new dna research

Omenn-Darling Bioengineering Institute

Researchers bend DNA strands with light, revealing a new way to study the genome

Researchers have developed a tool that can bend DNA strands using light. The work represents a new way to probe the genome. Shown here, from an unrelated study, are chromosomes (blue) inside a human cell nucleus. Image by Steve Mabon and Tom Misteli, NCI Center for Cancer Research, National Cancer Institute, National Institutes of Health

With the flick of a light, researchers have found a way to rearrange life’s basic tapestry, bending DNA strands back on themselves to reveal the material nature of the genome.

Scientists have long debated about the physics of chromosomes — structures at the deepest interior of a cell that are made of long DNA strands tightly coiled around millions of proteins. Do they behave more like a liquid, a solid, or something in between?

Much progress in understanding and treating disease depends on the answer.

A Princeton team has now developed a way to probe chromosomes and quantify their mechanical properties: how much force is required to move parts of it around and how well it snaps back to its original position. The answer to the material question, according to their findings, was that in some ways the chromosome acts like an elastic material and in other ways it acts like a fluid. By leveraging that insight in exacting detail, the team was able to physically manipulate DNA in new and precisely controlled ways.

They published their findings in the journal Cell on August 20.

“What’s happening here is truly incredible,” said Cliff Brangwynne, the June K. Wu ’92 Professor of Chemical and Biological Engineering, director of Princeton’s Omenn-Darling Bioengineering Institute and principal investigator of the study. “Basically we’ve turned droplets into little fingers that pluck on the genomic strings within living cells.”

The key to the new method lies in the researchers’ ability to generate tiny liquid-like droplets within a cell’s nucleus. The droplets form like oil in water and grow larger when exposed to a specific wavelength of blue light. Because the droplets are initiated at a programmable protein — a modified version of the protein used in the gene editing tool known as CRISPR — they can also attach the droplet to DNA in precise locations, targeting genes of interest.

With their ability to control this process using light, the team found a way to grow two droplets stuck to different sequences, merge the two droplets together, and finally shrink the resulting droplet, pulling the genes together as the droplet recedes. The entire process takes about 10 minutes.

Using condensates (green), the researchers pulled two sections of a DNA strand together, enabling them to touch. Illustration by Wright Seneres

Physically repositioning DNA in this way represents a completely new direction for engineering cells to improve health, and could lead to new treatments for disease, according to the researchers. For example, they showed that they could pull two distant genes toward each other until the genes touch. Established theory predicts this could lead to greater control over gene expression or gene regulation — life’s most fundamental processes.

The material science of our genome

A DNA molecule is structured like a long double strand. In living cells, this long strand is wrapped around specialized proteins to form a material called chromatin, which in turn coils on itself to form the structures we know as chromosomes. If uncoiled and stretched end-to-end, all of a person’s chromosomes would measure about six-and-a-half feet long. Human cells must fit 23 pairs of these chromosomes, collectively called the genome, into each cell’s nucleus. Hence the need for tight coiling.

Since DNA is both a carrier of information and a physical molecule, the cell needs to unfurl the tightly coiled parts of the DNA to copy its information and make proteins. The areas along the genome that are more likely to be expressed are less rigid physically and easier to open up. The areas that are silenced are physically more coiled and compact and therefore harder for the cell to open up and read. Like an instruction manual that opens more easily to some pages than others.

Amy R. Strom, Yoonji Kim and Cliff Brangwynne. Photo of Strom by Monica Khanna, photo of Kim by Wright Seneres, photo of Brangwynne by the Princeton University Office of Communications

The research team, including postdoctoral scholar Amy R. Strom and recently graduated Ph.D. student Yoonji Kim, turned to blobs of liquid known as condensates to do the work of bending the DNA strands and moving them around.

While some cellular components known to science are like soap bubbles, with a distinct membrane keeping the insides separated from the outside, condensates are liquid-like droplets that fuse together more like raindrops, with no membrane holding them together. After forming and carrying out a cellular function, they can break apart and disperse again.

To study chromatin in more detail, Strom and Kim built upon previous research from the Brangwynne lab that engineered condensates from biological molecules in the cell using laser light to create and fuse droplets together. In this new work, they utilized an additional component that attaches the condensate to specific locations on the DNA strands and direct their movement quickly and precisely via surface tension-mediated forces also known as capillary forces, which Princeton researchers had suggested could be ubiquitous in living cells. Previously, moving DNA like this relied on random interactions over a period of hours or even days.

“We haven’t been able to have this precise control over nuclear organization on such quick timescales before,” said Brangwynne.

Like CRISPR but different

Now that they can move the strands around in this controlled way, they can start to look at whether the genes in their new positions are expressed differently. This is potentially important for furthering our understanding of the physical mechanisms and material science of gene expression.

Strom said that scientists have looked at the stiffness of the nucleus by poking at it from the outside, and taking a measurement of the whole nucleus. Scientists can also look at one gene and see if it is turned on or off. But the space in between is not well understood.

“We can use this technology to build a map of what’s going on in there and better understand when things are disorganized like in cancer,” said Strom.

This new tool is poised to help researchers understand gene expression better, but it is not intended to edit the DNA. “Our tool does not actually cleave the DNA sequences like CRISPR does,” said Kim.

“CRISPR is really good for diseases that are related to the need to cut and actually change the DNA sequence,” said Strom. This technology could work for a different class of diseases, especially those related to protein imbalances such as cancer.

“If we can control the amount of expression by repositioning the gene,” said Strom, “there is a potential future for something like our tool.”

The paper “Condensate interfacial forces reposition DNA loci and probe chromatin viscoelasticity” was published with support from the Howard Hughes Medical Institute, the Princeton Biomolecular Condensate Program, the Princeton Center for Complex Materials, a MRSEC (NSF DMR-2011750), the St. Jude Collaborative on Membraneless Organelles and the Air Force Office of Scientific Research Multidisciplinary Research Program of the University Research Initiative (AFOSR MURI) (FA9550-20-1-0241). In addition to Brangwynne, Strom and Kim, contributing authors include Cornelis Storm of Eindhoven University of Technology, and Hongbo Zhao, Yi-Che Chang, Natalia D. Orlovsky and Andrej Košmrlj, all from Princeton University.

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September 10, 2024

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Researchers bend DNA strands with light, revealing a new way to study the genome

by Wright Seneres, Princeton University

With the flick of a light, researchers have found a way to rearrange life's basic tapestry, bending DNA strands back on themselves to reveal the material nature of the genome.

Scientists have long debated about the physics of chromosomes—structures at the deepest interior of a cell that are made of long DNA strands tightly coiled around millions of proteins. Do they behave more like a liquid, a solid, or something in between?

Much progress in understanding and treating disease depends on the answer.

The answer to the material question, according to their findings, is that in some ways the chromosome acts like an elastic material and in other ways it acts like a fluid. By leveraging that insight in exacting detail, the team was able to physically manipulate DNA in new and precisely controlled ways.

They published their findings in the journal Cell on August 20.

"What's happening here is truly incredible," said Cliff Brangwynne, the June K. Wu '92 Professor of Chemical and Biological Engineering, director of Princeton's Omenn-Darling Bioengineering Institute and principal investigator of the study. "Basically we've turned droplets into little fingers that pluck on the genomic strings within living cells."

The key to the new method lies in the researchers' ability to generate tiny liquid-like droplets within a cell's nucleus. The droplets form like oil in water and grow larger when exposed to a specific wavelength of blue light.

Because the droplets are initiated at a programmable protein—a modified version of the protein used in the gene editing tool known as CRISPR—they can also attach the droplet to DNA in precise locations, targeting genes of interest.

Established theory predicts this could lead to greater control over gene expression or gene regulation—life's most fundamental processes.

The material science of our genome

If uncoiled and stretched end-to-end, all of a person's chromosomes would measure about six-and-a-half feet long. Human cells must fit 23 pairs of these chromosomes, collectively called the genome, into each cell's nucleus. Hence the need for tight coiling.

In this new work, they utilized an additional component that attaches the condensate to specific locations on the DNA strands and directs their movement quickly and precisely via surface tension-mediated forces also known as capillary forces, which Princeton researchers had suggested could be ubiquitous in living cells.

Previously, moving DNA like this relied on random interactions over a period of hours or even days.

"We haven't been able to have this precise control over nuclear organization on such quick timescales before," said Brangwynne.

Like CRISPR but different

"We can use this technology to build a map of what's going on in there and better understand when things are disorganized like in cancer," said Strom.

This new tool is poised to help researchers understand gene expression better, but it is not intended to edit the DNA. "Our tool does not actually cleave the DNA sequences like CRISPR does," said Kim.

"CRISPR is really good for diseases that are related to the need to cut and actually change the DNA sequence," said Strom. This technology could work for a different class of diseases, especially those related to protein imbalances such as cancer.

"If we can control the amount of expression by repositioning the gene," said Strom, "there is a potential future for something like our tool."

Journal information: Cell

Provided by Princeton University

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Fastest DNA sequencing technique helps undiagnosed patients find answers in mere hours

A research effort led by Stanford scientists set the first Guinness World Record for the fastest DNA sequencing technique, which was used to sequence a human genome in just 5 hours and 2 minutes.

January 12, 2022 - By Hanae Armitage

Euan Ashley and John Gorzynski were part of a team that devised a method for genome sequencing so speedy it produced results for one study participant in just over five hours. Steve Fisch

A new ultra-rapid genome sequencing approach developed by Stanford Medicine scientists and their collaborators was used to diagnose rare genetic diseases in an average of eight hours — a feat that’s nearly unheard of in standard clinical care.

“A few weeks is what most clinicians call ‘rapid’ when it comes to sequencing a patient’s genome and returning results,” said Euan Ashley , MB ChB, DPhil, professor of medicine, of genetics and of biomedical data science at Stanford.

Genome sequencing allows scientists to see a patient’s complete DNA makeup, which contains information about everything from eye color to inherited diseases. Genome sequencing is vital for diagnosing patients with diseases rooted in their DNA: Once doctors know the specific genetic mutation, they can tailor treatments accordingly.

Now, a mega-sequencing approach devised by Ashley and his colleagues has redefined “rapid” for genetic diagnostics: Their fastest diagnosis was made in just over seven hours. Fast diagnoses mean patients may spend less time in critical care units, require fewer tests, recover more quickly and spend less on care. Notably, the faster sequencing does not sacrifice accuracy.

A paper describing the researchers’ work published Jan. 12 in The New England Journal of Medicine . Ashley, associate dean of the Stanford School of Medicine and the Roger and Joelle Burnell Professor in Genomics and Precision Health, is the senior author of the paper. Postdoctoral scholar John Gorzynski , DVM, PhD, is the lead author.

Setting out to set a record

Over the span of less than six months, the team enrolled and sequenced the genomes of 12 patients, five of whom received a genetic diagnosis from the sequencing information in about the time it takes to round out a day at the office. (Not all ailments are genetically based, which is likely the reason some of the patients did not receive a diagnosis after their sequencing information was returned, Ashley said.) The team’s diagnostic rate, roughly 42%, is about 12% higher than the average rate for diagnosing mystery diseases.

In one of the cases, it took a snappy 5 hours and 2 minutes to sequence a patient’s genome, which set the first Guinness World Records title for fastest DNA sequencing technique. The record was certified by the National Institute of Science and Technology’s Genome in a Bottle group and is documented by Guinness World Records.

Euan Ashley

“It was just one of those amazing moments where the right people suddenly came together to achieve something amazing,” Ashley said. “It really felt like we were approaching a new frontier.”

The time it took to sequence and diagnose that case was 7 hours and 18 minutes, which, to Ashley’s knowledge, is about twice as fast as the previous record for a genome sequencing-based diagnosis (14 hours) held by the Rady Children’s Institute. Fourteen hours is still an impressively quick turnaround, Ashley said. Stanford scientists plan to offer a sub-10-hour turnaround to patients in intensive care units at Stanford Hospital and Lucile Packard Children’s Hospital Stanford — and, eventually, to other hospitals.

Speeding up

To achieve super-fast sequencing speeds, the researchers needed new hardware. So Ashley contacted colleagues at Oxford Nanopore Technologies who had built a machine composed of 48 sequencing units known as flow cells. The idea was to sequence just one person’s genome using all flow cells simultaneously. The mega-machine approach was a success — almost too much. Genomic data overwhelmed the lab’s computational systems.

“We weren’t able to process the data fast enough,” Ashley said. “We had to completely rethink and revamp our data pipelines and storage systems.” Graduate student Sneha Goenka found a way to funnel the data straight to a cloud-based storage system where computational power could be amplified enough to sift through the data in real time. Algorithms then independently scanned the incoming genetic code for errors that might cause disease, and, in the final step, the scientists conducted a comparison of the patient’s gene variants against publicly documented variants known to cause disease.

From start to finish, the team sought to hasten every aspect of sequencing a patient’s genome. Researchers literally ran samples by foot to the lab, new machines were rigged to support simultaneous genome sequencing, and computing power was escalated to efficiently crunch massive datasets. Now, the team is optimizing its system to reduce the time even further. “I think we can halve it again,” Ashley said. “If we’re able to do that, we’re talking about being able to get an answer before the end of a hospital ward round. That’s a dramatic jump.”

Long-read sequencing

Perhaps the most important feature of the diagnostic approach’s ability to quickly spot suspicious fragments of DNA is its use of something called long-read sequencing. Traditional genome-sequencing techniques chop the genome into small bits, spell out the exact order of the DNA base pairs in each chunk, then piece the whole thing back together using a standard human genome as a reference. But that approach doesn’t always capture the entirety of our genome, and the information it provides can sometimes omit variations in genes that point to a diagnosis. Long-read sequencing preserves long stretches of DNA composed of tens of thousands of base pairs, providing similar accuracy and more detail for scientists scouring the sequence for errors.

“Mutations that occur over a large chunk of the genome are easier to detect using long-read sequencing. There are variants that would be almost impossible to detect without some kind of long-read approach,” Ashley said. It’s also much faster: “That was one of the big reasons we went for this approach.”

Only recently have companies and researchers honed the accuracy of the long-read approach enough to rely on it for diagnostics. That and a drop from its once-hefty price tag created an opportunity for Ashley’s team. To his knowledge, this study is the first to demonstrate the feasibility of this type of long-read sequencing as a staple of diagnostic medicine.

During the study, Ashley’s team offered the accelerated genome sequencing technique to undiagnosed patients in Stanford hospitals’ intensive care units. They provided established standard of care testing to the study patients along with the experimental rapid gene sequencing, with which they sought answers to two important questions: Are genetics to blame for the patient’s ailment? If so, what specific DNA errors are stirring up trouble?

Researchers were able to quickly determine that Matthew Kunzman's heart failure was the result of a genetic condition — a finding that cleared the way for him to be placed on a heart transplant list immediately. Courtesy of Jenny Kunzman

Heart mystery

Those were the key questions in the case of Matthew Kunzman of Oregon. About a year ago, when Matthew was 13, an irksome cough and high fever landed him at a local doctor’s office. “We thought it was the flu, or maybe COVID,” said Jenny Kunzman, Matthew’s mother. It turned out the cough was the first sign of a heart condition known as myocarditis — inflammation of the heart — that makes it hard for the organ to pump blood to the rest of the body. Subsequent tests at Matthew’s local hospital revealed a dire situation: His heart was failing. His doctor recommended the family fly immediately to Stanford Hospital for care.

