Huntington’s Disease (HD) is an inherited neurodegenerative disorder, in which a trinucleotide CAG expansion mutation in the huntingtin (HTT) gene results in the production and accumulation of a toxic protein. The resulting neuronal dysfunction gives rise to cognitive, behavioral and motor symptoms, and the disease progresses until death. There are currently no disease-modifying therapies for HD and today’s approved drugs only focus on improving clinical symptoms.
Considering the mutation results in a toxic gain-of-function, the depletion of the mutant HTT (mHTT) expression is a promising therapeutic approach. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is a gene-modifying technology ubiquitously used, whereby an endonuclease (CRISPR associated protein9 - Cas9) specifically targets the gene of interest leading to its inactivation. Consequently, cells react to the cut with a DNA repair mechanism inserting or deleting a few nucleotides. This provokes a frame shifting of the gene coding sequence, giving rise to premature stop codons, inactivating the gene. In order for the technology to work it is essential that once translated, the Cas9 protein is enabled to enter the nucleus, where the target gene is found. For nuclear transportation of large proteins, specialized sequences called Nuclear Localization Sequences (NLS) are needed to tag the specific protein cargo destined to the nucleus and facilitate its passage.
The aim of the project was to improve HTT gene-editing efficiency by enhancing Cas9 nuclear- localization through the use of a more efficient NLS sequence. A cloning strategy was put in place, introducing the bi-partite NLS (BPNLS) in the Cas9 gene. The editing efficiency of both original (Cas9-SV40NLS) and the new construct (Cas9-BPNLS) was compared in Human Embryonic Kidney 293T (HEK293T) cells, HD-derived neural precursor cells (HD-NPCs) and HD- derived neurons. High editing levels were found from both constructs in mitotic cells, yet no significant gene editing efficiency improvement was found using the BPNLS sequence.