The scientist also employed machine learning “to gauge how easily accessible genes are for transcription” in research that could lead to new clinical laboratory diagnostic tests
Anatomic pathologists and clinical laboratories are of course familiar with the biological science of genomics, which, among other things, has been used to map the human genome. But did you know that a three-dimensional (3D) map of a genome has been created and that it is helping scientists understand how DNA regulates its organization—and why?
The achievement took place at St. Jude Children’s Research Hospital (St. Jude) in Memphis, Tenn. Scientists there created “the first 3D map of a mouse genome” to study “the way cells organize their genomes during development,” a St. Jude news release noted.
Some experts predict that this new approach to understanding how changes happen in a genome could eventually provide new insights that anatomic pathologists and clinical laboratory scientists could find useful when working with physicians to diagnose patients and using the test results to identify the most appropriate therapy for those patients.
In addition to 3D modeling, the researchers applied machine learning to data from multiple sources to see how the organization of the genome changed at different times during development. “The changes are not random, but part of the developmental program of cells,” Dyer said in the news release.
The St. Jude study focused on the rod cells in a mouse retina. That may seem like a narrow scope, but there are more than 8,000 genes involved in retinal development in mice, during which those genes are either turned on or off.
To see what was happening among the cells, the researchers used HI-C analysis, an aspect of ultra-deep chromosome conformation capture, in situ. They found that the loops in the DNA bring together regions of the genome, allowing them to interact in specific ways.
Until this study, how those interactions took place was a
mystery.
“Understanding the way cells organize their genomes during development will help us to understand their ability to respond to stress, injury and disease,”Michael Dyer, PhD (above), Chair of St. Jude’s Developmental Neurobiology Department, co-leader of the Developmental Biology and Solid Tumor Program, and Investigator at Howard Hughes Medical Institute (HHMI), said in the news release. (Photo copyright: St. Jude Children’s Research Hospital.)
The scientists also discovered there were DNA promoters, which encourage gene expression, and also DNA enhancers that increase the likelihood gene expression will occur.
“The research also included the first report of a powerful regulator of gene expression, a super enhancer, that worked in a specific cell at a specific stage of development,” the news release states. “The finding is important because the super enhancers can be hijacked in developmental cancers of the brain and other organs.”
St. Jude goes on to state, “In this study, the scientists determined that when a core regulatory circuit super-enhancer for the VSX2 gene was deleted, an entire class of neurons (bipolar neurons) was eliminated. No other defects were identified. Deletion of the VSX2 gene causes many more defects in retinal development, so the super-enhancer is highly specific to bipolar neurons.”
The St. Jude researchers developed a genetic mouse model of
the defect that scientists are using to study neural circuits in the retina,
the news release states.
Research Technologist Victoria Honnell (left); Developmental Neurobiologist Jackie Norrie, PhD (center); and Postdoctoral Researcher Marybeth Lupo, PhD (right), work in the St. Jude clinical laboratory of Michael Dyer, PhD, using 3D genomic mapping to study gene regulation during development and disease. (Photo copyright: St. Jude Children’s Research Hospital.)
DNA Loops May Matter to Pathology Sooner Rather than
Later
Previous researcher studies primarily used genomic sequencing technology to locate and investigate alterations in genes that lead to disease. In the St. Jude study, the researchers examined how DNA is packaged. If the DNA of a single cell could be stretched out, it would be more than six feet long. To fit into the nucleus of a cell, DNA is looped and bundled into a microscopic package. The St. Jude scientists determined that how these loops are organized regulates how the cell functions and develops.
Scientists around the world will continue studying how the loops in DNA impact gene regulation and how that affects the gene’s response to disease. At St. Jude Children’s Research Hospital, Dyer and his colleagues “used the same approach to create a 3D genomic map of the mouse cerebellum, a brain structure where medulloblastoma can develop. Medulloblastoma is the most common malignant pediatric brain tumor,” noted the St. Jude’s news release.
In addition to providing an understanding of how genes
function, these 3D studies are providing valuable insight into how some
diseases develop and mature. While nascent research such as this may not impact
pathologists and clinical laboratories at the moment, it’s not a stretch to
think that this work may lead to greater understanding of the pathology of
diseases in the near future.
