REENGINEERING LIFE

Fresh Off Her Nobel Prize Win, Jennifer Doudna Predicts What’s Next for CRISPR

The new Nobel laureate chats with ‘Future Human’ about what her gene-editing companies are up to.


Emily Mullin

Photo illustration; Image source: picture alliance/Getty Images

Reengineering Life is a series from Future Human about the astonishing ways genetic technology is changing humanity and the world around us.

When the Royal Swedish Academy of Sciences announced on October 7 that she had won the 2020 Nobel Prize in chemistry, Jennifer Doudna was still fast asleep at home in California. It was just before 3 a.m. when a phone call woke her up. It was a reporter from Nature, asking if she could comment on the award.

“Well, who won it?” Doudna asked.

Doudna, PhD, of the University of California, Berkeley, and Emmanuelle Charpentier, PhD, of the Max Planck Institute in Germany, share the award for the discovery of the gene-editing technology CRISPR. The two biochemists began collaborating in 2011 and just a year later published a groundbreaking paper on CRISPR, which has revolutionized our ability to edit genes.

Short for clustered regularly interspaced short palindromic repeats, CRISPR is actually a naturally occurring bacterial immune system. When viruses attack bacteria, bacteria in turn grab snippets of genetic material from their viral invaders and incorporate these bits into their own DNA. This helps bacteria recognize viruses later on and thwart future invaders. Bacteria do this by producing an RNA molecule that acts as a guide, which cuts up the viral genome.

Doudna and Charpentier realized they could harness this cutting ability to edit genes in just about any living thing. In their 2012 paper, they described how this bacterial system could be used as “DNA scissors,” and the gene-editing technology CRISPR was born.

Declared as one of the most important discoveries of the 21st century, CRISPR is faster, cheaper, and more accurate than previous gene-editing systems and has since become ubiquitous in labs around the world. Scientists are using it in an attempt to treat serious genetic diseases, restore eyesight in people with a type of inherited blindness, engineer crops that are more resilient to disease and climate change, and eliminate disease-carrying pests like mosquitoes and mice. And researchers are already working on newer and improved versions of CRISPR that are even more precise.

The power to edit genes also opens up many ways for CRISPR to be abused. In 2018, Chinese scientist He Jiankui was widely condemned after revealing that he used CRISPR to make the world’s first known gene-edited babies. He is now serving a prison sentence, but the revelation has raised fears that CRISPR could lead to genetically enhanced “designer babies.”

After the Nobel announcement last week, I talked with Doudna about what’s next for CRISPR, the field of gene editing, and her own scientific work.

This interview has been lightly edited for grammar and clarity.

Future Human: First off, congratulations. What an incredible honor. How surprised were you to find out that you had won a Nobel prize?

Jennifer Doudna: Oh, total shock! I mean really. Coming out of a deep sleep and getting news like that — I couldn’t believe it. I said to the reporter who had called me, “I can’t talk to you right now. I have to call somebody and find out if this is official.”

We’re seeing a handful of clinical trials for CRISPR-based treatments get underway right now. What diseases do you see CRISPR being most promising for in the near future?

Certainly, diseases that are caused by single genes or genetic mutations. A great example, and we’ve already seen early results from one trial, is for sickle cell disease. But I think going forward, we’ll see opportunities to use CRISPR for other kinds of blood disorders, genetic diseases of the eye, and then, maybe in the longer term, cystic fibrosis and muscular dystrophy, which are also genetic diseases.

What do you think is going to be the biggest obstacle to getting these treatments to patients?

It’s probably delivery. One of the reasons why blood disorders have been some of the early targets of CRISPR is that the genome editing that’s used to correct those mutations can be done in cells that are taken out of a patient. The editing is done in the laboratory before reintroducing them versus a disease like cystic fibrosis or muscular dystrophy, where the editing would actually need to be done inside the body, in the right cells, to have a clinical benefit. That’s a hard challenge right now.

Your company, Mammoth Biosciences, is working on a rapid CRISPR-based test for Covid-19. What role do you think CRISPR diagnostic tests will play in the future?

There are several efforts underway to develop CRISPR diagnostics in comparable companies and academic labs. I think we’re going to see everything from high-throughput laboratory tests that require robotic equipment and experts to point-of-care tests that can be run in a research lab, a doctor’s office, or an emergency room. Down the road, we hope to have an at-home test that would work like a pregnancy test for Covid. What’s exciting with the CRISPR technology is that it’s potentially a faster and more direct way to detect the presence of the virus and also relies on a different supply chain than what’s necessary for the PCR (polymerase chain reaction) test.

What’s the status of your company’s Covid-19 test?

