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Posts Tagged ‘gene editing’


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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Advances in Gene Editing and Gene Silencing | September 20-21, 2016 | Boston, MA

Kinase Inhibitor Discovery September 21-22, 2016 Boston

KEYNOTE SESSION: GENOME EDITING FOR IN VIVO APPLICATIONS

Part 1 (of a two-part conference) will cover the use of CRISPR/Cas9 and RNAi for identifying new drug targets and therapies. It will bring together experts from all aspects of basic science and clinical research to talk about how and where gene editing and RNAi can be best applied. What are the different tools that can be used and what are their strengths and limitations? How does the CRISPR/Cas system compare to RNAi and other gene editing tools, such as Transcription Activator-like Effector Nucleases (TALENs) and zinc finger nucleases (ZFNs), and do they have any complementary uses? Scientists and clinicians from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of gene editing for target discovery, disease modeling, and for creating cell and viral therapies. Learn more atDiscoveryOnTarget.com/RNAi-screens-functional-genomics

Advance Registration Discount Available!
Register by August 12 Week to Save up to $200

Keynote Session: Genome Editing for In Vivo Applications

AAV for Gene Therapy and Genome Editing
James Wilson, M.D., Ph.D., Professor, Department of Pathology and Laboratory Medicine, Perelman School of Medicine; Director, Orphan Disease Center and Director, Gene Therapy Program, University of Pennsylvania
In vivo delivery of nucleic acid therapeutics remains the primary barrier to success. My lab has focused on the use of vectors based on adeno-associated virus (AAV) for achieving success in pre-clinical and clinical applications of gene replacement therapy. Most of the current academic and commercial applications of in vivo gene replacement therapy are based on endogenous AAVs we discovered as latent viral genomes in primates. These vectors are reasonably safe and efficient for application of gene replacement therapy. The emergence of genome editing methods has suggested more precise and effective methods to treat inherited diseases in which genes are silenced or mutations are corrected. AAV vectors have been the most efficient platform for achieving genome editing in vivo. We will review our attempts to achieve therapeutic genome editing in animal models of liver disease using AAV.

Using CRISPR/Cas to Target and Destroy Viral DNA Genomes
Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
A number of pathogenic human DNA viruses, including HBV, HIV-1 and HSV1, cause chronic diseases in humans that remain refractory to cure, though these diseases can be controlled by antivirals. In addition the DNA virus HPV causes tumors that depend on the continued expression of viral genes. Here, I will present data demonstrating that several of these viruses can be efficiently cleaved and destroyed using viral vectors that express Cas9 and virus-specific guide RNAs, thus providing a potential novel approach to treatment.

Targeted Endonucleases as Antiviral Agents: Promises and Pitfalls
Keith R. Jerome, M.D., Ph.D., Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington
Genome editing offers the prospect of cure for infections such as HIV, hepatitis B virus, herpes simplex, and human papillomavirus, by disruption of essential viral nucleic acids or the human genes encoding receptors needed for viral entry. This talk will highlight the most recent laboratory data and the challenges still ahead in bringing this technology to the clinic.

Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel 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
High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our high throughput methods for developing and characterizing RNA delivery and gene editing systems. Libraries of degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to deliver RNA, both in vitro and in vivo. A number of delivery formulations have been developed with in vivo efficacy, and show potential applications for the treatment of genetic diseases, viral infections and cancers.

PANEL DISCUSSION: CRISPR/Cas: A Realistic and Practical Look at What the Future Could Hold
Moderator: Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
Participants: Session Speakers
Each speaker will spend a few minutes sharing their viewpoints and experiences on where things stand with using the CRISPR/Cas system for in vivo applications. Attendees will have an opportunity to ask questions and share their opinions.

About the Conference

Cambridge Healthtech Institute’s 13th annual two-part conference on Advances in Gene Editing and Gene Silencing will cover the latest in the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-based gene editing and RNA interference (RNAi) for use in drug discovery and for developing novel drug therapies.


