Scientists at Harvard’s Wyss Institute for Biologically Inspired Engineering have transformed CRISPR into a powerful mutation surveillance and disease prevention tool.
In a feat of mutation-sensing might straight from the pages of an X-Men comic book, scientists at Harvard’s Wyss Institute for Biologically Inspired Engineering have adapted CRISPR/Cas-9 gene editing to detect and destroy point mutations linked to diseases like cancer and drug-resistant infections.
The breakthrough is a step toward novel methods for preventing some of the most vexing genetic diseases. In an unintentional nod to the Marvel Comics Universe, the work evokes the mutant-hunting prowess of the Sentinels – an army of giant robots deployed solely to target and kill super-powered mutants like the X-Men.
The X-Men and their ilk are said to derive their special abilities from the X-gene, a fictitious mutation that alters the normal progression of human development in spectacular ways. But the X-gene is also a mutant’s Achilles’ heel. The Sentinels’ powerful remote sensors are attuned to the gene’s unique biological signal, enabling the malevolent androids to isolate a lone mutant in a crowd of people with lethal accuracy.
That’s the general idea behind the Wyss Institute’s recent CRISPR breakthrough, which essentially transforms the technique into a genomic surveillance tool for random single-nucleotide polymorphisms (SNPs) occurring at a single point on a DNA sequence. But unlike the Sentinels, whose purpose was to kill off the entire mutated being, the institute’s new method destroys only the abnormal gene so that the host organism remains healthy.
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Point mutations occur spontaneously but can also be magnified by exposures to chemicals, X-ray or UV radiation, or other environmental stressors. They can trigger the replication of abnormal cells that give rise to a host of debilitating and difficult-to-treat diseases, including certain forms of cancer, color blindness, cystic fibrosis, neurofibromatosis, sickle-cell anemia, and Tay-Sachs disease. They are also a major reason why certain bacteria develop resistance to antibiotics, making it harder for doctors to thwart infectious diseases. But as tiny and random as point mutations may be, researchers like Wyss’s George Church and James Collins – senior investigators on the current study – have shown it may be possible to hunt them down Sentinel-style and stop them in their tracks before they pose a disease risk.
Increased Specificity
In the standard CRISPR/Cas9 method, a small sequence of synthetic RNA is used to guide the Cas9 enzyme to a complementary genetic sequence at a site of interest. There, the enzyme chemically cuts apart the sequence at exactly the desired location. However, one of the ongoing challenges of CRISPR/Cas9 is its occasional propensity to make secondary random cuts at unwanted locations.
The unspecific activity that causes these problems has also hindered the technique’s usefulness in targeting point mutations. The new study sidestepped these issues by focusing on an improved method of guiding the Cas9 enzyme to its genomic target. Fine-tuning the features of the RNA guide helped the Cas9 enzyme to correctly differentiate between two genomic target sites that differed by only one nucleotide, cutting at the undesired site while leaving the normal site intact.
"Our approach dramatically enhances Cas9's specificity up to a level where single nucleotide polymorphisms can be clearly distinguished and unwanted genetic variants erased," Church says. "Our method opens up an entirely new way to think about disease prevention in the future."
Underlying their approach is the evidence that a guide RNA sequence and its target sequence don’t necessarily have to match identically for Cas9 to find the correct site in the DNA. Previous studies show certain mismatches to have no effect on cutting accuracy. That led the group to ponder whether some strategic combination of mismatched guide RNA sequences might direct Cas9 to go after dangerous point mutations while largely ignoring normal sequences.
Beginning with several known point mutations linked to antibiotic resistance in bacterial enzymes, the group then screened multiple guide RNA variants to identify specific guide sequences. The team tested their process in strains of antibiotic-resistant E. coli bacteria, both in controlled laboratory conditions and in mice, which were found to be responding to antibiotic therapy over several days of study.
The Future
As with all new genetic engineering breakthroughs, the mutation prevention approach will require years of further refinement and testing before it is ready for human testing and clinical use. But the belief that the lower cost and wider adoption of next-generation DNA sequencing technology will accelerate its development and scale-up. In the nearer term, the researchers suggest it may find use as a research tool in the study of microbial evolution, or as a means of improving the efficiency of processing large-scale bacteria cultures in the biotechnology industry where point mutations often cause contamination and waste.
Michael MacRae is a technical writer based in Portland, OR.
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