Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene. SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5-NNGRRT-3, than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. Adeno-Associated Virus-DJ (AAV-DJ) is a synthetic serotype made from eight different wild-type AAV serotypes (AAV2, 4, 5, 8, 9, avian, bovine, and goat AAV) using DNA shuffling. These modifications give the AAV-DJ serotype improved transduction efficiency in vitro and in vivo compared to wild-type serotypes. Consequently, AAV-DJ can infect a broad range of cell types.