Mammoth Biosciences is planning on rolling out its test to a few partner labs for initial beta testing in November. Depending on how those experiments go and how those results turn out, we’ll expand to other labs after that. We want to see how it compares to the PCR test.

You recently just launched another CRISPR company, Scribe Therapeutics. What’s the focus of this new startup?

This is the thing about CRISPR: There’s so many different ways that it can be deployed. For clinical applications, the reason we’re seeing a lot of early efforts focused on blood disorders like sickle cell disease and, to some extent, diseases of the eye or even the liver is because those tissues are easier to introduce gene-editing molecules into. With Scribe Therapeutics, we’re looking at opportunities to use CRISPR for neurodegenerative diseases. For those disorders, the technology obviously needs to be very robust and very safe. It also has to get into brain cells and neural tissue where it can have an impact. We want to make sure that the editing tools are the best they can be and then figure out the best way to introduce them into the brain. That’s really the focus of the company.

What do you think is going to be the next big CRISPR advance?

That’s always a hard question. We’ve so much going on in the field. I think one interesting possibility is that we’ll see CRISPR being used not to edit genomes, or at least not to make permanent changes to genomes, but instead to regulate them, to control levels of human proteins that are produced from different genes. This is a newer way of using the CRISPR technology. I think it has a lot of potential to allow control of cells that doesn’t require actual permanent chemical changes being made to the DNA.

After the birth of the CRISPR babies in China in 2018, there’s been a lot of talk around the idea of germline or heritable genome editing. Do you think that should be off-limits to scientists right now?

I don’t think it needs to be completely off-limits. I was pretty pleased with the recent report that came out from the National Academies and the U.K. Royal Society that recommends a kind of a measured approach to developing the technology for use in the human germline. They’re encouraging research to understand how the technology works in embryos. First, the technology will need to be proven safe. Secondly, any clinical use [to establish a pregnancy] would need to be restricted to cases of serious genetic disease where there are few or no other options to treat the disease. I think those are both pretty high bars. Those situations are pretty rare. I personally think there are more viable strategies today, like embryo screening and selection in an IVF (in vitro fertilization) clinic, rather than using genome editing.

The report you mentioned also calls for “extensive societal dialogue” before countries decide to permit the use of heritable human genome editing. You’ve talked in the past about public engagement around CRISPR. How do we educate and engage the public about CRISPR?

Yeah, that’s really critical. I think the media has an important role to play in terms of large-scale education. Interactive media, like videos and documentaries, can also help. The challenge of course is making sure that the science is right.

How do we make sure that the public’s voice is heard in regard to how CRISPR is used?

It’s a really important question. It’s a challenge because, on one hand, I think it’s critical to have more public engagement in important decisions like this about how technology is used. On the other hand, that requires a level of understanding about the technology that the average person might not have or maybe doesn’t want to have. So, I think it’s important to have different formats and forums for encouraging discussion. We’ve already seen this with CRISPR in some way. On the one hand, there are highly technical meetings that include discussion of ethical and societal issues from a pretty detailed technical standpoint. But there are also increasingly conferences and events that don’t get into the weeds of the science per se, but they spend a lot more effort thinking through CRISPR’s implications and its different uses. Inviting people who are nonspecialists to engage in those meetings has been really effective.

Other than medicine, where else do you think CRISPR could be transformative?

Agriculture is the other area where it’s going to be impactful. We’re already seeing a lot of use of CRISPR in making plants that have genetic changes that can enable things like better crop yield, resistance to drought, higher levels of nutritional value, things like that. I think that’s really exciting, and there’s clearly a lot more to be done there. That’s likely to be the area where we’ll see a broader impact in the near term.

Given CRISPR’s potential for misuse, how do you think it should be regulated?

Fortunately, I think there’s quite a good regulatory framework in the United States and in most places that have major research operations that can serve to regulate the use of CRISPR. That really goes back to the 1970s [at the Asilomar Conference on Recombinant DNA], when voluntary guidelines were put in place for using some of these early tools of molecular biology, like molecular cloning. That being said, as we discussed, there are certain applications of CRISPR like in human embryos, where I think there needs to be special attention.

Who are the CRISPR scientists you really admire?

Many. It’s become a huge field. It used to be tiny, and now it’s vast. One is Luciano Marraffini. He’s a scientist at Rockefeller University. He works on the fundamentals of CRISPR biology and understanding how it works in bacteria as an immune system. Jill Banfield at Berkeley is continuing to do work on bacteria that are not cultured in the lab but are growing in various environmental niches. She was one of the very early discoverers of CRISPR systems and bacteria, and she continues to find a lot of new ones. In plant biology, I really like the work of Pamela Ronald at the University of California, Davis. She’s doing work primarily in rice, where they are using CRISPR to make modifications to the rice genome that I think are going to be really important as rice farmers face the challenges of climate change. On the biomedical side, I’m really excited about the work of Charles Gersbach at Duke University.

