Information

Can CRISPR also remove DNA viruses?


If I'm not mistaken only RNA viruses insert themselves into the host genome. As an example of DNA viruses, herpes viruses for example do not insert themselves in the host genome.

Can CRISPR cut DNA that isn't in a chromossome, like a DNA virus?


Yes, this should in principle work, and a number of groups have shows that it works in cultured cells:

We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection… Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

--Inhibition of HSV-1 Replication by Gene Editing Strategy

There's also been at least one trial in an animal model:

Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

--The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo


Could gene editing tools such as CRISPR be used as a biological weapon?

James Revill received funding from the Economic and Social Research Council (ESRC) for his work on the Strategic Governance of Science and Technology (ES/K011324/1) and Science and Technology Reviews under the Biological Weapons Convention (RES-062-23-1192)

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The gene editing technique CRISPR has been in the limelight after scientists reported they had used it to safely remove disease in human embryos for the first time. This follows a “CRISPR craze” over the last couple of years, with the number of academic publications on the topic growing steadily.

There are good reasons for the widespread attention to CRISPR. The technique allows scientists to “cut and paste” DNA more easily than in the past. It is being applied to a number of different peaceful areas, ranging from cancer therapies to the control of disease carrying insects.

Some of these applications – such as the engineering of mosquitoes to resist the parasite that causes malaria – effectively involve tinkering with ecosystems. CRISPR has therefore generated a number of ethical and safety concerns. Some also worry that applications being explored by defence organisations that involve “responsible innovation in gene editing” may send worrying signals to other states.

Concerns are also mounting that gene editing could be used in the development of biological weapons. In 2016, Bill Gates remarked that “the next epidemic could originate on the computer screen of a terrorist intent on using genetic engineering to create a synthetic version of the smallpox virus”. More recently, in July 2017, John Sotos, of Intel Health & Life Sciences, stated that gene editing research could “open up the potential for bioweapons of unimaginable destructive potential”.

An annual worldwide threat assessment report of the US intelligence community in February 2016 argued that the broad availability and low cost of the basic ingredients of technologies like CRISPR makes it particularly concerning.

Smallpox virus. CDC/ Fred Murphy

However, one has to be careful with the hype surrounding new technologies and, at present, the security implications of CRISPR are probably modest. There are easier, cruder methods of creating terror. CRISPR would only get aspiring biological terrorists so far. Other steps, such as growing and disseminating biological weapons agents, would typically be required for it to become an effective weapon. This would require additional skills and places CRISPR-based biological weapons beyond the reach of most terrorist groups. At least for the time being.

This does not mean that the hostile exploitation of CRISPR by non-state actors can be ignored. Nor can one ignore the likely role of CRISPR in any future state biological weapons programme.


Scientists Used Crispr To Edit HIV-Like Viruses In Monkey DNA

Scientists have used Crispr gene-editing to remove an HIV-like virus from monkey DNA, a major step towards a cure for HIV infection in humans.

In a study led by neurovirologist Kamel Khalili of Temple University in Philadelphia, researchers constructed a modified adenovirus containing a Crispr-Cas9 gene-editing system. That 'construct' (called 'AAV9-CRISPR-Cas9') was then injected into rhesus macaque monkeys to deliver the Crispr system into cells.

The monkey cells were infected with SIV (Simian Immunodeficiency Virus), a close relative of HIV (Human Immunodeficiency Virus). Both are retroviruses — viral parasites that cut-and-paste their genetic material into a host's DNA. SIV infects macaques and other non-human primates in the same way that HIV infects people, making it a good model for studying retroviral infection — and testing how to remove those viruses from the human genome.

The gene-editing construct was designed to target specific sites where the retrovirus was integrated into the macaque genome. It was able to reach tissues where viruses like SIV and HIV can hide for years without being detected, known as reservoirs, such as bone marrow, lymph nodes, T cells of the immune system and the brain. According to the study, the construct was precise and has a low risk of cutting the wrong places in DNA ('off-target' sites).

The research has obvious implications for preventing or treating AIDS (Acquired Immunodeficiency Syndrome) in humans by curing a patient of HIV infection.


Could CRISPR be used as a biological weapon?

