The U.S. Food and Drug Administration’s recent authorization of the inaugural CRISPR-Cas9–based gene therapy signifies a significant achievement in biomedicine, affirming genome editing as a viable treatment approach for conditions such as sickle cell disease, muscular dystrophy, and particular types of cancers.
CRISPR-Cas9, frequently compared to “molecular scissors,” enables researchers to cleave DNA at specific locations to cut, mend, or substitute genes. Yet, in spite of its impressive capabilities, Cas9 presents a substantial safety concern: the active enzyme may persist in cells and induce unintended DNA disruptions — known as off-target effects — potentially instigating detrimental mutations in healthy genes.
Currently, scientists in the laboratories of Ronald T. Raines, an MIT chemistry professor, and Amit Choudhary, a medicine professor at Harvard Medical School, have devised a meticulous method to deactivate Cas9 once its task is complete — considerably minimizing off-target effects and enhancing the clinical safety of gene editing. Their discoveries are articulated in a new study published in the Proceedings of the National Academy of Sciences (PNAS).
“To ‘deactivate’ Cas9 after it fulfills its intended genome-editing purpose, we created the first cell-permeable anti-CRISPR protein system,” remarks Raines, the Roger and Georges Firmenich Professor of Natural Products Chemistry. “Our innovation diminishes the off-target activity of Cas9 and augments its genome-editing precision and clinical applicability.”
The innovative tool — named LFN-Acr/PA — employs a protein-based delivery mechanism to swiftly and effectively transport anti-CRISPR proteins into human cells. While natural Type II anti-CRISPR proteins (Acrs) are known to inhibit Cas9, their therapeutic application has been restricted due to their often excessive size or charge, making them difficult to enter cells, along with the conventional delivery techniques being too slow or ineffective.
LFN-Acr/PA surmounts these challenges utilizing a component derived from anthrax toxin to introduce Acrs into cells in just minutes. Even at picomolar concentrations, the system rapidly and accurately ceases Cas9 activity — enhancing genome-editing specificity by up to 40 percent.
Bradley L. Pentelute, a chemistry professor at MIT, specializes in the anthrax delivery system and is also a co-author of the paper.
The ramifications of this breakthrough are extensive. With patent applications underway, LFN-Acr/PA signifies a quicker, safer, and more controllable method of utilizing CRISPR-Cas9, paving the way for more refined gene therapies with fewer unforeseen consequences.
The research received support from the National Institutes of Health and a Gilliam Fellowship from the Howard Hughes Medical Institute awarded to lead author Axel O. Vera, a graduate student in the Department of Chemistry.