For sponsorship and exhibit sales information including podium presentation opportunities, contact:
Jon Stroup | T: +1 781-972-5483 | E: jstroup@healthtech.com


Recommended All Access Package:
Includes access to 1 Symposium and 2 Conferences

September 19 Symposium: 
Understanding CRISPR: Mechanisms and Applications

September 20-21 Conference:
Advances in Gene Editing and Gene Silencing – Part 1

September 21-22 Conference: 
Advances in Gene Editing and Gene Silencing – Part 2


Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA, USA

Tel: 781-972-5400 | Fax: 781-972-5425 | www.healthtech.com
This email is being sent to sjwilliamspa@comcast.net for marketing purposes. If it is not of interest to you, please disregard and we apologize for any inconvenience this may have caused.

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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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AACR2016 – Cancer immunotherapy

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

AACR 2016: Cancer Immunotherapy and Beyond

At this year’s meeting there was a palpable buzz around subjects ranging from microbiomics to the tumor microenvironment and cancer vaccines, big data to in vitro and in vivo modeling and drug delivery (to name just a few).

By all accounts, this year’s American Association of Cancer Research’s 2016 annual meeting (AACR) was dominated by immunotherapy writ large. Which is not to say the meeting was devoid of other topics – there was certainly palpable buzz around subjects ranging from microbiomics to the tumor microenvironment and cancer vaccines, big data to in vitro and in vivo modeling and drug delivery (to name just a few), that kept the meeting’s 19,500 attendees rapt in New Orleans April 16-20. Yet in the 12-13 years that David R. Soto-Pantoja, Ph.D., has been attending AACR, the Wake Forest University School of Medicine assistant professor of Cancer Biology has “never seen anything take over so much like immunotherapy.”

That being said, it’s not always easy to put cancer research into neat little boxes. Researchers interested in cell signaling, and oncologists concerned with genomics, may have found themselves in sessions dedicated to finding and exploiting neo-antigens (one of immunotherapy’s buckets).

Immunotherapy Buckets

For years there have been certain pillars used to target cancer: chemotherapy, surgery, radiation therapy, and more recently targeted therapies. “And now immunotherapy,” says Wafik El-Deiry, M.D., Ph.D., Deputy Director for Translational Research at the Fox Chase Cancer Center. “This is an emerging and expanding field that is going to be very intensely explored in every direction you can imagine. I wouldn’t even try to pretend that we know what all the buckets are at this point.”

One known bucket that  isfast being filled is the area of checkpoint inhibitors (checkpoint blockades). Over the past few years several therapeutics have been developed that had to do with getting the immune system to either better recognize, better target, or better fight, a tumor, with some success. Recall that last year former president Jimmy Carter, earlier diagnosed with metastatic melanoma, was declared “cancer free” following treatment with radiation and an antibody targeted against the immune programmed cell death – 1 (PD-1) antigen. PD-1 keeps the immune cells in check; blockading PD-1 de-inhibits, or “releases the brakes” of these cells and allow them free rein to attack the cancer. Encouraging results from several trials and follow-ups of PD-1 and other checkpoint inhibitors, such as anti-PDL-1 and anti-CTLA-4, were presented. These are in some cases being combined for even greater efficacy.

In addition to trials, “a lot of people, whether in posters or in other smaller talks, delve into the scientific mechanism at the cellular and immunological level of how those worked,” remarked Emil Lou, M.D., Ph.D., assistant professor of Medicine in the Division of Hematology, Oncology and Transplantation at the University of Minnesota.

Another bucket  receiving many contributions is the area of CAR-T cells: T cells imbued with a chimeric antigen receptor. T cells that have been harvested from a patient are given a receptor that will recognize a new protein – CD-19, found on B cells, for example – expanded, and re-infused (adoptively transferred) back into the patient to attack the cancer — B cell lymphomas, in the present example. There were many hurdles and pitfalls to be overcome – from finding the right antigen and designing the CAR, to controlling the CAR-T cell’s response – yet the field “has gone at such a rapid pace that it’s very clinically relevant,” says Dr. Lou.

Much familiar (and not-so-familiar) technology, such as high content (phenotypic) screening platforms like the IntelliCyt, is being leveraged to help with array testing, selection, and even manufacturing of CAR-T cells, says Janette Phi, the company’s CBO.

Miltenyi Biotec touted their CliniMACS Prodigy, a benchtop-sized automated cell processing and separation platform, for manufacturing CAR-T cells. It can take a patient’s cells from apheresis and selection on beads, through viral transduction to final product “in 8-10 days,” says clinical instrument specialist Kevin Longin. “It’s a GMP lab without a GMP lab.”

