I'm working on a project using CRISPR/dead Cas9 for gene expression. I've seen a lot of discussion and recommendations here for using CRISPR in gene editing or knockouts, but not so much regarding gene expression and silencing. Does anyone have advice for plasmid design or introduction?
We will be using:
-AAV
-HEK293 cells
- Muscle and brain tissue in vivo
I am particularly interested in any design drawbacks you've experienced, as well as recommended transfection methods. Has electroporation been useful? We may also be using ChIP assay on transfected cells and tissues at a later date.
Thanks!
Rachel
hi,
all your answers are in these 2 papers:
For all in vitro methods, lipofectamine 2000/3000 works great.. For in vivo you can use AAv or in vivo transfection (described in the supplementary materials of the Cas9 mouse paper).
You can get the empty plasmids from addgene and design your own gRNAs using the zhang's lab online tool. You can also order the premade plasmids from several companies.
Not sure if you have more specific questions but these papers are a great way to start!
http://www.ncbi.nlm.nih.gov/pubmed/24157548
http://www.ncbi.nlm.nih.gov/pubmed/25748654
Good luck with the AAV vehicle. Cas9 by itself with guide is already at the limit of AAV packaging....so if you plan on making Cas9 fusions with either enhancers/suppressors, you may run into packaging problems down the line....not so much an issue if you're using HEK cells as you can readily transfect them...but moving from HEK to tissue and viral infection will be a bit harder as you may run into expression problems given the constrains of AAV
Thanks Aubin! I'll check them out.
Hi Nicolas - yes, my PI and I are actually attempting to use the staph aureus Cas9 in the hopes that it will be easier to package. We shall see... We're hoping that muscle, at least, will be easier to transfect given its multinucleation.
If you use saCas9 you need to remember that it has a different PAM requirement than the spCas9. Most gRNA design tools will default to spCas9 even if they have other options. We have found that for gene expression we usually need several gRNAs targeting the same promoter in order to get an effect which is not a huge problem in culture but may complicate thing if you want to replicate it in vivo.
Yes, I had to design the gRNA "by hand" to ensure that the gRNA sequence was in a promoter or an enhancer and matched the saCas9 PAM. We've also planned for multiple gRNAs.
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