In an ideal world, after the CRISPR-Cas9 gene-editing tool enters a cell’s nucleus and cuts its targeted slice of DNA, it would disappear.
“You don’t want the Cas9 protein to stick around too long,” says Krishanu Saha, a professor of biomedical engineering at the University of Wisconsin-Madison who uses gene editing-based tools to develop precision therapies. “The longer Cas9 is in the system, the higher the chance it has to bind to unintended sites in the genome. These off-target sites are areas you didn’t want to edit, causing serious collateral damage we don’t want.”
Currently, there’s no easy way to turn off gene-editing activity. But CRISPR researchers like Saha are experimenting with “molecular-glue degraders” (aka, degrons) to tame the Cas9 protein responsible for cutting DNA—like a dimmer switch for a light.
In a recent paper in the journal Molecular Therapy Nucleic Acids, a UW-Madison research team led by Saha and Namita Khajanchi (PhD BME ’24) demonstrated the effectiveness of one such system in a range of cell lines, including induced pluripotent stem cell-derived neurons. Saha and Khajanchi recently spoke about their work on a podcast produced by the American Society of Gene and Cell Therapy (listen below).
Importantly, their system uses pomalidomide, a drug that’s already approved by the U.S. Food and Drug Administration, to trigger degradation of the Cas9 enzyme. Pomalidomide is usually used to slow the growth of cancerous cells in multiple myeloma, a blood cancer that originates in bone marrow.
The degradation system could be particularly useful for treating heterozygous mutations, in which a patient inherits two different alleles of a given gene, one being a so-called “poison pill mutation” that needs to be edited. That’s the case in two inherited retina diseases that Saha and collaborators are investigating through the National Institutes of Health-funded CRISPR Vision Program.
Conditions that are caused by a repeated segment of DNA, such as Huntington’s disease, are another potential target. And, Saha notes, the ability to turn off gene-editing activity is crucial for controlling genomic instability—which can lead to cancerous cells. These cells pose problems in manufacturing cell therapies and are also a challenge for the precise editing required in CRISPR-enabled drug discovery.
“The degron approach is a platform technology, in that it can be adapted to many types of editors and many types of editing outcomes you might want,” says Saha, whose lab is helping lead a UW-Madison collaboration with the manufacturer Cellares to produce CRISPR-edited CAR-T cells.
The degradation system involves adding a degron tag to the Cas9 protein, so that the addition of pomalidomide triggers machinery in the cell to gobble up the Cas9.
Namita Khajanchi
“The degradation system is kind of like Pac-Man,” says Khajanchi, who now works for the gene-sequencing company Plasmidsaurus in Boston.
The researchers tested their system in human embryonic kidney cells, induced pluripotent stem cells (iPSC), hepatic (liver) cells and, finally, iPSC-derived neurons. They reported a three- to five-fold decrease in editing activity within four hours of induction—a timescale that Saha says is therapeutically relevant—as well as the ability to turn editing back on.
“This platform has the potential to ‘dim’ genome editing in a wide variety of contexts,” says Saha, “not only inside the body, but outside the body, and also has implications for fundamental studies of how genome editing occurs in cells, in tissues, and in animals.”
Kris Saha holds the Retina Research Foundation Kathryn and Latimer Murfee Chair through the McPherson Eye Research Institute at UW-Madison. Funding for this research came from the National Institutes of Health (grants T32 HG002760/HG/NHGRI and R35 GM119644-01), the National Institute of Neurological Disorders and Stroke (grant 1U19NS132296) and the Retina Research Foundation.
Other authors on the paper include Vrusha Patel (BS cell and molecular biology ’25); Ronak Dua (BS microbiology and immunology ’25); Bikash Pattnaik, an associate professor of pediatrics; and Meha Kabra, a scientist in the Pattnaik lab.
Top photo by Joel Hallberg