Bioterrorism exercise. Credit: Oregon National Guard/Flickr, CC BY-SA

The gene editing technique CRISPR has been in the limelight after scientists reported they had used it to safely remove disease in human embryos for the first time. This follows a "CRISPR craze" over the last couple of years, with the number of academic publications on the topic growing steadily.

There are good reasons for the widespread attention to CRISPR. The technique allows scientists to "cut and paste" DNA more easily than in the past. It is being applied to a number of different peaceful areas, ranging from cancer therapies to the control of disease carrying insects.

Some of these applications – such as the engineering of mosquitoes to resist the parasite that causes malaria – effectively involve tinkering with ecosystems. CRISPR has therefore generated a number of ethical and safety concerns. Some also worry that applications being explored by defence organisations that involve "responsible innovation in gene editing" may send worrying signals to other states.

Concerns are also mounting that gene editing could be used in the development of biological weapons. In 2016, Bill Gates remarked that "the next epidemic could originate on the computer screen of a terrorist intent on using genetic engineering to create a synthetic version of the smallpox virus". More recently, in July 2017, John Sotos, of Intel Health & Life Sciences, stated that gene editing research could "open up the potential for bioweapons of unimaginable destructive potential".

An annual worldwide threat assessment report of the US intelligence community in February 2016 argued that the broad availability and low cost of the basic ingredients of technologies like CRISPR makes it particularly concerning.

However, one has to be careful with the hype surrounding new technologies and, at present, the security implications of CRISPR are probably modest. There are easier, cruder methods of creating terror. CRISPR would only get aspiring biological terrorists so far. Other steps, such as growing and disseminating biological weapons agents, would typically be required for it to become an effective weapon. This would require additional skills and places CRISPR-based biological weapons beyond the reach of most terrorist groups. At least for the time being.

This does not mean that the hostile exploitation of CRISPR by non-state actors can be ignored. Nor can one ignore the likely role of CRISPR in any future state biological weapons programme.

International efforts

Fortunately, most states around the world regard biological warfare with particular abhorrence. There are already measures in place to prohibit and prevent the development of biological weapons. At the international level, this includes the Biological and Toxin Weapons Convention. Under this convention, states have agreed "never under any circumstances to acquire or retain biological weapons".

This convention is imperfect and lacks a way to ensure that states are compliant. Moreover, it has not been adequately "tended to" by its member states recently, with the last major meeting unable to agree a further programme of work. Yet it remains the cornerstone of an international regime against the hostile use of biology. All 178 state parties declared in December of 2016 their continued determination "to exclude completely the possibility of the use of (biological) weapons, and their conviction that such use would be repugnant to the conscience of humankind".

These states therefore need to address the hostile potential of CRISPR. Moreover, they need to do so collectively. Unilateral national measures, such as reasonable biological security procedures, are important. However, preventing the hostile exploitation of CRISPR is not something that can be achieved by any single state acting alone.

As such, when states party to the convention meet later this year, it will be important to agree to a more systematic and regular review of science and technology. Such reviews can help with identifying and managing the security risks of technologies such as CRISPR, as well as allowing an international exchange of information on some of the potential benefits of such technologies.

Most states supported the principle of enhanced reviews of science and technology under the convention at the last major meeting. But they now need to seize the opportunity and agree on the practicalities of such reviews in order to prevent the convention being left behind by developments in science and technology.

Biological warfare is not an inevitable consequence of advances in the life sciences. The development and use of such weapons requires agency. It requires countries making the decision to steer the direction of life science research and development away from hostile purposes. An imperfect convention cannot guarantee that these states will always decide against the hostile exploitation of biology. Yet it can influence such decisions by shaping an environment in which the disadvantages of pursuing such weapons outweigh the advantages.

This article was originally published on The Conversation. Read the original article.


New genetic method of using CRISPR to eliminate COVID-19 virus genomes in cells

It is predicted the development of a safe and effective vaccine to prevent COVID-19 will take 12 to 18 months, by which time hundreds of thousands to millions of people may have been infected. With a rapidly growing number of cases and deaths around the world, this emerging threat requires a nimble and targeted means of protection.

Could CRISPR be the next virus killer?