Hours later, Matthew and his father, Matthew Kunzman, Sr., arrived at Stanford Hospital. Jenny Kunzman arrived a day later and found that her son's condition had worsened. Matthew was on life support.

There are two reasons a mostly healthy 13-year-old experiences this kind of heart failure, Ashley said. One is known as myocarditis, and it happens when immune cells swarm the heart, often triggered by a virus or some other bodily stress. The other is a genetic cause, a mutation in a gene involved in heart function.

Knowing the difference, Ashley said, is crucial. “Myocarditis is often reversible,” he said. “With treatment, the heart can go back to normal. But a genetic condition is not. If Matthew’s condition was genetic, likely the only solution would be a heart transplant.”

Gorzynski approached Matthew’s parents, explaining the rapid sequencing research, and asked if they would like to enroll the boy in the study. “They told us there’s this brand-new research that they were working on to try to speed up the process of diagnosis,” Jenny Kunzman said. “They asked if we would be willing to participate, and we said, ‘Absolutely.’ We wanted as much information as possible to try and figure out what the cause was.”

With a few milliliters of Matthew’s blood, the team began the rapid-genetic-sequencing process. “In a matter of hours, sequencing data showed the condition was rooted in genetics,” Ashley said.

Armed with that information, Matthew was immediately put on a heart transplant list. Twenty-one days later, he received a new heart; today, about a year later, his mom says he’s doing “exceptionally well.”

Suspicious seizures

In another case, a 3-month-old patient came to Stanford’s pediatric emergency department with unexplained seizures. It was clear the infant was suffering from a form of epilepsy, but exactly what was causing the symptoms was unknown.

The researchers sequenced the patient’s genome, running the data through mutation-detecting algorithms and cross-referencing public genomic and disease data. They simultaneously requested standard clinical diagnostic testing for blood biomarkers associated with seizures of genetic origin. Just over eight hours later, thanks to the rapid sequencing data, the team had their answer: The young patient’s convulsions were due to a mutation in a gene called CSNK2B.

In a matter of hours, sequencing data showed the condition was rooted in genetics.

If the team had relied only on the standard testing, no diagnosis would have been made at the time, though it’s likely that further tests would have surfaced the correct diagnosis for the patient eventually, Ashley said. “We would have been in the dark for many weeks,” he said.

Standard tests screen a patient’s blood for markers associated with disease, but they scan for only a handful of well-documented genes. Commercial labs, which often run these tests, are slow to update the molecules for which they screen, meaning it can take a long time before newly discovered disease-causing mutations are integrated into the test. And that can lead to missed diagnoses.

That's why rapid genome sequencing could be such a game-changer for patients ailing from rare genetic diseases, Ashley said. Scientists can scan a patient's entire genome for all gene variants suggested by the scientific literature, even if that variant was discovered only the day before. Furthermore, if a patient doesn't initally receive a genetic diagnosis, there's still hope that scientists will find a new gene variant linked to the patient's disease down the line.

Interest from other clinicians is already starting to pour in. “I know people at Stanford have heard we can make a genetic diagnosis in a few hours, and they’re excited about it,” Ashley said. “Genetic tests just aren’t thought of as tests that come back quickly. But we’re changing that perception."

Other Stanford authors of the study are clinical data scientist Dianna Fisk, PhD; graduate student Tanner Jensen; Jonathan Bernstein, MD, PhD, professor of pediatrics; clinical exercise physiologist Jeffrey Christle, PhD; software engineer Karen Dalton; genetic counselor Megan Grove; Maura Ruzhnikov, MD, clinical assistant professor of neurology and neurological sciences; Elizabeth Spiteri, MD, clinical assistant professor of pathology; and pediatric resident Katherine Xiong, MD.

Researchers from Google, UC Santa Cruz, Oxford Nanopore Technology and Baylor College of Medicine also contributed to this study.

This study was supported by Oxford Nanopore Technologies, Google and NVIDIA.

About Stanford Medicine

Stanford Medicine is an integrated academic health system comprising the Stanford School of Medicine and adult and pediatric health care delivery systems. Together, they harness the full potential of biomedicine through collaborative research, education and clinical care for patients. For more information, please visit med.stanford.edu .

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“Using twin prime editing is like using two word processors at the same time to simultaneously write different parts of the same paragraph,” explains Harvard Professor David Liu, the paper’s senior author.

Stephanie Mitchell/Harvard file photo

Twin gene-editing system gives twice the efficiency

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New technique will allow programmable manipulation of large DNA segments

A team of researchers led by Harvard and Broad Institute scientists has developed twin prime editing, a new, CRISPR-based gene-editing strategy that enables manipulation of gene-sized chunks of DNA in human cells without cutting the DNA double helix.

Because it can make larger edits than previously possible, the new technique could make it possible to study and treat genetic diseases arising from loss of gene function or complex structural mutations, such as hemophilia or Hunter syndrome.

In the paper , published in the journal Nature Biotechnology, the researchers detail how they developed twin prime editing (PE), which uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites without requiring double-stranded DNA cuts.

When combined with a site-specific recombinase, twin PE allowed integration of gene-sized DNA of more than 5,000 base pairs into the human genome at sites chosen by the researchers. Because large structural variants are found in many human disease-causing alleles, this technique may help treat genetic diseases. The study used twin PE plus recombinase to edit genes linked to Hunter syndrome, phenylketonuria (PKU), Duchenne muscular dystrophy, and hemophilia.

“A major remaining challenge in mammalian cell gene editing is our inability to make targeted gene-sized insertions at sites of our choosing,” said David Liu, the paper’s senior author, Thomas Dudley Cabot Professor of the Natural Sciences, and a core faculty member of the Broad Institute. “Such a capability could advance gene therapy by enabling genes to be restored in their native sequence locations, without increased risk of cancer from semi-random or uncontrolled integration at other locations in the genome.”

This work was performed by members of Liu’s lab, including former postdoctoral fellow Andrew Anzalone, former graduate student Chris Podracky, postdoctoral fellow Xin (Daniel) Gao, and current graduate student Andrew Nelson.

This new method is designed to overcome some of the limitations of existing gene-editing techniques, and build on the strengths of prime editing.

In 2016 the Liu group developed base editing, an efficient and precise genome-editing method that functions like a genetic pencil, chemically rewriting one DNA base to another without completely breaking the DNA backbone. The Liu group developed two classes of base editors that can correct four of the most common kinds of single-letter mutations, which collectively account for about 30 percent of known disease-associated human genetic errors. Three years later, the group developed prime editing, in which a reverse transcriptase directly copies edited DNA sequences into a specified target site from an extended guide RNA, again without requiring double-stranded DNA breaks. Like a genetic word processor, prime editing lets researchers search for one DNA segment and swap it for another.

Now, twin PE lets researchers search and replace larger DNA segments, again without breaking double-stranded DNA.

“Using twin prime editing is like using two word processors at the same time to simultaneously write different parts of the same paragraph,” said Liu, who is also a Howard Hughes Medical Institute Investigator. “In the process, you can create that paragraph more efficiently.”

The researchers developed twin PE by using simultaneous prime edits to create both strands of the edited DNA. By itself, twin PE enables efficient deletions, insertions, and substitutions of hundreds of base pairs, achieving larger edits than simple prime editing. Seeking the ability to make even larger edits of up to thousands of base pairs — a size large enough to include many whole genes — the team installed Bxb1 recombinase “landing sites” at targets site in human cells. The recombinase then mediated the insertion of large DNA cargo sequences into the landing sites.

The team tested twin PE plus Bxb1 recombinase by inserting large DNA into a variety of target sites in the human genome, including at “safe harbor loci” thought to be ideal for gene therapy because inserting genes there has been found to not induce cancer or other apparent toxicities.

The researchers also applied twin PE-mediated deletions to target DMD. Pathogenic DMD alleles, which cause Duchenne muscular dystrophy, commonly contain large deletions in exonic regions that result in frame-shifted transcripts. These experiments show that twin PE can generate large deletions at therapeutically relevant loci in humans with far fewer potentially harmful byproducts than paired Cas9 nuclease strategies.

The team also used twin PE and a recombinase to install a pathogenic 40,000 bp inversion that causes Hunter syndrome. Their success suggests that the combination might eventually work as a therapeutic strategy for correcting other large or complex pathogenic gene variants.

Going forward, Liu is optimistic that other recombinases can work effectively with twin PE.

“We hope to apply twin PE to some basic science and some therapeutic questions, while continuing to develop this strategy to increase the efficiency of large gene size integrations,” Liu said.

Podracky, who joined the lab in 2015, said he hopes that the twin PE plus recombinase technique will both be studied and be an inspiration for scientists across the country.

“We could see other academic labs take the technology and put it put it to good use,” Podracky said. “If this enables [scientists] to realize their gene-editing ideas more readily, that would be a success.”

This study was supported by the Merkin Institute of Transformative Technologies in Healthcare, the National Institutes of Health, and the Howard Hughes Medical Institute.

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The Complete Human Y Chromosome Marks an Opportunity to Move Away from Stigma

The Y chromosome was once used to label people as criminal. A new complete Y chromosome sequence just might combat this dangerous myth

Christopher R. Donohue, Anna Rogers

New method uses light to bend DNA strands for better disease understanding

Princeton researchers developed a new tool that enables them to physically move dna around to study gene expression to never before seen depths..

Maria Mocerino

New method uses light to bend DNA strands for better disease understanding

Steve Mabon,Tom Misteli, NCI Center for Cancer Research

A Princeton research team has developed a groundbreaking tool to study chromosomes by physically moving DNA strands around.

Having found and turned a key, they can access the deepest mechanisms of gene expression to find new solutions to diseases such as cancer.

According to the study, they first had to solve a longstanding scientific mystery by identifying that chromosomes behave like elastic and liquid. They leveraged this finding to manipulate DNA physically, bending strands back to probe the genome.

Building on previous research with condensates, a class of membrane-less organelles that carry out functions within the cell and then disperse, the team figured out a way to bioengineer condensates that respond to laser light. This enables them to pull back “the curtains,” the strands of DNA, as the study explains.

Now, researchers can use these liquid-like forms of matter to manipulate the structure of the DNA to assess how that might change gene expression.

“What’s happening here is truly incredible,” said Cliff Brangwynne, director of Princeton’s Omenn-Darling Bioengineering Institute and study lead . “Basically, we’ve turned droplets into little fingers that pluck on the genomic strings within living cells.”

Princeton researchers have created a new tool to understand gene expression like never before.

Getting to the deepest level of a cell

Scientists are studying gene expression to increasingly new depths, which holds promise for locating disease before it starts or the precise mechanism that’s causing dysfunction in the first place.

However, Princeton researchers have figured out a way to play with DNA’s very structure. They can even pull a couple of strands together until they touch by directing condensation to specific spots on the DNA strands.

Using laser light, principally, they could “direct their movement quickly and precisely via surface tension-mediated forces also known as capillary forces.”

“We haven’t been able to have this precise control over nuclear organization on such quick timescales before,” Brangwynne said in the Princeton press release . This tool provides a way to investigate gene expression in new, stunning detail and the material science of gene expression.

Could scientists play our DNA like physical symphonies?

Whereas this function may happen randomly, with this tool, they can control the strands and observe how genes react, thereby studying the physical material of chromosomes , a structure of DNA of thread-like strands tightly coiled around millions of proteins in the nucleus of every cell.

They compare the new tool to CRISPR technology, except it doesn’t edit the gene but opens up a new way to understand and possibly treat certain classes of disease, specifically related to protein imbalances, such as cancer.

With this genome -probing technology, they can “build a map of what’s going on…and better understand when things are disorganized, like in cancer,” as per postdoctoral scholar Amy R. Strom .

What they don’t know yet, and what might be the next phase in their research, is whether or not they can “control the amount of expression by repositioning the gene.” In a groundbreaking approach that seems almost sci-fi, researchers may soon be able to manipulate the material of genes to address dysfunctions at their very core.

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ABOUT THE EDITOR

Maria Mocerino Originally from LA, Maria Mocerino has been published in Business Insider, The Irish Examiner, The Rogue Mag, Chacruna Institute for Psychedelic Plant Medicines, and now Interesting Engineering.

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Scientists Have Confirmed a New DNA Structure Inside Human Cells

Earlier this year, scientists identified the existence of a brand new DNA structure never before seen in living cells. That's right, it's not just the double helix.

The discovery of what's described as a 'twisted knot' of DNA in living cells confirms our complex genetic code is crafted with more intricate symmetry than just the double helix structure everybody associates with DNA. Importantly, the forms these molecular variants take affect how our biology functions.

"When most of us think of DNA, we think of the double helix," said antibody therapeutics researcher Daniel Christ from the Garvan Institute of Medical Research in Australia back in April when the discovery was made.

"This new research reminds us that totally different DNA structures exist – and could well be important for our cells."

The DNA component the team identified is called the intercalated motif (i-motif) structure, which was first discovered by researchers in the 1990s , but up until now had only ever been witnessed in vitro , not in living cells.

Thanks to Christ's team, we now know the i-motif occurs naturally in human cells, meaning the structure's significance to cell biology – which has previously been called into question, given it had only been demonstrated in the lab – demands new attention from researchers.

019 dna i motif structure living cells 2

If your only familiarity with DNA shapes is the dual helical spirals made famous by Watson and Crick, the configuration of the intercalated motif could come as a surprise.

"The i-motif is a four-stranded 'knot' of DNA," explained genomicist Marcel Dinger , who co-led the research.

"In the knot structure, C [cytosine] letters on the same strand of DNA bind to each other – so this is very different from a double helix, where 'letters' on opposite strands recognise each other, and where Cs bind to Gs [guanines]."

According to Garvan's Mahdi Zeraati, the first author of the new study, the i-motif is only one of a number of DNA structures that don't take the double helix form – including A-DNA, Z-DNA, triplex DNA and Cruciform DNA – and which could also exist in our cells.

Another kind of DNA structure, called G-quadruplex (G4) DNA, was first visualised by researchers in human cells in 2013 , who made use of an engineered antibody to reveal the G4 within cells.

In the April study, Zeraati and fellow researchers employed the same kind of technique, developing an antibody fragment (called iMab) that could specifically recognise and bind to i-motifs.

In doing so, it highlighted their location in the cell with an immunofluorescent glow.

"What excited us most is that we could see the green spots – the i-motifs – appearing and disappearing over time, so we know that they are forming, dissolving and forming again," said Zeraati .

While there's still a lot to learn about how the i-motif structure functions, the findings indicate that transient i-motifs generally form late in a cell's 'life cycle' – specifically called the late G1 phase , when DNA is being actively 'read'.

The i-motifs also tend to appear in what are known as 'promoter' regions – areas of DNA that control whether genes are switched on or off – and in telomeres , genetic markers associated with ageing.

"We think the coming and going of the i-motifs is a clue to what they do," said Zeraati .

"It seems likely that they are there to help switch genes on or off, and to affect whether a gene is actively read or not."

Now that we definitively know this new form of DNA exists in cells, it'll give researchers a mandate to figure out just what these structures are doing inside our bodies.

As Zeraati explains, the answers could be really important – not just for the i-motif, but for A-DNA, Z-DNA, triplex DNA, and cruciform DNA too.

"These alternative DNA conformations might be important for proteins in the cell to recognise their cognate DNA sequence and exert their regulatory functions," Zeraati explained to ScienceAlert.

"Therefore, the formation of these structures might be of utmost importance for the cell to function normally. And, any aberration in these structures might have pathological consequences."