What’s next for you?

CRISPR is going to keep us busy for a while. There are still a lot of fundamental questions about how these pathways operate that I really like to try to answer. Jill Banfield is a close collaborator of ours, and she continues to supply us with many, many new CRISPR pathways that we’re excited to investigate. We’re also really interested in genome editing in natural microbial communities. I think there’s a really interesting opportunity to be able to manipulate certain microbes. I think those are areas where I’ll be focusing my efforts in the near term.


WRITTEN BY
Emily Mullin
Staff writer at OneZero and Future Human, where I cover biotechnology and genetic privacy. I also teach science writing at Johns Hopkins.

Future Human is a science publication from Medium about the survival of our species. It is run by the OneZero editorial team, which also publishes stories about major forces in technology through its namesake publication and stories about gadgets on Debugger.

Scientists Program CRISPR to Fight Viruses in Human Cells

A common gene-editing enzyme could be used to disable RNA viruses such as flu or Ebola

By Tanya Lewis on October 23, 2019

Researchers modified the enzyme Cas13 to target and inactivate viruses such as influenza (shown here). Credit: Kateryna Kon Getty Images

CRISPR is usually thought of as a laboratory tool to edit DNA in order to fix genetic defects or enhance certain traits—but the mechanism originally evolved in bacteria as a way to fend off viruses called bacteriophages. Now scientists have found a way to adapt this ability to fight viruses in human cells.

In a recent study, Catherine Freije, Cameron Myhrvold and Pardis Sabeti at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, and their colleagues programmed a CRISPR-related enzyme to target three different single-stranded RNA viruses in human embryonic kidney cells (as well as human lung cancer cells and dog kidney cells) grown in vitro and chop them up, rendering them largely unable to infect additional cells. If further experiments show this process works in living animals, it could eventually lead to new antiviral therapies for diseases such as Ebola or Zika in humans.

Viruses come in many forms, including DNA and RNA, double-stranded and single-stranded. About two thirds of the ones that infect humans are RNA viruses, and many have no approved treatment. Existing therapies often use a small molecule that interferes with viral replication—but this approach does not work for newly emerging viruses or ones that are evolving rapidly.

“CRISPR” refers to a series of DNA sequences in bacterial genomes that were left behind from previous bacteriophage infections. When the bacteria encounter these pathogens again, enzymes called CRISPR-associated (Cas) proteins recognize and bind to these sequences in the virus and destroy them. In recent years, researchers (including study co-author Feng Zhang) have reengineered one such enzyme, called Cas9, to cut and paste DNA in human cells. The enzyme binds to a short genetic tag called a guide RNA, which directs the enzyme to a particular part of the genome to make cuts. Previous studies have used Cas9 to prevent replication of double-stranded DNA viruses or of single-stranded RNA viruses that produce DNA in an intermediate step during replication. Only a small fraction of RNA viruses that infect humans produce such DNA intermediates—but another CRISPR enzyme, called Cas13, can be programmed to cleave single-stranded RNA viruses.

“The nice thing about CRISPR systems and systems like Cas13 is that their initial purpose in bacteria was to defend against viral infection of bacteria, and so we sort of wanted to bring Cas13 back to its original function—and apply this to mammalian viruses in mammalian cells,” says Freije, who is a doctoral student in virology at Harvard. “Because CRISPR systems rely on guide RNAs to specifically guide the CRISPR protein to a target, we saw this as a great opportunity to use it as a programmable antiviral.”

Freije and her colleagues programmed Cas13 to target three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV) and vesicular stomatitis virus (VSV). LCMV is an RNA virus that mostly infects mice—but it is in the same family as the virus that causes Lassa fever, which is found in West Africa and is much more dangerous to study in the lab. IAV is a flu virus; although some antiviral medications for flu already exist, such viruses evolve rapidly, so there is a need for better options. Finally, VSV is a model for many other single-stranded RNA viruses.

To determine how effective Cas13 was at destroying the viruses, the researchers also used it as a diagnostic tool to see how much viral RNA was being released from infected cells. They saw a twofold to 44-fold reduction in RNA, depending on which virus they were looking at and the time point. They also looked at how well the released RNA was able to go on and infect new cells. In most cases, they saw a 100-fold reduction in infectivity—and in some cases, more than 300-fold—according to Freije. The findings were published online on October 10 in Molecular Cell.

