Can I transduce HEK293T with Cas9 lentivirus followed by transfection with sgRNAs?

Question

[louisflower1999]

Hi everyone,

Our lab currently has several sgRNAs ordered from IDT, but we are running low on Cas9 protein, and ordering more is relatively costly. To address this, we are considering transducing our cells with lentivirus encoding Cas9 before sgRNA transfection.

Has anyone tried this workflow before—delivering Cas9 via lentivirus followed by sgRNA transfection? If so, I would greatly appreciate any protocols, tips, or references you can share.

Thanks in advance!

[GenoMax]

While someone may answer, this post does not fit the theme of this forum. Please ask this question on biology stackexchange or a relevant reddit.

Answer 1

[jared.andrews07]

You might be interested in the MuLE system, which I've had some success with in the past. It's flexible and pretty easy to use, has multiple selection options/markers, etc.

We made lines with dox-inducible Cas9 and then used this system to introduce the guides, select, induce Cas9 for a few days, then do whatever. You could do it all in one vector if you were okay with constitutive Cas9 expression long term.

The more parts you try to cram in, the more trouble the cloning is, but it was manageable.

There's got to be a billion examples of this in the literature by this point as well. Or just browse the Addgene CRISPR page for options.