Single-AAV Delivery of the SpCas9 Nickase Improves CAG/CTG Repeat contraction in a mouse model of Huntington's disease

The best studied Cas9 enzyme, from Streptococcus pyogenes, is thought to be too large to be efficiently packaged into a single adeno-associated virus (AAV) together with a single-guide (sg) RNA cassette, requiring a dual-vector system. Here we have circumvented this limitation and packaged the full CRISPR system into a single AAV by using a single promoter driving the expression of both the Cas9 enzyme and a sgRNA. We compared the ability of the dual and single vector systems to induce contraction of CAG/CTG repeats in the striatum of a Huntington's disease model. We show that the all-in-one system led to larger contractions of the CAG/CTG repeats. This approach provides a more efficient and less costly approach for in vivo gene editing and improves the translational potential of AAV-mediated delivery of Cas9.