The ability for scientists to edit human genomic DNA has been around for decades. Such research has revealed how a single mutation in just one of the billions of base pairs in the human genome can cause destructive diseases. In the past decade, advances in CRISPR-Cas9 technology have taken DNA editing a major step farther and promise to revolutionize regenerative medicine. One of the biggest challenges for therapeutic application is the efficiency of precise genome editing. A new study by Junior Associate Professor Akitsu Hotta and colleagues reports a simple but key step in the delivery of CRISPR-Cas9 into iPS cells that enhances this efficiency.

The 2020 Nobel Prize in Chemistry was awarded for the discovery of the CRISPR-Cas9 system. In nature, CRISPR-Cas9 has evolved as a primitive immune system found in bacteria. Biotechnologists have harnessed the power of CRISPR-Cas9 for various genome editing applications beyond medicine, including farming and biofuels.

To edit the genome, Cas9 protein introduces a DNA break at a desired site with the help of a guide RNA (gRNA) molecule. The cut is subsequently repaired by the cell's natural DNA repair system. Scientists can design the CRISPR-Cas9 system so that the repair results in a functional gene.

"To make Cas9 cut at the right place, we deliver it into the cell using plasmids or as a RNP [ribonucleoprotein] complexed with gRNA," explained Dr. Mandy Lung, one of the lead authors of the study. However, the delivery module - plasmids or RNP - was a major determinant of the efficiency.

"Compared to Cas9 and gRNA expressed from plasmids, the cleavage efficiency with RNP was much higher. That led us to think there was some kind of factor blocking the Cas9 activity in the cell," Lung added.

While it is not technically difficult to introduce CRISPR-Cas9 into an iPS cell, the delivery mode results in Cas9 encountering the cell environment differently.

When using RNP, the gRNA and Cas9 bind to form a complex before being delivered. Not so when using plasmids, where the two interact inside the cell. Lung discovered that RNA endogenous to the cell was somehow preventing the gRNA-Cas9 interaction when plasmids were used.

"We are not sure how, but the RNA seems to structurally block the binding site of gRNA on Cas9," she said.

Because RNA is so plentiful in the cell, no matter how much Cas9 was delivered, unless it was already complexed with the gRNA as an RNP, the genome editing was poor. On the other hand, she also found that as an RNP, Cas9 was no longer inhibited by RNA.

Realizing RNP provided better results, the research team applied this finding to two muscle disorders, Miyoshi myopathy and Duchenne muscular dystrophy, both congenital disorders that lead to muscle degeneration. Indeed, RNP was beneficial to correcting the mutations in patient iPS cells.

They also applied this approach to generate universal donor iPS cells that reduce the risk of an immunological response. These cells are being tested as part of a larger project at the CiRA Foundation that will make iPS cells available to the general public for regenerative medicine.

"The observation that RNP delivery leads to higher genome editing efficiency than plasmid delivery is not novel. However, no one had a clear reason for the difference. Finding that RNA can disrupt genome editing will help optimize CRISPR-Cas9 for future therapies," said Hotta.

• doi: 10.1016/j.stemcr.2021.02.013