Clinical laboratories involved in genetic testing may find this welcomed news, after a pair of studies conducted in 2019 raised concerns about CRISPR base editing. The researchers of those studies observed that it “causes a high number of unpredictable mutations in mouse embryos and rice,” Chemical and Engineering News (C&EN) reported, adding, “Other groups have raised concerns about off-target mutations caused when the traditional CRISPR-Cas9 form of gene editing cuts DNA at a location that it wasn’t supposed to touch. The results of the new studies are surprising, however, because scientists have lauded base editors as one of the most precise forms of gene editing yet.”
Nevertheless, UC Berkeley’s latest breakthrough is expected to drive development of new and more accurate CRISPR-Cas genome-editing tools, which consist of base editors as well as nucleases, transposases, recombinases, and prime editors.
Understanding CRISPR Base Editors At a ‘Deeper Level’
Harvard University Chemistry and Chemical Biology Professor David Liu, PhD, who co-authored the study, explained the significance of this latest discovery.
“While base editors are now widely used to introduce precise changes in organisms ranging from bacteria to plants to primates, no one has previously observed the three-dimensional molecular structure of a base editor,” he said in a UC Berkeley news release. “This collaborative project reveals the beautiful molecular structure of a state-of-the-art highly-active base editor—ABE8e—caught in the act of engaging a target DNA site.”
UC Berkeley Professor Jennifer Doudna, PhD (above), who served as senior author of the study, says scientists may now have the information necessary to refine base editors and improve their precision and genome-targeting ability. “This structure helps us understand base editors at a much deeper level,” she said in the UC Berkeley statement. “Now that we can see what we’re working with, we can develop informed strategies to improve the system.” (Photo copyright: UC Berkeley.)
Jennifer Doudna, PhD, UC Berkeley Professor, Howard Hughes Medical Institute Investigator, and senior author of the study, has been a leading figure in the development of CRISPR-Cas9 gene editing. In 2012, Doudna and Emmanuelle Charpentier, PhD, Founding, Scientific and Managing Director at Max Planck Unit for the Science of Pathogens in Berlin, led a team of researchers who “determined how a bacterial immune system known as CRISPR-Cas9 is able to cut DNA, and then engineered CRISPR-Cas9 to be used as a powerful gene editing technology.” In a 2017 news release, UC Berkeley noted that the work has been described as the “scientific breakthrough of the century.”
Viewing the Base Editor’s 3D Shape
CRISPR-Cas9 gene editing allows scientists to permanently edit the genetic information of any organism, including human cells, and has been used in agriculture as well as medicine. A base editor is a tool that manipulates a gene by binding to DNA and replacing one nucleotide with another.
According to the recent UC Berkeley news release, the research team used a “high-powered imaging technique called cryo-electron microscopy” to reveal the base editor’s 3D shape.
Genetic Engineering and Biotechnology News notes that, “The high-resolution structure is of ABE8e bound to DNA, in which the target adenine is replaced with an analog designed to trap the catalytic conformation. The structure, together with kinetic data comparing ABE8e to earlier ABEs [adenine base editors], explains how ABE8e edits DNA bases and could inform future base-editor design.”
The graphic above, taken from the UC Berkeley news release, shows the “3D structure of a base editor, comprised of the Cas9 protein (white and gray), which binds to a DNA target (teal and blue helix) complementary to the RNA guide (purple), and the deaminase proteins (red and pink), which switch out one nucleotide for another.” (Image and caption copyright: UC Berkeley.)
Knowing the Cas9 fusion protein’s 3D structure may help eliminate unintended off-target effects on RNA, extending beyond the targeted DNA. However, until now, scientists have been hampered by their inability to understand the base editor’s structure.
“If you really want to design truly specific fusion protein, you have to find a way to make the catalytic domain more a part of Cas9, so that it would sense when Cas9 is on the correct target and only then get activated, instead of being active all the time,” study co-first author Audrone Lapinaite, PhD, said in the news release. At the time of the study, Lapinaite was a postdoctoral fellow at UC Berkeley. She is now an assistant professor at Arizona State University.