“The results are very impressive,” says Chen Liang, a professor at the Lady Davis Institute at Jewish General Hospital and the department of microbiology and immunology at McGill University in Montreal, who was not involved in the study. His own laboratory has used the Cas9 enzyme to deactivate DNA viruses. The concept is very similar, but Cas13 has a few advantages, he says. For one, Cas13 can be used to target one virus using several guide RNAs, making it difficult for the virus to “escape.” Secondly, the new study also used Cas13 to detect how much viral RNA was left over to infect cells. The amount of viral knockdown the group achieved is “very significant,” Liang says. “If you can target and inactivate all three [of these] viruses, in principle, you can inactivate any virus.

As with any approach, there are limitations. One is the question of how to deliver the Cas13 to target a virus in a living person, Liang notes, and the researchers have not yet done any animal studies. Another is the fact that viruses will eventually develop resistance. But Cas13 has an advantage here: when Cas9 cuts viral DNA, mammalian cells repair it and can cause mutations that make the virus more resistant. Yet with Cas13, these cells do not have the mechanism to repair the RNA and introduce errors that would help the virus escape being destroyed. Even if a virus does evolve resistance, or if a new virus is encountered, the method could be quickly adapted.

“One of the things that’s most exciting about this approach is the programmability,” says Myhrvold, a postdoctoral fellow at Harvard. “Once you figure out how to do this well for one virus it’s not that hard to design sequences against another virus—or another one. Furthermore, if the virus changes its own sequence—as viruses are known to do, just during an outbreak or in response to therapy—you can very easily update the CRISPR RNA sequence and keep up with the virus.”

Freije agrees. “We are definitely excited about future prospects of optimizing the system and trying it out in mouse models,” she says. Beyond therapeutics, the team hopes to understand more about how viruses operate—how they replicate and what parts of their genomes are most important. Using approaches like this, “you can really start to get a better picture of what parts of these viruses are and, most importantly, what really makes them tick.”

How do bacteria defend themselves against viruses?

The CRISPR-Cas system in some bacteria helps to form an effective barrier to invading viruses.

DIGEST Apr 3, 2019



A transmission electron microscopy image of bacteriophages taken at The University of Alabama’s Optical Analysis Facility. Image credit: Chou-Zheng and Hatoum-Aslan, 2019 (CC BY 4.0)

Just as humans are susceptible to viruses, bacteria have their own viruses to contend with. These viruses – known as phages – attach to the surface of bacterial cells, inject their genetic material, and use the cells’ enzymes to multiply while destroying their hosts.

To defend against a phage attack, bacteria have evolved a variety of immune systems. For example, when a bacterium with an immune system known as CRISPR-Cas encounters a phage, the system creates a ‘memory’ of the invader by capturing a small snippet of the phage’s genetic material. The pieces of phage DNA are copied into small molecules known as CRISPR RNAs, which then combine with one or more Cas proteins to form a group called a Cas complex. This complex patrols the inside of the cell, carrying the CRISPR RNA for comparison, similar to the way a detective uses a fingerprint to identify a criminal. Once a match is found, the Cas proteins chop up the invading genetic material and destroy the phage.

There are several different types of CRISPR-Cas systems. Type III systems are among the most widespread in nature and are unique in that they provide a nearly impenetrable barrier to phages attempting to infect bacterial cells. Medical researchers are exploring the use of phages as alternatives to conventional antibiotics and so it is important to find ways to overcome these immune responses in bacteria. However, it remains unclear precisely how Type III CRISPR-Cas systems are able to mount such an effective defense.

Chou-Zheng and Hatoum-Aslan used genetic and biochemical approaches to study the Type III CRISPR-Cas system in a bacterium called Staphylococcus epidermidis. The experiments showed that two enzymes called PNPase and RNase J2 played crucial roles in the defense response triggered by the system. PNPase helped to generate CRISPR RNAs and both enzymes were required to help to destroy genetic material from invading phages.

Previous studies have shown that PNPase and RNase J2 are part of a machine in bacterial cells that usually degrades damaged genetic material. Therefore, these findings show that the Type III CRISPR-Cas system in S. epidermidis has evolved to coordinate with another pathway to help the bacteria survive attack from phages. CRISPR-Cas immune systems have formed the basis for a variety of technologies that continue to revolutionize genetics and biomedical research. Therefore, along with aiding the search for alternatives to antibiotics, this work may potentially inspire the development of new genetic technologies in the future.

Phage-Encoded Anti-CRISPR Defenses
Annual Review of Genetics

Vol. 52:445-464 (Volume publication date November 2018)
First published as a Review in Advance on September 12, 2018
https://doi.org/10.1146/annurev-genet-120417-031321

Sabrina Y. Stanley1 and Karen L. Maxwell2
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
2Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1M1, Canada; email: karen.maxwell@utoronto.ca

Abstract

The battle for survival between bacteria and bacteriophages (phages) is an arms race where bacteria develop defenses to protect themselves from phages and phages evolve counterstrategies to bypass these defenses. CRISPR-Cas adaptive immune systems represent a widespread mechanism by which bacteria protect themselves from phage infection. In response to CRISPR-Cas, phages have evolved protein inhibitors known as anti-CRISPRs. Here, we describe the discovery and mechanisms of action of anti-CRISPR proteins. We discuss the potential impact of anti-CRISPRs on bacterial evolution, speculate on their evolutionary origins, and contemplate the possible next steps in the CRISPR-Cas evolutionary arms race. We also touch on the impact of anti-CRISPRs on the development of CRISPR-Cas-based biotechnological tools.