To address this global pandemic challenge, we are developing a genetic vaccine that can be used rapidly in healthy and patients to greatly reduce the coronavirus spreading. We developed a safe and effective CRISPR system to precisely target, cut and destroy COVID-19 virus and its genome, which stops coronavirus from infecting the human lung.

We’ve shown that the CRISPR system can reduce 90% of coronavirus load in human cells. It can also protect humans against essentially 90% of all current and emerging coronaviruses. The project is ongoing, and we are working around the clock towards getting an actual product by combing our CRISPR method with an inhaler-based delivery device.

The project will likely to result in a potential therapeutics towards COVID-19, which can help slow down or eliminate the outbreak.


CRISPR may help neuroscientists unlock genetics of psychiatric disease

The brain is one of the most complex entities in biology. For thousands of years, humans have wondered how the human brain works, but only in the past few years has technology evolved so that scientists can actually answer some of the many questions we have. What are the causes of brain disorders? How do our brains develop? How does the brain heal after a head injury? While we still have a long way to go before we can understand the many facets of the human brain, one technology – CRISPR – has allowed us to start answering these questions on a genetic level.

What is CRISPR?

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is the latest in a long line of genome editing techniques. Over the past few years, CRISPR has swept through the biology community and into mainstream news. The technology, which can be used to make specific changes in the DNA of plants and animals, has become instrumental to studying disease systems in the lab because of its low cost, precision, and ease of use. Unlike other genome editing methods, scientists can use it to change any stretch of DNA in a genome, as long as they know the sequence to target. They can make multiple changes in one fell swoop.

The CRISPR system has two parts: 1) a protein borrowed from bacteria that cuts DNA, and 2) a guide RNA that tells the protein where to cut. Once the DNA of the gene being targeted is cut, the cell will try to repair the DNA but will often make a mistake, causing the gene to be disrupted and no longer function. Sometimes, scientists will also introduce a third component of the system: a DNA template that tells the cell how to repair the cut DNA and introduce a very specific mutation that changes the gene in some way. Either way, scientists can use CRISPR to alter the DNA inside cells (Figure 1). (Learn more specifics about CRISPR and how CRISPR works here.)

Figure 1: How CRISPR works. A guide RNA that matches the genomic DNA sequence of interest helps direct a CRISPR protein toward this site in the DNA. The protein cuts the strands of DNA. The cell will try to repair this break, but during this process may introduce random mutations that render the gene non-functional – the gene is thus silenced. However, if scientists introduce a DNA template that is slightly different from the original DNA sequence, the cell can use it to guide the repair and introduce a specific mutation into the gene sequence.

Why is CRISPR So Important for Neuroscience?

The advent of CRISPR has been timed perfectly with the recent neuroscience research boom. Over the past few years, scientists have been using gene sequencing to uncover genes that are important in brain development and in neurological diseases, like Alzheimer’s disease and schizophrenia. Whereas some neurodevelopmental disorders, like Fragile X Syndrome, are known to be caused by mutations in a single gene, diseases like schizophrenia involve many genes and are very complicated, so thousands of people had to be sequenced before scientists could figure out which genetic differences might be linked to the disease. Today, there are clues to the genes that might affect a number of disorders, like OCD, autism, and major depression. The next step is to figure out if disrupting these genes can cause any of these diseases. CRISPR seems to be the perfect technology to make this happen.

Because the brain is the product of millions of connections between neurons, it’s important to see what these genetic changes do in an actual animal brain. If we think that a specific mutation in the Huntingtin gene causes Huntington’s disease, we can introduce that mutation into the embryo of a mouse via the CRISPR system. These mice and their offspring will contain this mutation and we can study their behavior and physical changes and see if they have the “mouse version” of Huntington’s disease. These mice can then be given potential drugs to see if those drugs help relieve symptoms (Figure 2).

Figure 2: Using CRISPR to study neurological diseases in model organisms. Once a possible genetic link has been found to a neurological disease like Huntington’s disease, Alzheimer’s disease, or Parkinson’s disease, scientists can use the CRISPR system to introduce the relevant genetic mutations into model organisms like mice. By understanding the differences between mice with the genetic mutation and mice without it, scientists can paint a clearer picture of how the disease might be affecting a human body. Then, the mice can be given potential drugs or treatments that can help to alleviate their symptoms or even help cure the disease.