The findings have been reported in Nature Chemistry .

A version of this story was first published in April 2018.

MIT Technology Review

Newsletters

The first gene-editing treatment: 10 Breakthrough Technologies 2024

Sickle-cell disease is the first illness to be beaten by CRISPR, but the new treatment comes with an expected price tag of $2 to $3 million.

Antonio Regalado archive page

a scientist looks at a tall strand of DNA in a suspension of liquid. A hose sends the liquid back to an IV and into the arm of a patient seated comfortably in a domestic chair with two nice plants and a happy, observant cat.

CRISPR Therapeutics, Editas Medicine, Precision BioSciences, Vertex Pharmaceuticals

The first gene-editing cure has arrived. Grateful patients are calling it “life changing.”

It was only 11 years ago that scientists first developed the potent DNA-snipping technology called CRISPR. Now they’ve brought CRISPR out of the lab and into real medicine with a treatment that cures the symptoms of sickle-cell disease.

Sickle-cell is caused by inheriting two bad copies of one of the genes that make hemoglobin. Symptoms include bouts of intense pain, and life expectancy with the disease is just 53 years. It affects 1 in 4,000 people in the US, nearly all of them African-American.

So how did this disease become CRISPR’s first success ? A fortuitous fact of biology is part of the answer. Our bodies harbor another way to make hemoglobin that turns off when we’re born. Researchers found that a simple DNA edit to cells from the bone marrow could turn it back on.

Many CRISPR treatments are in trials, but in 2022, Vertex Pharmaceuticals, based in Boston, was first to bring one to regulators for approval. That treatment was for sickle-cell. After their bone marrow was edited, nearly all the patients who volunteered in the trial were pain free.

Good news. But the expected price tag of the gene-editing treatment is $2 to $3 million. And Vertex has no immediate plans to offer it in Africa—where sickle-cell disease is most common, and where it still kills children.

The company says this is because the treatment regimen is so complex. It involves a hospital stay; doctors remove the bone marrow, edit the cells, and then transplant them back. In countries that still struggle to cover basic health needs, the procedure remains too demanding. So simpler, cheaper ways to deliver CRISPR could come next.

Biotechnology and health

A controversial Chinese CRISPR scientist is still hopeful about embryo gene editing. Here’s why.

He Jiankui, who went to prison for three years for making the world’s first gene-edited babies, talked to MIT Technology Review about his new research plans.

Zeyi Yang archive page

screenshot from a session of Roundtables with HE Jiankui in the frame

Controversial CRISPR scientist promises “no more gene-edited babies” until society comes around

In a public interview, Chinese biophysicist He Jiankui said he is receiving offers of financial support from figures in the US.

horse running with a snippet of DNA linked to a nexus diagram of lines and circles

How our genome is like a generative AI model

Our genetic code works a bit like DALL-E, apparently

Jessica Hamzelou archive page

A photo illustration showcasing a ball and chain with the chain as a DNA helix,

Why we need safeguards against genetic discrimination

There's a lot of genetic data out there. We're still figuring out how to use and protect it.

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Scientists Finish the Human Genome at Last

The complete genome uncovered more than 100 new genes that are probably functional, and many new variants that may be linked to diseases.

By Carl Zimmer

Two decades after the draft sequence of the human genome was unveiled to great fanfare, a team of 99 scientists has finally deciphered the entire thing. They have filled in vast gaps and corrected a long list of errors in previous versions, giving us a new view of our DNA.

The consortium has posted six papers online in recent weeks in which they describe the full genome. These hard-sought data, now under review by scientific journals, will give scientists a deeper understanding of how DNA influences risks of disease, the scientists say, and how cells keep it in neatly organized chromosomes instead of molecular tangles.

For example, the researchers have uncovered more than 100 new genes that may be functional, and have identified millions of genetic variations between people. Some of those differences probably play a role in diseases.

For Nicolas Altemose, a postdoctoral researcher at the University of California, Berkeley, who worked on the team, the view of the complete human genome feels something like the close-up pictures of Pluto from the New Horizons space probe.

“You could see every crater, you could see every color, from something that we only had the blurriest understanding of before,” he said. “This has just been an absolute dream come true.”

Experts who were not involved in the project said it will enable scientists to explore the human genome in much greater detail. Large chunks of the genome that had been simply blank are now deciphered so clearly that scientists can start studying them in earnest.

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Graduate School

#GradStudentResearch: Researchers bend DNA strands with light, revealing a new way to study the genome

Researchers have developed a tool that can bend DNA strands using light. The work represents a new way to probe the genome. Shown here, from an unrelated study, are chromosomes (blue) inside a human cell nucleus. Steve Mabon, Tom Misteli, NCI Center for Cancer Research, National Cancer Institute, National Institutes of Health

Yoonji Kim *24 was member of research team that developed tool to help researchers better understand gene expression

With the flick of a light, researchers have found a way to rearrange life’s basic tapestry, bending DNA strands back on themselves to reveal the material nature of the genome.

Much progress in understanding and treating disease depends on the answer.

They published their findings in the journal Cell on August 20.

Using condensates (green), the researchers pulled two sections of a DNA strand together, enabling them to touch. Illustration by Wright Seneres

Physically repositioning DNA in this way represents a completely new direction for engineering cells to improve health and could lead to new treatments for disease, according to the researchers. For example, they showed that they could pull two distant genes toward each other until the genes touch. Established theory predicts this could lead to greater control over gene expression or gene regulation — life’s most fundamental processes.

The material science of our genome

Amy R. Strom, Yoonji Kim and Cliff Brangwynne. Photo of Strom by Monica Khanna, photo of Kim by Wright Seneres, photo of Brangwynne by the Princeton University Office of Communications

To study chromatin in more detail, Strom and Kim built upon previous research from the Brangwynne lab that engineered condensates from biological molecules in the cell using laser light to create and fuse droplets together. In this new work, they utilized an additional component that attaches the condensate to specific locations on the DNA strands and directs their movement quickly and precisely via surface tension-mediated forces also known as capillary forces, which Princeton researchers had suggested could be ubiquitous in living cells. Previously, moving DNA like this relied on random interactions over a period of hours or even days.

“We haven’t been able to have this precise control over nuclear organization on such quick timescales before,” said Brangwynne.

Like CRISPR but different

“We can use this technology to build a map of what’s going on in there and better understand when things are disorganized like in cancer,” said Strom.

“If we can control the amount of expression by repositioning the gene,” said Strom, “there is a potential future for something like our tool.”

The paper “ Condensate interfacial forces reposition DNA loci and probe chromatin viscoelasticity ” was published with support from the Howard Hughes Medical Institute, the Princeton Biomolecular Condensate Program, the Princeton Center for Complex Materials, a MRSEC (NSF DMR-2011750), the St. Jude Collaborative on Membraneless Organelles, and the Air Force Office of Scientific Research Multidisciplinary Research Program of the University Research Initiative (AFOSR MURI) (FA9550-20-1-0241). In addition to Brangwynne, Strom and Kim, contributing authors include Cornelis Storm of Eindhoven University of Technology, and Hongbo Zhao, Yi-Che Chang, Natalia D. Orlovsky and Andrej Košmrlj, all from Princeton University.

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PMC10376292

Next-Generation Sequencing Technology: Current Trends and Advancements

Heena satam.

1 miBiome Therapeutics, Mumbai 400102, India; moc.emoibim@aneeh (H.S.); moc.liamg@ofnioibpradnak (K.J.); ni.emoiborcim@anasapu (U.M.); ni.emoiborcim@rebonas (S.W.); ni.emoiborcim@zanlug (G.Z.); moc.emoibim@inavarhs (S.R.)

Kandarp Joshi

Upasana mangrolia, sanober waghoo, gulnaz zaidi, shravani rawool, ritesh p. thakare.

2 Department of Molecular Cell and Cancer Biology, UMass Chan Medical School, Worcester, MA 01605, USA; [email protected] (R.P.T.); [email protected] (S.B.); [email protected] (A.K.M.)

Shahid Banday

Alok k. mishra, sunil k. malonia, associated data.

Not applicable.

Simple Summary

Next-generation sequencing (NGS) is a powerful tool used in genomics research. NGS can sequence millions of DNA fragments at once, providing detailed information about the structure of genomes, genetic variations, gene activity, and changes in gene behavior. Recent advancements have focused on faster and more accurate sequencing, reduced costs, and improved data analysis. These advancements hold great promise for unlocking new insights into genomics and improving our understanding of diseases and personalized healthcare. This review article provides an overview of NGS technology and its impact on various areas of research, such as clinical genomics, cancer, infectious diseases, and the study of the microbiome.

The advent of next-generation sequencing (NGS) has brought about a paradigm shift in genomics research, offering unparalleled capabilities for analyzing DNA and RNA molecules in a high-throughput and cost-effective manner. This transformative technology has swiftly propelled genomics advancements across diverse domains. NGS allows for the rapid sequencing of millions of DNA fragments simultaneously, providing comprehensive insights into genome structure, genetic variations, gene expression profiles, and epigenetic modifications. The versatility of NGS platforms has expanded the scope of genomics research, facilitating studies on rare genetic diseases, cancer genomics, microbiome analysis, infectious diseases, and population genetics. Moreover, NGS has enabled the development of targeted therapies, precision medicine approaches, and improved diagnostic methods. This review provides an insightful overview of the current trends and recent advancements in NGS technology, highlighting its potential impact on diverse areas of genomic research. Moreover, the review delves into the challenges encountered and future directions of NGS technology, including endeavors to enhance the accuracy and sensitivity of sequencing data, the development of novel algorithms for data analysis, and the pursuit of more efficient, scalable, and cost-effective solutions that lie ahead.

1. Introduction

Next-generation sequencing (NGS) has revolutionized genomics, expanding our knowledge of genome structure, function, and dynamics. This groundbreaking technology has enabled extensive research and allowed scientists to explore the complexities of genetic information in unprecedented ways. With its high-throughput capacity and cost-effectiveness, NGS has become a fundamental tool for researchers across diverse disciplines, from basic biology to clinical diagnostics [ 1 ]. NGS has not only enabled comprehensive genome sequencing but also facilitated transcriptomics, epigenomics, metagenomics, and other omics studies [ 2 ]. The advent of advanced NGS platforms, such as Illumina, Pacific Biosciences, and Oxford Nanopore, has transformed the field of genomics by allowing for the parallel sequencing of millions to billions of DNA fragments [ 3 , 4 ]. This capability has unlocked new opportunities for understanding genetic variation, gene expression, epigenetic modifications, and microbial diversity. NGS has been instrumental in identifying disease-causing variants, uncovering novel drug targets, and shedding light on complex biological phenomena, including the heterogeneity of tumors and developmental processes [ 3 , 4 , 5 ]. This review provides a comprehensive overview of NGS technology, highlighting its transformative impact in various fields, including clinical genomics, cancer research, infectious disease, surveillance, and microbiome analysis. We also discuss the future prospects of NGS, including emerging technologies, its potential for advancing genomics research, and its applications in the biomedical sciences.

2. Generations of Sequencing Technologies

Technologies for “reading” DNA sequences have evolved rapidly over the past two decades [ 6 , 7 , 8 , 9 , 10 ]. This rapid progress has paved the way for significant breakthroughs in the field of DNA sequencing, leading to the emergence of three generations of sequencing technologies ( Figure 1 ).

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Object name is biology-12-00997-g001.jpg

Evolution of sequencing technologies. The development of sequencing technologies over the past four decades can be categorized into three generations. The first generation was represented by Sanger sequencing, providing the foundation for DNA sequencing. The second generation introduced massively parallel sequencing with platforms such as Illumina and Ion Torrent, enabling high-throughput sequencing. The current third generation includes PacBio and Nanopore, offering long-read and single-molecule sequencing capabilities.

2.1. First-Generation Sequencing Technology

The first attempts at sequencing DNA and RNA involved chemical degradation or enzymatic cleavage of the molecules to generate fragments that could be analyzed individually. Robert Holley was the first to sequence a nucleic acid molecule, Alanine tRNA, in 1964 using ribonuclease from S. cerevisiae [ 11 ]. Similarly, Walter Gilbert and Allan Maxam developed a chemical degradation technique that allowed the sequencing of complete bacteriophage PhiX174 [ 12 ]. However, the real breakthrough came with the introduction of the chain termination-based sequencing method by Fredrick Sanger [ 13 ]. This technique used dideoxynucleotides, which terminate the chain elongation of DNA strands during replication, and allowed for the production of sequence reads of up to a few hundred nucleotides in length. Sanger’s method was widely adopted and revolutionized the field of molecular biology by allowing for the rapid sequencing of DNA and RNA [ 12 ]. In 1987, the first commercial automated sequencing machine, the Applied Biosystems ABI 370, was launched in the United States. This machine used fluorescently labeled dideoxynucleotides and capillary electrophoresis to automate the Sanger sequencing method, significantly increasing the speed and accuracy of DNA sequencing [ 14 , 15 ]. The ABI 370 quickly became the industry standard, and subsequent improvements in the technology led to the development of higher-throughput sequencers capable of producing longer reads [ 15 , 16 ]. While the first-generation technology has been largely superseded by newer, higher-throughput sequencing technologies, it remains an important historical milestone in the development of sequencing techniques. The ability to sequence DNA and RNA has revolutionized many areas of biology and medicine and has led to numerous discoveries and advancements in the understanding of genetics and molecular biology.

2.2. Second-Generation Sequencing Technologies

Second-generation sequencing methods have revolutionized DNA sequencing by enabling the simultaneous sequencing of thousands to millions of DNA fragments. These methods differ from traditional Sanger sequencing in their ability to perform parallel sequencing. Several widely used second-generation sequencing platforms have emerged, one of which is Roche’s 454 sequencing method, which relies on pyrosequencing, where the sequence is determined by detecting the release of pyrophosphate when nucleotides are added to the DNA template. Another platform is Ion Torrent sequencing, which detects the release of hydrogen ions during DNA synthesis to determine the sequence. The widely used Illumina sequencing platform utilizes a sequencing-by-synthesis method based on reversible dye terminators. Another upcoming technology, SOLiD sequencing (Sequencing by Oligonucleotide Ligation and Detection), employs a ligation-based approach using reversible terminators to determine the DNA sequence. These second-generation sequencing technologies have significantly increased the throughput and speed of DNA sequencing, enabling a wide range of applications in genomics research and clinical diagnostics [ 17 ]. These platforms have enabled whole-genome sequencing, transcriptome analysis, and targeted sequencing, leading to breakthroughs in genetic variation, disease research, and personalized medicine. Many developments in the second generation of sequencing methods have been achieved over the years and are represented in Figure 2 and briefly described in Table 1 .

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Object name is biology-12-00997-g002.jpg

Overview of various NGS technologies with different platforms and principles.

Different generations of NGS platforms.

