dual vector system - in vivo genes editing

I prepare to use Cas9 to do genes knockout in vivo. And I find the dual-vector system that packages SpCas9 (AAV-SpCas9) and sgRNA expression cassettes (AAV-SpGuide) in paper "in vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9" _which use a truncated version of the mouse _Mecp2 promoter in AAV-SpCas9. My questions as below:

1. If my target tissues are not brain, can I replace Mecp2 promoter as other universal promoters like CMV to use this dual vector system in other tissues?

2. Or if I use pX600-AAV-CMV:: NLS-SaCas9NLS-3xHA-bGHpA with AAV-SpGuide together, this dual vector system will work or not?

Please let me know your thoughts and better suggestions for this experiment.

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Pearl Kelpieover 2 years ago

1. You can replace the promoter according to your target tissue, however you have to make sure it is a small promoter similar to the size of the truncated Mecp2 promoter. AAV can only successfully package 4.8kb construct and SpCas9 is very large so you would need a small promoter that can express in your target tissue.

2. The dual vector can work and has been shown to work. In this case you would have to consider whether your can target your sequence of interest successfully using SaCas9 since it has a different PAM sequence to SpCas9.

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