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

Tay-Sachs disease is a fatal neurodegenerative disorder caused by mutations inactivating the metabolic enzyme HexA. The most common mutation is c.1278insTATC, a tandem 4-bp duplication disrupting expression by frameshift. In an engineered cell model, we explore the use of CRISPR-Cas9 for therapeutic editing of c.1278insTATC. Within genomic microduplications, the microhomology-mediated end joining (MMEJ) pathway is favored to repair double-stranded breaks with collateral deletion of one repeat. Protospacer adjacent motif (PAM) constraints on Cas9 endonuclease activity prevented cleavage at the duplication center, the optimal position for MMEJ initiation. Rather, cleavage 1 bp from the c.1278insTATC duplication center spontaneously reconstructed the wild-type sequence at ∼14.7% frequency, with concomitant restoration of normal cellular HexA activity. As an alternative to perfect correction, short insertions or deletions were serially introduced to restore an open reading frame across a 19-bp sequence encompassing c.1278insTATC. Frame-restored variants did not recover significant HexA function, presumably due to structural incompatibility of incurred amino acid insertions. Hence, precise correction of c.1278insTATC is the only therapeutically relevant outcome achieved in this study, with MMEJ highlighted as a potential template-free CRISPR-Cas9 modality to that end.