Hi everyone,
If I have 3 gRNAs, will you suggest to screen in 293 first to identify the "best" one and then only use that one to generate KO in our interest cells? Or the result in one cell line may be not necessarily applied to another? On the other hand, if the knockdown is not evident in bulk cells after transfection, does that mean the chance of generating KO by that gRNA may be low? Thanks for your time.
It is recommended to verify the 3 gRNAs efficiency directly in the target cells. Finding a suitable transfection method for targeting cells may increase transfection efficiency. This may reflect the efficiency of your gRNA with the true efficiency.
1. Check this discussion on RG. She did in vitro testing to find out the 'best' one first, then use the best one for her interested cells..
"Has anyone tested crRNA and tracrRNA in vitro (cell culture)?"
https://www.researchgate.net/post/Has_anyone_tested_crRNA_and_tracrRNA_in_vitro_cell_culture#view=58608ce048954cbb992527e4
2. To your question: "Can a good gRNA efficiency in one cell be necessarily applied to other cells?", the answer is probably NO. According to this attached 2016 paper, the efficiency is different in different cells (Figure 1B).
Hi Jinxin and Yuan-Yeu,
Thanks for your time and suggestion!
In addition, knockout may be partially validated in bulk sorted cells. I should also test that next time.
Best,
Wen An
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