Previous methods for making mouse models can take up to two years from design of the mutated gene to multiple rounds of mouse breeding to make sure that the mouse offspring have the correct genetic mutation. In contrast, it takes only about two months to create a mouse model using CRISPR because the components are more easily introduced into the embryo and multiple breeding steps are not required. In addition, if we think that more than one gene is contributing to a disease like schizophrenia, we can easily introduce two or more mutations into a mouse embryo at the same time simply by introducing multiple guide RNAs (and template DNAs), something that is not easy to do with other genome editing technologies (Figure 3).

Figure 3: Timeline of CRISPR compared to a traditional genome editing technique. For diseases like OCD that are known to be caused by more than one genetic mutation, making model organisms with multiple genetic mutations is crucial to understanding the disease. With traditional genome editing methods, it might take up to three years to create a mouse model with two genetic mutations, with CRISPR, scientists can create a mouse model with one, two, or even more genetic mutations in as little as six weeks!

With CRISPR, dozens of mouse models and other animal models have been made to study neuroscience. For example, the Zhang lab at the Broad Institute in Cambridge, MA have used CRISPR to make mouse models of OCD and autism. Mice with OCD-related genetic mutations groom themselves more and seem to be anxious about their environmental cleanliness and mice with autism-related genetic mutations are generally less sociable than other mice. The Zhang lab is currently working on making mouse models with particular mutations in a gene called Shank3 which might be important in both autism and schizophrenia.

The Challenges of CRISPR

CRISPR is still a relatively new technology and it’s not perfect. The human genome is large and sometimes, multiple stretches of DNA are similar enough that the CRISPR system will make unintended cuts in the DNA. In this way, unintended mutations might arise which might affect the health or even survival of the animal and can confuse the results of any experiments. Many researchers are currently studying ways of making the CRISPR system more specific so that only the genes one intends to target are affected.

Right now, it is easy to inject CRISPR components into mouse embryos, but if scientists want to introduce CRISPR into an adult rat brain (perhaps with therapeutic intent), they’re out of luck. It is very difficult to get the CRISPR components to cross the blood-brain barrier. Some progress has been made by stripping the CRISPR components down and stuffing them into a modified non-disease-causing virus that can easily cross the blood-brain barrier. Think of this like organizing your carry-on bag (the virus) with only your essentials (the CRISPR components) such that it just makes the size cutoff at the airport. However, although the virus package has no disease-causing power, the long term effects of using such a virus and the ramifications of stripping down the CRISPR components on their effectiveness in the brain are still being investigated.

Finally, there are ethical issues to consider when proposing CRISPR as a gene therapy for humans or even using CRISPR in primate animal models. While primates are used in a number of brain imaging studies, the ethics of genetic manipulation in these animals, and potentially in humans, is still being hotly contested.

The Future of CRISPR

Even as research is being done to improve the specificity and efficiency of CRISPR itself, neuroscientists are finding more inventive ways to use the technique, developing model organisms not previously used in neuroscience, such as social insects like locusts, more sophisticated social mammals like bats. Many neurological disorders cause people to behave unusually in social situations, and studying other social animals might help us to understand why that is. Many scientists are also using CRISPR in human induced pluripotent stem cells (see this special edition article) or in neurons derived from these stem cells to study the effects of genetic changes on human neurons in a dish. As neuroscientists create more animal and cell models with CRISPR, we will be able to unravel more about what makes the brain tick and how to fix it if it breaks.

Angela She is a PhD student studying neurodegenerative diseases at Harvard. She is also one of the co-producers of the SITN podcast, SIT’N Listen. Figures were prepared by Shannon McArdel. This article is part of the was originally published as part of Science in the News’s April 2016 Special Edition on Neurotechnology. You can follow Science in the News on twitter @SITNBoston.


Gene therapy: past and present

Traditionally scientists use viruses - from which dangerous disease-causing genes have been removed - as vehicles to transport new genes to specific organs. These genes then produce a product that can compensate for the faulty genes that are inherited genetically. This is how gene therapy works.

