Deletion in the AAV plasmid from Ran et al.(2015)

[chissay23]

Hello, 

I'd like to use the plasmid (Addgene plasmid # 61591) from Ran et al.(2015) for AAV production (pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA).

I found an 11 bp-deletion within the 5’-ITR of AAV2 plasmid comparing to the wt AAV-2 ITR.

The part is missing in the electronic sequence and the deletion is confirmed by sequencing.

I'd like to know if this could affect the virus titer?

It would be great if you could tell me how high were the titers you obtained using this plasmid with the deletion?

Thank you in advance for any information.

Cheers,

Natasa

[Winston Yan]

Hi Natasa,

Thank you for your question and interest! We referenced the 5' and 3' ITRs from here:

http://www.cellbiolabs.com/sites/default/files/VPK-410-aav-expression-vector.pdf

As you can see the L-ITRs and R-ITRs are slightly different. We don't believe it interfered with packaging or transduction.

With our AAV8 packaging plasmids we were able to obtain titers of ~2-8 * 10^12 viral particles  (e.g., 800uL of 5*10^12 GC/mL) made using 10x 10cm dishes of triple transfected HEK 293FT cells. I should add that this was made for us by a local viral core, the kind folks from the Vandenberghe Lab at the Mass Eye and Ear Infirmary (http://www.harvardvector.org/)

Please let us know if you have any other questions,

Winston

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[chissay23]

Hi Winston,

Thanks a lot for the prompt and detailed answers to my questions.

All the best,

Natasa