Precise Genome Editing in vivo with Single, Canonical AAV Vectors. Encoding Guide RNA, Cas9, and Homologous Repair Donor

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes. Overall design: This study consists of 59 samples total. FAH and HPD were the gene targets for our samples, which include multiple replicates for each sample and also negative controls (PBS, scramble guide RNA, and scramble donor DNA). Libraries were made for the following mice: 3 PBS treated Fahneo/neo, 8 dual-sgRNA Design 1 treated Fahneo/neo, 8 dual-sgRNA Design 4 treated Fahneo/neo, 3 healthy C57BL/6, 4 dual-sgRNA Design 1 treated C57BL/6, 4 dual-sgRNA Design 4 treated C57BL/6, 3 PBS treated FahPM/PM, 3 rAAV:HDR:FahDonor:ncSpacer treated FahPM/PM, 3 rAAV:HDR:ncDonor:FahSpacer treated FahPM/PM, 10 rAAV:HDR:uncleaved treated FahPM/PM, and 10 rAAV:HDR:cleaved treated FahPM/PM