Sr No.	Platform	Use	Sequencing Technology	Amplification Type	Principle	Read Length (bp)	Limitations	Ref.
1	454 pyrosequencing	Short read sequencing	Seq by synthesis	Emulsion PCR	Detection of pyrophosphate released during nucleotide incorporation.	400–1000	May contain deletion and insertion sequencing errors due to inefficient determination of homopolymer length.	[ , , ]
2	Ion Torrent	Short read sequencing	Seq by synthesis	Emulsion PCR	Ion semiconductor sequencing principle detecting H ion generated during nucleotide incorporation.	200–400	When homopolymer sequences are sequenced, it may lead to loss in signal strength.	[ , , ]
3	Illumina	Short read sequencing	Seq by synthesis	Bridge PCR	Solid-phase sequencing on immobilized surface leveraging clonal array formation using proprietary reversible terminator technology for rapid and accurate large-scale sequencing using single labeled dNTPs, which is added to the nucleic acid chain.	36–300	In case of sample overloading, the sequencing may result in overcrowding or overlapping signals, thus spiking the error rate up to 1%.	[ , , ]
4	SOLiD	Short read sequencing	Seq by ligation	Emulsion PCR	An enzymatic method of sequencing using DNA ligase. 8-Mer probes with a hydroxyl group at 3′ end and a fluorescent tag (unique to each base A, T, G, C) at 5′ end are used in ligation reaction.	75	This platform displays substitution errors and may also under-represent GC-rich regions. Their short reads also limit their wider applications.	[ , ]
5	DNA nanoball sequencing	Short read sequencing	Seq by ligation	Amplification by Nanoball PCR	Splint oligo hybridization with post-PCR amplicon from libraries helps in the formation of circles. This circular ssDNA acts as the DNA template to generate a long string of DNA that self-assembles into a tight DNA nanoball. These are added to the aminosilane (positively charged)-coated flow cell to allow patterned binding of the DNA nanoballs. The fluorescently tagged bases are incorporated into the DNA strand, and the release of the fluorescent tag is captured using imaging techniques.	50–150	Multiple PCR cycles are needed with a more exhaustive workflow. This, combined with the output of short-read sequencing, can be a possible limitation.	[ , ]
6	Helicos single-molecule sequencing	Short-read sequencing	Seq by synthesis	Without Amplification	Poly-A-tailed short 100–200 bp fragmented genomic DNA is sequenced on poly-T oligo-coated flow cells using fluorescently labeled 4 dNTPS. The signal released upon adding each nucleotide is captured.	35	Highly sensitive instrumentation required. As the sequence length increases, the percentage of strands that can be utilized decreases.	[ , ]
7	PacBio Onso system	Short-read sequencing	Seq by binding	Optional PCR	Sequencing by binding (SBB) chemistry uses native nucleotides and scarless incorporation under optimized conditions for binding and extension ( , accessed on 1 July 2023).	100–200	The higher cost compared to other sequencing platforms.
8	PacBio Single-molecule real-time sequencing (SMRT) technology	Long-read sequencing	Seq by synthesis	Without PCR	The SMRT sequencing employs SMRT Cell, housing numerous small wells known as zero-mode waveguides (ZMWs). Individual DNA molecules are immobilized within these wells, emitting light as the polymerase incorporates each nucleotide, allowing real-time measurement of nucleotide incorporation	average 10,000–25,000	The higher cost compared to other sequencing platforms.	[ , ]
9	Nanopore DNA sequencing	Long-read sequencing	Sequence detection through electrical impedance	Without PCR	The method relies on the linearization of DNA or RNA molecules and their capability to move through a biological pore called “nanopores”, which are eight nanometers wide. Electrophoretic mobility allows the passage of linear nucleic acid strand, which in turn is capable of generating a current signal.	average 10,000–30,000	The error rate can spike up to 15%, especially with low-complexity sequences. Compared to short-read sequencers, it has a lower read accuracy.	[ , , ]

2.3. Third-Generation Sequencing

Third-generation sequencing technologies represent the latest advancements in DNA sequencing, offering new approaches that overcome the limitations of previous generations. These technologies provide long-read sequencing capabilities, enabling the sequencing of much larger DNA fragments compared to earlier methods. Examples include PacBio Sequencing, which uses a single-molecule, real-time (SMRT) approach with fluorescently labeled nucleotides, enabling long-read sequencing of DNA fragments up to tens of kilobases in length. Another technology is Oxford Nanopore sequencing, based on nanopore technology, where a single-stranded DNA molecule passes through a nanopore, and changes in electrical current are measured to determine the DNA sequence. Oxford Nanopore sequencing provides long-read lengths, portability, and real-time analysis. Third-generation sequencing methods have been summarized in Table 1 . Figure 3 describes technologies available on NGS and the type of data generated in each type of NGS assay and their brief application.

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Object name is biology-12-00997-g003.jpg

Various approaches used for genome analysis and applications of NGS, including technological platforms, data analysis, and applications. WGS, whole-genome sequencing; WES, whole-exome sequencing; Seq, sequencing; ITS, internal transcribed spacer; ChIP, chromatin immunoprecipitation; ATAC, assay for transposase-accessible chromatin; AMR, anti-microbial resistance.

Long-Read and Short-Read Sequencing

The basic principle for short-read sequencing involves sequencing by synthesis based on enrichment through hybridization, amplification, or fragmentation. Whereas long-read sequencing works on sequence detection either by synthesis or by electrical voltage change/impedance, generating the current as a single base is passed through the biological membrane pore. Long-read sequencing can generate reads up to 25–30 kb, whereas short-read sequencing can generate reads around 600–700 bp. Furthermore, the amplification bias is eliminated in long-read sequencing as opposed to short-read sequencing. As the library preparation is PCR-free, the base modification such as DNA methylation can be easily detected by long-read sequencing. The introduction of high-throughput sequencing platforms has significantly reduced error rates and notably improved the accuracy of long-read sequencing technologies [ 29 , 31 ]. Short-read sequencing is useful for determining the abundance of specific sequences, profiling transcript expression, and identifying variants. However, long-read sequencing technologies excel in providing comprehensive genome coverage, enabling researchers to identify complex structural variants such as large insertions, deletions, inversions, duplications, and more [ 8 , 29 , 31 ].

3. Next-Generation Sequencing-Based Omics

Understanding complex human diseases requires data integration from multiple omics techniques such as genomics, transcriptomics, epigenomics, and proteomics. Here, we briefly describe various omics technologies that are implemented on the NGS platform:

3.1. Genomics

Genomics studies using NGS profoundly analyze DNA using various approaches such as whole-genome sequencing, whole-exome sequencing, and targeted sequencing.

3.1.1. Whole-Genome Sequencing

Whole-genome sequencing (WGS) is a powerful and comprehensive genomic analysis technique that involves determining the complete DNA sequence of an individual’s genome. It provides a detailed blueprint of an individual’s genetic makeup, encompassing all the genes, regulatory regions, and non-coding elements present in their genome. It finds its application mainly in discovery science, such as plant and animal research, cancer research, rare genetic diseases, patients with complex disease symptoms, population genetics, and novel genome assembly of eukaryotes and prokaryotes [ 32 ]. By sequencing all the DNA in an organism’s genome, WGS enables the identification of genetic variations, ranging from single-nucleotide polymorphisms (SNPs) to larger structural changes such as insertions, deletions, and rearrangements. This wealth of information obtained through WGS offers a multitude of applications in various fields [ 33 ]. WGS has two types of sequencing approaches on the basis of genome size viz. (1) large whole-genome sequencing deciphering larger genomes of >5 Mb such as eukaryotes, and (2) small whole-genome sequencing deciphering smaller genomes of <5 Mb mainly of prokaryotes. Short-read sequencing is preferred for mutation calling, while long-read sequencing is preferred for genome assemblies. Combining short and long-read sequencing for sequencing novel genomes has been successfully applied for accurate genome assembly without a reference sequence.

3.1.2. Whole-Exome Sequencing

Whole-exome sequencing (WES) is a sequencing approach that focuses on capturing and sequencing the protein-coding regions of the genome, known as the exome. The exome represents approximately 1–2% of the entire genome but contains the majority of known disease-causing variants. By sequencing the exome, WES enables the identification of genetic variations, including single-nucleotide variants (SNVs), insertions, deletions, and copy number variations (CNVs), within protein-coding genes [ 34 , 35 ]. WES is a cost-effective alternative to WGS for rare clinical diseases with clusters of symptoms, as well as in identifying variants for population and cancer genetics [ 36 ]. WES involves the enrichment of exonic regions using hybrid capture or target-specific amplification techniques, followed by high-throughput sequencing. Various exome capture assays from NimbleGen, Agilent, Illumina, Twist, and IDT are available that are compatible with the Illumina NGS platform [ 37 ]. The bioinformatic approach used for WES data analysis is the same as that of WGS since WES is a part of WGS.

3.1.3. Targeted Sequencing

Targeted sequencing, as the name suggests, has less exploratory power than WGS or WES as it targets specific regions of the gene and is able to pick up various types of genetic variations from SNVs to small gene deletions, duplications, insertions, or gene rearrangements associated with disease phenotypes. However, advantages include cost-effectiveness and manageable data for clinicians, making clinical decisions easier with more specific disease-relevant information. It can give much deeper coverage up to 5000× for rare alleles in genetic diseases, as well as for low-abundant evolving mutant clones arising as a result of tumor heterogeneity or disease evolution in cancer [ 38 ]. The candidate gene approach or commercially available targeted panels is the result of WGS/WES projects carried out at the population scale. The germline, as well as somatic variants, can be tested using targeted NGS panels, few examples of which are listed in Table 2 . Targeted panels work on a simple approach of enrichment by amplification using pools of region-specific oligonucleotide primers. Specific size libraries that are produced are then sequenced and analyzed bioinformatically [ 39 ].

Examples of targeted panels available in research and diagnostic settings.

Disease Condition	Available Panel	Type of Inheritance	Specimen Type
Inherited cardiovascular defects	Cardiovascular research panel	Germline	Blood
Arrhythmias and cardiomyopathies	Arrhythmias and cardiomyopathy research panel	Germline	Blood
Sensitivity to pharmacological drugs	Pharmacogenomics research panel (PGex Seq panel)	Germline	Blood
Antimicrobial treatment efficacy testing	Antimicrobial resistance research panel	Microbial gene testing	Bacterial culture
Infertility conditions	Infertility research panel	Germline	Blood
Homologous recombination defect analysis	HRR gene panel	Somatic	Tumor tissue
myeloid cancers	Myeloid cancer panel	Somatic	Blood
HIV speciation and drug resistance	HIV-Xgene panel	Pathogen detection	HIV-positive plasma
Antimicrobial resistance in MTB	TB research panel	Pathogen detection	MTB-positive specimen
Inborn errors of metabolism	Error of metabolism research panel	Germline	DBS/blood
Hereditary cancers	BRACA and extended breast and ovarian cancer research panel, inherited cancer research panel	Germline	Blood

3.2. Transcriptomics

Next-generation sequencing (NGS) has had a transformative impact on transcriptomics, revolutionizing our ability to study the transcriptome—the complete set of RNA molecules in an organism or specific cell population. NGS technologies offer high-throughput and cost-effective methods for profiling and analyzing RNA molecules, allowing researchers to gain deep insights into gene expression, alternative splicing, non-coding RNA regulation, and various biological processes and diseases [ 40 , 41 , 42 , 43 ]. Here are some key roles of NGS in transcriptomics:

(a) mRNA Sequencing (RNA-Seq): RNA-seq is a widely used NGS application in transcriptomics. It involves the sequencing and quantification of mRNA molecules, providing a comprehensive snapshot of the expressed genes in a biological sample. By generating millions of short sequencing reads, NGS allows researchers to identify and quantify gene expression levels accurately. RNA-seq data can be analyzed to detect differential gene expression between different conditions, discover novel transcripts, assess alternative splicing events, and study gene expression dynamics over time or across different tissues or cell types [ 44 , 45 ].
(b) Alternative Splicing Analysis: Alternative splicing, a process in which a single gene can generate multiple mRNA isoforms, significantly contributes to transcriptome complexity. NGS provides the ability to study alternative splicing patterns comprehensively. By aligning RNA-seq reads to the reference genome, researchers can identify splice junctions and detect alternative splicing events. This information allows for the quantification and characterization of transcript isoforms, providing insights into isoform diversity, tissue-specific expression, and the functional implications of alternative splicing [ 46 ].
(c) Long Non-Coding RNA (lncRNA) and Small-RNA Analysis: NGS facilitates the study of non-coding RNAs, which play critical roles in gene regulation. Techniques such as small-RNA sequencing and long non-coding RNA sequencing enable the identification and characterization of various classes of non-coding RNAs. Small-RNA sequencing allows the profiling of small regulatory RNAs, including microRNAs, piRNAs, and snoRNAs, providing insights into their roles in post-transcriptional gene regulation. Long non-coding RNA sequencing enables the identification and analysis of long non-coding RNA transcripts, which have been implicated in diverse biological processes and diseases [ 47 , 48 , 49 ]. Long RNA-seq reads can inform about the connectivity between multiple exons and reveal sequence variations (SNPs) in the transcribed region [ 50 ]. Small-RNA sequencing is a non-targeted approach that allows the detection of novel miRNA and other small RNAs [ 51 ]. The transcriptome with ChIP-seq studies in cancer biology has helped to understand the emerging role of ncRNAs such as sncRNAs and lncRNA in gene regulation mechanisms during carcinogenesis/cancer progression [ 52 , 53 , 54 ].
(d) Transcriptome Assembly and Annotation: NGS data can be utilized to reconstruct and annotate the transcriptome of an organism. By aligning RNA-seq reads to a reference genome or using de novo assembly approaches, researchers can identify novel transcripts, splice variants, untranslated regions, and other transcript features. This information enhances our understanding of the transcriptome’s complexity and improves the annotation of reference genomes, enabling the discovery of previously unknown genes and regulatory elements [ 55 ].
(e) Single-Cell Transcriptomics: NGS has facilitated the emergence of single-cell transcriptomics, enabling the study of gene expression profiles at the individual cell level. Single-cell RNA-seq (scRNA-Seq) technologies allow the profiling of transcriptomes from individual cells, providing insights into cellular heterogeneity, cell type identification, cell lineage analysis, and gene expression dynamics in complex tissues or developmental processes [ 56 , 57 ].
(f) Integrative Transcriptomics: NGS data from transcriptomics can be integrated with other omics data, such as genomics, epigenomics, and proteomics, to gain a comprehensive understanding of gene regulation and biological processes. Integrative approaches provide a system-level view of molecular interactions and enable the identification of key regulatory mechanisms underlying cellular processes and diseases [ 56 ].