FULL ARTICLE HERE



Biological Sciences
RESEARCH ARTICLE
Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria
Ido Yosef, Miriam Manor, Ruth Kiro, and View ORCID Profile Udi Qimron
PNAS June 9, 2015 112 (23) 7267-7272; first published May 18, 2015 https://doi.org/10.1073/pnas.1500107112

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 28, 2015 (received for review January 25, 2015)

Significance

Antibiotic resistance of pathogens is a growing concern to human health, reviving interest in phage therapy. This therapy uses phages (natural bacterial enemies) to kill pathogens. However, it encounters many obstacles such as delivery barriers into the tissues and bacterial resistance to phages. Here, we use phages for delivering a programmable DNA nuclease, clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas), to reverse antibiotic resistance and eliminate the transfer of resistance between strains. This approach combines CRISPR-Cas delivery with lytic phage selection of antibiotic-sensitized bacteria. The strategy may reduce the prevalence of antibiotic-resistant bacteria in treated surfaces and on skin of medical personnel, as it uses phages in a unique way that overcomes many of the hurdles encountered by phage therapy.

Abstract

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

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   Genetically Engineered Phages: a Review of Advances over the Last Decade

Diana P. Pires, Sara Cleto, Sanna Sillankorva, Joana Azeredo, Timothy K. Lu

DOI: 10.1128/MMBR.00069-15

 

SUMMARY

Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

INTRODUCTION

Bacteriophages (phages) are among the most abundant biological particles on earth. They are also highly versatile and adaptable to a great number of applications. Phages are viruses that infect bacteria; their self-replication depends on access to a bacterial host. Phages were discovered independently by Frederick Twort in 1915 (1) and by Félix d'Hérelle in 1917 (2), and they were used early on as antimicrobial agents. Although the initial results of phage therapy were promising (3, 4), poorly controlled trials and inconsistent results generated controversy within the scientific community about the efficacy and reproducibility of using phages to treat bacterial infections (5–7). The discovery of penicillin in 1928 and the subsequent arrival of the antibiotic era further cast a shadow on phage therapy (5, 6). As a result, phage therapy was discontinued in Western countries, even as its use continued in Eastern Europe and the former Soviet Union (8–10).

Despite the important success of antibiotics in improving human health, antibiotic resistance has emerged with steadily increasing frequency, rendering many antibiotics ineffective (11–14). Multidrug-resistant bacteria currently constitute one of the most widespread global public health concerns (15–17). More than 2 million people are sickened every year in the United States alone as a result of antibiotic-resistant infections, resulting in at least 23,000 deaths per year (16). The rising tide of antibiotic resistance coupled with the low rate of antibiotic discovery (17, 18) has revived interest in phages as antibacterial agents (19–21).

Unlike most antibiotics, phages are typically highly specific for a particular set of bacterial species or strains and are thus expected to have fewer off-target effects on commensal microflora than antibiotics do (22). The self-replicating nature of phages and the availability of simple, rapid, and low-cost phage production processes are additional advantages for their use as antimicrobials (22). Phages have been used not only to treat and prevent human bacterial infections (9, 23–25) but also to control plant diseases (26–29), detect pathogens (30–33), and assess food safety (34–37).

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REVIEW ARTICLE
Microbiol., 03 May 2019 | https://doi.org/10.3389/fmicb.2019.00954

Genetic Engineering of Bacteriophages Against Infectious Diseases

Yibao Chen1,2, Himanshu Batra3, Junhua Dong1,2, Cen Chen1,2, Venigalla B. Rao3 and Pan Tao1,2,3*

Bacteriophages (phages) are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. The therapeutic use of phages to treat bacterial infections (“phage therapy”) was conceived by Felix d’Herelle nearly a century ago. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases. In addition to phage therapy, phages have also been developed as vaccine platforms to deliver antigens as part of virus-like nanoparticles that can stimulate immune responses and prevent pathogen infections. Phage engineering promises to generate phage variants with unique properties for prophylactic and therapeutic applications. These approaches have created momentum to accelerate basic as well as translational phage research and potential development of therapeutics in the near future.

