CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.
Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.
CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.
But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.
Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.
“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.
The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 via the journal Science’s fast-track service, First Release.
The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.
The UC Berkeley researchers tested this strategy using patient samples containing human papillomavirus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.
“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said.
“We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”
The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.
The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.
These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.
The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.
“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.
How Do we Use MRSA Against Itself?
Antibiotic-resistant infections cause more than 30,000 deaths annually in the U.S. alone. The majority of those are caused by methicillin-resistant Staphylococcus aureus, more commonly known as MRSA, which can turn routine medical operations into near-death battles.
MRSA evolved to become a deadly killer because it’s wily and resilient. A new Michigan State University study, however, is figuring out how to turn one of its strengths against it.
“Attacking the cell membrane and inhibiting its ability to produce lipids, or fats, could be an effective treatment protocol,” said Neal Hammer, MSU assistant professor of microbiology and molecular genetics, and senior author of the study that appears in the current issue of the Journal of Bacteriology. “MRSA, though, bypasses the effects of fatty acid inhibitors by absorbing human lipids.”
Antibiotic resistance is a significant challenge in modern medicine. Pathogens encode genes to stay one step ahead, and scientists conduct research that will hopefully stop them, Hammer added.
The study began with the already established fact that MRSA has a genetic hardwired, fat-absorbing pathway. In an evolutionary arms race, MRSA’s ability to absorb human fat and use it as a shield of sorts gives it an advantage. The scientists asked what was the source of the fatty acids in humans?
The answer lay in a simple test that many of us take each year.
Human blood is filled with lipids — good and bad cholesterol — that everyone knew existed but didn’t connect to MRSA. The scientists suggest that MRSA steals these fatty acids and then integrates the lipids into its own cell membrane. This allows it to resist antimicrobials that target fatty acid synthesis. And since there’s plenty of these fatty acids in the blood and liver, MRSA has a veritable endless buffet on which to feast.
“MRSA secretes enzymes, called ‘lipases,’ that free the fatty acids in human LDLs, or bad cholesterol,” Hammer said. “We used mass spectrometry to identify how MRSA was able to perform this feat — the first time this process has been observed.”
Past research laid the groundwork for this discovery. Many of those studies focused on fatty sources found on human skin. This emphasis was due in part to knowing that as much as 30 percent of the world’s population carry MRSA on their skin — without any detrimental health effects.
Now that Hammer’s team is shining the scientific spotlight on how MRSA consumes fatty acids present in the host, future research can focus on these new targets and preventing MRSA from obtaining host fatty acids. This could be a strategy to improve the efficacy of triclosan, an antibacterial agent used in hospitals and found in many household products, as well as other bacterial fatty acid synthesis inhibitors.
The interdisciplinary team of MSU scientists who were part of the study includes: Phillip Delekta, John Shook, Todd Lydic and Martha Mulks.
Does Body Odour Point the Way to Malaria?
Typhoid Mary may have infected a hundred or more people, but asymptomatic carriers of malaria infect far more people every year. An international team of researchers is working toward a way to identify malaria patients including infected individuals who show no malaria symptoms.
People who have malaria but are not symptomatic abound in the heaviest areas of malaria infestation. Even blood tests do not necessarily pick up infection with the plasmodium parasite, especially at low parasite densities. DNA tests for the parasite usually show infection, but they are far from rapid.
“Our previous work in a mouse model found that malaria infection altered the odors of infected mice in ways that made them more attractive to mosquitoes, particularly at a stage of infection where the transmissible stage of the parasite was present at high levels,” said Consuelo De Moraes, adjunct professor of biology, Penn State, and professor of environmental systems science, ETH Zurich. “We also found long-term changes in the odor profiles of infected mice.”
The researchers wanted to see if they could identify changes in human odors associated with malaria infection that might be useful for diagnosing infected individuals. They were particularly interested in identifying those who were infected, but had no symptoms. The researchers initially used microscopy and an SD Bioline Rapid Diagnostic Test to identify patients with malaria. Because these methods have limited sensitivity, particularly when parasite loads are low, infections were confirmed by DNA tests. They identified 333 people who unambiguously were either infected with malaria or were not infected with malaria.
