Selection for bigger tomatoes has made the fruits less sweet, but now it has been shown that gene editing can make them sweeter without decreasing yields

Patrick Doherty volunteered for a new medical intervention of gene-editor infusions for the treatment of genetically-based diseases.; Credit: /Patrick Doherty

Patrick Doherty had always been very active. He trekked the Himalayas and hiked trails in Spain.

But about a year and a half ago, he noticed pins and needles in his fingers and toes. His feet got cold. And then he started getting out of breath any time he walked his dog up the hills of County Donegal in Ireland where he lives.

"I noticed on some of the larger hill climbs I was getting a bit breathless," says Doherty, 65. "So I realized something was wrong."

Doherty found out he had a rare, but devastating inherited disease — known as transthyretin amyloidosis — that had killed his father. A misshapen protein was building up in his body, destroying important tissues, such as nerves in his hands and feet and his heart.

Doherty had watched others get crippled and die difficult deaths from amyloidosis.

"It's terrible prognosis," Doherty says. "This is a condition that deteriorates very rapidly. It's just dreadful."

So Doherty was thrilled when he found out that doctors were testing a new way to try to treat amyloidosis. The approach used a revolutionary gene-editing technique called CRISPR, which allows scientists to make very precise changes in DNA.

"I thought: Fantastic. I jumped at the opportunity," Doherty says.

On Saturday, researchers reported the first data indicating that the experimental treatment worked, causing levels of the destructive protein to plummet in Doherty's body and the bodies of five other patients treated with the approach.

"I feel fantastic," Doherty says. "It's just phenomenal."

The advance is being hailed not just for amyloidosis patients but also as a proof-of-concept that CRISPR could be used to treat many other, much more common diseases. It's a new way of using the innovative technology.

"This is a major milestone for patients," says Jennifer Doudna of the University of California, Berkeley, who shared a Nobel Prize for her work helping develop CRISPR.

"While these are early data, they show us that we can overcome one of the biggest challenges with applying CRISPR clinically so far, which is being able to deliver it systemically and get it to the right place," Doudna says.

CRISPR has already been shown to help patients suffering from the devastating blood disorders sickle cell disease and beta thalassemia. And doctors are trying to use it to treat cancer and to restore vision to people blinded by a rare genetic disorder.

But those experiments involve taking cells out of the body, editing them in the lab, and infusing them back in or injecting CRISPR directly into cells that need fixing.

The study Doherty volunteered for is the first in which doctors are simply infusing the gene-editor directly into patients and letting it find its own way to the right gene in the right cells. In this case, it's cells in the liver making the destructive protein.

"This is the first example in which CRISPR-Cas9 is injected directly into the bloodstream — in other words systemic administration — where we use it as a way to reach a tissue that's far away from the site of injection and very specifically use it to edit disease-causing genes," says John Leonard, the CEO of Intellia Therapeutics, which is sponsoring the study.

Doctors infused billions of microscopic structures known as nanoparticles carrying genetic instructions for the CRISPR gene-editor into four patients in London and two in New Zealand. The nanoparticles were absorbed by their livers, where they unleashed armies of CRISPR gene-editors. The CRISPR editor honed in on the target gene in the liver and sliced it, disabling production of the destructive protein.

Within weeks, the levels of protein causing the disease plummeted. Researchers reported at the Peripheral Nerve Society Annual Meeting and in a paper published in The New England Journal of Medicine.

"It really is exciting," says Dr. Julian Gillmore, who is leading the study at the University College London, Royal Free Hospital.

"This has the potential to completely revolutionize the outcome for these patients who have lived with this disease in their family for many generations. It's decimated some families that I've been looking after. So this is amazing," Gillmore says.

The patients will have to be followed longer, and more patients will have to be treated, to make sure the treatment's safe, and determine how much it's helping, Gillmore stresses. But the approach could help those struck by amyloidosis that isn't inherited, which is a far more common version of the disease, he says.

Moreover, the promising results potentially open the door for using the same approach to treatment of many other, more common diseases for which taking cells out of the body or directly injecting CRISPR isn't realistic, including heart disease, muscular dystrophy and brain diseases such as Alzheimer's.

"This is really opening a new era as we think about gene-editing where we can begin to think about accessing all kinds of different tissue in the body via systemic administration," Leonard says.

Other scientists who are not involved in the research agree.

"This is a wonderful day for the future of gene-editing as a medicine,"
agree Fyodor Urnov, a professor of genetics at the University of California, Berkeley. "We as a species are watching this remarkable new show called: our gene-edited future."

Doherty says he started feeling better within weeks of the treatment and has continued to improve in the weeks since then.

"I definitely feel better," he told NPR. "I'm speaking to you from upstairs in our house. I climbed stairs to get up here. I would have been feeling breathless. I'm thrilled."

This content is from Southern California Public Radio. View the original story at SCPR.org.

CRISPR Immune Cells Not Only Survive, They Thrive After Infusion Into Cancer Patients

CRISPR–Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR–Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.

No, CRISPR gene editing technology is not going to “cure” heart disease. But a New York Times story by Gina Kolata on an extremely early study in animals prominently plays up just this extremely unlikely claim. The Times story is based on a press release issued by Verve Therapeutics, a new biotechnology company founded by Sekar Kathiresan, an influential cardiologist and genomic...

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Researchers have developed a CRISPR-based diagnostic tool that can rapidly and accurately detect gene fusions associated with acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML).