But making bespoke CAR-Ts is a “very expensive approach,” notes Dr. Soto-Pantoja. There were discussions at the meeting about generating off-the-shelf CAR-T cells.

Gene Editing

One way to do this is to use gene editing techniques such as CRISPR/Cas9 to knock out the proteins that could cause rejection of these foreign cells by the patient’s own immune system, or graft-versus-host disease (in which the introduced cells treat the host as foreign).

CRISPR has really become a hot buzz word, says Dr. Lou. “More from the basic science side, and slowly making its way into the clinical talks – it’s not ready for prime time.” While gene editing spins off a different conversation about ethics – people are hesitant about its capacity to create designer babies — he notes that “in cancer it’s helpful to be able to study and reproduce the genes that are driving cancer, in the lab.”

CRISPR is not just for knocking genes in or out, either. In his plenary talk, MIT’s W. M. Keck Career Development Professor of Biomedical Engineering Feng Zhang, Ph.D., discussed a host of other uses to which his lab and others have put the CRISPR/Cas9 system and its relatives. They can be used to create selective transcriptional activation or repression, for example, to recruit epigenetic writers, erasers, and readers, to edit RNA transcripts, and even to identify non-coding regions of the genome.

Neo-antigens and Other Biomarkers

Gene therapy and gene editing are not the only ways in which the complement of expressed proteins is altered. Among the hallmarks of cancer is genetic mutation of oncogenes, tumor suppressor genes, and others – whether as point mutations, duplications, insertions and deletions (indels), or fusions. Because they’re mutated and encode other amino acids, they are “foreign” and may be recognized by the immune system as such, Dr. El-Deiry points out. “One needs to analyze these neo-antigens — to figure out what they are – as well as to analyze the various immune cell subsets that may react with those neo-antigens.” There are many tools that researchers are using to do just that, and there was no shortage of vendors from instrument and assay manufacturers and software developers to service providers – in areas like next-generation genomic sequencing (for both genomics and transcriptomics) and its associated preparatory and analytics, flow cytometry, and antibody development, to name just a few – vying for the attention of AACR conventioneers.

Neo-antigens are, of course, only one mark of a cancerous cell or tissue. In fact, most biomarkers used to diagnose or track cancers in the lab or the clinic rely on “signatures” — collections of multiple markers, each of which in and of themselves may be considered within the normal range but taken together (at the levels expressed) correlate with disease, prognosis, or likely response to treatment. Many panels, available on different platforms, are currently approved for clinical testing and others are working toward that goal.

It’s important to realize that cancers are often not static – they evolve, often as the result of treatment, sometimes selecting for a resistant population. “We’re trying to overcome the mistake of treating a patient’s cancer, after their tumor has grown despite different types of chemotherapies, based on what the information was at the time of diagnosis, when in reality the tumor has potentially transformed into something different,” relates Dr. Lou. Serial biopsies under the auspices of clinical trials, mostly in lung cancer, have revealed the evolution of cancer genomics and an understanding of how to better target therapy to the patient’s tumor at the time of recurrence and progression.

But taking a biopsy is an invasive and sometimes risky procedure.

Liquid Biopsy

It has been known for many years that cells, nucleic acids, and even vesicles derived from tumors can be found in blood and other fluids. “The idea that we can biopsy less invasively by using blood-based biomarkers is really coming to maturity just in the last two years or so,” says Dr. Lou. There is currently a lot of excitement about the possibility of using these as liquid biopsies to “provide a window into the cancer,” says Shane Booth, D. Phil., CTO of Angle LLC. They can look for the presence or evolution of a tumor, for example to monitor the effect of therapy.

Only a single circulating tumor cell (CTC) platform, CELLSEARCH, has thus far been approved for clinical use. Dr. Booth estimates that there are currently perhaps 20-30 different companies with technologies to isolate CTCs, most (like CELLSEARCH) based on affinity capture or (like Angle’s Parsortix platform) on “very sophisticated, bleeding-edge filtration techniques”. These differ from each other in a variety of ways including whether the platform performs analysis, whether it is specific or agnostic to the type of cancer, whether cells can be recovered (and whether they can be recovered alive) for downstream use, the purity of CTCs, and the sample preparation required.

