Hi,
I have the plasmid 41815 from addgene and for in vivo experiment, I have to transfer the whole hCas9 cassette including CMV promoter,Cas9 ORF, NLS and polyadenylation signal in AAV. I am transferring gRNA in separate AAV vector. Can someone suggest me good cloning strategy for transferring the whole hCas9 cassette in AAV?
Feng Zhang's Lab has already created AAV-cas9 vectors See https://www.addgene.org/crispr/zhang/
His lab has created SpCas9 and SaCas9 versions in AAV
Also due to AAV packaging limits the entire CMV promoter will not fit with SpCas9 and poly A signal into AAV. You'd need to truncate it to 250bps or less and your poly A signal/3’UTR needs to be less than 50bps. If you use SaCas9 than you can use the whole CMV promoter, but you need to use gRNAs compatible with SaCas9.
Thank you very much for the reply. I have already gone through the AAV vectors catalogues on the Addgene website but they didn't mention the cloning strategy being used. My professor have his own capsid modified AAV2 vector which shows >90% transduction efficiency in vitro. I want to use the same AAV2 vector construct for my experiment and hence I am looking for strategy to transfer the entire assembly of hCas9 in AAV2 backbone. I already did the in vitro experiment with the SpCas9 and it yielded good result, hence I can't switch to SaCas9 for the in vivo experiment. I have already restricted the CMV sequence to 280 bp including the poly A signal. I will try to assemble the whole cassette of hCas9 in bits may be by golden gate assembly and at the same time will make sure that the transgene size won't exceed the packaging capacity of the AAV. Thanks once again for the reply.
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