Nobel Prize-winning inventors of CRISPR, awarded in 2020, have seen their gene-editing tool gain approval in the UK for the first-ever therapy targeting

Assistant Prof. Ibrahim, Bitar, Department of Microbiology, Faculty of Medicine and University Hospital in Plzen, Charles University in Prague, Plzen,

Researchers at the University of Toronto have created an RNA-targeting technology that uses the CRISPR system to precisely manipulate human gene parts

In a monumental leap forward in the battle against medlinkHIV/AIDS/medlink, researchers at the University of Amsterdam have reported a success in

Joined by CRISPR luminaries Feng Zhang and J. Keith Joung, the Harvard chemist raises up to $87 million in funding

Joined by CRISPR luminaries Feng Zhang and J. Keith Joung, the Harvard chemist raises up to $87 million in funding

Emmanuelle Charpentier, Jennifer Doudna, and Virginijus Siksnys win Kavli Prize in nanoscience, renewing Nobel Prize speculation and resurfacing a forgotten name in the CRISPR patent battle

Award resurfaces a forgotten name in the CRISPR patent battle amid Nobel Prize speculation

News reports linking the gene-editing technology to cancer are the latest in a series of hyped alarms due to be tested in humans soon

Baylis, Françoise, 1961- author

Seizures often herald the clinical appearance of gliomas or appear at later stages. Dissecting their precise evolution and cellular pathogenesis in brain malignancies could inform the development of staged therapies for these highly pharmaco-resistant epilepsies. Studies in immunodeficient xenograft models have identified local interneuron loss and excess glial glutamate release as chief contributors to network disinhibition, but how hyperexcitability in the peritumoral microenvironment evolves in an immunocompetent brain is unclear. We generated gliomas in WT mice via in utero deletion of key tumor suppressor genes and serially monitored cortical epileptogenesis during tumor infiltration with in vivo electrophysiology and GCAMP7 calcium imaging, revealing a reproducible progression from hyperexcitability to convulsive seizures. Long before seizures, coincident with loss of inhibitory cells and their protective scaffolding, gain of glial glutamate antiporter xCT expression, and reactive astrocytosis, we detected local Iba1+ microglial inflammation that intensified and later extended far beyond tumor boundaries. Hitherto unrecognized episodes of cortical spreading depolarization that arose frequently from the peritumoral region may provide a mechanism for transient neurological deficits. Early blockade of glial xCT activity inhibited later seizures, and genomic reduction of host brain excitability by deleting MapT suppressed molecular markers of epileptogenesis and seizures. Our studies confirmed xenograft tumor–driven pathobiology and revealed early and late components of tumor-related epileptogenesis in a genetically tractable, immunocompetent mouse model of glioma, allowing the complex dissection of tumor versus host pathogenic seizure mechanisms.

The study, originally published in June, contained an error that its authors caught months later.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Source: www.extremetech.com - Friday, May 08, 2020
Public health officials universally agree that the world needs much more coronavirus testing before we can safely ease current lock-down restrictions. Even at the low end, experts say we’ll need to do hundreds of thousands more daily tests, but the equipment and resources to make that happen are in short supply. An MIT spin-off company called Sherlock Biosciences has gotten FDA approval to begin using its CRISPR-based COVID-19 test , which promises to be faster and easy to perform without access to a full lab. Current coronavirus testing is based on PCR (polymerase chain reaction), the same technology used in DNA tests. This involves repeatedly heating the sample to amplify the genetic material so technicians can detect viral RNA. Sequencing those samples to hunt for viral genes requires expensive machines that many facilities don’t have, but the Sherlock method relies on a device similar to a pregnancy test. MIT’s Broad Institute developed Sherlock as a way to identify diseases with the clever addition of a reporter molecule with a DNA segment. Sherlock Biosciences now develops tests with this technology for specific diseases like COVID-19. CRISPR/Cas9 has gained fame as a powerful tool for genetic engineering, but that’s slightly different than the system devised by Sherlock. CRISPR is the sequence that guides Cas9 to the specific genetic code where you want to make a cut (known as cleaving), but scientists can also pair CRISP

Title: CRISPR Used Inside Person's Body For First Time
Category: Health News
Created: 3/4/2020 12:00:00 AM
Last Editorial Review: 3/4/2020 12:00:00 AM

ABSTRACT

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.

IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

ABSTRACT

Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity.

IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long host-virus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPR-Cas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

Alexion Pharmaceuticals CEO Ludwig Hantson has made no secret that he wants to diversify his company’s drug portfolio and he has shown willingness to open the corporate checkbook to accomplish that goal. Last October, Alexion (NASDAQ: ALXN) struck a $930 million deal to acquire Achillion Pharmaceuticals, a biotech whose lead drug complements the Boston company’s […]

An inexpensive assay based on the technique can provide yes or no answers in under an hour—perhaps even in the home soon

-- Read more on ScientificAmerican.com

Using CRISPR/Cas9, a gene editing tool gene, the FREP1 gene can be inactivated to reduce mosquitoes vulnerability to Plasmodium parasite, a genus that causes malaria in humans.

CRISPR-Cas9 protein was found to help increase the targeting accuracy in the genome editing process, revealed a team of researchers from City University of Hong Kong (CityU) and Karolinska Institutet.

New genetic screening platform using CRISPR technology for targeting thousands of genes in a massively-parallel fashion give an accurate and fast method

CRISPR-Cas9, the gene editing technology helps better understand facioscapulohumeral muscular dystrophy (FSHD) and explore potential treatments, found new study.

Modified CRISPR gene editing tool could aid to develop fast-track therapies for HIV, sickle cell disease and, potentially, other immune conditions, according

Her gene-editing tool could cure disease and change the human race. But what happens if it falls into the wrong hands?

One of the teams of scientists that first developed the gene-editing tool has altered it so that it can search out viral RNA instead of human DNA for a test that could even eventually be run at home.