3.3. Epigenomics

Epigenomics refers to the study of epigenetic modifications, which are heritable changes in gene expression patterns that do not involve alterations in the DNA sequence [ 58 , 59 ]. The most common types of epigenetic modifications studied are DNA methylation [ 60 ], histone modification, and RNA methylation (epi-transcriptome). These chemical tags in turn alter DNA accessibility, chromatin remodeling, and nucleosome positioning [ 61 ]. These modifications are influenced by environmental factors such as nutrients, pollutants, toxicants, and inflammation [ 62 , 63 ]. The knowledge and data generated through whole-genome-wide sequencing in humans, plants, and animals [ 64 ] have helped scientists to gain better insights into these epigenetic alterations, especially DNA methylation and hydroxymethylation. Epigenetic alterations have attracted researchers’ and clinicians’ interest in complex disorders such as behavioral disorders, memory, cancer, autoimmune disease, addiction, neurodegenerative, and psychological disorders [ 65 ]. There are various platforms and assays developed to study epigenetic modifications, which have been very well described elsewhere [ 66 ]. NGS has been utilized for investigating epigenomics, as discussed below:

(a) DNA Methylation Profiling: DNA methylation is a crucial epigenetic modification that plays a critical role in gene regulation and cellular processes. NGS enables genome-wide profiling of DNA methylation patterns at single-nucleotide resolution [ 67 ]. Several strategies, such as whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS), leverage NGS to identify methylated cytosines [ 68 ]. However, RRBS is based on enriching methylated genomic regions using restriction enzymatic digestion [ 66 , 69 ]. These methods allow researchers to study DNA methylation dynamics, uncover differentially methylated regions (DMRs) associated with diseases, and understand the impact of methylation on gene expression.
(b) Chromatin Accessibility Mapping: NGS-based techniques, such as assay for transposase-accessible chromatin using sequencing (ATAC-seq) and DNase-seq, enable the genome-wide profiling of chromatin accessibility. These methods identify regions of the genome that are accessible to DNA-binding proteins and transcription factors, providing insights into gene regulatory elements, enhancers, and promoters. By combining chromatin accessibility data with other epigenetic modifications, gene expression data, and transcription factor binding data, researchers can unravel the functional elements within the genome [ 70 , 71 ].
(c) Histone Modification Analysis: Histone modifications, including acetylation, methylation, phosphorylation, and more, are critical epigenetic marks that regulate chromatin structure and gene expression. Chromatin immunoprecipitation sequencing (ChIP-seq) enables genome-wide profiling of histone modifications by antibody-based pull down of the protein followed by enrichment of DNA bound to the protein and sequencing. This technique finds application in many different areas of research, such as transcription factor (TF) binding site identification, histone modification analysis of the DNA, and DNA methylation. For studying histone modifications, antibodies targeted to histone modifications are used to pull down the DNA and sequenced using the NGS technique. The resulting reads are aligned to the reference genome, enabling the identification of histone modification patterns at specific genomic regions. ChIP-Seq can provide insights into the epigenetic regulation of gene expression, chromatin states, and the identification of enhancers and other regulatory elements [ 72 , 73 , 74 , 75 ].
(d) Chromatin Conformation Analysis: NGS-based techniques, such as Hi-C and 4C-seq, allow the investigation of 3D chromatin organization and interactions. These methods capture long-range chromatin interactions and enable the construction of chromatin interaction maps [ 76 , 77 ]. By integrating 3D chromatin conformation data with epigenetic modifications, gene expression data, and functional annotations, researchers can gain insights into the spatial organization of the genome and understand how it influences gene regulation.
(e) In addition to these standalone approaches, NGS data from epigenomics can be integrated with transcriptomics data to unravel the relationship between epigenetic modifications and gene expression. Integration of DNA methylation profiles with RNA-seq data can identify differentially methylated regions (DMRs) associated with gene expression changes. Integration of histone modification and chromatin accessibility data with RNA-seq allows the identification of regulatory elements associated with specific gene expression patterns and the exploration of epigenetic regulatory mechanisms.

3.4. Metagenomics

Metagenomics deals with direct genetic analysis of the prokaryotic genome including bacteria, fungi, and viruses contained in a sample [ 78 ] either by targeted approach or adaptor ligation PCR approach for shotgun sequencing in a culture-independent manner. The hypervariable region in 16S or 18S ribosomal RNA genes of bacteria and fungi is used in the targeted approach. A blend of conserved and hypervariable regions helps in the identification of each bacterial species from the sample. Similarly, for fungal species identification, ITS1 and ITS2 regions spanning the 5.8S rRNA gene of the fungal genome are selected for amplification [ 79 ]. For viral genome sequencing, reads generated from NGS (shotgun) are again the culture-independent method for studying viral diversity, abundance, and functional potential of viruses in the environment. All filtered reads are mapped with the human reference sequence, and remaining, unmapped reads are mapped against the NCBI RefSeq viral genomic database ( Table 3 ) [ 80 ]. The targeted viral and bacterial genome panels are also available, e.g., ChapterDx for HR HPV and microbial infection detection, the HIV drug resistance panel, the AMR panel, the gastrointestinal disorder panel, etc.

Based on the nucleotide sequence similarities, pre-processed sequences are clustered at 97% similarity into operational taxonomic units (OTUs). OTUs are compared with the database to identify the microorganisms [ 81 ]. Several analysis pipelines are used for the analysis of 16S amplicon reads ( Table 3 ) [ 82 ]. For shotgun metagenomics samples, taxonomic and functional profiles can be obtained by different approaches, as elaborated in Table 3 [ 83 , 84 , 85 , 86 , 87 , 88 , 89 ]. Microbiome sequencing can identify the full spectrum of microbial species present in the sample. The results are highly quantitative, and one can study the bacterial communities over a specific interval of conditions. The NGS platform can also generate reads for low-abundance species in a sample.

4. Bioinformatic Approaches for NGS Data Analysis

NGS generates vast amounts of DNA or RNA sequences, necessitating computational methods to handle, analyze, and interpret these data. Raw sequencing data produced by NGS instruments need to be processed, analyzed, and interpreted to derive biological insights. This is where bioinformatic approaches come into play. These approaches encompass a wide range of computational methods, algorithms, and tools that handle preprocessing, alignment, variant calling, gene expression quantification, differential expression analysis, and other specialized analyses. Once processed, various computational techniques, such as de novo assembly, reference-based mapping, and transcriptome analysis, are employed to extract meaningful biological information. Furthermore, advanced bioinformatic tools facilitate the identification of genetic variations, including single-nucleotide polymorphisms (SNPs), copy number variations (CNVs), and structural variants. Integrative analyses, combining NGS data with other genomic and functional data sources, enable the exploration of gene expression and regulatory networks. The various bioinformatics tools used in NGS analysis are listed in Table 3 .

Bioinformatic steps and tools used for NGS data analysis.

Analysis	Commonly Used Tools

Quality check of sequences	FastQC [ ], FASTX-toolkit [ ], MultiQC [ ]
Trimming of adaptors and low-quality bases	Trimmomatic [ ], Cutadapt [ ], fastp [ ]
Alignment of sequence reads to reference genome	BWA [ ], Bowtie [ ], dragMAP [ ]
Reports visualization	MultiQC [ ]

Removal of duplicate reads	Picard [ ], Sambamba [ ]
Variant calling (single-nucleotide polymorphisms and indels)	GATK [ ], freeBayes [ ], Platypus [ ], VarScan [ ], DeepVariant [ ], Illumina Dragen [ ]
Filter and merge variants	bcftools [ ]
Variant annotation	ANNOVAR [ ], ensemblVEP [ ], snpEff [ ], NIRVANA [ ]
Structural variant calling	DELLY [ ], Lumpy [ ], Manta [ ], GRIDDS [ ], Wham [ ], Pindel [ ]
Copy number variation (CNV) calling	CNVnator [ ], GATK gCNV [ ], cn.MOPS [ ], cnvCapSeq(targeted sequencing) [ ], ExomeDepth (CNVs from Exome) [ ]

Alignment of reads to reference	Splice-aware aligner such as TopHat2 [ ], HISAT2 [ ], and STAR [ ]
Transcript quantification	featureCounts [ ], HTSeq-count [ ], Salmon [ ], Kallisto [ ]
Differential gene expression analysis enrichment of gene categories	DESeq2 [ ], EdgeR [ ], DAVID [ ], clusterProfiler [ ], Enrichr [ ]

Sequence aligners	Bwameth [ ], BS-Seeker2 [ ], Bismark [ ]
Methylation level quantification	MethylDackel *
Differential methylation	Metilene [ ], BSsmooth [ ], methylKit [ ]

Removal of PCR duplicates	Samtools [ ]
Peak calling	MACS2 [ ], SICER2 [ ], SPP [ ]
Peak filtering	Bedtools [ ]
Enrichment quality control	ChipQC [ ], Phantompeakqualtools [ ]
Enrichment comparison	diffBind [ ], MAnorm [ ], MMDiff [ ]
Motif analysis	MemeCHiP [ ], Homer [ ], RSAT [ ]

16S rRNAseq analysis pipelines	QIIME2 [ ], mothur [ ], USEARCH [ ]
Ribosomal RNA databases	Greengenes [ ], Silva [ ], RDP [ ]

Taxonomic classification	MetaPhlAn4 [ ], Kaiju [ ], Kraken [ ]
Assembly of metagenomic reads	metaSPAdes [ ], metaIDBA [ ]
Protein databases for taxonomic classification	NCBI non-redundant protein database [ ]
Gene annotation	Prokka [ ], MetaGeneMark [ ]
Databases for functional annotation of genes	COG [ ], KEGG [ ], GO [ ]

Footnote: ANNOVAR—ANNOtate VARiation; BWA—Burrows Wheeler Aligner; cn.mops Copy Number Estimation by a Mixture Of PoissonS; COG—Clusters of Orthologous Groups of Proteins; DAVID—A Database for Annotation, Visualization and Integrated Discovery; Ensembl VEP—Ensembl Variant Effect Predictor; Fastp—Fsatq Preprocessor; GATK—Genome Analysis Tool Kit; GO—Gene Ontology; HISAT2—Hierarchical Indexing for Spliced Alignment of Transcripts; HOMER—Hypergeometric Optimization of Motif EnRichment; Htseq-count—High-Throughput Sequence Analysis in Python; KEGG: Kyoto Encyclopedia of Genes and Genomes; NCBI—National Center for Biotechnology Information; MACS: Model-Based Analysis for ChIP-Seq; MEME—Multiple EM for Motif Elicitation; Meta-IDBA—Meta-Iterative De Bruijn Graph De Novo Short-Read Assembler; MetaPhlAn—Metagenomic Phylogenetic Analysis; metaSPAdes—meta St Petersburg Genome Assembler; QIIME—Quantitative Insights Into Microbial Ecology; RDP—Ribosomal Database Project; RSAT—Regulatory Sequence Analysis tools; SICER—Spatial Clustering Approach for the Identification of ChIP-Enriched regions; SPP—The Signaling Pathways Project; STAR—Spliced Transcripts Alignment to a Reference. * Available at: https://github.com/dpryan79/MethylDackel/ (accessed on 1 June 2023). Bold represents the categories of analysis and commonly used bioinformatics tools used for NGS data analysis.

5. NGS Applications in Research and Diagnostics

NGS has revolutionized the field of scientific research and clinical genomics due to high-throughput multiplexing. This power of NGS in translation medicine lies not only in its advanced multiplexing efficiency but also in the equally smart bioinformatic tools used for data curation followed by various reference databases that help researchers, medical practitioners, and drug designers to understand the genetic basis of the disease. Different population genome sequencing projects such as 1000 G, ExAC, ESP6500, UK 100 K, Indigenome, and gnomAD generated vast amounts of data on NGS [ 162 ]. Among the reference population databases, gnomAD is the largest and most widely used database generated from harmonized sequencing data incorporating exome and genome sequencing data from 140,000 humans. This has been widely used as a resource for estimating allele frequency in rare diseases, disease gene discovery, and the biological effect of variation [ 163 ]. This has led to the creation of knowledge bases and in turn large and small sequencing panels for major applications in clinical research and diagnostics [ 164 ]. The large gene panels find their major application in clinical research mainly in cancer genetics.

5.1. Role of NGS in Research

5.1.1. microbiome research.

Given the ubiquitous nature of microbes, their symbiotic, pathogenic, and commensal characteristics are of importance to humans by forming a highly functioning ecosystem. The microbiome community became an obligatory factor in our survival through evolution [ 165 ]. However, a close monitoring and comprehensive understanding of the host–microbiome and microbiome–intercommunity interactions are vital to healthy survival. The approaches include pathogen surveillance, functional dysbiosis, and therapeutic potential. Metagenomic studies have linked the gut microbiome to disorders affecting mental health [ 166 ], autoimmune diseases (rheumatoid arthritis) [ 167 ], and metabolic disorders (diabetes and obesity) [ 168 ], thus instrumental in evaluating the functional potential of the microbiome. This opens doors for more therapeutic approaches and options. Designing targeted panels to pick up mutations (aiding in antibiotic resistance tracking) or identifying the pathogenic genes followed by sequencing can help in detecting pathogens with known antimicrobial resistance. Research is also underway for the pharmacomicrobiomics of individuals requiring drug treatment. This would aid in identifying the effect of drugs on an individual’s microbiome and drug disposition by the microbiome.

5.1.2. Human Disease Research

The focus of NGS-based research is now extended from genomic research to the study of transcriptome, epi-transcriptome, and epigenome. Human genome-based research through WGS and WES has provided novel insights into the biological processes and has found application in wellness research; agriculture and food research; genome-wide association research studies uncovering the wide range of population genetic variants; their genetic linkage and molecular basis to various diseases, including cancer; and the study of new pathogenic/emerging variants such as SARS-CoV-2 variants in human diseases. The redefinition of the mutational landscapes in tumors has resulted in translating this information into clinical research through the ever-growing list of targeted large gene panels such as the 261 gene panel, the 400 gene panel, the TSO 500 panel from Illumina, IDT, Agilent, and Thermo Fisher. These panels assess not only SNVs but also clinically relevant CNVs and RNA fusion transcripts, TMB, and microsatellite instability (MSI) for lung cancer, breast cancer, colorectal cancer, and even for difficult cancers such as ovarian, pancreatic, renal, urothelial cancers, etc.

RNA-seq finds its application mainly in research for analyzing pathogen transcriptomic signatures [ 169 ], metastatic biomarkers, therapeutic resistance, immune microenvironment, immunotherapy, and neoantigen research in cancer [ 170 , 171 ]. With NGS, it is now possible to study single-cell behavior with respect to its differentiation, de-differentiation, proliferation, and tumorigenesis in cancer using single-cell RNA-sequencing strategies such as Smart-seq2, MATQ-seq, SUPeR-seq, Drop-seq, Seq-Well, Chromium, DroNC-seq, STRT-seq, etc. [ 172 ]. The recent new development of the RiboSeq technique can plot potential ongoing events of translation in the cytosol, which is useful in identifying potentially functional micro-peptides. This is how thousands of sORFs (small open-reading frames) were discovered in lncRNA. Thus, with transcriptomics, Ribo-seq, and MS proteomics, the bifunctional potential of RNA molecules is identified [ 173 , 174 ].

The role of epigenomics in gene regulation, the maintenance of tissue-specific expression, and developmental processes is evident from X chromosome inactivation, embryonic development, genomic imprinting, epigenetic reprogramming, cell identity establishment, and lineage specification studies. Epigenetic signatures are important biomarkers that have promise not only in cancer, malignant transformation, and metastasis but also for their clinical applicability in other disease conditions such as diabetes, neurological conditions, infectious diseases, and immune disorders [ 175 , 176 ]. The reversible nature of epigenetic changes makes them promising candidates for precision medicine in cancer and other conditions [ 164 , 176 ]. Pharmacoepigenomics is an emerging research area, where the relationship between variable drug response and epigenetic status is being studied [ 59 ]. Epi-drugs have been developed over the last 40 years, and few are in clinical practice, whereas some are in clinical trials [ 177 ]. Non-coding RNAs (ncRNAs) are gene expression regulators apart from epigenetic modifications that are being explored as drug targets. Numerous lncRNAs are subsequently identified and found to be aberrantly expressed in various tumors [ 58 ]. Increasing studies have shown miRNAs as biomarkers of multiple cancers as their abnormal quantity has been correlated with the stage of pathology and prognosis [ 178 ]. The applications of miRNA analog or anti-miRNAs have shown promising outcomes in vitro and in vivo cancer studies, suggesting that miRNA-based drugs are emerging as a novel strategy for cancer therapy [ 179 ]. Apart from cancer, multiple FDA-approved drugs exist for DMD, SMA, familial hypercholesterolemia, CMV retinitis, etc. [ 178 ].