“As a structural biologist, I really want to look at a molecule and think about ways to rationally improve it. This structure and accompanying biochemistry really give us that power,” added UC Berkeley postdoctoral fellow Gavin Knott, PhD, another study co-author. “We can now make rational predications for how this system will behave in a cell, because we can see it and predict how it’s going to break or predict ways to make it better.”
Clinical laboratory leaders and pathologists will want to monitor these new advances in CRISPR technology. Each breakthrough has the power to fuel development of cost-effective, rapid point-of-care diagnostics.
‘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.
With $191 million in startup capital, the genomics startup will draw on existing genetic databases to create personalized medicine therapies for chronic diseases
Why do some people get sick while others do not? That’s what genetic researchers at Maze Therapeutics want to find out. They have developed a new approach to using tools such as CRISPR gene editing to identify and manipulate proteins in genetic code that may be the key to providing personalized protection against specific diseases.
If viable, the results of Maze’s research could mean the development of specific drugs designed to mimic genetic code in a way that is uniquely therapeutic to specific patients. This also would create the need for clinical laboratories to sequence and analyze patients’ DNA to determine whether a patient would be a candidate for any new therapies that come from this line of research.
Based in San Francisco, Maze Therapeutics (Maze) is studying modifier genes—genes that affect the phenotype or physical properties of other genes—and attempting to create drugs that replicate them, reported MIT Technology Review. Maze believes that genetic modifiers could afford a “natural form of protection” against disease.
“If you have a disease-causing gene, and I have the disease-causing gene, why is it that you may be healthy and I may be sick? Are there other genes that come into play that provide a protective effect? Is there a drugging strategy to recover normal phenotype and recover from the illness?” Maze Chief Executive Officer Jason Coloma, PhD, asked in an interview with FierceBiotech.
In 2019, Maze received $191 million in financing from Third Rock Ventures, ARCH Venture Partners, and others, to find ways to translate their findings into personalized medicines, according to a news release. And with the availability of international public genetic databases and CRISPR gene editing, now may be good timing.
“This was the perfect time to get into this space with the tools that were being developed and the amount of data that has been accumulated on the human genetic side,” Charles Homcy, MD, Third Rock Ventures Partner and Maze Scientific Founder, told Forbes, which noted that Maze is tapping existing population-wide genetic databases and large-scale studies, including the United Kingdom’s Biobank and Finland’s Finngen.
To help find genetic modifier drug targets, Maze is accessing CRISPR gene editing capabilities. Jonathan Weissman, PhD, Maze Scientific Founder and Professor of Cellular Molecular Pharmacology at University of California, San Francisco (UCSF), told MIT Technology Review: “You take a cell with a disease-causing gene and then see if you can turn it back to normal. We can do 100,000 experiments at once because each cell is its own experiment.”
“At Maze, we are focused on expanding our understanding of the natural disease protection provided by genetic modifiers through an integrated approach that combines studying natural human genetic variation across the globe and conducting large-scale experiments of gene perturbations,” Charles Homcy, MD (above), Founder and interim CEO of Maze and a partner at Third Rock Ventures, said in a news release. “Through our integrated approach, we believe we will create novel medicines based around those modifiers to treat a number of diseases.” (Photo copyright: Forbes.)
Using CRISPR to Identify the Cause of Disease
One drug research program reportedly progressing at Maze involves developing gene therapy for the neurogenerative disease amyotrophic lateral sclerosis (ALS). The program borrows from previous research conducted by Aaron Gitler, PhD, Professor of Genetics at Stanford University and Maze co-founder, which used CRISPR to find genetic modifiers of ALS. The scientists found that when they removed the protein coding gene TMX2 (Thioredoxin Related Transmembrane Protein 2), the toxicity of proteins building the disease was reduced, reported Chemical and Engineering News.
“We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells,” Gitler and colleagues wrote in Nature Genetics. “Together, our results demonstrate the promise of using CRISPR-Cas9 screens in defining the mechanisms of neurodegenerative diseases.”
“We have the flexibility to think differently. We like to
think of ourselves as part of this new breed of biotech companies,” Coloma told
FierceBiotech.
