New gene-editing systems could provide markedly improved accuracy for DNA and RNA editing leading to new precision medicine tools and genetic therapies
In what may turn out to be a significant development in genetic engineering, researchers from three institutions have identified nearly 200 new systems that can be used for editing genes. It is believed that a number of these new systems can provide comparable or better accuracy when compared to CRISPER (Clustered Regularly Interspaced Short Palindromic Repeats), currently the most-used gene editing method.
CRISPR-Cas9 has been the standard tool for CRISPR gene editing and genetic engineering. However, publication of these new research findings are expected to give scientists better, more precise tools to edit genes. In turn, these developments could lead to new clinical laboratory tests and precision medicine therapies for patients with inherited genetic diseases.
“Best known as a powerful gene-editing tool, CRISPR actually comes from an inbuilt defense system found in bacteria and simple microbes called archaea. CRISPR systems include pairs of ‘molecular scissors’ called Cas enzymes, which allow microbes to cut up the DNA of viruses that attack them. CRISPR technology takes advantage of these scissors to cut genes out of DNA and paste new genes in,” according to Live Science.
In its article, New Atlas noted that the researchers looked to bacteria because “In nature, CRISPR is a self-defense tool used by bacteria.” They developed an algorithm—called FLSHclust—to conduct “a deep dive into three databases of bacteria, found in environments as diverse as Antarctic lakes, breweries, and dog saliva.”
In their paper, the researchers wrote, “We developed fast locality-sensitive hashing–based clustering (FLSHclust), a parallelized, deep clustering algorithm with linearithmic scaling based on locality-sensitive hashing. FLSHclust approaches MMseqs2, a gold-standard quadratic-scaling algorithm, in clustering performance. We applied FLSHclust in a sensitive CRISPR discovery pipeline and identified 188 previously unreported CRISPR-associated systems, including many rare systems.”
“In lab tests [the newfound CRISPR systems] demonstrated a range of functions, and fell into both known and brand new categories,” New Atlas reported.
“Some of these microbial systems were exclusively found in water from coal mines,” Soumya Kannan, PhD (above), a Graduate Fellow at MIT’s Zhang Lab and co-first author of the study, told New Atlas. “If someone hadn’t been interested in that, we may never have seen those systems.” These new gene-editing systems could lead to new clinical laboratory genetic tests and therapeutics for chronic diseases. (Photo copyright: MIT McGovern Institute.)
Deeper Look at Advancement
The CRISPR-Cas9 made a terrific impact when it was announced in 2012, earning a Nobel Prize in Chemistry.
Though CRISPR-Cas9 brought huge benefits to genetic research, the team noted in their Science paper that “existing methods for sequence mining lag behind the exponentially growing databases that now contain billions of proteins, which restricts the discovery of rare protein families and associations.
“We sought to comprehensively enumerate CRISPR-linked gene modules in all existing publicly available sequencing data,” the scientist continued. “Recently, several previously unknown biochemical activities have been linked to programmable nucleic acid recognition by CRISPR systems, including transposition and protease activity. We reasoned that many more diverse enzymatic activities may be associated with CRISPR systems, many of which could be of low abundance in existing [gene] sequence databases.”
Among the previously unknown gene-editing systems the researchers found were some belonging to the Type 1 CRISPR systems class. These “have longer guide RNA sequences than Cas9. They can be directed to their targets more precisely, reducing the risk of off-target edits—one of the main problems with CRISPR gene editing,” New Atlas reported.
“The authors also identified a CRISPR-Cas enzyme, Cas14, which cuts RNA precisely. These discoveries may help to further improve DNA- and RNA-editing technologies, with wide-ranging applications in medicine and biotechnology,” the Science paper noted.
Testing also showed these systems were able to edit human cells, meaning “their size should allow them to be delivered in the same packages currently used for CRISPR-Cas9,” New Atlas added.
Another newfound gene-editing system demonstrated “collateral activity, breaking down nucleic acids after binding to the target, New Atlas reported. SHERLOCK, a tool used to diagnose single samples of RNA or DNA to diagnose disease, previously utilized this system.
Additionally, New Atlas noted, “a type VII system was found to target RNA, which could unlock a range of new tools through RNA editing. Others could be adapted to record when certain genes are expressed, or as sensors for activity in cells.”
Looking Ahead
The strides in science from the CRISPR-Cas9 give a hint at what can come from the new discovery. “Not only does this study greatly expand the field of possible gene editing tools, but it shows that exploring microbial ecosystems in obscure environments could pay off with potential human benefits,” New Atlas noted.