5.2. NGS in Diagnostics

A decisive approach is important when selecting an NGS assay. Type of variant, disease symptoms, and probable genetic associations are important aspects when selecting NGS-based tests in clinical decision making, as per recommendations by the National Comprehensive Cancer Network (NCCN), the College of American Pathologists (CAP), the American Society of Clinical Oncology (ASCO), the Association of Molecular Pathology (AMP), the American College of Medical Genetics (ACMG), and the European Society of Medical Oncology (ESMO).

5.2.1. Infectious Diseases

The identification of the exact etiological agent in microbial infections is important for precision medicine, which has driven the approach of syndromic testing/multiple pathogen testing assays such as BioFire or multiplex PCRs. However, with the limitations of multiplexing, NGS panels are being developed that can detect any pathogen using a shotgun approach or a targeted approach (16S) from various diseased specimens or clinical isolates. These panels can not only pick up causative pathogens but can be used to identify drug-resistant mutations such as antimicrobial drug-resistant mutations and antiviral drug-resistant mutations [ 180 ]. The useful data generated through NGS on microbial identification and drug resistance genotyping, e.g., in MTB, HIV, and SARS-CoV-2 [ 181 ], have proven important for disease surveillance, disease containment, public health epidemiological studies, policy making, and rapid therapeutic interventions, as evident during the COVID-19 outbreak [ 182 ]. However, with the need for fast diagnosis, NGS, in its current form for infectious pathogen detection, cannot replace current standard point-of-care testing such as PCR, multiplex BioFire panel testing, or multiplex QPCR commercial kits.

5.2.2. Inherited Genetic Diseases

The association of multiple genes in multifactorial disorders such as diabetes, hypercholesterolemia, infertility, etc., has been discovered in the rapidly emerging field of genomics. For example, the classical approach to comprehending the genes participating in infertility, gametogenesis, the hormonal cycle, fecundation, and embryo development would have been difficult and time-consuming. Targeted NGS panels have evolved as a result of WGS, and WES has enabled the simultaneous evaluation of multiple genes and their variants explaining the complexity of various disorders, including infertility, inherited genetic diseases, and reproductive genome testing, including NIPT (non-invasive prenatal testing), PGS/PGD (preimplantation genetic disease testing), and pediatric disorders such as developmental delay disorders, metabolic syndromes [ 183 ]. This has enabled disease treatment through personalized genome testing for the betterment of human health, preventive testing, and disease management.

5.2.3. HLA Typing

NGS-based HLA typing using WGS or targeted panels over conventional HLA typing methods for organ transplant or HSCT provides more unambiguous, high-throughput, high-resolution typing results from a single platform. This approach provides complete information on all the HLA loci involved in (1) the etiopathogenesis of immune disorders such as coeliac disease, psoriasis, rheumatoid arthritis, type I diabetes, SLE, lung diseases (e.g., asthma or sarcoidosis) [ 184 ], infectious disease predispositions (e.g., HIV, hepatitis, leprosy, tuberculosis), and other conditions such as malignancies and neuropathies [ 185 ]) generating population/ancestry-based database.

Epigenetics study through methylation profiling was in fact first studied using the HLA gene, which has its epigenetic regulators located in the non-coding region such as enhancers, promoters, and UTR regions that regulate HLA gene expression. Bioinformatically, the sequence data obtained are analyzed using commercial HLA-specific software such as NGSengine or exome-data-based software such as OptiType [ 186 ], Polysolver [ 187 ], xHLA [ 188 ], and HLAminer [ 189 ] to determine the HLA types [ 190 ].

5.2.4. Cancer

The comprehensive human genome sequencing project, WGS and WES, has identified cancer as the disease of the genome and is a multifactorial disease with non-mendelian (Somatic) origin in the majority of cases and mendelian origin in inherited cancers. Through the efforts of TCGA (The Cancer Genome Atlas) and ICGC (International Cancer Genome Consortium), the understanding of cancer and the comprehensive gene alteration data in protein-coding regions for all types of human cancers are now readily available [ 191 ].

Different enterprises, such as FoundationOne by Foundation Medicine (Cambridge, MA, USA), Oncomine by Thermo Fisher (Waltham, MA, USA), CANCERPLEX by KEW (Cambridge, MA, USA), MSK-IMPACT by the Memorial Sloan Kettering Cancer Center (New York, NY, USA), OmniSeq Advance by the Roswell Park Cancer Institute (Buffalo, NY, USA), the CC Onco Panel by Sysmex (Kobe, Japan), and the Todai Onco Panel by Riken Genesis (Tokyo, Japan) have come up with multigene panels using TCGA and ICGC data for different NGS platforms that are now frequently used in cancer prognosis and therapeutics [ 191 ]. Figure 4 summarizes the various data integration methods for cancer diagnosis, prognosis, and therapeutics [ 192 ]. Though all alterations picked up in NGS may not find immediate application in translation medicine, they help discover the different pathways operating in cancer pathogenesis and build on the cancer genomics database. Lung cancer biomarkers have been developed for almost over a decade for the development of a commercial NGS panel of 15–21 genes for precision oncology in lung cancer, picking up all types of structural variants (SVs) on a single platform [ 193 , 194 ]. This landmark study of precision oncology in lung cancer opened the doors for various solid tumors such as CRC, breast, ovarian, endometrial, pancreatic, and even liquid tumors such as myeloid and lymphoid malignancies to use NGS panels effectively with limited sample requirement, infrastructure, and different technical and analytical expertise [ 98 ]. Thus, a comprehensive gene testing approach in cancer provides maximum treatment efficacy and reduces the window period of disease progression in a cancer patient, resulting in improved QOL (quality of life), PFS (progression-free survival), and OS (overall survival).

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Role of NGS technology in cancer diagnosis, prognosis, and therapeutics using an integrative omics approach. FFPE, formalin-fixed paraffin-embedded; Bx, biopsy; AI, artificial intelligence; Ml, machine learning.

One important aspect of somatic mutation testing in cancer is tumor heterogeneity. It needs to be clearly and carefully dealt with by setting the variant calling cutoff thresholds to avoid false-positive or false-negative variant calling and reporting [ 195 ]. Being the most sensitive method of mutation detection, evolving mutant clones, the allelic burden of mutation and thus the disease progression can be determined through NGS. Liquid biopsy testing in cancer has become a very handy tool in tracking disease progression and treatment monitoring in clinical oncology using the circulating tumor DNA in a metastatic setting [ 196 ]. NGS plays a crucial role in identifying biomarkers associated with hereditary/germline cancers. For example, in the case of hereditary breast and ovarian cancer syndrome (HBOC), the understanding of its genetic basis has evolved beyond the BRCA1 and BRCA2 mutations. The inclusion of other genes involved in the homologous recombination repair (HRR) pathway, known as BRACAness genes, has reshaped our understanding of HBOC. These additional genes include CDH1, PTEN, TP53, STK11, PALB2, ATM, CHEK2, MUTYH, BARD1, MRE11A, NBN, RAD50, RAD51C, RAD51D, and NF1, in addition to BRCA1 and BRCA2. NGS has facilitated the identification and characterization of these extended sets of genes associated with HBOC, expanding our knowledge of hereditary cancer predisposition [ 197 ].

5.3. NGS in Forensics

Ever since 1984, when Sir Alec Jeffreys first proposed the application of DNA profiling to distinguish between different samples at a crime site, DNA analysis has emerged as a prime investigative tool in forensic science [ 198 ]. This field is now being dominated by NGS, keeping behind the old methods of DNA fingerprinting such as restriction fragment length polymorphism (RFLP), mitochondrial DNA, variable number of tandem repeat (VNTR) profiling, and short tandem repeat (STR) typing to solve an array of criminal mysteries [ 199 ]. NGS has gained rapid importance in this domain due to its ability to deliver highly accurate, reproducible, and results of the highest sensitivity from highly contaminated and degraded sample qualities received in forensic labs [ 200 ]. NGS is being applied to solve different categories of criminal cases: mtDNA for the investigation of maternal lineage [ 201 ], Y chromosome STR analysis for the identification of male DNA in a contaminated sample [ 202 ], animal and plant DNA analysis to identify important clues in poisoning cases [ 203 ], ancestry tracing [ 204 ], predicting phenotypes based on the genes [ 205 ], epigenetic analysis to identify the age of the donor DNA [ 206 ], and microRNA analysis for identifying body fluids and post-mortem interval [ 207 ]. The application of NGS in biodefense and bioterrorism involving the detection of microbial signatures at crime sites is another discipline gaining rapid attraction [ 208 , 209 ]. The major providers of NGS technology dominating the forensic domain are Illumina’s MiSeq FGx, Thermo Fisher’s Ion Torrent PGM, and Ion S5 [ 210 , 211 ]

6. Future Prospects and Conclusions

The future scope of NGS holds tremendous potential for advancements and applications in various fields. The progress in bioinformatics, robotics, liquid handling, and nucleic acid preparation will revolutionize NGS sequencing methods, making them faster and more precise. These forthcoming sequencing platforms will necessitate smaller amounts of input DNA and reagents, scaling down to zeptoliters and even a few molecules. Additionally, they will become increasingly portable, enabling their utilization in diagnostic applications across various fields such as medical, agricultural, ecological, and other field-based settings. Taken together, NGS holds immense potential for transformative advancements across multiple domains. NGS has already revolutionized fields such as clinical diagnostics, cancer genomics, and microbial genomics, providing unprecedented insights into the genetic underpinnings of diseases and driving personalized medicine. As technology progresses, NGS is expected to play a pivotal role in areas such as single-cell genomics, long-read sequencing, epigenomics, and multi-omics integration, enabling a deeper understanding of cellular processes, disease mechanisms, and personalized treatment strategies. The development of real-time sequencing and point-of-care applications will further extend the reach of NGS, empowering rapid diagnostics and monitoring in various settings. Additionally, advancements in bioinformatics and data analysis will be crucial for extracting meaningful insights from the vast amount of NGS data generated. The higher order multiplexing will enable more samples to be processed in a shorter time and at a reduced cost supported by the advances in robotics, liquid handling, and sample processing will contribute to these advancements. Equally important will be advanced in faster and more accurate bioinformatic data analysis, as well as data transfer and storage. With ongoing technological improvements and cost reduction, NGS will become more accessible and widespread, facilitating its integration into routine clinical practice, research, agriculture, and environmental studies. The future of NGS is promising, promising to unlock new frontiers of knowledge and catalyze advancements that will have a profound impact on human health, agriculture, environmental conservation, and beyond.

Acknowledgments

The authors express their heartfelt gratitude and tribute to the late Professor Michael Green from the Department of Molecular Cell and Cancer Biology, UMass Chan Medical School, for his invaluable support and remarkable contributions to the field of molecular genetics and cancer genomics.

Funding Statement

This research received no external funding.

Author Contributions

Conceptualization: H.S., G.D. and S.K.M.; original draft preparation: H.S., K.J., G.D. and S.K.M.; literature search, analysis, writing, review, and editing: H.S., K.J., U.M., S.W., G.Z., S.R., R.P.T., S.B., A.K.M., G.D. and S.K.M. visualization: H.S., S.W. and R.P.T. Supervision: A.K.M., G.D. and S.K.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Informed consent statement, data availability statement, conflicts of interest.

The authors declare no conflict of interest.

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Four U.S. CRISPR Trials Editing Human DNA to Research New Treatments

Breaking down how the gene editing technology is being used, for the first time in the United States, to treat patients with severe medical conditions

Lila Thulin

Former Associate Editor, Special Projects

Last fall, the birth of genetically edited twin girls in China —the world’s first “designer babies”—prompted an immediate outcry in the medical science community. The change to the twins’ genomes, performed using the gene editing technology CRISPR, was intended to make the girls more resistant to H.I.V. But the edited genes may result in adverse side effects , and the International Commission on the Clinical Use of Human Germline Genome Editing is currently working on stricter and less ambiguous guidelines for editing the DNA of human embryos as a response to the rogue experiment.

Human genetic engineering has also witnessed more regulated advances. In the past 12 months, four clinical trials launched in the United States to use CRISPR to treat and potentially cure patients of serious medical conditions.

CRISPR-Cas9 is a technology derived from single-celled prokaryotic microorganisms and is composed of guide strands of RNA as well as the Cas9 enzyme, which does the "cutting." It allows scientists to make changes at highly specific locations in a cell’s genetic code by removing or replacing parts of the genome . Even tiny changes to individual genes can fundamentally alter the function of a cell. CRISPR has been used to edit all types of organisms, from humans to corn , but clinical trials represent a stride toward turning the technology into a drug or medical treatment.

The clinical trials in the U.S. are Phase 1 and 2 trials, small studies designed to demonstrate the safety and efficacy of a potential treatment. Essentially, these make-or-break trials take a drug from the laboratory to test on real patients. They’re “the first requirement for a product to end up on the market,” says Saar Gill, an assistant professor at the University of Pennsylvania’s medical school who works on genetically-edited immune cells.

While some of the diseases CRISPR therapies aim to tackle have other treatments available, part of gene editing’s allure lies in the possibility of a more effective or even permanent fix. The four U.S. clinical trials involving CRISPR have the potential to tackle cancers such as melanoma and lymphoma, sickle cell disease, and even blindness.

“As complicated and expensive as [genetic editing] is, you really are talking about the potential to cure a disease or essentially halt its progress or its adverse effect on the body forever,” Gill says.

Editing Patients’ T Cells to Fight Cancer

The first clinical trial in the U.S. to use CRISPR in a treatment began last September. Led by University of Pennsylvania professor of medicine Edward Stadtmauer, it consists of genetically modifying patients’ own T cells—a type of immune cell that circulates in the blood—to make them more efficient at fighting certain kinds of cancer cells. The 18 patients will have types of relapsed cancer, like multiple myeloma or melanoma, that tend to overproduce an antigen called NY-ESO-1.

Once the T cells have been extracted from the patients’ blood, scientists will make several edits using CRISPR as well as a genetic modification technique derived from viruses like H.I.V. An added gene will cause the modified T cells to target cells with NY-ESO-1 as if it were a microscopic signal flare.

Another edit will stop T cells from producing proteins that could distract the cells from targeting NY-ESO-1. And researchers will also aim to turbo-boost the T cells by eliminating a protein called PD-1 that can prevent the T cells from killing cancer cells.

Patients will undergo chemotherapy to deplete their natural reserve of T cells, and then they’ll receive an infusion of the edited cells to replace them. The specific chemotherapy isn’t likely to affect the patients' cancers, so that step of the trial won’t complicate the study’s assessment of the usefulness of T cell therapy.

According to a spokesperson for Penn Medicine, two patients—one with multiple myeloma and one with sarcoma—have already begun treatment. The trial is scheduled to conclude in 2033 , and it will assess both safety (whether the edited T cell treatment leads to any negative side effects) and also efficacy (measured by outcomes such as whether the cancer disappears, the length of remission, and overall patient survival).

Boosting Fetal Hemoglobin in Patients With Sickle Cell Disease

A trial helmed by Massachusetts-based Vertex Pharmaceuticals and CRISPR Therapeutics is the first CRISPR-based clinical trial in the U.S. for a condition with a clear, heritable genetic basis: sickle cell disease. The recessive condition is caused by a single base-pair change, meaning that both copies of a patient’s affected gene differ by just one genetic “letter” from a normally functioning gene. Victoria Gray, a 34-year-old woman from Mississippi who was recently profiled by NPR , was the first patient to receive CRISPR-edited stem cells as part of the trial.