Half of the genes identified were found to be singletons, unique to specific individuals, offering the possibility of developing precision medicine therapies targeted to specific patients, as well as clinical laboratory tests
Microbiologists and other medical laboratory scientists may soon have more useful biomarkers that aid in earlier, more accurate detection of disease, as well as guiding physicians to select the most effective therapies for specific patients, a key component of Precision Medicine.
The scientists also found that more than half of the bacterial genes examined occurred only once (called “singletons”) and were specific to each individual. A total of 11.8 million of these singletons came from oral samples and 12.6 million of them derived from gut samples, a Harvard news release noted.
In a paper published in Cell Host and Microbe the researchers state, “Despite substantial interest in the species diversity of the human microbiome and its role in disease, the scale of its genetic diversity, which is fundamental to deciphering human-microbe interactions, has not been quantified.”
To determine this quantity, the researchers conducted a meta-analysis of metagenomes from the human mouth and gut among 3,655 samples from 13 unique studies. Of their findings, they wrote, “We found staggering genetic heterogeneity in the dataset, identifying a total of 45,666,334 non-redundant genes (23,961,508 oral and 22,254,436 gut) at the 95% identity level.”
The scientists also found that while genes commonly found in
all the samples seemed to drive the basic functions of a microbe’s survival,
the singletons perform more specialized functions within the body, such as
creating barriers to protect the micro-organisms from external onslaughts and
helping to build up resistance to antibiotics.
“Some of these unique genes appear to be important in solving evolutionary challenges,” said Braden Tierney, a PhD student at Harvard Medical School and one of the authors of the study, in the news release. “If a microbe needs to become resistant to an antibiotic because of exposure to drugs, or suddenly faces a new selective pressure, the singleton genes may be the wellspring of genetic diversity the microbe can pull from to adapt,” he concluded.
‘More Genes in the Human Microbiome than Stars in the
Universe’
According to their published paper, the team of microbiologists and bioinformaticians pinpointed more than 46 million bacterial genes contained within 3,655 Deoxyribonucleic acid (DNA) samples. They identified 23,961,508 non-redundant genes in the oral samples and 22,254,436 non-redundant genes in the intestinal samples.
While similar research in the past has targeted bacteria in
either the gut or the mouth, the scientists believe their study is the first
that analyzed DNA collected from both areas simultaneously.
The graphic above, taken from the Harvard Medical School study, illustrates the ratio of singleton vs. non-singleton bacteria contained in human microbiome. The sheer amount of diversity seems to have impressed the scientists. “There may be more genes in the collective human microbiome than stars in the observable universe, and at least half of these genes appear to be unique to each individual,” the Harvard news release states. This diversity could lead to new precision medicine treatments and clinical laboratory diagnostics. (Graphic copyright: Harvard Medical School.)
“Just like no two siblings are genetically identical, no two bacterial strains are genetically identical, either,” said study co-author Chirag Patel, PhD, Assistant Professor of Biomedical Informatics at Harvard’s Blavatnik Institute. “Two members of the same bacterial strain could have markedly different genetic makeup, so information about bacterial species alone could mask critical differences that arise from genetic variation.”
The scientists also endeavored to determine the number of
genes that reside in the human microbiome but found the precise number difficult
to identify. One calculation estimated that number to be around 232 million,
while another suggested the number could be substantially higher.
“Whatever it may be, we hope that our catalog, along with a
searchable web application, will have many practical uses and seed many directions
of research in the field of host-microbe relationships,” stated Patel in the
news release.
New Diagnostics for Clinical Laboratories?
This type of research could have lasting effects on clinical
laboratories. As the volume of data generated by diagnostic testing of microbes
in patients opens new understanding of how these factors affect human disease
and create differences from one individual to another, the increased number of
genes and gene mutations mean that microbiology laboratories will increase
their use of information technology and analytical software tools.
“Ours is a gateway study, the first step on a what will
likely be a long journey toward understanding how differences in gene content
drive microbial behavior and modify disease risk,” said Tierney in the Harvard
news release.
That’s good news, because new biomarkers derived from such
research will help microbiologists and other clinical laboratory scientists
more accurately detect disease and identify the best therapies for individual
patients.
