Dear All,
I was wondering if anyone has any experience using store bought CRISPR-Cas9-gRNA complexes such as the one listed here;
https://www.scbt.com/scbt/browse/signaling-intermediate-crispr-knockout-and-activation-products/_/N-1e3aawx
I've done some research into it, and my goal is to establish a hiPSC-KO line with a gene of interest. The idea is that these should be optimal already so I can transform the plasmid to last longer. Then later transfect plasmid to hiPSCs, single cell sort for GFP, grow up and later check for presence of gene as one can generate both heterozygous and homozygous lines.
Has anyone done this with these particular products? Any advice about how to approach this?
Many thanks,
Cengiz
The CRISPR plasmids they provide can be transfected using basic lipofection. They do have their own lipofection reagent you can purchase, or you can use one that you already know works well with iPSCs (for example for siRNA I had the most success with the RNAiMAX reagent in stem cells). The other option they have is to purchase the plasmids pre-packaged in lentiviral particles for delivery by infection/transduction (add the virus directly to your cells with or without a cationic surfactant like polybrene and incubate for 2 hours to overnight).
If you are using a CRISPR cutting (which the SCBT seems to use), then the gene should be knocked out. It will not go away (it is not a transient KO). By selecting with Puromycin or GFP, you should have cells that have the KO. CRISPR using active Cas9 to cut the target, so the only way to lose the KO is to have some sort of mutation which causes the indels from your CRISPR to start producing functional protein again. You can also titrate the cells to attain single cells and propagate monoclonal lineages to screen and test to ensure it does have the KO. This will prevent you from having a mixed population in case the selection did not work efficiently.
Thanks for the answers!
I have decided to design gRNA's myself using freeware Benchling, and will go about electroporating hiPSCs and selecting via Puromyocin.
Good luck to you all!
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