Introduction

Bacteriophages (phages), discovered in the early 20th century independently by Frederick Twort and Felix d’Herelle, are the most abundant organisms on earth with up to 2.5 × 108 phages per ml in natural waters (Bergh et al., 1989). It is well accepted that phages specifically infect bacteria and, therefore, were considered for the development of natural approaches to treat bacterial infections since their discovery (Wittebole et al., 2014; Salmond and Fineran, 2015). However, due to the discovery of antibiotics that provided greater breadth and potency, phage therapy lagged behind although research continued in some Eastern European countries (Chanishvili, 2012, 2016; Wittebole et al., 2014). Therefore, in the following several decades, phages were mainly used as model organisms to explore the basic mechanisms of life and led to the birth of modern molecular biology. One classical example is the demonstration of a central biological question in the early 20th century, the nature of a gene, by “Hershey-Chase experiment” (also called “Waring blender experiment”) (Salmond and Fineran, 2015). This elegant experiment demonstrated that DNA, not protein, is the genetic material of T2 phage.

Recently, the emergence of multi-antibiotic resistant bacterial pathogens and the low rate of new antibiotic discovery brought new urgency to develop phage-based therapies (Lu and Koeris, 2011; Viertel et al., 2014; Domingo-Calap and Delgado-Martinez, 2018). A striking example is the recent San Diego patient who was infected by multi-drug resistant Acinetobacter baumannii stain during travelling to Egypt. The patient went into a coma for nearly 2 months but awoke 2 days after intravenous injection of a phage cocktail that lyses this bacterium and finally completely recovered (Schooley et al., 2017). With recent advances, particularly the genome engineering (Martel and Moineau, 2014; Ando et al., 2015; Lemay et al., 2017; Tao et al., 2017b; Kilcher et al., 2018), the applications of phages have greatly expanded. In addition to its use in antibacterial therapy, phages were used in synthetic biology (Lemire et al., 2018), material science (Cao et al., 2016), and biomedical fields (Cao et al., 2018; Tao et al., 2018c). Considering the abundance and diversity, there is vast potential to engineer phages for different applications. In this review, we will focus on the applications of phages in infectious disease, in particular, vaccine development and phage therapy. We will discuss the phage engineering strategies and how these can equip the phages with the ability to advance the vaccine and phage therapy fields.

1College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
2The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, China
3Department of Biology, The Catholic University of America, Washington, DC, United States

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Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
Yan-ru Cui,
Shao-jie Wang,
Jun Chen,
Jie Li,
Wenzhang Chen,
Shuyue Wang,
Bing Meng,
Wei Zhu,
Zhuhong Zhang,
Bei Yang,
Biao Jiang,
Guang Yang,
Peixiang Ma &
Jia Liu 

Genome Biology volume 21, Article number: 51 (2020) Cite this article
Research
Open Access
Published: 26 February 2020

Abstract

Background

CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.

ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.

Conclusion

G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

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Heterogeneous Diversity of Spacers within CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Article (PDF Available) in Physical Review Letters 105(12):128102 · September 2010 with 231
Download full-text PDF

DOI: 10.1103/PhysRevLett.105.128102 · Source: PubMedCite this publication

Jiankui he
Southern University of Science and Technology


Michael Deem
Rice University

Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) in bacterial and archaeal DNA have recently been shown to be a new type of antiviral immune system in these organisms. We here study the diversity of spacers in CRISPR under selective pressure. We propose a population dynamics model that explains the biological observation that the leader-proximal end of CRISPR is more diversified and the leader-distal end of CRISPR is more conserved. This result is shown to be in agreement with recent experiments. Our results show that the CRISPR spacer structure is influenced by and provides a record of the viral challenges that bacteria face.
https://www.researchgate.net/publication/46424214_Heterogeneous_Diversity_of_Spacers_within_CRISPR_Clustered_Regularly_Interspaced_Short_Palindromic_Repeats

Volume 366
Issue 9
May 2019

Article Contents
ABSTRACT
INTRODUCTION
BIOLOGICAL RELEVANCE OF ANTI-CRISPR PROTEINS
MECHANISMS AND STRUCTURES OF ANTI-CRISPR PROTEINS
APPLICATIONS OF ANTI-CRISPR PROTEINS
OUTLOOK
FUNDING
REFERENCES


MINI REVIEW

Keeping CRISPR in check: diverse mechanisms of phage-encoded anti-CRISPRS 
Despoina Trasanidou, Ana Sousa Gerós, Prarthana Mohanraju, Anna Cornelia Nieuwenweg, Franklin L Nobrega, Raymond H J Staals

FEMS Microbiology Letters, Volume 366, Issue 9, May 2019, fnz098, https://doi.org/10.1093/femsle/fnz098

Published: 11 May 2019

ABSTRACT

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.

