A modified version of the genome-editing CRISPR-Cas9 system may prevent the production of faulty huntingtin protein, moving closer to treatment for Huntington’s disease.
The study, “Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases,” was published at Frontiers in Neuroscience.
The new gene-editing strategy can remove the CAG (cytosine, adenine, and guanine) repeats in the HTT gene that cause Huntington’s disease and reduce the levels of the faulty protein by about 70 percent.
“In our study we further improve the CRISPR/Cas9 approach by using a nickase version of Cas9,” Marta Olejniczak, PhD, said in a press release. She is associate professor at the Institute of Bioorganic Chemistry, in Poland, and senior author of the study. “Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for Huntington’s disease.”
CRISPR-Cas9 is an innovative method that uses enzymes to introduce breaks in a specific DNA sequence. This can be used to induce the destruction of the sequence, or promote its replacement for a healthy version of the targeted gene.
In the new strategy researchers used paired Cas9 nickases (enzymes) that specifically cut one single DNA strand, increasing its specificity in comparison with previously used gene-editing methods. Using paired enzymes allows researchers to target both DNA strains, flanking and excising the CAG repeat tract in the HTT gene.
The team tested its Cas9 nickases approach in skin cells collected from three patients with different CAG repeat lengths in the HTT gene. They found that the enzymes specifically targeted the CAG repeats, while normal length sequences (smaller than 36 repeats) were not affected.
As a result, the levels of the faulty protein were found to be significantly reduced by 82%, 68%, and 71% in each cell line, respectively.
Importantly, the length of the CAG repeat tract did not influence the excision efficiency of the Cas9 nickases and the strategy was proven specific, as potential off-targets were not affected by the activity of the enzymes.
“We demonstrated that excision of the repeat tract with the use of a Cas9 nickase pair resulted in inactivation of the huntingtin gene and abrogation of toxic protein synthesis in cellular models of Huntington’s disease,” Olejniczak said. “Our strategy is safe and efficient, and no sequence-specific side effects were observed.”
These findings add new knowledge on gene-editing possibilities to achieve enhanced effectiveness and specificity to prevent the production of the toxic versions of the huntingtin protein.
Although this is not yet a cure and further studies are warranted, it may bring researchers one step closer to a potential treatment for Huntington’s and similar genetic diseases.