Though there have been examples showing that gene therapy was helpful in some genetic diseases, they are still not perfect. Sometimes, a faulty gene is so big that you can’t simply fit the healthy replacement in the viruses commonly used in gene therapy.

Another problem is that when the immune system sees a virus, it assumes that it is a disease-causing pathogen and launches an attack to fight it off by producing antibodies and immune response – just as happens when people catch any other infectious viruses, like SARS-CoV-2 or the common cold.

Recently, though, with the rise of a gene editing technology called CRISPR, scientists can do gene therapy differently.

CRISPR can be used in many ways. In its primary role, it acts like a genetic surgeon with a sharp scalpel, enabling scientists to find a genetic defect and correct it within the native genome in desired cells of the organism. It can also repair more than one gene at a time.

Scientists can also use CRISPR to turn off a gene for a short period of time and then turn it back on, or vice versa, without permanently changing the letters of DNA that makes up or genome. This means that researchers like me can leverage CRISPR technology to revolutionize gene therapies in the coming decades.

But to use CRISPR for either of these functions, it still needs to be packaged into a virus to get it into the body. So some challenges, such as preventing the immune response to the gene therapy viruses, still need to be solved for CRISPR-based gene therapies.

Being trained as a synthetic biologist, I teamed up with Ebrahimkhani to use CRISPR to test whether we could shut down a gene that is responsible for immune response that destroys the gene therapy viruses. Then we investigated whether lowering the activity of the gene, and dulling the immune response, would allow the gene therapy viruses to be more effective.


CRISPR meets Pac-Man: New DNA cut-and-paste tool enables bigger gene edits

Gene editing for the development of new treatments, and for studying disease as well as normal function in humans and other organisms, may advance more quickly with a new tool for cutting larger pieces of DNA out of a cell's genome, according to a new study by UC San Francisco scientists.

Publication of the UCSF study on Oct. 19, 2020 in the journal Nature Methods comes less than two weeks after two researchers who first used the genetic scissors known as CRISPR-Cas9 were selected to receive this year's Nobel Prize in Chemistry.

Though now employed as a research tool in laboratories around the world, CRISPR evolved eons ago in bacteria as a means to fight their ancient nemeses, a whole host of viruses known as bacteriophages. When bacteria encounter a phage, they incorporate a bit of the viral DNA into their own DNA, and it then serves as a template to make RNA that binds to the corresponding viral DNA in the phage itself. The CRISPR enzymes then target, disable and kill the phage.

In his latest work exploring this ancient and strange arms race, principal investigator Joseph Bondy-Denomy, PhD, associate professor in the UCSF Department of Microbiology and Immunology, joined scientists Bálint Csörg?, PhD, and Lina León to develop and test a new CRISPR tool.

The already renowned CRISPR-Cas9 ensemble is like a molecular chisel that can be used to rapidly and precisely excise a small bit of DNA at a targeted site. Other methods can then be used to insert new DNA. But the new CRISPR-Cas3 system adapted by the UCSF scientists employs a different bacterial immune system. The key enzyme in this system, Cas3, acts more like a molecular wood chipper to remove much longer stretches of DNA quickly and accurately.

"Cas3 is like Cas9 with a motor -- after finding its specific DNA target, it runs on DNA and chews it up like a Pac-Man," Bondy-Denomy said.

This new capability to delete or replace long stretches of DNA will enable researchers to more efficiently assess the importance of genomic regions that contain DNA sequences of indeterminate function, according to Bondy-Denomy, an important consideration in understanding humans and the pathogens that plague them.

"Previously, there was no easy and reliable way to delete very large regions of DNA in bacteria for research or therapeutic purposes," he said. "Now, instead of making 100 different small DNA deletions we can just make one deletion and ask, 'What changed?'"

Because bacteria and other types of cells are commonly used to produce small molecule or protein-based pharmaceuticals, CRISPR-Cas3 will enable biotechnology industry scientists to more easily remove potentially pathogenic or useless DNA from these cells, according to Bondy-Denomy.

"Large swathes of bacterial DNA are poorly understood, with unknown functions that in some cases are not necessary for survival," Bondy-Denomy said. "In addition, bacterial DNA contains large stretches of DNA imported from other sources, which can cause disease in the bacterium's human host, or divert bacterial metabolism."

