Precise template-free correction restores gene function in Tay-Sachs disease while reframing is ineffective.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Tay-Sachs disease is a fatal neurodegenerative disorder caused by mutations in the HEXA gene.
• The c.1278insTATC mutation, a 4-bp duplication, is a common cause of Tay-Sachs disease, leading to frameshift and loss of HexA enzyme function.

Purpose of the Study:

• To investigate the therapeutic potential of CRISPR-Cas9 gene editing for correcting the c.1278insTATC mutation in Tay-Sachs disease.
• To explore the role of microhomology-mediated end joining (MMEJ) in repairing CRISPR-induced double-strand breaks within genomic microduplications.

Main Methods:

• Utilized CRISPR-Cas9 technology in an engineered cell model to target the c.1278insTATC mutation.
• Analyzed repair outcomes, including precise correction and indel formation, following Cas9 endonuclease activity.
• Assessed HexA enzyme activity in cells with edited sequences.

Main Results:

• Cleavage near the c.1278insTATC duplication center spontaneously reconstructed the wild-type sequence with approximately 14.7% frequency.
• Restoration of the wild-type sequence led to the recovery of normal cellular HexA activity.
• Alternative editing strategies aiming to restore the open reading frame without precise correction did not yield significant HexA function.

Conclusions:

• Precise correction of the c.1278insTATC mutation is the only therapeutically relevant outcome observed.
• Microhomology-mediated end joining (MMEJ) is highlighted as a potential template-free CRISPR-Cas9 strategy for precise gene correction in Tay-Sachs disease.