Need advice on RNP setup

[Complete_Designer451]

New to CRISPR and I'm curious what worked well for you when doing RNP transfection in stem cells? should the crRNA and tracrRNA before adding Cas9? Anyone doing something different or have tips to share?

 

[nucLe01]

Welcome to the world of CRISPR!
For RNP transfection in stem cells, I've had good success pre-complexing the crRNA and tracrRNA at a 1:1 ratio and incubating for about 5 minutes at room temp before adding the Cas9 protein.
Then, incubate the full RNP complex for another 10-15 minutes.
We use Lipofectamine RNAiMAX, and keeping the cell density precise has been key.
Also, gentle handling during media changes post-transfection seems to improve viability and editing efficiency!

 

[TieProfessional28]

Welcome to the world of CRISPR!
For RNP transfection in stem cells, I've had good success pre-complexing the crRNA and tracrRNA at a 1:1 ratio and incubating for about 5 minutes at room temp before adding the Cas9 protein.
Then, incubate the full RNP complex for another 10-15 minutes.
We use Lipofectamine RNAiMAX, and keeping the cell density precise has been key.
Also, gentle handling during media changes post-transfection seems to improve viability and editing efficiency!

Cool! Question though, for the RNAiMAX, did you have better results using reverse transfection or standard protocols?