Single-AAV Delivery of SpCas9 Efficiently Edits CAG/CTG Repeats In An In Vivo Huntington Disease Model.
The best studied Cas9 enzyme, from Streptococcus pyogenes, is too large to be efficiently packaged into a single adeno-associated virus together with a single-guide (sg) RNA cassette, requiring a dual-vector system. To circumvent this limitation, here we packaged the full CRISPR system in a single vector by using a single promoter driving the expression of both the Cas9 enzyme and a sgRNA. We compared striatal injections of a dual vector system composed of the Cas9 nickase and a sgRNA against the repeat tract to our all-in-one system in their ability to contract CAG/CTG repeats that cause Huntington disease. We show that the all-in-one system led to more contractions of the CAG/CTG repeat in medium spiny neurons of a R6/1 mouse model compared to the dual system. This approach, therefore, provides a more efficient and less costly approach for in vivo gene editing and improves the translational potential of AAV-mediated delivery of Cas9.