Several companies are offering platforms or services to look at cell free DNA (cfDNA, aka circulating tumor DNA, ctDNA). Trovagene, for example, has assays to examine DNA in blood or urine for common BRAF, KRAS, and EGFR mutations. Meanwhile Nanostring Technologies uses digital molecular barcoding to multiplex hundreds of assays from single molecules without amplification.

Caris Life Sciences’ ADAPT Biotargeting System can profile the different proteins, miRNAs, and DNA found in exosomes. “It’s still early times, but these kinds of test will in the future be used to try and make some predictions about prognosis or response to therapy,” says Dr. El-Deiry.

Personalized Medicine

If there was a theme (implicitly) pervading AACR 2016 more than that of immunotherapy it was that of personalized medicine (aka precision medicine, individualized medicine). From genomic sequencing to determine whether a patient will benefit from (or be harmed by) a given therapy, to examining the microenvironment in which a tumor is found, one size no longer fits all.

The challenge, notes Dr. Soto-Pantoja, is to take the results seen in cases such as checkpoint inhibitor therapy, in which about one third of patients are seen to benefit, and figure out how to extend that to other patients and apply that to other types of cancer.

 

 

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CRISPR/Cas9 and HIV1

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus

Hirotaka EbinaNaoko MisawaYuka Kanemura & Yoshio Koyanagi

Scientific Reports 2013; 2510(3)   http://dx.doi.org:/10.1038/srep02510

Even though highly active anti-retroviral therapy is able to keep HIV-1 replication under control, the virus can lie in a dormant state within the host genome, known as a latent reservoir, and poses a threat to re-emerge at any time. However, novel technologies aimed at disrupting HIV-1 provirus may be capable of eradicating viral genomes from infected individuals. In this study, we showed the potential of the CRISPR/Cas9 system to edit the HIV-1 genome and block its expression. When LTR-targeting CRISPR/Cas9 components were transfected into HIV-1 LTR expression-dormant and -inducible T cells, a significant loss of LTR-driven expression was observed after stimulation. Sequence analysis confirmed that this CRISPR/Cas9 system efficiently cleaved and mutated LTR target sites. More importantly, this system was also able to remove internal viral genes from the host cell chromosome. Our results suggest that the CRISPR/Cas9 system may be a useful tool for curing HIV-1 infection.

 

Integration of reverse transcribed viral DNA into the host cell genome is an essential step during the HIV-1 life cycle1. The integrated retroviral DNA is termed a provirus, which serves as the fundamental source of viral protein production. HIV-1 gene expression is regulated by LTR promoter and enhancer activities, where cellular transcription factors such as NF-κB, SP-1 and TBP bind to promote RNA polymerase II processivity. Subsequently, Tat protein is expressed from early double-spliced transcripts and binds to the trans activation response (TAR) region of HIV-1 RNA for its efficient elongation2.

Latent infection occurs when the HIV-1 provirus becomes transcriptionally inactive, resulting in a latent reservoir that has become the main obstacle in preventing viral eradication from HIV-1 infected individuals. However, the mechanisms of viral silencing and reactivation remain incompletely understood3. Previous studies have suggested that the position of the integration site strongly influences viral gene expression and may be one of the determinants of HIV-1 latency4. While highly active anti-retroviral therapy (HARRT) has dramatically decreased mortality from HIV-1 infection, there is currently no effective strategy to target the latent form of HIV-1 proviruses5.