“This study introduces FLSHclust as a tool to cluster millions of sequences quickly and efficiently, with broad applications in mining large sequence databases. The CRISPR-linked systems that we discovered represent an untapped trove of diverse biochemical activities linked to RNA-guided mechanisms, with great potential for development as biotechnologies,” the researchers wrote in Science.
How these newfound gene-editing tools and the new FLSHclust algorithm will eventually lead to new clinical laboratory tests and precision medicine diagnostics is not yet clear. But the discoveries will certainly improve DNA/RNA editing, and that may eventually lead to new clinical and biomedical applications.
CRISPR-Cas9 connection to cancer prompts research to investigate different approaches to gene editing
Dark Daily has covered CRISPR-Cas9 many times in previous e-briefings. Since its discovery, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, has been at the root of astonishing breakthroughs in genetic research. It appears to fulfill precision medicine goals for patients with conditions caused by genetic mutations and has anatomic pathologists, along with the entire scientific world, abuzz with the possibilities such a tool could bring to diagnostic medicine.
All of this research has contributed to a deeper understanding of how cells function. However, as is often the case with new technologies, unforeseen and problematic questions also have arisen.
“Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors, and a human differentiated cell line,” wrote the authors in their introduction.
Another study, this one conducted by biomedical researches at Cambridge, Mass., and published in Nature, describes possible toxicity caused by Cas9.
“Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR-Cas9 for genome engineering and screening in hPSCs [human pluripotent stem cells]. Moreover, as hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR-Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.”
Essentially what both groups of researchers found is that CRISPR-Cas9 cuts through the double helix of DNA, which the cell responds to as it would any injury. A gene called p53 then directs a cellular “first-aid kit” to the “injury” site that either initiates self-destruction of the cell or repairs the DNA.
Therefore, in some instances, CRISPR-Cas9 is inefficient because the repaired cells continue to function. And, the repair process involves the p53 gene. P53 mutations have been implicated in ovarian, colorectal, lung, pancreatic, stomach, liver, and breast cancers.
Though important, some experts are downplaying the significance of the findings.
Erik Sontheimer, PhD (above), Professor, RNA Therapeutics Institute, at the University of Massachusetts Medical School, told Scientific American that the two studies are important, but not show-stoppers. “This is something that bears paying attention to, but I don’t think it’s a deal-breaker,” he said. (Photo copyright: University of Massachusetts.)
“It’s something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous,” Sam Kulkarni, PhD, CEO of CRISPR Therapeutics, told Scientific American.
Both studies are preliminary. The implications, however, is in how genes that have become corrupted are used.
A team from the Salk Institute may have found a solution. They are investigating a different enzyme—Cas13d—which, in conjunction with CRISPR would target RNA rather than DNA. “DNA is constant, but what’s always changing are the RNA messages that are copied from the DNA. Being able to modulate those messages by directly controlling the RNA has important implications for influencing a cell’s fate,” Silvana Konermann, PhD, a Howard Hughes Medical Institute (HHMI) Hanna Gray Fellow and member of the research team at Salk, said in a news release.
The Salk team published their findings in the journal Cell. The paper describes how “scientists from the Salk Institute are reporting for the first time the detailed molecular structure of CRISPR-Cas13d, a promising enzyme for emerging RNA-editing technology. They were able to visualize the enzyme thanks to cryo-electron microscopy (cryo-EM), a cutting-edge technology that enables researchers to capture the structure of complex molecules in unprecedented detail.”
The researchers think that CRISPR-Cas13d may be a way to make the process of gene editing more effective and allow for new strategies to emerge. Much like how CRISPR-Cas9 led to research into recording a cell’s history and to tools like SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), a new diagnostic tool that works with CRISPR and changed clinical laboratory diagnostics in a foundational way.
Each discovery will lead to more branches of inquiry and, hopefully, someday it will be possible to cure conditions like sickle cell anemia, dementia, and cystic fibrosis. Given the high expectations that CRISPR and related technologies can eventually be used to treat patients, pathologists and medical laboratory professionals will want to stay informed about future developments.
SHERLOCK makes accurate, fast diagnoses for about 61-cents per test with no refrigeration needed; could give medical laboratories a new diagnostic tool
The tool is called SHERLOCK, which stands for (Specific High-sensitivity Enzymatic Reporter unLOCKing). And it is causing excitement in the scientific community for several reasons:
It can detect pathogens in extremely small amounts of genetic matter;
Tests can be performed using urine and/or saliva rather than blood;
The tests are extremely sensitive; and they
Cost far less than the diagnostic tests currently in use.