The disease, which occurs most frequently in people of African descent, affects a protein called hemoglobin, which plays a critical role in helping red blood cells carry oxygen to different tissues in the body. Sickle cell causes hemoglobin proteins to clump into long fibers that warp disc-shaped red blood cells into sickle shapes. The irregularly shaped blood cells are short-lived and can’t flow smoothly through blood vessels, causing blockages, intense pain and anemia.

Like the University of Pennsylvania T cell study, the sickle cell trial involves editing a patient’s own cells ex-vivo, or outside of the body in a lab. Stem cells are collected from the bloodstream and edited with CRISPR so they will pump out high levels of fetal hemoglobin, a protein that typically dwindles to trace levels after infancy. Fetal hemoglobin (HbF) is encoded by an entirely different gene than beta-globin, the part of hemoglobin that can cause red blood cells to sickle. Adults with sickle cell whose bodies naturally make more HbF often experience less severe symptoms. Fetal hemoglobin can take one or both of sickle hemoglobin’s spots in the four-part hemoglobin molecule, substantially lowering a cell’s likelihood of adopting a sickle shape.

The trial, slated to conclude in May 2022 , will destroy participants’ unedited bone marrow cells with chemotherapy and then inject edited stem cells through a catheter in a onetime infusion. Doctors will look for the treatment to generate 20 percent or more HbF in the bloodstream for at least three months. Fetal hemoglobin normally constitutes only around 1 percent of adults’ hemoglobin supply, but previous studies have shown that proportions of fetal hemoglobin above 20 percent can keep enough cells from sickling to significantly reduce symptoms, including severe pain episodes.

If successful, the therapy would offer another option for a disease with few available treatments. The only current cure for sickle cell disease is a bone marrow transplant, but, according to the National Heart, Blood, and Lung Institute , such transplants work best in children and the likelihood of finding a marrow donor match is low. Just two FDA-approved drugs for sickle cell currently exist, aimed at ameliorating the worst of patients’ symptoms, and one of them, hydroxyurea , also works by increasing fetal hemoglobin.

Editing Donor T Cells to Fight Lymphoma

The same companies behind the sickle cell treatment have also begun a trial to use CRISPR-edited T cells to treat non-responsive or relapsed non-Hodgkin’s lymphoma. This cancer of the lymphatic system plays a major role in the body’s immune response. Unlike the University of Pennsylvania trial, the study involves editing T cells from donors. The cells will be edited using CRISPR to target CD-19, a protein that marks B cells, which become malignant in some types of non-Hodgkin’s lymphoma. The edits also remove two proteins to stop a patient’s immune system from rejecting the donated T cells and to prevent the edited T cells from attacking non-cancerous cells.

A 2019 poster from the researchers explains that a prototype treatment in mice with acute leukemia stalled tumor growth for about 60 days. Additionally, lab tests showed that modified human T cells were successfully able to target and kill CD-19-marked cancer cells. For the clinical trial, which will eventually include a maximum of 95 participants, researchers will track how patients tolerate different doses of the T cell treatment and how many patients see their cancers shrink or disappear entirely. After the treatment is complete, scientists will keep tabs on patients and their survival and recurrence rates over the course of five years.

Editing Photoreceptor Cells to Treat Inherited Blindness

At the end of July, Cambridge, Massachusetts-based Editas Medicine, working with Irish company Allergan, announced that they’d begun enrollment in a clinical trial for EDIT-101, a treatment for a type of inherited childhood blindness known as Leber Congenital Amaurosis (LCA). It will be the first instance of a CRISPR clinical trial that conducts cellular editing within a human body, or in vivo. The trial will include about 18 participants, including patients as young as age 3, with a particular subset of LCA caused by a single genetic mutation that impairs photoreceptors. These cells in the eye convert light into signals for the brain to process.

The treatment comes in the form of an injection into the space behind the retina . A type of virus known as an adenovirus will “infect” the photoreceptor cells with DNA instructions to produce Cas9, the CRISPR enzyme , to cut the photoreceptor genome in specified locations. The edits change the photoreceptors’ DNA to fix the blindness-causing mutation, spurring the cells to regrow previously faulty light-sensing components, which should improve the patients’ vision.

Medical researchers aim to affect 10 percent or more of the targeted photoreceptor cells, the threshold that other research suggests is required to make a leap in visual acuity. Medical staff will measure patients’ vision in various ways, including an obstacle course featuring barriers with different contrast levels, a color vision test, the pupil’s response to light, and the person’s own assessment of visual change.

The EDIT-101 treatment has been tested in non-human primates and also in tiny samples of a donated human retina. In the human retina, the desired edit was made about 17 percent of the time, and scientists detected no unintended “off-target” changes.

The method of injecting a virus subretinally to treat LCA has been successful before. Jean Bennett and Albert Maguire’s treatment Luxturna doesn’t involve CRISPR, but it does use a similar viral injection to deliver a working copy of a malfunctioning gene to pigment cells in the retina. The work was recognized by Smithsonian magazine’s 2018 Ingenuity Award for life sciences.

The Future of CRISPR in Medicine

Early clinical trials are not without risks. In 1999, an 18-year-old participant named Jesse Gelsinger died in a Phase 1 gene therapy trial—a tragedy that still lingers over the field. Gelsinger had inherited a metabolic disorder, and like other patients in the trial, received an injection straight to his liver of the ammonia-digesting gene his body lacked. Four days later, multiple organs failed , and Gelsinger was taken off life support. After his death, investigations uncovered a tangle of ethical lapses . Critics said inadequate information had been provided about the study’s risks and pointed out that a key administrator at the University of Pennsylvania center behind the study had a financial conflict of interest.

Mildred Cho, a bioethicist and professor at the Stanford School of Medicine, sits on NExTRAC , the panel that advises the National Institutes of Health (NIH) on emerging biotechnologies. She says she’s “concerned that the factors at play in Jesse Gelsinger’s death have not actually been eliminated.” Specifically, Cho is wary of the risks of clinical trials moving too quickly in an environment where patients, physician-scientists and pharmaceutical companies alike are anxious to alleviate devastating medical conditions. “I think there’s a lot of pressuring pushing these new technologies forward, and at the same time, there’s more reluctance to regulate,” she says.

In the U.S., the current scientific consensus is that CRISPR is worth the risk, particularly to treat serious diseases with few alternative options. Other gene therapies have been successful before, like the cancer treatments Kymriah and Yescarta . But unlike most other gene editing techniques, CRISPR is relatively easy to engineer and use, opening up the floodgates for possible applications. The potential of tools like CRISPR to cure currently unfixable diseases represents a “massive paradigm shift from taking a pill for the rest of your life,” Gill says.

CRISPR is no miracle cure, yet. Larger trials must follow this preliminary work before the FDA can approve any new treatment. James Wilson, the former director of the University of Pennsylvania center that ran the trial in which Jesse Gelsinger died, said in a recent interview : “It’s going to be a long road before we get to the point where editing would be deemed safe enough for diseases other than those that have really significant morbidity and mortality.”

But for conditions that often prove deadly or debilitating, a little genetic engineering, done properly, could go a long way.

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Lila Thulin | | READ MORE

Lila Thulin is the former associate web editor, special projects, for Smithsonian magazine and covers a range of subjects from women's history to medicine.

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Beyond DNA: How scientists are learning to control gene function

NSF Emerging Frontiers in Research and Innovation program funds advancements in tissue regeneration, gene therapy, DNA mobility and epigenetic editing

Nearly every cell in your body contains the exact same DNA, from your skin cells to your brain cells. But how does a cell know how and when to turn into skin, muscle or brain?

Imagine that the DNA in cells is a long, twisted ladder made of billions of tiny building blocks. This DNA ladder carries all the instructions that tell your body how to grow, function and repair itself. When stretched out, the ladder in each human cell is 2 meters long, and it is difficult to imagine how it fits inside. Chromatin is how life solves this problem. Think of chromatin as a way of organizing DNA to fit within the nucleus (the control center of a cell). Chromatin is made of DNA wrapped around special proteins called histones to form a structure that looks like beads on a string, which is then looped and tightly compacted into chromosomes. This way DNA can be packed into a small space and unpacked whenever the cell needs access to genetic information.

DNA contains both coding and non-coding sequences. Proteins, which are essential cellular building blocks and mediators, are built using instructions contained in specific segments of coding DNA known as genes. Non-coding DNA plays a crucial supporting role by controlling when and how these genes are turned on or off for expression into proteins. Many non-coding regions enable chromatin interactions that regulate its structure and dynamics. For cells to become distinct tissues, many genes must be turned on and off across different DNA regions and over time. Chromatin organization can control this process — tightly packed chromatin restricts access to genes, keeping them off, whereas loosely packed chromatin allows genes to be turned on and expressed. Chromatin organization is influenced by chemical modifications of DNA and histone proteins, which thereby affect gene expression.

Thus, chromatin not only solves the problem of fitting DNA into a cell, it also provides a mechanism for regulating how the information in DNA is used.

Even though people inherit a fixed set of genes, their expression can be influenced by many factors throughout their lives, including environmental factors such as diet, stress and exposure to pollution. This phenomenon, called epigenetics, controls the identity and function of cells, in addition to the genetic sequence in DNA. To fully understand and potentially manipulate a cell's destiny, researchers must understand both its genetics and epigenetics.

EFMA and EFRI

Every two years, the Office of Emerging Frontiers and Multidisciplinary Activities (EFMA) in the Directorate for Engineering at the U.S. National Science Foundation identifies out-of-the-box research topics for the NSF Emerging Frontiers in Research and Innovation (EFRI) program. Under four-year grants, interdisciplinary teams work on transformative, high-risk, high-reward projects and to tackle the biggest challenges facing the nation.

In 2018 and 2019, EFRI focused on chromatin and epigenetic engineering to find new ways to control how genes are turned on and off. Through deeper knowledge and novel tools, researchers can engineer gene expression for many applications, including combatting disease, boosting crop plant performance or developing organisms that can remediate environmental damage.

Turning cancer cells off

Vadim Backman focuses on understanding and controlling chromatin organization. His team developed a high-resolution genome imaging platform to visualize chromatin in 3D, enabling more accurate predictions for genome engineering outcomes.

Backman’s interdisciplinary team combines genome biology with physics to model genome functions. They classify cellular features, like DNA structure and accessibility to predict the likelihood of gene activity from chromatin edits. This precise manipulation has applications in cancer treatment, organ regeneration, injury prevention, and reversing aging.

The team is developing drugs and interventions targeting cells affected by cancer or oxygen loss from strokes or heart attacks. For example, they developed an electromagnetic simulation technique that alters chromatin and gene expression, enabling heart cells to quickly repair damaged tissue.

Revealing how DNA gets rearranged inside the cell

Megan King 's goal was to understand the relationship between chromatin structure and its functions and to engineer a device to measure changes in chromatin mobility.

King and her team discovered that a special protein complex called INO80 is an important driver of chromatin movements inside the nucleus and are engineering a device to watch chromatin interactions happening in real time inside living cells. Previous methods analyzed millions of cells in aggregate at a single time. The new device can look at what is happening in a single cell over many time points. This is crucial for understanding the complexity of tissues of many different cell types, like the brain or immune system.

Folding genetic information

Carlos Castro has made important advances with his team in delivering DNA into cells using nanostructures . Using principles from origami paper folding to create intricate designs, researchers can package genetic information very tightly within these nanostructures, enabling the delivery of even the longest genes into the nucleus. This new technology offers a safer, more cost-efficient alternative to traditional viral gene therapy, with potential applications in treating diseases and improving live cell imaging.

Additionally, DNA origami structures can control how gene products interact with cell components , enabling the manipulation of cell properties and functions. This capability could be used in tissue engineering to create artificial tissues and organs.

Epigenetic editing to control gene expression and combat disease

Charles Gersbach's project uses the cancer-associated gene called MYC as a case study to test how changes in chromatin architecture lead to changes in gene expression and tumor characteristics. The team developed new genome-editing technologies to specifically target the non-coding regulatory regions of DNA that turn genes on or off.

This approach can add or remove chemical modifications (epigenetic marks), mimicking changes that might occur in nature in response to the environment. This epigenome engineering approach, which addresses variations in the non-coding genome linked to disease susceptibilities, can improve disease interventions. An epigenetic editing company, Tune Therapeutics , was founded to develop new therapies based on this research.

Empowering Future Innovators

Many EFRI teams leverage the NSF Research Experience and Mentoring program to provide paid research experiences and mentoring to broaden participation and include more diverse talents in engineering. Backman's team offers an opportunity for high school students and undergraduates to participate in research. King supports undergraduates from underrepresented minority groups and/or first-generation, low-income college students to begin their careers. Castro enables undergraduates to experience research merged with technology development and entrepreneurship.

The EFRI projects have yielded groundbreaking advancements in the understanding and manipulation of gene expression. Supported by interdisciplinary research and mentoring programs, these collaborative efforts have advanced scientific knowledge and fostered a new generation of scientists equipped to tackle complex challenges in genetic and epigenetic engineering.

About the Author

NSF 101: EPSCoR Graduate Fellowship Program

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Browsing: DNA

DNA, or deoxyribonucleic acid, is the molecule that carries the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses. Structurally, DNA is composed of two strands that coil around each other to form a double helix, with each strand consisting of a sugar-phosphate backbone attached to nitrogenous bases. These bases—adenine (A), thymine (T), cytosine (C), and guanine (G)—pair specifically (A with T, and C with G) to encode the genetic information. DNA’s role is pivotal in heredity and gene expression, guiding the synthesis of proteins via processes like transcription and translation. Since the discovery of its structure by Watson and Crick in 1953, DNA has been central to the fields of genetics and molecular biology, enabling advancements such as genetic engineering, forensic science, and medical diagnostics.

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New Approach to DNA Research Could Be Key to Solving Mysteries of Deadly Diseases

By Russ Bahorsky , [email protected]

Plasmodium falciparum, the parasite that causes malaria in humans, forms protrusions called “knobs” on the surface of its host red blood cell which enable it to avoid destruction and cause inflammation. (Photo courtesy of National Institutes of Health)

To analyze the genome or the genetic characteristics of a living organism, scientists typically rely on samples of millions of cells. The problem is that the DNA in each of our cells is not identical.

Until recently, the amount of DNA that could be extracted from a single cell couldn’t provide enough material for genetic analysis, but advances in single-cell genomics could be the key to solving some of the mysteries of diseases like cancer, which is the result of damage to individual cells. It could also help researchers better understand complex bodily systems like the brain and the immune system that are composed of a variety of cell types, each with their own unique genetic characteristics.

As a means to solving the problems posed by single-cell genomics, a process called whole-gene amplification is providing researchers with ways to generate sufficient quantities of DNA necessary for analysis by replicating the genetic material extracted from each cell. The process is not without its challenges, but a paper by Shiwei Liu, a Ph.D. candidate in biology in the University of Virginia’s College of Arts & Sciences; UVA biology professor Jennifer L. Güler; and others, published recently in the journal Genome Medicine, outlines an approach to whole-genome amplification resulting from a collaboration with neuroscientists in UVA’s School of Medicine that could provide an effective framework for creating new and more effective treatments for a variety of diseases.

