All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo.

BACKGROUND: Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.

RESULTS: Here, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded > 35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.

CONCLUSIONS: Our findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.