I have been trying to knock down my protein in A549 cells using CRISPR CAS9 system ,i did exactly as the protocol within the minimum range of both the plasmid and Transfecting reagent
but the cells are dying, around 70% mortality
Did anyone experience this problem? if so? what was the solution?
I am using Santacruz CRISPR System Transfection Protocol:
-The cells are A549 lung cancer, they were around 80 % confluence grown in antibiotic free normal media
-I diluted the plasmid using supplied deionized water " Plasmid DNA: Resuspend the lyophilized Plasmid DNA in 200µl of deionized water (Final concentration 0.1µg/µl)
-I used 2µg (around 20µl) Plasmid/DNA ,and 7µl of the UltraCruz transfection reagent, both diluted separately in the supllied Transfection media
Thank you Andrei Nikonov for you advice, actually i have already started the "mock transfection" to determine whether it is the Transfection agent or the plasmid
In my lab, we have ever tried KO cell lines using CRISPR/CAS9 as well as TALEN. Similar to you, we faced massive cell death after transfection. In control transfection, we had no observation for massive cell death. However, transfection of CRISPR/Cas9 or TALEN against the specific gene gave rise to uncontrolled cell death, nearly 80~90%. After all, we concluded that the specific gene of our interests is very essential for cell survival and then its complete knocking out leads to inevitable cell death.
So, if your problem is not due to the purity of DNA or to transfection reagents, it may result from your own gene of interest.
Hi Muataz, can you update us? I have also tried CRISPR from Santa Cruz. My cells are also dying after 2-3 days, CRISPR negative control cells were also dead. I also used lung cancer cell line. I wonder you were able to fix the problem. I would be happy if you can share your protocol. Thanks!
yes update seriously needed! I'm facing the same dilemma now although with glioblastoma cells not lung cancer cells. I'l try the mock transfection and see how that goes.
Same problem, same reagents, but with pancreatic cancer cells instead, anyone have an update?
Why are you guys using a kit? Make your own vectors (super easy) and transfect your cells as you normally would.
hello Muataz,
I was reading the comments and problems with santacruz crispr kit. I have been recently using the kit too. Could you figure out the solution for dying cells post transfection as I have same problem with 70% mortality.
hello Fatema Khambati
Actually i have contacted santacruz regarding this problem , and they did not provide me neither with an explanation nor a solution, and even after i changed the conditions and the cell lines, i got the same results ( 70 % mortality ) .
Finally , i changed my approach and started using siRNA, my advice is either to change the kit ( look for other source) or change the method
Surprising !
I suggest you to titrate the Ultracruz transfection reagent from 5 - 15 ul while keeping the plasmid DNA conc. 1 ug. Wait for 72 h, before actually select in puromycin medium. Again, puromycin conc. to be titrated as well ranging from 1-10 µg/ml. If you still have problem, then i suggest you to try Fugene 6 transfection reagent. All the best !!!
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