Assistant professor of biology Jennifer Güler studies the genetic and metabolic mechanisms that allow malaria to adapt to and survive anti-malarial medications. (Photo by Molly Angevine)

Güler and Liu study the single-celled protozoan parasite, called Plasmodium, that causes malaria, a disease that kills nearly half a million people every year. There are no effective vaccines in widespread use for the disease, and one of the problems the medical community faces is that the organism can rapidly develop resistance to the drugs that have been developed to wipe it out. Güler’s team has been working to understand cellular mechanisms that allow it to survive and how genetic diversity within the parasite population affects its resistance to drugs.

“If you take them on a single-cell level, we start to appreciate that individual cells in a population of cells actually have small differences, and those small differences might not be noticeable, but they can have an impact if they disrupt how drugs or other treatments work,” Güler said.

In recent years, scientists have been finding ways to capture and extract DNA from single cells, which makes it possible to identify the small but critical differences between individual cells. However, the process requires a series of steps that create additional problems for researchers attempting to amplify the DNA, a process that involves reproducing enough identical copies of that DNA to be able to identify, or sequence, its component parts. The amplification process is especially challenging for malaria researchers.

Shiwei Liu, a UVA Ph.D. candidate in biology, was lead author on a paper published recently in the journal Genome Medicine on single-cell sequencing. (Contributed photo)

“The genome of the malaria parasite is really small, almost 300 times smaller than the human genome, so if we capture one genome from the malaria parasite, we’re starting at a much lower level than we need to be able sequence it, so we have to use a really sensitive, highly specific method to be able to amplify it,” Güler explained. “Then you sequence it, and presumably everything in that sequence of all those different copies is going to reflect that first genome, and this is where a big challenge comes in. When you make those many copies, you introduce errors, and you can’t always assume that those many copies reflect the initial genome. That’s been a big problem in single-cell genomics.”

Because the Plasmodium parasite lives in the human bloodstream, Güler and her team also needed a method that would allow them to preferentially amplify the genome of the protozoan over its host, a problem that is unique to studying organisms that live inside the cells of other organisms.

The solution came as the result of a collaboration with Mike McConnell, a neuroscientist who works as an investigator at the Lieber Institute for Brain Development Maltz Research Laboratories in Baltimore. Güler met McConnell when he worked in the UVA School of Medicine’s Department of Biochemistry and Molecular Genetics.

McConnell specializes in single-cell genome analysis for human brain cells and had already developed strategies for capturing single cells. He had also worked with Ian Burbulis, an assistant professor of biochemistry and molecular genetics at UVA, to use a method called multiple annealing and looping based amplification cycles, or MALBAC, to solve some of the problems inherent in the process of single-cell genome amplification.

Güler recognized the similarities in the challenges they were facing, and her team was able to use McConnell’s method for capturing single cells and was also able to adapt the MALBAC method for use in reproducing the Plasmodium DNA accurately while limiting the contamination that can be caused by its host’s DNA.

“The collaboration with Mike McConnell’s lab helped build the basis for our single-cell sequencing project. They not only provided the original standard protocol of the whole genome amplification method called MALBAC, but they also offered instructions to conduct the essential steps of the single-cell sequencing pipeline, including single-cell isolation, whole- genome amplification and data analysis,” Liu said.

“We had worked out all of the molecular biology steps, the enzymes to use and how to analyze it and that sort of thing, and we made some improvements to make it better, and Jenny was able to start there,” McConnell said. He gave credit to Güler and Liu for seeing the potential that his research offered.

“Jenny and Shiwei did the heavy lifting to take what we had done and make it work for malaria,” McConnell said.

“I think our collaboration with his lab from the very beginning of his project was absolutely instrumental because we could go to his lab and learn what they were doing instead of starting from ground zero, so we started at a much higher level,” Güler said. “It was a team effort that ended up being very successful because we had that head start.”

“A lot of this single-cell technology is really focused on human cells, and that’s great; we want to learn more about human health,” Güler said. “But when you have these microbes or other organisms that have more challenging genomes, we need to be able to apply these methods to those genomes, too. This is one of the first studies to suggest that we can overcome those challenges.

“It’s a start for us to understand the biology of the malaria parasite, but it’s also a start for understanding other organisms with challenging genomes.”

Media Contact

Russ Bahorsky

UVA College and Graduate School of Arts & Sciences

[email protected] (434) 924-5357

Article Information

June 1, 2021

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Latest Research and Reviews

Genome-wide discovery and integrative genomic characterization of insulin resistance loci using serum triglycerides to HDL-cholesterol ratio as a proxy

Here the authors use UK Biobank data to identify 251 genetic loci associated with serum triglycerides to HDL-cholesterol ratio, a surrogate marker for insulin resistance. Key genes, including PLA2G12A , PLA2G6, and TNFAIP8 , offer potential therapeutic targets for metabolic diseases.

Natalie DeForest
Amit R. Majithia

Sequence variants influencing the regulation of serum IgG subclass levels

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. Here the authors describe 14 genetic variants that affect IgG levels in blood. The data provide new insight into the regulation of humoral immunity that could be useful in the development of antibody-based therapeutics.

Thorunn A. Olafsdottir
Gudmar Thorleifsson
Kari Stefansson

Population suppression by release of insects carrying a dominant sterile homing gene drive targeting doublesex in Drosophila

CRISPR homing gene drives can suppress pest populations by targeting female fertility genes, converting wild-type alleles into drive alleles in the germline of drive heterozygotes. Here the authors demonstrate a genetic pest suppression system based on dominant female-sterile doublesex alleles and show that releases of transgenic males eliminated Drosophila cage populations, with modelling showing improved performance compared to similar systems.

Weizhe Chen
Jialiang Guo
Jackson Champer

Evaluating the cost-effectiveness of polygenic risk score-stratified screening for abdominal aortic aneurysm

Kelemen et al. find that leveraging related traits improves polygenic score performance for abdominal aortic aneurysm. Health-economic modelling suggests that combining smoking and genetic risk information may improve cost-effectiveness of screening.

Sexually dimorphic renal expression of mouse Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Identification and deletion of the mouse Klotho gene enhancer reveals HNF1b-driven, sexually dimorphic regulation of Klotho expression in the kidney and shows the effects of Klotho depletion on phenotype.

Jakub Jankowski
Hye Kyung Lee
Lothar Hennighausen

Prenatal cannabis exposure is associated with alterations in offspring DNA methylation at genes involved in neurodevelopment, across the life course

Alexandra J. Noble
Alex T. Adams
Amy J. Osborne

News and Comment

Polygenic basis for seedless grapes, x chromosome dosage shapes renal cell carcinoma risk.

Tiago Faial

The genomics of childhood T-lineage acute lymphoblastic leukemia

Safia Danovi

Why does heart disease affect so many young South Asians?

Geneticists are trying to understand the elevated risks of heart and metabolic disease among people of South Asian ancestry, but some question whether a purely biological approach is best.

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DNA
DNA (deoxyribonucleic acid) is the nucleic acid polymer that forms the genetic code for a cell or virus. ... Research Highlights 17 May 2023 Nature Structural & Molecular Biology. Volume: 30, P ...
Researchers bend DNA strands with light, revealing a new way to study
Researchers have developed a tool that can bend DNA strands using light. The work represents a new way to probe the genome. Shown here, from an unrelated study, are chromosomes (blue) inside a human cell nucleus. Steve Mabon, Tom Misteli, NCI Center for Cancer Research, National Cancer Institute, National Institutes of Health
Researchers bend DNA strands with light, revealing a new way to study
In this new work, they utilized an additional component that attaches the condensate to specific locations on the DNA strands and directs their movement quickly and precisely via surface tension-mediated forces also known as capillary forces, which Princeton researchers had suggested could be ubiquitous in living cells. Previously, moving DNA ...
Researchers bend DNA strands with light, revealing a new way to study
Researchers have developed a tool that can bend DNA strands using light. The work represents a new way to probe the genome. Shown here, from an unrelated study, are chromosomes (blue) inside a ...
Scientists Unveil a More Diverse Human Genome
Published May 10, 2023 Updated May 12, 2023. More than 20 years after scientists first released a draft sequence of the human genome, the book of life has been given a long-overdue rewrite. A more ...
Scientists sequence the complete human genome for the first time
The new research introduces 400 million letters to the previously sequenced DNA - an entire chromosome's worth. The full genome will allow scientists to analyze how DNA differs between people ...
Fastest DNA sequencing technique helps undiagnosed patients find
A research effort led by Stanford scientists set the first Guinness World Record for the fastest DNA sequencing technique, which was used to sequence a human genome in just 5 hours and 2 minutes. ... "They told us there's this brand-new research that they were working on to try to speed up the process of diagnosis," Jenny Kunzman said ...
Revolutionizing DNA repair research and cancer therapy with ...
CRISPR-based genetic screens are providing new insights into the consequences of deficiencies in DNA damage response and repair pathways. These include insights into the regulation of homologous ...
Human Molecular Genetics and Genomics
In 1987, the New York Times Magazine characterized the Human Genome Project as the "biggest, costliest, most provocative biomedical research project in history." 2 But in the years between the ...
New technique enables manipulation of large DNA segments
New technique will allow programmable manipulation of large DNA segments. A team of researchers led by Harvard and Broad Institute scientists has developed twin prime editing, a new, CRISPR-based gene-editing strategy that enables manipulation of gene-sized chunks of DNA in human cells without cutting the DNA double helix.
DNA
A new complete Y chromosome sequence just might combat this dangerous myth. Learn and share the most exciting discoveries, innovations and ideas shaping our world today. DNA coverage from ...
New method uses light to bend DNA strands for better disease understanding
A Princeton research team has developed a groundbreaking tool to study chromosomes by physically moving DNA strands around. Having found and turned a key, they can access the deepest mechanisms of ...
Scientists Have Confirmed a New DNA Structure Inside Human Cells
According to Garvan's Mahdi Zeraati, the first author of the new study, the i-motif is only one of a number of DNA structures that don't take the double helix form - including A-DNA, Z-DNA, triplex DNA and Cruciform DNA - and which could also exist in our cells. Another kind of DNA structure, called G-quadruplex (G4) DNA, was first ...
The first gene-editing treatment: 10 Breakthrough Technologies 2024
The first gene-editing cure has arrived. Grateful patients are calling it "life changing.". It was only 11 years ago that scientists first developed the potent DNA-snipping technology called ...
Scientists Finish the Human Genome at Last
They ended up with 99 scientists working directly on sequencing the human genome, and dozens more pitching in to make sense of the data. The researchers worked remotely through the pandemic ...
Grad students at work: Researchers bend DNA strands with light
Yoonji Kim *24 was member of research team that developed tool to help researchers better understand gene expression With the flick of a light, researchers have found a way to rearrange life's basic tapestry, bending DNA strands back on themselves to reveal the material nature of the genome.Scientists have long debated about the physics of chromoso
DNA and RNA
A molecular jack-of-all-trades. DNA is much more than the genetic information it carries. It is a versatile material for creating systems with tailor-made functionalities that are having an ...
Next-Generation Sequencing Technology: Current Trends and Advancements
Next-generation sequencing (NGS) is a powerful tool used in genomics research. NGS can sequence millions of DNA fragments at once, providing detailed information about the structure of genomes, genetic variations, gene activity, and changes in gene behavior. Recent advancements have focused on faster and more accurate sequencing, reduced costs ...
Now fully complete, human genome reveals new secrets
A research team has finally completed the sequence of the human genome, filling in the last 8 percent of the genome's 3 billion nucleotides. ... The new DNA sequences in and around the centromere ...
Four U.S. CRISPR Trials Editing Human DNA to Research New Treatments
Human genetic engineering has also witnessed more regulated advances. In the past 12 months, four clinical trials launched in the United States to use CRISPR to treat and potentially cure patients ...
CRISPR Gene Editing News
Precise Genetics: New CRISPR Method Enables Efficient DNA Modification. July 30, 2024 — A research group has developed a new method that further improves the existing CRISPR/Cas technologies: it ...
New DNA Research Changes Origin of Human Species
Credit: Brenna Henn/UC Davis. "This uncertainty is due to limited fossil and ancient genomic data, and to the fact that the fossil record does not always align with expectations from models built using modern DNA," she said. "This new research changes the origin of species.". Research co-led by Henn and Simon Gravel of McGill University ...
Beyond DNA: How scientists are learning to control gene function
DNA contains both coding and non-coding sequences. Proteins, which are essential cellular building blocks and mediators, are built using instructions contained in specific segments of coding DNA known as genes. ... An epigenetic editing company, Tune Therapeutics, was founded to develop new therapies based on this research. Empowering Future ...
DNA News
DNA, or deoxyribonucleic acid, is the molecule that carries the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses. Structurally, DNA is composed of two strands that coil around each other to form a double helix, with each strand consisting of a sugar-phosphate ...
Genetics research
Research Open Access 10 Sept 2024 Scientific Reports Volume: 14, P: 21119 Genomic Balancing Act: deciphering DNA rearrangements in the complex chromosomal aberration involving 5p15.2, 2q31.1, and ...
New Approach to DNA Research Could Be Key to Solving Mysteries of
New Approach to DNA Research Could Be Key to Solving Mysteries of Deadly Diseases. By Russ Bahorsky, [email protected]. June 1, 2021. Plasmodium falciparum, the parasite that causes malaria in humans, forms protrusions called "knobs" on the surface of its host red blood cell which enable it to avoid destruction and cause inflammation.
DNA Research
DNA Research is the official journal of Kazusa DNA Research Institute, published by Oxford University Press and supported by funding from Chiba Prefecture, Japan. Growing Impact Factor, fully open access journal, low open access charges, and more. Volume 26, Issue 6:
National Human Genome Research Institute Home
About the National Human Genome Research Institute. At NHGRI, we are focused on advances in genomics research. Building on our leadership role in the initial sequencing of the human genome, we collaborate with the world's scientific and medical communities to enhance genomic technologies that accelerate breakthroughs and improve lives.
Genetics
RSS Feed. Genetics is the branch of science concerned with genes, heredity, and variation in living organisms. It seeks to understand the process of trait inheritance from parents to offspring ...
A DNA Aptamer for 2‐Aminopurine: Binding‐induced Fluorescence Quenching
Its fluorescence property probes local dynamics in DNA and RNA because the surrounding bases quench its fluorescence. 2AP-labeled probes that can bind to specific DNA or RNA sequences, enabling the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases.

Omenn-Darling Bioengineering Institute

Researchers bend DNA strands with light, revealing a new way to study the genome

The material science of our genome

Like CRISPR but different

Researchers bend DNA strands with light, revealing a new way to study the genome

The material science of our genome

Like CRISPR but different

Saturday Citations: Permian-Triassic mystery solved; cute baby sighted; the nine-day 2023 seismic event

Researchers develop novel covalent organic frameworks for precise cancer treatment delivery

Flowers use adjustable 'paint by numbers' petal designs to attract pollinators, researchers discover

Astronomers discover new planet in Great Bear constellation

Device malfunctions from continuous current lead to discovery that can improve design of microelectronic devices

Soil pH drives microbial community composition: Study shows how bacteria work together to thrive in difficult conditions

Gravity study gives insights into hidden features beneath lost ocean of Mars and rising Olympus Mons

Technique to study how proteins bind to DNA is easily misused: Researchers offer a solution

Findings from experimental setup demonstrate potential for compact and portable nuclear clocks

Global warming is driving rapid evolutionary response in fruit flies, research suggests

Epothilone B study connected to 'Hard Problem of Consciousness' Model

Any stereo audio learning resources for other languages?

Too much fluoride might lower IQ in kids?

The predictive brain (Stimulus-Specific Error Prediction Neurons)

Any suggestions to dampen the sounds of a colostomy bag?

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