‘Prime editing’ is what researchers are calling the proof-of-concept research that promises improved diagnostics and more effective treatments for patients with genetic defects
Known as Prime Editing, the scientists developed this technique as a more accurate way to edit Deoxyribonucleic acid (DNA). In a paper published in Nature, the authors claim prime editing has the potential to correct up to 89% of disease-causing genetic variations. They also claim prime editing is more powerful, precise, and flexible than CRISPR.
The research paper describes prime editing as a “versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit.”
And a Harvard Gazette article states, “Prime editing differs from previous genome-editing systems in that it uses RNA to direct the insertion of new DNA sequences in human cells.”
Assuming further research and clinical studies confirm the
viability of this technology, clinical laboratories would have a new diagnostic
service line that could become a significant proportion of a lab’s specimen
volume and test mix.
In that e-briefing we wrote that Liu “has led a team of scientists in the development of a gene-editing protein delivery system that uses cationic lipids and works on animal and human cells. The new delivery method is as effective as protein delivery via DNA and has significantly higher specificity. If developed, this technology could open the door to routine use of genome analysis, worked up by the clinical laboratory, as one element in therapeutic decision-making.”
Now, Liu has taken that development even further.
“A major aspiration in the molecular life sciences is the ability to precisely make any change to the genome in any location. We think prime editing brings us closer to that goal,” David Liu, PhD (above), Director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, told The Harvard Gazette. “We’re not aware of another editing technology in mammalian cells that offers this level of versatility and precision with so few byproducts.” (Photo copyright: Broad Institute.)
Cell Division Not Necessary
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is considered the most advanced gene editing technology available. However, it has one drawback not found in Prime Editing—CRISPR relies on a cell’s ability to divide to generate desired alterations in DNA—prime editing does not.
This means prime editing could be used to repair genetic mutations in cells that do not always divide, such as cells in the human nervous system. Another advantage of prime editing is that it does not cut both strands of the DNA double helix. This lowers the risk of making unintended, potentially dangerous changes to a patient’s DNA.
The researchers claim prime editing can eradicate long lengths of disease-causing DNA and insert curative DNA to repair dangerous mutations. These feats, they say, can be accomplished without triggering genome responses introduced by other forms of CRISPR that may be potentially harmful.
“Prime editors are more like word processors capable of
searching for targeted DNA sequences and precisely replacing them with edited
DNA strands,” Liu told NPR.
The scientists involved in the study have used prime editing to perform over 175 edits in human cells. In the test lab, they have succeeded in repairing genetic mutations that cause both Sickle Cell Anemia (SCA) and Tay-Sachs disease, NPR reported.
“Prime editing is really a step—and potentially a significant step—towards this long-term aspiration of the field in which we are trying to be able to make just about any kind of DNA change that anyone wants at just about any site in the human genome,” Liu told News Medical.
Additional Research Required, but Results are Promising
Prime editing is very new and warrants further
investigation. The researchers plan to continue their work on the technology by
performing additional testing and exploring delivery mechanisms that could lead
to human therapeutic applications.
“Prime editing should be tested and optimized in as many cell types as researchers are interested in editing. Our initial study showed prime editing in four human cancer cell lines, as well as in post-mitotic primary mouse cortical neurons,” Liu told STAT. “The efficiency of prime editing varied quite a bit across these cell types, so illuminating the cell-type and cell-state determinants of prime editing outcomes is one focus of our current efforts.”
Although further research and clinical studies are needed to
confirm the viability of prime editing, clinical laboratories could benefit
from this technology. It’s worth watching.
This is not the first time genetic-testing company Orig3n has been scrutinized by state and federal investigators over its business practices
It’s not often that multiple employees of a clinical laboratory company go public with criticism about the quality of their lab company’s tests. But that is what is happening at Orig3n. Problems at the Boston-based genetic testing company were the subject of an investigative report published by Bloomberg Businessweek (Bloomberg).
In September, Bloomberg reported that 17 former Orig3n employees said the company’s Deoxyribonucleic acid (DNA) tests sometimes failed to deliver the intended results or were often contaminated or inaccurate. The individuals had been employed by the company as managers, lab technicians, software engineers, marketers, and salespeople between 2015 and 2018.
