Tackling CRISPRs Biggest Challenge Through Novel Delivery System

Gene-editing tool shows promise treating genetic diseases.

Scientists have developed a novel nanoparticle-based delivery system designed to aid CRISPR/Cas9 technology in overcoming difficulties crossing the cell membrane and penetrating its nucleus.

Although this process may seem simple, it can trigger cell defenses that entrap CRISPR/Cas9, ultimately reducing the system’s treatment potential.

This gene editing tool has gained significant interest throughout the research and medical world, with the end goal of treating incurable genetic diseases by manipulating the disease’s genes.

“However, to achieve this biotech and pharmaceutical companies are constantly searching for more efficient CRISPR delivery methods,” said Rubul Mout, lead investigator of a study published in ACS Nano.

The novel delivery method entails engineering the Cas9En protein and carrier nanoparticles, according to the authors.

“By finely tuning the interactions between engineered Cas9En protein and nanoparticles, we were able to construct these delivery vectors,” said investigator Vincent Rotello. “The vectors carrying the Cas9 protein and sgRNA come into contact with the cell membrane, fuse, and release the Cas9:sgRNA directly into the cell cytoplasm.

“Cas9 protein also has a nuclear guiding sequence that ushers the complex into the destination nucleus. The key is to tweak the Cas9 protein. We have delivered this Cas9 protein and sgRNA pair into the cell nucleus without getting it trapped on its way. We have watched the delivery process live in real time using sophisticated microscopy.”

Using the nanoparticle-based delivery system, the investigators say they can deliver the Cas9 protein and sgRNA pair into approximately 90% of cells grown in a culture dish, with an editing efficacy of approximately 30%.

“Ninety percent cytosolic/nuclear delivery is a huge improvement compared to other methods,” Mout said.

In addition to improving CRISPR/Cas9, the investigators believe the Cas9En could also serve as a platform for delivering other materials, such as polymers, lipid nanoparticles, or self-assembling peptides.

“Now that we have achieved efficient gene editing in cultured cells, we are aiming to edit genes in pre-clinical animal models,” Rotello said. “We are also interested in gene editing for adoptive therapies, where a diseased cell is isolated from a patient, corrected by CRISPR in the lab, and delivered back to the patient.”

Investigator Moumita Ray added, “Our method allows the precise monitoring of Cas9 protein movement inside a cell, opening new opportunities in genomic research.”