Over the last decade, novel genome-editing methods that utilize artificial nucleases such as zinc finger nucleases (ZFNs)6 and transcription activator like-effector nucleases (TALENs)7 have been developed. These molecularly engineered nucleases recognize and cleave specific nucleotide sequences in target genomes for digestion, resulting in various mutations such as substitutions, deletions and insertions induced by host DNA repair machinery. These technologies have enabled the production of genome-manipulated animals in a wide range of species such as Drosophila8, Zebrafish9 and Rat10. However, ZFNs or TALENs remain somewhat difficult and time-consuming to design, develop, and empirically test in a cellular context11. Recently, a third genome-editing method was developed based on clustered regularly interspaced short palindromic repeat (CRISPR) systems. CRISPR systems were originally identified in bacteria and archaea12 as part of an adaptive immune system, dependent on a complex consisting of CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) proteins to degrade complimentary sequences of invading viral and plasmid DNA. Mali et al. created a novel version of the genome-editing tool applicable to mammalian cells, termed the CRISPR/Cas9 system, which is based on modifications of the Streptococcus pyogenes type II CRISPR system in crRNA fused to trans-encoded tracrRNA13. This CRISPR/Cas9 system is composed of guide RNA (gRNA) and a human codon-optimized Cas9 nuclease that forms an RNA-protein complex to digest unique target sequences matching those of gRNA. The CRISPR/Cas9 system can be utilized by simple transfection of designed gRNA and a humanized Cas9 (hCas9) expression plasmid into target mammalian cells, making it a promising tool for various applications.

In this study, we tested the ability of the CRISPR/Cas9 system to suppress HIV-1 expression by editing HIV-1 integrated proviral DNA. Cas9 and gRNA, designed to target HIV-1 LTR, were transfected and significantly inhibited LTR-driven expression under the control of Tat. This LTR-targeted CRISPR/Cas9 system can also excise provirus from the cellular genome.

 

http://www.nature.com/article-assets/npg/srep/2013/130826/srep02510/images_hires/w926/srep02510-f2.jpg

CRISPR/Cas9 system can target the latent form of HIV-1 provirus in Jurkat cell

Because the putative latently infected cells are CD4+ T cells, we next tested the genome editing potential of the CRISPR/Cas9 system in these cells.

…..

 

In this study, we successfully disrupted the expression of HIV-1 provirus utilizing the CRISPR/Cas9 system (Fig. 1). Importantly, this disruption not only restricted transcriptionally active provirus, it also blocked the expression of latently integrated provirus (Fig. 3). Cas9 proteins are predicted to contain RuvC and HNH motifs15, which possess autonomous ssDNA cleavage activity. Interestingly, mutants lacking one of the motifs become nicking endonucleases16. It is plausible that the independent nicking activity of each domain may enhance efficient access to the heterochromatin state of latently integrated provirus. Another possibility is that Cas9 has a highly efficient target surveillance system similar to what has been previously reported for the Cas3 system17.

T6 gRNA that targeted the NF-κB binding site, also strongly suppressed the LTR promoter activity (Fig. 1). However, the effect was weaker than that of T5 gRNA. In this study we used an LTIG vector modified from the LTR of HIV-1 strain NL4-3 that possesses two adjacent NF-κB binding sites18. The T6 target site is at the end of the 5′ NF-κB binding site, meaning that mutations may not completely render transcription inactive since the 3′ NF-κB binding site may remain functional. On the other hand, T5 gRNA that targeted TAR, is profoundly effective in disrupting HIV-1 gene expression. The putative cleavage site was positioned at the neck of the stem loop region of TAR, which is critical for Cyclin T1-Tat-TAR ternary complex formation19. Therefore, the TAR sequence may be one of the best targets for blocking HIV-1 provirus expression. Target specificity of the CRISPR/Cas system is very high and a single mutation can disrupt targeting20, meaning that some provirus may escape from this genome-editing machinery if mutations arise in target sequences. However, given that the TAR region is relatively conserved and there is little variation among HIV-1 subtypes21, it could still be an appropriate target for the elimination of latently infected provirus.

Perhaps the most important finding in this study is that we could excise provirus from the host genome of HIV-1 infected cells, which may provide a ray of hope to eradicate HIV-1 from infected individuals. However, there are numerous hurdles that must be cleared before utilizing genome editing for HIV-1 eradication therapies such as gene therapy. First, the efficiency of genome-editing and/or proviral excision should be quantified in HIV infected primary cells, including latently infected CD4+ quiescent T cells. Second, an efficient delivery system must be developed. Fortunately, the CRISPR/Cas9 system has the advantage in size compared with TALENs22. Thus, the CRISPR system has the potential to be delivered by lentivirus vectors, whereas TALENs do not because of their large size and repeat sequences23. The final hurdle concerns possible off-target effects, which are pertinent concerns for all genome-editing strategies that may lead to nonspecific gene modification events. If Cas9 has off-target effects, then removal of the off-target activity may be the best approach before utilizing CRISPR/Cas system for anti-HIV treatment.