The former employees claimed that Orig3n “habitually cut
corners, tampered with or fabricated results, and failed to meet basic
scientific standards,” Bloomberg reported. The individuals also stated
that advice intended to be personalized to individual consumers’ genetic
profiles was often just generic information or advice that had no scientific
basis.
According to Bloomberg, the individuals also alleged
that Orig3n’s lab was careless in its handling of genetic samples in several
ways, including:
Multiple samples being labeled with the same
barcode;
DNA and blood samples for stem cell bank
misplaced or mixed up;
No controls to ensure accuracy;
Handling methods that could lead to
contamination; and
Fabricating results when a test outcome was
unclear.
The former employees also stated that “Orig3n ran tests without proper authorization in its lab at the 49ers’ stadium, and that managers regularly compelled them to write positive reviews of Orig3n’s tests on Amazon.com and Google to offset waves of negative feedback,” Bloomberg reported.
“Accurate science didn’t seem to be a priority. Marketing
was the priority,” said a former lab technician who spoke with Bloomberg
on the condition of anonymity. Orig3n denied the accusations in a statement,
describing them as “grossly inaccurate,” and claimed the former employees were
simply disgruntled.
“In some cases, former employees are former employees for a reason,” Orig3n Chief Executive Officer Robin Smith told Bloomberg. “We’ve found after employees are gone that they have not done things appropriately.”
In 2018, NBC Chicago(NBC) conducted an investigation into various consumer DNA testing kits. NBC sent DNA samples to several different testing companies. This included non-human samples, which NBC’s investigators had obtained from a female Labrador Retriever.
With the exception of Orig3n, all of companies identified
the DNA as non-human and did not process the kits. Orig3n did, however, process
the canine DNA. It then returned a seven-page analysis that suggested the
subject of the sample “would probably be great for quick movements like boxing
and basketball, and that she has the cardiac output for long endurance bike
rides or runs,” NBC reported.
This would be funny if it weren’t so concerning.
Following reports that it had processed dog DNA, Orig3n stated
it had made changes and improvements to the company’s testing methodologies. Smith
also stated Orig3n’s lab protocols had been improved as well.
“Sometimes we look at the accuracy of things and go, ‘Man,
that’s not working,’” Smith told Bloomberg. “Our approach and our
philosophy is [sic] to constantly improve the products.”
Serious Accusations of Clinical Laboratory Malfeasance
Founded in 2014 with the intent of creating the world’s largest stem cell bank, by 2016, Boston-based Orig3n had refocused its attention on the burgeoning field of direct-to-consumer DNA testing. On its website, Orig3n sells several DNA-testing kits with varying costs.
Orig3n’s attempt to offer free genetic tests to large numbers of people at a professional sporting event in the fall of 2017 may be what caught the attention of federal investigators and led to a deeper investigation. Dark Daily previously covered this controversy, which centered around Orig3n’s plan to distribute free genetic testing kits to fans at a Baltimore Ravens football game.
In that situation, state and federal healthcare regulators blocked the giveaway over concerns about protected health information (PHI). Now, Orig3n is being accused of questionable business practices by 17 of its former employees.
The former employees’ statements that the company’s genetic
testing lab did not follow appropriate test protocols—and that it allegedly
mishandled specimens and even reported false test results—are serious
allegation of malfeasance and warrants an investigation.
Pathologists and clinical laboratory managers know that patient
harm can potentially result from inaccurate genetic test results if used for
clinical purposes. Dark Daily will continue to follow the investigation
into Orig3n.
Use of synthetic genetics to replicate an infectious disease agent is a scientific accomplishment that many microbiologists and clinical laboratory managers expected would happen
Microbiologists and infectious disease doctors are quite familiar with Escherichia coli (E. coli). The bacterium has caused much human sickness and even death around the globe, and its antibiotic resistant strains are becoming increasingly difficult to eradicate.
Now, scientists in England have created a synthetic “recoded” version of E. coli bacteria that is being used in a positive way—to fight disease. Their discovery is being heralded as an important breakthrough in the quest to custom-alter DNA to create synthetic forms of life that one day could be designed to fight specific infections, create new drugs, or produce tools to diagnose or treat disease.
