Since its initial use as a gene editing tool less than a decade ago, CRISPR has become a very attractive prospect to researchers looking to investigate or alter the structure of an organism’s genome. The technology’s potential in treating a host of genetic diseases mean that it has been of particular interest within the medical community, but as many people have found out, CRISPR in practice is not always that easy. In truth, there are many problems that have to be overcome before CRISPR can be considered as a therapeutic option.

Delivering CRISPR To Cells

CRISPR-Cas9 /Ernesto del Aguila III, National Human Genome Research Institute, National Institutes of Health

One of these problems is our limited ability to introduce the components of the CRISPR-Cas9 system to the nucleus of the cell so that it can perform the desired edits. The system is made up of two different molecular components: a protein called Cas9 which can cleave DNA, and an RNA molecule called gRNA (or sgRNA) that guides the protein to the right locus in the genome. If either of these components are not present in the nucleus, gene editing cannot be achieved and the reaction will fail. In the past, attempts at introducing them both have triggered the natural defences of the cell, ‘trapping’ the components before they can reach the nucleus and significantly reducing the treatment potential.

New research published in ACS Nano from the University of Massachusetts Amherst suggests that the solution might lie in nanochemistry. A group of researchers from Vincent Rotello’s laboratory have designed a novel delivery system that utilises nanoparticles to move CRISPR-Cas9 components across the cell membrane and to the nucleus without triggering defensive cellular machinery.

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The delivery method involves engineering the Cas9 protein, dubbed Cas9En, and nanoparticles to act as carriers.

“By finely tuning the interactions between engineered Cas9En protein and nanoparticles, we were able to construct these delivery vectors. 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,” Rotello, a nanochemistry expert and co-author of the paper, has said. “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.”

The team report that their delivery system can deliver Cas9 and sgRNA into roughly 90% of cells grown in a culture dish, achieving an editing efficiency of around 30%. Rubul Mout, the experiment leader, said, “Ninety percent cytosolic/nuclear delivery is a huge improvement compared to others methods.”

While this investigation focussed on introducing CRISPR components cells, the technique may have implications in other areas of biology, such as delivery platforms for polymers or self-assembling peptides. The research team intend to further develop their system in subsequent work.

“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.”