Only if both microscopy and DNA studies were negative were subjects considered malaria-free. Infected patients for the initial studies were both microscopy and DNA positive for malaria. In some later analyses, the researchers included 77 people who were positive for malaria according to DNA, but showed no parasites in the microscopic tests.Malaria infection does not create new volatile chemicals in the body, but alters the amounts — up or down — of volatile chemicals that are already present in the odors of healthy people.
“It is interesting that the symptomatic and asymptomatic infections were different from each other as well as from healthy people,” said Mark C. Mescher, adjunct professor of biology, Penn State, and professor of environmental systems science, ETH Zurich.
This difference among infected, infected asymptomatic, and healthy individuals may eventually lead to tests capable of rapidly and accurately identifying infected people, even those without symptoms.
The researchers report in today’s (May 14) issue of Proceedings of the National Academy of Sciences that predictive models using machine learning reliably identify infection status based on volatile biomarkers. They state “our models identified asymptomatic infections with 100 percent sensitivity, even in the case of low-level infections not detectable by microscopy.” These results far exceed any currently available rapid diagnostic tests.
“But, we should emphasize that we are a long way away from developing a practical diagnostic assay based on odor cues,” said De Moraes.
For a test to succeed it would need to be rapidly and cheaply deployable under field conditions, but still detect infections with high sensitivity.
“In the near term, our goal is to refine the current findings to find the most reliable and effective biomarkers we can,” said Mescher. “This is really basic science to identify the biomarkers of malaria. There is still a lot more work to be done to develop a practical diagnostic assay.”
The Common Cold: Could we be Close to a Cure
The appropriately named common cold strikes the average adult two to three times per year, and children even more regularly.
Currently, there is no way to prevent a common cold, and once it has arrived, there is no way to get rid of it.
Despite the impressively high-tech world we are living in, medical research cannot yet defeat this foe. All we can do is treat its symptoms and hold tight until it has passed.
Why is the common cold difficult to tackle?
The common cold has evaded medical science’s advances for two primary reasons. The first issue is that there is not just one single culprit. Colds are most often caused by a rhinoviruses — a large family of viruses with hundreds of variants. This makes vaccination an impossibility and gives our immune system a challenging task.
Secondly, these viruses evolve rapidly — so even if we could produce vaccines to cover the full spectrum of rhinoviruses, they would quickly become resistant.
Although dealing with a cold is not a huge issue for most people, there are good reasons to keep hunting for ways to fight it. One person involved in the hunt is Prof. Ed Tate, of Imperial College London in the United Kingdom. He explains the importance of battling the common cold:
“The common cold is an inconvenience for most of us, but can cause serious complications in people with conditions like asthma and [chronic obstructive pulmonary disease].”
A new approach
The scientists were initially looking for a compound that would target a protein in malaria parasites. They found two likely molecules and discovered that they were most effective when they were combined.
Using advanced techniques, they combined the two molecules and produced a new compound that blocks an enzyme found in human cells, called N-myristoyltransferase (NMT).
Viruses normally steal NMT from human cells and use it to create a protective shell around their genetic information, known as the capsid. NMT is vital for the survival of cold viruses; without it, they cannot replicate and spread.
All strains of the common cold virus use this technique, so inhibiting NMT would scupper all strains of common cold virus. In fact, it should also work against the related viruses that cause foot-and-mouth disease and polio.
Also, because the molecule targets human cells rather than the virus, resistance would not be an issue. The team’s findings were recently published in the journal Nature Chemistry.
The researchers have high hopes for the drug, which currently goes under the codename of IMP-1088.
“A drug like this could be extremely beneficial if given early in infection, and we are working on making a version that could be inhaled so that it gets to the lungs quickly.”
Prof. Ed Tate
Though other drugs that target human cells in this way have been trialed before, IMP-1088 is “more than 100 times more potent” than its predecessors.
Also, earlier drugs designed to block NMT were too toxic to be of benefit. This new drug, however, did not damage cultured human cells. Of course, more research will be needed to confirm that the drug is safe for use.
Another concern is outlined by Prof. Tate, who explains, “The way the drug works means that we would need to be sure it was being used against the cold virus, and not similar conditions with different causes, to minimize the chance of toxic side effects.”
So, we are not there yet, but we are as close as we have ever been to a cure for the common cold.
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