It’s an exciting time. Clinical laboratories can look
forward to new precision medicine diagnostic tests to detect disease and
monitor the effects of patient therapies. And the research initiatives by Maze
and other genetic companies represent a new approach in the use of genetic code
to create specific drug therapies targeted at specific diseases that work best
for specific patients.
The companion diagnostics that may come from this research would
be a boon to anatomic pathology.
These “off-target” genetic alterations demonstrate that certain CRISPR base editors need further refinement in a research finding of interest to pathologists
Could CRISPR
DNA-editing technology unintentionally effect RNA as well? A new study conducted
at Massachusetts General Hospital
(MGH) suggests that it can. Clinical
laboratories doing genetic testing will want to understand why this
research implies that refinements to CRISPR may be needed for it to be accurate
in therapeutic applications.
For years, a huge value of CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) base editors have been their ability to
edit genes or convert a specific DNA base without breaking the DNA. Now, the MGH
scientists have discovered that certain CRISPR base editors may extend beyond
the targeted DNA and perform unwanted edits to RNA, according to a news release.
“Most investigation of off-target base editing has focused
on DNA, but we have found that this technology can induce large numbers of RNA
alterations as well. This surprising finding suggests the need to look at more
than just genetic alterations when considering unintended off-target effects of
base editors in cells,” J. Keith Joung, MD,
PhD, MGH Pathologist and Professor of Pathology at Harvard Medical School, stated in the news release.
The MGH scientists published their study in Nature.
How the MGH Researchers
Found Off-Target Effects on RNA
The researchers had set their sights on developing a base
editor that targets cytosine,
according to the study.
“Previous studies of cytosine base editor specifically have
identified off-target DNA edits in human cells. Here, we show that a cytosine
base editor with rat APOBEC1
[rAPOBEC1] enzyme can cause extensive transcriptome-wide RNA
cytosine deamination in
human cells,” the scientists wrote in Nature.
According to the news
release, when the researchers put base editors into human liver and kidney cells,
they found their technology induced efficient edits at the target DNA site.
However, they also discovered tens of thousands of cytosine-to-uracil edits in the cells. They
found that deaminases, an enzyme that acts as a catalyst, which they used in
their base editor to change DNA, also altered the RNA in the cells, Science reported.
“Base editors are still incredibly powerful tools. This is just another parameter we need to understand,” J. Keith Joung, MD, PhD (above), MGH Pathologist and Professor of Pathology at Harvard Medical School, told Science. (Photo copyright: Massachusetts General Hospital.)
The researchers developed a way to reduce the unwanted RNA
edits, while maintaining the targeted DNA effects. They came up with cytosine
base editor variants, which they dubbed SElective Curbing of Unwanted RNA
Editing (SECURE).
“We engineered two cytosine base editor variants bearing
rAPOBEC1 mutations that substantially decreased the number of RNA edits in
human cells,” the researchers wrote in their study.
However, they also
called for changes to how base editors are used. “For research applications,
scientists using base editors will need to account for potential RNA off-target
effects in their experiments,” the MGH news release notes. “For therapeutic
applications, our results further argue for limiting the duration of base-editor
expression to the shortest length of time possible and the importance of
minimizing and accounting for potential impacts of these effects in safety
assessments.”
Other Studies Explore CRISPR
Other studies published earlier this year on mice and on rice also suggested
that “modified CRISPR-Cas9 technology will need to be further refined before it
can safely be used for research and therapeutic applications,” The Scientist reported.
Clinical laboratory leaders and pathologists recognize
CRISPR technology is changing the way research is done for diagnosing disease
as well as guiding treatment. Dark Daily has reported on key
CRISPR developments over many years.
And now, though the MGH study may appear to be a set-back
for CRISPR, it also may propel further research into possible therapeutic
applications of CRISPR base editing. It’s a development worth watching.
Researchers at UC Berkley developed new ways to use CRISPR as a genetic “search engine” in addition to a cut and paste tool
Clinical pathology laboratory professionals have long been aware of the potential diagnostic properties related to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. Now, new tests using the gene-editing tool show that potential is being realized.