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The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Melia E Bonomo1,3 and Michael W Deem1,2,3,4

Published 30 April 2018 • © 2018 IOP Publishing Ltd
Physical Biology, Volume 15, Number 4

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Article information

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.

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1. Introduction


In 1987, Ishino and colleagues had set out to identify the encoded protein and primary structure of a particular gene in Escherichia coli by analyzing its chromosomal DNA segment and flanking regions [1]. They found an interesting sequence structure at the gene's 3'-end flanking region, in which five homologous sequences of 29 nucleotides were arranged as direct repeats with 32-nucleotide sequences spaced between them. Little did they know that their discovery would prove to have critical immunological significance. It was not until 2000 that these mysterious repeated genomic elements were revisited when Mojica and colleagues searched the available microbial genome database and found many organisms that contained partially palindromic sequences of 24–40 basepairs with 20–58 basepair sequences spaced between them [2]. These were found in almost all archaea, about half of bacteria, no viruses, and no eukaryotes. Related and unrelated species had nearly identical structure in these repeat sequence units. The sequences in between, called 'spacers', were unique to an individual locus and were not found in other genomes [3]. After many suggested abbreviations, including SRSRs, short regularly spaced repeats, and SPIDR, spacers interspersed direct repeats, the scientific community settled on calling these elements clustered regularly interspaced short palindromic repeats, or CRISPR.

SEE 
https://plawiuk.blogspot.com/search?q=BACTERIOPHAGES

https://plawiuk.blogspot.com/search?q=PHAGES

 

CRISPR-copies: New tool accelerates and optimizes genome editing

CABBI researchers publically share a new tool to revolutionize CRISPR gene editing

UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN INSTITUTE FOR SUSTAINABILITY, ENERGY, AND ENVIRONMENT

 

IMAGE: 

CRISPR-COPIES HAS APPLICATIONS IN SYNTHETIC BIOLOGY TOOLKIT CHARACTERIZATION, GENE THERAPY, AND METABOLIC ENGINEERING.

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CREDIT: AASHUTOSH BOOB ET AL.

CRISPR/Cas systems have undergone tremendous advancement in the past decade. These precise genome editing tools have applications ranging from transgenic crop development to gene therapy and beyond. And with their recent development of CRISPR-COPIES, researchers at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) are further improving CRISPR’s versatility and ease of use.

“CRISPR-COPIES is a tool that can quickly identify appropriate chromosomal integration sites for genetic engineering in any organism,” said Huimin Zhao, CABBI Conversion Theme Leader and Steven L. Miller Chair of Chemical and Biomolecular Engineering (ChBE) at the University of Illinois. “It will accelerate our work in the metabolic engineering of non-model yeasts for cost-effective production of chemicals and biofuels.”

Gene editing has revolutionized scientists’ capabilities in understanding and manipulating genetic information. This form of genetic engineering allows researchers to introduce new traits into an organism, such as resistance to pests or the ability to produce a valuable biochemical.

With CRISPR/Cas systems, researchers can make precise, targeted genetic edits. However, locating optimal integration sites in the genome for these edits has been a critical and largely unsolved problem. Historically, when researchers needed to determine where to target their edits, they would typically manually screen for potential integration sites, then test the site by integrating a reporter gene to assess its cellular fitness and gene expression levels. It’s a time- and resource-intensive process.

To address this challenge, the CABBI team developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool can identify genome-wide neutral integration sites for most bacterial and fungal genomes within two to three minutes.

“Finding the integration site in the genome manually is like searching for a needle in a haystack,” said Aashutosh Boob, a ChBE Ph.D. student at the University of Illinois and primary author of the study. “However, with CRISPR-COPIES, we transform the haystack into a searchable space, empowering researchers to efficiently locate all the needles that align with their specific criteria.”

In their paper published in Nucleic Acids Research, the researchers demonstrated the versatility and scalability of CRISPR-COPIES by characterizing integration sites in three diverse species: Cupriavidus necator, Saccharomyces cerevisiae, and HEK 293T cells. They used integration sites found by CRISPR-COPIES to engineer cells with increased production of 5-aminolevulinic acid, a valuable biochemical that has applications in agriculture and the food industry.

In addition, the team has created a user-friendly web interface for CRISPR-COPIES. This incredibly accessible application can be used by researchers even without significant bioinformatics expertise.

A primary objective of CABBI is the engineering of non-model yeasts to produce chemicals and fuels from plant biomass. Economically producing biofuels and bioproducts from low-cost feedstocks at a large scale is a challenge, however, due to the lack of genetic tools and the cumbersome nature of traditional genome-editing methods. By enabling researchers to swiftly pinpoint genomic loci for targeted gene integration, CRISPR-COPIES provides a streamlined pipeline that facilitates the identification of stable integration sites across the genome. It also eliminates the manual labor involved in designing components for CRISPR/Cas-mediated DNA integration.