CRISPR-Cas3 also should also allow entire genes to be inserted into the genome in industrial, agricultural or even in human gene therapy applications, Bondy-Denomy said.

The UCSF researchers selected and modified the CRISPR-Cas3 system used by the bacterium Pseudomonas aeruginosa, and demonstrated in this species and in three others, including bacteria that cause disease in humans and plants, that their more compact version functions well to remove selected DNA in all four species. Other CRISPR-Cas3 systems have been made to work in human and other mammalian cells, and that also should be achievable for the modified P. aeruginosa system, Bondy-Denomy said.

Bondy-Denomy studies a range of bacteria, bacteriophage, and CRISPR systems to learn more about how they work and to find useful molecular tools. "CRISPR-Cas3 is by far the most common CRISPR system in nature," he said. "About 10 times as many bacterial species use a Cas3 system as use a Cas9 system. It may be that Cas3 is a better bacterial immune system because it shreds phage DNA."

Unlike Cas9, when Cas3 binds to its precise DNA target it begins chewing up one strand of the double-stranded DNA in both directions, leaving a single strand exposed. The deletions obtained in the UCSF experiments ranged in size, in many cases encompassing as many as 100 bacterial genes. The CRISPR-Cas3 mechanism should also allow for easier replacement of deleted DNA with a new DNA sequence, the researchers found.

For DNA deletion and editing in the lab, scientists program CRISPR systems to target specific DNA in the genome of an organism of interest using any guide sequence they choose.

In the new CRISPR-Cas3 study, by manipulating the sequences of DNA provided to the bacteria for repairing the deletions, the researchers were able to precisely set the boundaries of these large DNA repairs, something they were unable to accomplish with CRISPR-Cas9.

Bondy-Denomy previously discovered anti-CRISPR strategies that phage evolved to fight back against bacteria, and these might prove useful for stopping the gene editing reactions driven by Cas enzymes used as human therapeutics before side effects arise, or in using phage to remove unwanted bacteria that have populated the gut, he said. Apart from E. coli and a couple of other species, relatively little is known about the 1,000 or so bacterial species that normally reside there.

"Non-model microbes have largely been left behind in the genetics world, and there is a huge need for new tools to study them," he said.


Cascade and CRISPR

PDB entry 4qyz captures Cascade in action. The structure includes the strand of CRISPR RNA (red) and a short piece of the viral DNA (yellow) after it has been unwound and recognized. The structure revealed a surprising but very logical structure for the RNA and DNA. The RNA is stretched open in a long spiral groove in Cascade, and the DNA binds side-by-side, instead of in the classical double helix. To explore this amazing structure in more detail, click on the image for an interactive JSmol.

Topics for Further Discussion

  1. When reading articles about CRISPR sequences, watch out for some of the terminology, because it can be confusing. For instance, the term "spacer" is often used to refer to the short pieces of viral DNA that are stored in the CRISPR, and "repeat" is used to refer to the short repeated sequences separating each piece of viral DNA.
  2. Many of these large CRISPR/Cas complexes have been characterized by electron microscopy. For instance, to see the structure of a type III complex (which is different from type I Cascade and type II Cas9) take a look at the EMDataBank.

Related PDB-101 Resources

References

  1. J. van der Oost, E. R. Westra, R. N. Jackson & B. Wiedenheft (2014) Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nature Reviews Microbiology 12, 479-492.
  2. H. Ebina, N. Misawa, Y. Kanemura & Y. Koyanagi (2013) Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Scientific Reports 3, 2510.
  3. 4qqw: Y. Huo, K. H. Nam, F. Ding, H. Lee, L. Wu, Y. Xiao, M. D. Farchione, S. Zhou, K. Rajashankar, I. Kurinov, R. Zhang & A. Ke (2014) Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nature Structural & Molecular Biology 21, 771-777.
  4. 4un3: C. Anders, O. Niewoehner, A. Duerst & M. Jinek (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569-573.
  5. 4tvx: R. N. Jackson, S. M. Golden, P. B. G. van Erg, J. Carter, E. R. Westra, S. J. J. Brouns, J. van der Oost, T. C. Terwilliger, R. J. Read & B. Wiedenheft. (2014) Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345, 1473-1479.
  6. 4qyz: S. Mulepati, A. Heroux & S. Bailey (2014) Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssFNA target. Science 345, 1479-1484.
  7. 4p6i: J. K. Nunez, P. J. Kranzusch, J. Noeske, A. V. Wright, C. W. Davies & J. A. Doudna (2014) Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nature Structural & Molecular Biology 21, 528-534.