One example involves using CRISPR to detect diseases in Nigeria, where a Lassa fever epidemic has already led to the death of 69 people this year alone. According the journal Nature, this diagnostic test “relies on CRISPR’s ability to hunt down genetic snippets—in this case, RNA from the Lassa virus—that it has been programmed to find. If the approach is successful, it could help to catch a wide range of viral infections early, so that treatments can be more effective and health workers can curb the spread of infection.”
Researchers in Honduras and California are working on similar projects to develop diagnostic tests for dengue fever, Zika, and the strains of human papillomavirus (HPV) that lead to cancer. There’s also a CRISPR-based Ebola test pending in the Democratic Republic of Congo.
These new genetic tests, which may be as simple as at-home
pregnancy tests to use, could save many lives throughout the world. They will
give medical laboratories new tools for diagnosing disease and guiding
therapeutic decisions.
Shift in How
Researchers View CRISPR
“We really think of CRISPR fundamentally as a kind of search engine for biology—like Google for biology—rather than [a kind of] word processing tool, although it’s really good at that too,” Trevor Martin, PhD, co-founder and CEO of Mammoth Biosciences, told CRISPR Cuts, a Synthego CRISPR podcast.
Professor of Biochemistry and Molecular Biology, and Li Ka
Shing Chancellor’s Professor in Biomedical and Health.
Martin’s statement represents a shift in how researchers are thinking about CRISPR. At first, CRISPR was seen as a tool for cutting and pasting genetic material. Scientists could tell it to find a target DNA sequence, make a cut, and paste in something different. However, by thinking of the tool as a search engine, CRISPR’s tremendous diagnostic potential becomes apparent.
“This is a very exciting direction for the CRISPR field to
go in,” Doudna told Nature.
Martin told CRISPR
Cuts that diagnostics is “fundamentally a search problem,” adding, “Now you
can program [CRISPR] to find something, and then tell you that result.”
Doudna notes in Technology Networks that, “Mammoth’s technology exemplifies some of the most urgent, impactful, and untapped potential in the CRISPR space.”
Fehintola Ajogbasile (above), a graduate student at the African Centre of Excellence for Genomics of Infectious Diseases in Nigeria, uses a CRISPR diagnostic test to look for Lassa virus in a blood sample. Similar clinical pathology laboratory tests are becoming available in the US as well. (Photo and caption copyright: Nature/Amy Maxmen.)
Investors See
Economic Benefits of CRISPR
The potential financial and economic impact of simple-to-use CRISPR-based diagnostic tools is considerable. Technology Networks notes that the diagnostics market is estimated at $45 billion, and that venture capital firms Mayfield, First Trust Mid Cap Core AlphaDEX Fund (NASDAQ:FNX), and 8VC have all invested in Mammoth Biosciences.
Although the diagnostics market is huge, a critical aspect
of the Lassa fever diagnostic test the Nigerian researchers are developing is
that it will be as accurate as conventional clinical laboratory testing
methods, but much simpler and less expensive.
Dhamari Naidoo, a technical officer at the World Health Organization (WHO) told Nature that researchers often fail to think about the fact that new technology must be affordable for use in low-income countries.
About a dozen diagnostic tests for Ebola have been
developed, according to Naidoo, but only two have been used recently in the
Democratic Republic of Congo, where the virus is resurging, due to economic
concerns. To be useful, medical laboratory tests in low-income countries must
be affordable to license and distribute, and critically, the manufacturers must
identify a market large enough to motivate them to make and distribute such
diagnostic tests.
Future Directions for
CRISPR and Clinical Pathology
Researchers first discovered what would come to be known as CRISPR in the early 1990s. However, it wasn’t until 2012 – 2013 that scientists used CRISPR and Cas9 for genome editing, a Broad Institute CRISPR timeline notes.
Now, researchers around the world are finding innovative
ways to employ the technology of CRISPR to detect disease in some of the most
remote, challenging areas where diseases such as Lassa fever, Zika, and dengue
fever among others, have devastated the populations, as Dark Daily has previously reported.
What’s next for clinical and pathology laboratories and
CRISPR? We’ll let you know.
