emphasized the importance of editing specificity, so that no other cellular function is affected by the change. The collaborators focused on three chemical modifications strategically placed at each end of sgRNAs that Agilent had previously tested to show they maintained sgRNA function. A number of other optimization strategies in cell culturing and transfection were explored to ensure high editing yields.

    “Primary cells are difficult to manipulate and edit in comparison with cell lines already adapted to cell culture,” maintains Dr. Bruhn. Widely varied cellular properties of primary cells may require experimental adaptation of editing techniques for each primary cell type.

    The resulting data showed that chemical modifications can greatly enhance efficiency of gene editing. The next step would translate these findings into animal models. Another advantage of chemical synthesis of RNA is that it can potentially be used to make large enough quantities for therapeutics.

    “We are working with Agilent’s Nucleic Acid Solution Division—a business focused on GMP manufacturing of oligonucleotides for therapeutics—to engage with customers interested in this capability and better understand how we might be able to help them accomplish their goals,” says Dr. Bruhn.

  • Customized Animal Models

    “Given their gene-knockout capabilities, zinc-finger-based technologies and CRISPR-based technologies opened the doors for creation of animal models that more closely resemble human disease than mouse models,” says Myung Shin, Ph.D., senior principal scientist, Merck & Co. Dr. Shin’s team supports Merck’s drug discovery and development program by creating animal models mimicking human genetics.

    For example, Dr. Shin’s team has worked with the Dahl salt-sensitive strain of rats, a widely studied model of hypertension. “We used zinc-finger nucleases to generate a homozygous knockout of a renal outer medullary potassium channel (ROMK) gene,” elaborates Dr. Shin. “The resulting model represents a major advance in elucidating the role of ROMK gene.”

    According to Dr. Shin, the model may also provide a bridge between genetics and physiology, particularly in studies of renal regulation and blood pressure. In one study, the model generated animal data that suggest ROMK plays a key role in kidney development and sodium absorption. Work along these lines may lead to a pharmacological strategy to manage hypertension.

    In another study, the team applied zinc-finger nuclease strategy to knockout the coagulation Factor XII, and thoroughly characterize them in thrombosis and hemostasis studies. Results confirmed and extended previous literature findings suggesting Factor XII as a potential target for antithrombotic therapies that carry minimal bleeding risk. The model can be further utilized to study safety profiles and off-target effects of such novel Factor XII inhibitors.

    “We use one-cell embryos to conduct genome editing with zinc-fingers and CRISPR,” continues Dr. Shin. “The ease of this genetic manipulation speeds up generation of animal models for testing of various hypotheses.”

    A zinc finger–generated knockout of the multidrug resistance protein MDR 1a P-glycoprotein became an invaluable tool for evaluating drug candidates for targets located in the central nervous system. For example, it demonstrated utility in pharmacological analyses.

    Dr. Shin’s future research is directed toward preclinical animal models that would contain specific nucleotide changes corresponding to those of humans. “CRISPR technology,” insists Dr. Shin, “brings an unprecedented power to manipulate genome at the level of a single nucleotide, to create gain- or loss-of-function genetic alterations, and to deeply understand the biology of a disease.”

  • Transcriptionally Active dCas9

    “Epigenome editing is important for several reasons,” says Charles Gersbach, Ph.D., an associate professor of biomedical engineering at Duke University. “It is a tool that helps us answer fundamental questions about biology. It advances disease modeling and drug screening. And it may, in the future, serve as mode of genetic therapy.”

    “One part of our research focuses on studying the function of epigenetic marks,” Dr. Gersback continues. “While many of these marks are catalogued, and some have been associated with the certain gene-expression states, the exact causal link between these marks and their effect on gene expression is not known. CRISPR technology can potentially allow for targeted direct manipulation of each epigenetic mark, one at a time.”

    Dr. Gersback’s team mutated the Cas9 nuclease to create deactivated Cas9 (dCas9), which is devoid of endonuclease activity. Although the dCas9 protein lacks catalytic activity, it may still serve as an anchor for a plethora of other important proteins, such as transcription factors and methyltransferases.

    In an elegant study, Dr. Gersbach and colleagues demonstrated that recruitment of a histone acetyltransferase by dCas9 to a genomic site activates nearby gene expression. Moreover, the activation occurred even when the acetyltransferase domain was targeted to a distal enhancer. Similarly, recruitment of KRAB repressor to a distant site silenced the target gene in a very specific manner. These findings support the important role of three-dimensional chromatin structures in gene activation.

    “Genome regulation by epigenetic markers is not static,” maintains Dr. Gersbach. “It responds to changes in the environment and other stimuli. It also changes during cell differentiation. We designed an inducible system providing us with an ability to execute dynamic control over the target genes.”

    In a light-activated CRISPR-Cas9 effector (LACE) system, blue light may be used to control the recruitment of the transcriptional factor VP64 to target DNA sequences. The system has been used to provide robust activation of four target genes with only minimal background activity. Selective illumination of culture plates created a pattern of gene expression in a population of cells, which could be used to mimic what is observed in natural tissues.

    Together with collaborators at Duke University, Dr. Gersbach intends to carry out the high-throughput analysis of all currently identified regulatory elements in the genome. “Our ultimate goal,” he declares, “is to assign function to all of these elements.”

 

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