Scientists worldwide working in the field of synthetic genomics are looking for ways to modify genomes in order to produce new weapons against infection and disease. This research could eventually produce methods for doctors—after diagnosing a patient’s specific strain of bacteria—to then use custom-altered DNA as an effective weapon against that patient’s specific bacterial infection.
This latest milestone is the result of a five-year quest by researchers at the Medical Research Council Laboratory of Molecular Biology (MRC-LMB) in Cambridge, England, to create a man-made version of the intestinal bacteria by redesigning its four-million-base-pair genetic code.
The MRC-LMB lab’s success marks the first time a living
organism has been created with a compressed genetic code.
The researchers published their findings in the journal Nature.
“This is a landmark in the emerging field of synthetic
genomics and finally applies the technology to the laboratory’s workhorse
bacterium,” they wrote. “Synthetic genomics offers a new way of life, while at
the same time moving synthetic biology towards a future in which genomes can be
written to design.”
All known forms of life on Earth contain 64 codons—a specific sequence of three consecutive nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis. Jason Chin, PhD, Program Lead at MRC-LMB, said biologists long have questioned why there are 20 amino acids encoded by 64 codons.
“Is there any function to having more than one codon to encode each amino acid?” Chin asked during an interview with the Cambridge Independent. “What would happen if you made an organism that used a reduced set of codons?”
The MRC-LMB research team took an important step toward
answering that question. Their synthetic E. coli strain, dubbed Syn61,
was recoded through “genome-wide substitution of target codons by defined
synonyms.” To do so, researchers mastered a new piece-by-piece technique that
enabled them to recode 18,214 codons to create an organism with a 61-codon
genome that functions without a previously essential transfer RNA.
“Our synthetic genome implements a defined recoding and refactoring scheme–with simple corrections at just seven positions–to replace every known occurrence of two sense codons and a stop codon in the genome,” lead author Julius Fredens, PhD, a post-doctoral research associate at MRC, and colleagues, wrote in their paper.
Science Alert reports that the laboratory-created version of E. coli (above) “isn’t quite a dead ringer for its ancestor. The cells are a touch longer, and they reproduce 1.6 times slower. But the edited E. coli seems healthy and produces the same range and quantity of proteins as the non-edited versions.” (Photo copyright: Jason Chin/STAT.)
Joshua Atkinson, PhD, a postdoctoral research associate at Rice University in Houston, labeled the breakthrough a “tour de force” in the field of synthetic genomics. “This achievement sets a new world record in synthetic genomics by yielding a genome that is four times larger than the pioneering synthesis of the one-million-base-pair Mycoplasma mycoides genome,” he stated in Synthetic Biology.
“Synthetic genomics is enabling the simplification of
recoded organisms; the previous study minimized the total number of genes and
this new study simplified the way those genes are encoded.”
Manmade Bacteria That are Immune to Infections
Researchers from the J.
Craig Venter Institute in Rockville, Maryland, created the first synthetic
genome in 2010. According to an article in Nature,
the Venter Institute successfully synthesized the Mycoplasma mycoides genome
and used it “reboot” a cell from a different species of bacterium.
The MRC-LMB team’s success may prove more significant.
“This new synthetic E. coli should not be able to decode DNA from any other organism and therefore it should not be possible to infect it with a virus,” the MRC-LMB stated in a news release heralding the lab’s breakthrough. “With E. coli already being an important workhorse of biotechnology and biological research, this study is the first time any commonly used model organism has had its genome designed and fully synthesized and this synthetic version could become an important resource for future development of new types of molecules.”
Because the MRC-LMB team was able to remove transfer RNA and
release factors that decode three codons from the E. coli bacteria,
their achievement may be the springboard to designing manmade bacteria that are
immune to infections or could be turned into new drugs.
“This may enable these codons to be cleanly reassigned and
facilitate the incorporation of multiple non-canonical amino acids. This
greatly expands the scope of using non-canonical amino acids as unique tools
for biological research,” the MRC-LMB news release added.
Though synthetic genomics impact on clinical laboratory diagnostics is yet to be known, medical laboratory leaders should be mindful of the potential for rapid innovation in this field as proof-of-concept laboratory innovations are translated into real-world applications.