How SHERLOCK and CRISPR Differ and Why That’s Important
Scientists have long suspected that CRISPR could be used to detect viruses. However, far more attention has been given to the its genome editing capabilities. And, there are significant differences between how CRISPR and SHERLOCK work. According to the Science article, when CRISPR is used to edit genes, a small strip of RNA directs an enzyme capable of cutting DNA to a precise location within a genome. The enzyme that CRISPR uses is called Cas9 (CRISPR associated protein 9). It works like scissors, snipping the strand of DNA, so that it is either damaged or replaced by a healthy, new sequence.
SHERLOCK, however, uses a different enzyme—Cas13a (originally dubbed C2c2 by the researchers who discovered it). Cas13a goes to RNA, rather than DNA, and once it starts cutting, it doesn’t stop. It chops through any RNA it encounters. The researchers who developed SHERLOCK describe these cuts as “collateral cleavage.” According to an article published by STAT, “All that chopping generates a fluorescent signal that can be detected with a $200 device or, sometimes, with the naked eye.”
The screenshot above is from a video in which Feng Zhang, PhD (center), a Core Member of the Broad Institute at MIT and one of the lead researchers working on SHERLOCK, and his research team, explain the difference and value SHERLOCK will make in the detection of diseases like Zika. Click on the image above to watch the video. (Video copyright: Broad Institute/MIT.)
Early Stage Detection in Clinical Laboratories
A research paper published in Science states that SHERLOCK can provide “rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity.” Attomolar equates to about one part per quintillion—a billion-billion. According to the article on the topic also published in Science, “The detection sensitivity of the new CRISPR-Cas13a system for specific genetic material is one million times better than the most commonly used diagnostic technique.” Such sensitivity suggests that clinical laboratories could detect pathogens at earlier stages using SHERLOCK.
The Stat article notes that, along with sensitivity, SHERLOCK has specificity. It can detect a difference of a single nucleotide, such as the difference between the African and Asian strains of Zika (for example, the African strain has been shown to cause microcephaly, whereas the Asian strain does not). Thus, the combination of sensitivity and specificity could mean that SHERLOCK would be more accurate and faster than other diagnostic tests.
Clinicians in Remote Locations Could Diagnose and Treat Illness More Quickly
Perhaps one of the most important aspects of SHERLOCK is the portability and durability of the test. It can be performed on glass fiber paper and works even after the components have been freeze dried. “We showed that this system is very stable, so you can really put it on a piece of paper and it will survive. You don’t have to refrigerate it all the times,” stated Feng Zhang, PhD, in an interview with the Washington Post. Zhang is a Core Member of the Broad Institute at MIT and was one of the scientists who developed CRISPR.
The researchers note that SHERLOCK could cost as little as 61-cents per test to perform. For clinicians working in remote locations with little or no power, such a test could improve their ability to diagnose and treatment illness in the field and possibly save lives.
“If you had something that could be used as a screening test, very inexpensively and rapidly, that would be a huge advance, particularly if it could detect an array of agents,” stated William Schaffner, MD, Professor and Chair of the Department of Preventive Medicine at Vanderbilt University Medical Center, in the Post article. Schaffner describes the Broad Institute’s research as being “very, very provocative.”
The test could radically change the delivery of care in more modern settings, as well. “It looks like one significant step on the pathway [that] is the Holy Grail, which is developing point-of-care, or bedside detection, [that] doesn’t require expensive equipment or even reliable power,” noted Scott Weaver, PhD, in an article on Big Think. Weaver is a Professor and Director at the Institute for Human Infections and Immunity University of Texas Medical Branch in Galveston, Texas.
Just the Beginning
Anatomic pathologists and clinical laboratories will want to follow SHERLOCK’s development. It could be on the path to fundamentally transforming the way disease gets diagnosed in their laboratories and in the field.
According to the Post article, “The scientists have filed several US patent applications on SHERLOCK, including for uses in detecting viruses, bacteria, and cancer-causing mutations.” In addition to taking steps to secure patents on the technology, the researchers are exploring ways to commercialize their work, as well as discussing the possibility of launching a startup. However, before this technology can be used in medical laboratory testing, SHERLOCK will have to undergo the regulatory processes with various agencies, including applying for FDA approval.