 

Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

Rafal KaminskiYilan ChenTracy FischerEllen TedaldiAlessandro Napoli,Yonggang ZhangJonathan KarnWenhui Hu & Kamel Khalili

Scientific Reports 6, Article number: 22555 (2016)   http://dx.doi.org:/10.1038/srep22555

 

We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.

 

AIDS remains a major public health problem, as over 35 million people worldwide are HIV-1-infected and new infections continue at steady rate of greater than two million per year. Antiretroviral therapy (ART) effectively controls viremia in virtually all HIV-1 patients and partially restores the primary host cell (CD4+ T-cells), but fails to eliminate HIV-1 from latently-infected T-cells1,2. In latently-infected CD4+ T cells, integrated proviral DNA copies persist in a dormant state, but can be reactivated to produce replication-competent virus when T-cells are activated, resulting in rapid viral rebound upon interruption of antiretroviral treatment3,4,5,6,7,8. Therefore, most HIV-1-infected individuals, even those who respond very well to ART, must maintain life-long ART due to persistence of HIV-1-infected reservoir cells. During latency HIV infected cells produce little or no viral protein, thereby avoiding viral cytopathic effects and evading clearance by the host immune system. Because the resting CD4+ memory T-cell compartment9is thought to be the most prominent latently-infected cell pool, it is a key focus of research aimed at eradicating latent HIV-1 infection.

Recent efforts to eradicate HIV-1 from this cell population have used primarily a “shock and kill” approach, with the rationale that inducing HIV reactivation in CD4+ memory T-cells may trigger elimination of virus-producing cells by cytolysis or host immune responses. For example, epigenetic modification of chromatin structure is critical for establishing viral reactivation. Consequently, inhibition of histone deacetylase (HDAC) by Trichostatin A (TSA) and vorinostat (SAHA) led to reactivation of latent virus in cell lines10,11,12. Accordingly, other HDACi, including vorinostat, valproic acid, panobinostat and rombidepsin have been tested ex vivo and have led, in the best cases, to transient increases in viremia13,14. Similarly, protein kinase C agonists, can potently reactivate HIV either singly or in combination with HDACi15,16. However, there are multiple limitations of this approach: (i) since a large fraction of HIV genomes in this reservoir are non-functional, not all integrated provirus can produce replication-competent virus17; (ii) total numbers of CD4+ T cells reactivated from resting CD4+ T cell HIV-1 reservoirs, has been found by viral outgrowth assays to be much smaller than the numbers of cells infected, as detected by PCR-based assays, suggesting that not all cells within this reservoir are reactivated18; (iii) the cytotoxic T lymphocyte (CTL) immune response is not sufficiently robust to eliminate the reactivated infected cells19 and (iv) uninfected T-cells are not protected from HIV infection and can therefore sustain viral rebound.

These observations suggest that a cure strategy for HIV-1 infection should include methods that directly eliminate the proviral genome from the majority of HIV-1-positive cells, including CD4+ T-cells, and protect cells from future infection, with little or no harm to the host. The clustered, regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease has wide utility for genome editing in a broad range of organisms including yeast, Drosophila, zebrafish, C. elegans, and mice, and has been applied in a broad range of in vivo and in vitro studies toward human diseases20,21,22,23,24. Recently we modified the CRISPR/Cas9 system to enable recognition of specific DNA sequences positioned within the HIV-1 promoter spanning the 5′ long terminal sequence (LTR)25,26. Using this modified system, we now demonstrate excision of integrated copies of the proviral DNA fragment from a latently HIV-1-infected human T-lymphoid cell line, completely eliminating HDAC inhibition-elicited viral production. Results of whole-genome sequencing and comprehensive bioinformatic analysis ruled out any genotoxicity to host cell DNA. Further, we found that lentivirally-delivered CRISPR/Cas9 reduces viral replication upon HIV-1 infection of primary cultured CD4+ T-cells. The results point toward this approach as a promising potential therapeutic avenue to eradicating HIV-1 from T reservoir cells of host patients, to prevent AIDS re-emergence.

 

Despite its remarkable therapeutic success and efficacy, ART treatment is unable to eradicate HIV-1 from infected patients who must therefore undergo life-long treatment. A new therapeutic strategy is thus needed in order to achieve permanent remission allowing patients to stop ART and reduce it’s attendant costs and potential long-term side effects. Our findings address key barriers to this goal, as we developed CRISPR/Cas9 techniques that eradicated integrated copies of HIV-1 from human CD4+ T-cells, inhibited HIV-1 infection in primary cultured human CD4+ T-cells, and suppressed viral replication ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells of HIV-1+ patients. They also address a further key issue, providing evidence that such gene editing effectively impedes viral replication without causing genotoxicity to host DNA or eliciting destructive effects via host cell pathways. Prior studies using gene editing based on zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems prompted much interest in their potential abilities to suppress viral infection, either by altering virus receptors or introducing mutations in the viral genome (for review see26,30). All these studies suggest that gene editing strategies can be engineered for targeting specific regions of the viral genome and once efficiently delivered to infected cells, their robust antiviral activity effectively suppresses viral replication. However, there are several important issues that require close attention including the careful design of the targeting strategy that achieves the highest levels of specificity and safety with optimum efficiency of editing.

In this study, due to the complexity associated with determination of the sites and numbers of randomly integrated proviral HIV-1 DNA in in vitro infected primary cell culture and the difficulty in full scale characterization of the InDel/Excision by Cas9/gRNAs in these cells, as a first step, we chose to use the clonal 2D10 cell line as a human T-cell latency model to establish: (i) the ability of Cas9/gRNA in removing the entire coding sequence of the integrated copies of the HIV-1 DNA using ultradeep whole genome sequencing and (ii) investigate its safely related to off-target effects and cell viability. Once these goals were accomplished, we shifted our attention to primary cell culture as well as patient samples to examine the efficiency of the CRISPR/Cas9 in affecting viral DNA load in a laboratory setting.

We found that CRISPR/Cas9 edited multiple copies of viral DNA scattered among the chromosomes. Combined treatment of latently-infected T cells with Cas9 plus gRNAs A and B that recognize specific DNA motifs within the LTR U3 region efficiently eliminated the entire viral DNA fragment spanning between the two LTRs. The remaining 5′ LTR and 3′ LTR cleavage sites by Cas9 and gRNA B in chromosome 1, and by Cas9 and gRNAs A and B in chromosome 16, were joined by host DNA repair at sites located precisely three nucleotides upstream of the PAM. Genome-wide assessment of CRISPR/Cas9-treated HIV-1-infected 2D10 cells clearly verified complete excision of the integrated copies of viral DNA from the second intron of RSBN1 and exon 2 of MSRB1 genes. To address the critical issue related to its specificity and potential off-target and adverse effects, we analyzed this comprehensively and at an unprecedented level of detail, by whole-genome sequencing and bioinformatic analyses. These revealed many naturally-occurring mutations in the genomes of control cells and gRNAs A- and B-mediated HIV-1 DNA eradication. The mutations discovered included naturally-occurring InDels, base excisions, and base substitutions, all of which are, more or less, expected in rapidly growing cells in culture, including Jurkat 2D10 cells. The critical issue is our discovery that none of these mutations resulted from our gene-editing system, as we identified no sequence identities with either gRNA A or B within 1200 nucleotides of any such mutation site. Further, our method of HIV-1 DNA excision had no adverse effects on proximal or distal cellular genes and showed no impact on cell viability, cell cycle progression or proliferation, and did not induce apoptosis, thus strongly supporting its safety at this translational phase, by all in vitromeasures assessed in cultured cells. We found that the expression levels of Cas9 and the gRNAs diminished after several passages and eventually disappeared, but as long as Cas9 and single or multiplex gRNAs were present, cells remained protected against new HIV-1infection.

