AAV2 SaCas9
Adeno-Associated Virus serotype 2 (AAV2) is the best characterized AAV serotype. Nearly all recombinant AAV serotypes utilize the AAV2 inverted terminal repeats (ITRs). AAV2 requires the expression of Heparan Sulfate Proteoglycan (HSPG) on the surface of host for cells for binding and internalization. Of nearly all the discovered AAV serotypes, AAV2 has the best transduction efficiency in cell culture and is the best tool for in vitro studies.
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. The protein contains a C-terminus HA tag.
AAV was produced in HEK293-AAV cells and is supplied in PBS-MK (PBS Magnesium-Potassium) buffer containing 0.01% Pluronic F68.
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