January 2015, David Goodsell

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PDB-101 helps teachers, students, and the general public explore the 3D world of proteins and nucleic acids. Learning about their diverse shapes and functions helps to understand all aspects of biomedicine and agriculture, from protein synthesis to health and disease to biological energy.

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Herpes's Achilles heel

The herpes simplex virus, commonly known as the cold sore virus, is a devious microbe.

It enters the body through regions lined with mucous membranes -- mouth, nose and genitals -- but quickly establishes lifelong viral hideouts inside nerve cells. After initial infection, the virus lurks dormant only to be reawakened periodically to cause outbreaks marked by the eruption of cold sores or blisters. In a handful of people, the consequences of viral reawaking can be devastating, including blindness and brain inflammation.

Antiviral medications can prevent recurrent outbreaks, but they are not always effective, so for decades, researchers have sought a solution that would quiet the virus for good.

Now, using human fibroblast cells infected with herpes simplex virus (HSV), researchers at Harvard Medical School have successfully used CRISPR-Cas9 gene editing to disrupt not only actively replicating virus but also the far-harder to reach dormant pools of the virus, demonstrating a possible strategy for achieving permanent viral control.

The team's findings are described Dec. 2 in eLife.

"This is an exciting first step -- one that suggests it is possible to permanently silence lifelong infections -- but much more work remains to be done," said study lead investigator David Knipe, the Higgins Professor of Microbiology and Molecular Genetics in the Blavatnik Institute at Harvard Medical School.

Notably, the research represents the first successful instance of disrupting latent viral reservoirs through gene editing. Latent reservoirs are notoriously impervious to antiviral medications and have also proven hard to gene-edit.

The experiments also identify the mechanisms by which actively replicating virus becomes uniquely vulnerable to gene editing. These very mechanisms may also explain why latent forms of the virus are less amenable to this technique.

Specifically, the experiments reveal that the DNA of an actively replicating virus is more exposed to the Cas9 enzyme -- the molecular scissors in the CRISPR-Cas9 gene-editing system. This is because actively replicating viruses have fewer protective histones that wrap around their DNA to shield it.

"The absence of protective histones makes the DNA more accessible and easier to cut, so it's essentially identified HSV's Achilles heel," Knipe said.

The new findings offer a model system for using gene editing in a localized way to disrupt active replication in specific sites. However, Knipe cautions, the arch-challenge of delivering gene-editing therapy to neurons -- where the virus hides and enters a state of dormancy -- remains to be solved, Knipe added.

More than two-thirds of the world population harbors the virus according to the World Health Organization. While most infections are asymptomatic, in a handful of people HSV can cause serious damage. It can infect the eyes, a condition known as herpes keratitis, and lead to blindness. In people with compromised immune systems, HSV can cause brain inflammation. In newborns, the virus can cause disseminated, systemic disease and brain inflammation and can be fatal in a quarter of infected babies.

Thus, one early therapeutic use of this technique could involve local and limited gene-editing of the epithelial cells in the mouth, eyes or genitals of people with established HSV infections as a way to prevent the virus from causing active outbreaks at vulnerable sites, Knipe said.

"If you want to prevent corneal infections, for example, you might be able to use CRISPR-Cas9 editing in the corneal cells to prevent new infections or prevent the virus from reactivating or reduce the reactivation," Knipe said. "People who have recurrent herpes keratitis infection of the cornea start to go blind after a while because of the reactivation and the resulting inflammation that causes clouding of the cornea."

The advantage of limited, localized gene-editing is avoiding the widespread, possible off-target effects that might inadvertently alter the DNA of cells other than those intended.

"We still have a long way to go in ensuring hyperprecision and safety of new gene-editing tools so local editing could offer a safer, more limited first step," Knipe said.