For crop engineering, the tool can be used to increase biomass yields, pest resistance, and/or environmental resilience. For converting biomass to valuable chemicals — for instance, by using the yeast S. cerevisiae — CRISPR-COPIES can be used to engineer cells with significantly greater yields.

This versatile software is designed to simplify and accelerate the strain construction process, saving researchers both time and resources. Researchers around the world in both academia and industry can benefit from its utility in strain engineering for biochemical production and transgenic crop development.

Co-authors on this study include ChBE Ph.D. student Zhixin Zhu, ChBE visiting student Pattarawan Intasian, and Bioengineering Ph.D. student Guanhua Xun; Carl R. Woese Institute for Genomic Biology (IGB) Software Developers Manan Jain and Vassily Petrov; IGB Biofoundry Manager Stephan Lane; and CABBI postdoc Shih-I Tan.

— Article by CABBI Communications Specialist April Wendling

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JOURNAL

Nucleic Acids Research

DOI

10.1093/nar/gkae062 

METHOD OF RESEARCH

Experimental study

SUBJECT OF RESEARCH

Not applicable

ARTICLE TITLE

CRISPR-COPIES: An in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

ARTICLE PUBLICATION DATE

13-Feb-2024

Not too late to repair: gene therapy improves advanced heart failure in animal model

Peer-Reviewed Publication

BAYLOR COLLEGE OF MEDICINE

Heart failure remains the leading cause of mortality in the U.S. During a heart attack blood stops flowing into the heart. Without oxygen, part of the heart muscle dies. The heart muscle does not regenerate, instead it replaces dead tissue with a scar made of cells called fibroblasts that do not help the heart pump. If there is too much scarring, the heart progressively enlarges, or dilates, weakens and eventually stops working.

“The current thought is that advanced or chronic heart failure, a stage in which the cardiac muscle has become too weak, is a point of no return. The present understanding is that it is not possible to stimulate a heart in this condition to generate new heart cells to repair itself and that only palliative treatment is available to patients,” said corresponding author Dr. Tamer M. A. Mohamed, associate professor of surgery and medicine and director of cardiac regeneration at Baylor College of Medicine. “In this study published in the journal Cardiovascular Research, we show that advanced heart failure can be treated to improve cardiac function in an animal model.”

In a previous study, Mohamed and his collaborators had successfully used gene therapy to improve acute cardiac dysfunction in animals. Their method effectively and specifically delivered genes that promote proliferation to heart cells, generating new heart muscle. This approach not only strengthened the heart improving its ability to keep the blood flowing, but also prevented typical subsequent congestion in the liver, kidneys and lungs in rats and pigs.

“In this study, we did something that had not been done before,” Mohamed said. “We intervened with the same gene therapy but not during acute heart failure or early in the disease as in our previous experiments, but late in the disease during the chronic phase four weeks after cardiac injury had severely damaged the heart.”

Four months after treating the animals, the researchers checked cardiac function and heart structure. “We were surprised to see evidence of significant heart cell proliferation, a marked reduction in scar size and a significant improvement in cardiac function,” said first author Dr. Riham R E Abouleisa, assistant professor of surgery- cardiothoracic surgery at Baylor. “Although heart dilation and lung congestion associated with chronic heart failure were not improved, the treatment partially improved liver and kidney functionality.”

The findings show for the first time that contrary to expectations, it is possible to induce heart cell proliferation during advanced states of heart failure and improve heart function, with some beneficial effects on the liver and kidneys’ functions.

“Our work has important implications for the large group of patients with advanced heart failure for whom there are currently no treatments to improve their condition,” Mohamed said. “This approach offers the possibility of developing future new therapies for this deadly disease.”

Other authors on this study include: Xian-Liang Tang, Qinghui O, Abou-Bakr M. Salama, Amie Woolard, Dana Hammouri, Hania Abdelhafez, Sarah Cayton, Sameeha K. Abdulwali, Momo Arai, Israel D. Sithu, Daniel J. Conklin and Roberto Bolli. The authors are affiliated with one or more of the following institutions: Baylor College of Medicine, University of Louisville, Zagazig University, Alfaisal University and University of Manchester.

This work was supported by the National Institutes of Health grants (F32HL149140, R01HL147921, P30GM127607, R15HL168688, R01HL166280 and HL78825) and by the American Heart Association grant 16SDG29950012.

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JOURNAL

Cardiovascular Research

DOI

10.1093/cvr/cvae002 

METHOD OF RESEARCH

Experimental study

SUBJECT OF RESEARCH

Animals

ARTICLE TITLE

Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats