Study Shows How DNA Repair Pathways Influence CRISPR Precision in Nondividing Cells

A recent study conducted by researchers at Gladstone Institutes, the Innovative Genomics Institute, and UC Berkeley has revealed how DNA repair responses affect CRISPR gene editing in nondividing cells like neurons. Published in Nature Communications, the research highlights that gene editing outcomes in nondividing cells differ substantially from those in dividing cells due to differences in how DNA damage is processed. 

The research team set out to investigate why it has been difficult to use CRISPR-Cas9 to correct genetic diseases in the brain. Their findings show that neurons and other nondividing cells respond differently to gene editing than dividing cells, affecting the way DNA is repaired after Cas9 cuts the genetic material.

According to Bruce Conklin, who led the study, understanding and controlling these DNA repair mechanisms is crucial for creating therapies that yield the right genetic outcomes. “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. Those DNA repair mechanisms are particularly understudied in nondividing cells.”

One approach the researchers used was to generate neurons from induced pluripotent stem cells, which allowed them to compare cells with identical genetic codes but different dividing abilities. They found that the Cas9 protein stays active much longer in neurons than in dividing stem cells—about a month compared to only days. This extended presence of Cas9 in neurons can increase the likelihood of both correct and unintended gene edits, presenting a unique challenge for therapeutic safety.

The outcomes of DNA repair after Cas9 editing were also notably different. In neurons, only a limited set of DNA repair outcomes occurred, making results potentially more controlled and uniform. Additionally, neurons activated specific DNA repair genes previously thought to be inaccessible to nondividing cells, giving scientists new targets for directing how edits are repaired.

To further understand and influence these responses, the researchers collaborated with colleagues to deliver gene editing molecules and accompanying regulators directly into cells using specialized nanoparticles. By combining the editing tools with molecules that modulate DNA repair, the team could guide cells toward more desirable editing results.

This research not only advances the development of gene therapies for neurological diseases but also establishes a framework that could be extended to other nondividing cell types. “Our ultimate goal is to precisely control the gene editing process to deliver life-changing therapies,” Conklin added. “And now, we have important new tools to make sure we get this right.”