Recent studies have revealed significant discrepancies in the functioning of CRISPR-Cas9 gene-editing technology. Yet we took for granted how differently it would function in neurons versus other nondividing cells. Gokul Ramadoss, a Ph.D. candidate in Bruce Conklin’s lab, was the lead on the study. These results illustrate that CRISPR-Cas9 is able to long term endure and maintain effective expression of its components within neurons, while rapidly losing its editing ability in dividing induced pluripotent stem (iPS) cells.
The research highlights the complex activities of the contentious gene-editing tool CRISPR in various cell types. Jennifer Doudna, a Nobel Prize recipient for her groundbreaking work developing CRISPR, was among the authors of this timely research. Most notably, researchers led by the Broad Institute demonstrated that CRISPR-Cas9 persists with active editing for up to a month in neurons. In actively dividing cells, its activity only extends a few days.
Mechanism of Delivery
The team did their experiments with LNPs. These nanoparticles are uniquely suited for delivering exact quantities of CRISPR-Cas9 molecules into neurons. This groundbreaking technique allows several gene editing tools to be delivered at once. Surrounding it are specialized molecules that target and inhibit DNA repair genes specifically in neurons.
Niren Murthy, a bioengineering professor at UC Berkeley who was instrumental in the research’s development. Together, they co-invented these lipid nanoparticles. Doudna’s lab was pivotal in fabricating the enveloped delivery vehicles. These vehicles make it safe and possible to deliver gene editing molecules accurately into target cells.
“Our findings could have a major impact on how gene editing therapies are designed.” – Bruce Conklin, MD
This unique flexibility is what enables the CRISPR-Cas9 system to make targeted cuts in DNA. It very intentionally oversees the way in which these cuts are restored. The implications of this new capability are profound. Recognizing and controlling DNA repair pathways is key to obtaining the desirable outcomes in genome editing.
Differences in Cell Types
The research reveals that the regulatory landscape for genome editing is very different in dividing versus nondividing cells. As Ramadoss points out, “Most people studying CRISPR have focused on dividing cells, but it turns out the rules of genome editing are different in nondividing cells like neurons.”
The study highlights that various cell types use different strategies to repair DNA damage induced by CRISPR-Cas9. This distinction is important to understand, especially with the growing body of gene editing therapies being developed for many different cell types.
“This is because different cell types repair DNA damage, such as the cuts made by Cas9, in completely distinct ways.” – Gokul N. Ramadoss
Conklin went into great detail about the need to control DNA repair processes following a cut to the genome. He likened the situation to a hypothetical scenario where someone damages a masterpiece: “Imagine if someone slashed the Mona Lisa with a razor. Don’t you want as many people as possible working on the challenge at once? Think of a carpenter with a toolbox that has duct tape, superglue, and a stapler in it. Or would you prefer one fully devoted specialist, who fixes it the same way every time?
Implications for Future Research
The results from this study may have a profound impact on the design of next generation gene editing therapies. This will help researchers understand how CRISPR-Cas9 functions uniquely across different cell types. Armed with this understanding, they’re better able to customize their techniques to create more precise, targeted gene edits in the process.
Ramadoss stated, “You can think of it almost like doing surgery on the genome. Before, we could make an incision but we couldn’t control how the DNA gets stitched up afterward.” Having the capacity to start designing repair tools would allow for increased success of gene therapy applications.
“If we want to ensure genome edits result in the right outcomes, we need to understand and control how the cell’s DNA is repaired after we cut it,” – Bruce Conklin, MD.
“Our ultimate goal is to precisely control the gene editing process to deliver life-changing therapies. And now, we have important new tools to make sure we get this right.”