Cre Inducible saCas9/CRISPR AAV

Cas9 is the nuclease guided by the crRNA and tracrRNA (or trans-activating crRNA) to cleave specific DNA sequences. A guide RNA (gRNA) can be designed to include a hairpin that mimics the tracrRNA-crRNA complex. Binding specificity is based on the gRNA and a three nucleotide NGG sequence called the protospacer adjacent motif (PAM) sequence. With its ease in designing guide sequences to target specific genomic loci, the CRISPR/Cas system is a much simpler, faster, and robust alternative to TALEN and Zinc finger nuclease platforms.

The main problem with Cas9 from Streptococcus pyogenes SF370 (SpCas9) is that this gene is too big to be packaged into AAV under most promoters, and there’s no extra room for packaging a sgRNA along with the spCas9 in AAV. A small Cas9 from Staphylococcus aureus subsp. aureus (SaCas9) with high efficiency for cleaving mammalian endogenous DNA becomes the ideal solution to package into AAV for effective gene modification in vivo.

This is an AAV-DJ expressing a Cre-inducible saCas9 under a small CMV7 promoter. The saCas9 is inserted in reverse orientation relative to the 5′ CMV7 promoter and is flanked by oppositely oriented loxP and lox2272 sites. This AAV-CMV-DIO-saCas9 can be package into other AAV serotypes upon request.

We also offer custom AAV production service to package AAV-CMV-DIO-saCas9 with a sgRNA in same AAV virus. Please contact us for detail.