Another key translational feasibility question we addressed is whether CRISPR/Cas9-mediated HIV-1 eradication can prevent or suppress HIV-1 infection in the most relevant human and patient target cell populations. We provide a critical new advance, by observing in PBMCs and CD4+ T-cells from HIV-1 infected patients that lentivirally-delivered Cas9/gRNAs A/B significantly decreased viral copy numbers and protein levels. Using primer sets directed within the LTR, we amplified and detected residual viral DNA fragments that were not completely deleted in these cells, yet were affected by Cas9/gRNAs and contained InDel mutants near the PAM sequence. These findings verified that CRISPR/Cas9 exerted efficacious antiviral activity in the PBMCs of HIV-1 patients. We also found that introducing Cas9/gRNAs A/B via lentiviral delivery into primary cultured human CD4+ HIV-1JRFL– or HIV-1NL4-3-infected T-cells significantly reduced viral copy numbers, corroborating earlier findings by us and others that stably-integrated HIV-1-directed Cas9 and gRNAs (distinct from our gRNAs A and B used presently) conferred resistance to HIV-1 infection in cell lines31,32. With the notion that CRISPR/Cas9 can target both integrated, as well as episomal DNA sequences, as evidenced by its editing ability of various human viruses as well as plasmid DNAs in either configuration31,32,33,34,35,36, it is likely that both the integrated as well as pre-integrated, free-floating intracellular HIV-1 DNA are edited by Cas9/gRNA.

As noted, during the course of our studies no ART was included prior to the treatment with CRISPR/Cas9 as our goal in this study was to determine the extent of viral suppression during the productive stage of viral infection. We observed a significant level of suppression suggesting that CRISPR/Cas9 may effectively disable expression of the functionally active integrated copies of HIV-1 DNA in the host chromosome. This notion is supported by our observations using 2D10 CD4+ T-cells where the latent copies of HIV-1 that are integrated in chromosomes 1 and 16 were effectively eliminated by CRISPR/Cas9. Our future studies are aimed to address the impact of CRISPR/Cas9 in in vitro infected CD4+ T-cells where the virus is controlled by ART and a cohort of naïve and ART-treated patient CD4+ T-cells. Results from these studies should determine whether or not, in the context of ART, the virus enters into the latent stage and remains responsive to CRISPR/Cas9. Of note, results from these ex vivo studies using ART treated patient PBMCs and CD4+ T-cells show that CRISPR/Cas9 effectively suppresses viral replication by introducing InDel mutations.

Our findings show comprehensively and conclusively that the entire coding sequence of host-integrated HIV-1 was eradicated in human 2D10 T cells, providing a strong first step of support for potential translatability of such a system to T-cell-directed HIV-1 therapies in patients. The complete absence of genomic and off-target functional effects in all assays also provides critical support for the promise of developing this approach for future therapeutic applications.

When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical to understand that not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection. Thus, there is a high likelihood that rebounding viral infections will be contained by the resistant cells. Still, some formidable challenges remain before this type of strategy can be implemented. First, it will be important to maximize elimination of viral sequences from patients. This will require analysis of the HIV-1 quasi-species harbored by patients’ CD4+ T-cells and design of suitable, i.e. personalized CRISPRs. Second, improved delivery of CRISPR/Cas9 will be required to target the majority of circulating T-cells. In summary, our novel ex vivo findings that our lentiviral delivery-based approach reduced HIV-1 DNA copy numbers and protein levels in PBMCs of HIV-1 infected patients provides strong proof-of-concept evidence that CRISPR/Cas9 can be effectively utilized as part of HIV Cure strategies.

 

The therapeutic application of CRISPR/Cas9 technologies for HIV    PreviewFull text HTML   PDF

Sheena SaaymanabStuart A AlibKevin V MorrisacMarc S Weinberg*abd

Expert Opinion on Biological Therapy 2015;  15(6): 819-830    http://dx.doi.org:/10.1517/14712598.2015.1036736

Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system.

Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus.

Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

Genetic therapy for HIV/AIDS

Ananthalakshmi PoluriMarc van Maanen & Richard E Sutton

Expert Opinion on Biological Therapy Sept 2003; 3(6):951-963

 

Towards a durable RNAi gene therapy for HIV-AIDS

Ben Berkhout & Olivier ter Brake

Expert Opinion on Biological Therapy  Feb 2009; 9(2): 161-170

 

 

 

 

 

 

 

 

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Translational Gene Editing – June 16-17, 2016 in Boston, MA

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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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From GEN News Highlights

Reposted from GEN News

Nov 18, 2015

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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