I am doing CRISPR/Cas9 experiment on E. coli. I am introducing recombinant plasmid BPK764 (which carries Cas9 + sgRNA designed and added later in that plasmid) into compentent E.coli cells which already carries another plasmid with GFP gene. sgRNA will be designed so that it corresponds to that GFP gene becouse my main point is to cut out GFP gene. The main result of this experiment will be E.coli cells that do/don't express GFP gene (depending on CRISPR/Cas9 efficacy), therefore, the resultant cells will or hoply won't light green. I will see that under UV lamp, and by fluorescent microscopy.

Now I'm getting to my question. Is there something on those resultant cells (some trait, like membrane structure, youngs modulus, that differs from CRISPR untreated cells) that can be visualized on AFM (atomic force microscopy)? Something that changes on cells after inserting plasmid that can confirm efficacy of this experiment?

Or maybe some other idea on this experiment using AFM?

  • 3
    $\begingroup$ Welcome to Biology.SE, Mila99. I think there is a good question here, but it's not entirely clear. Are you asking for a suggestion for a control that you can use to evaluate whether the plasmid has been successfully incorporated? Does it need to involve AFM? $\endgroup$ – De Novo Mar 21 '19 at 17:52

Unless you're specifically targeting genes involved in the physical morphology of the cell, I doubt you would find any differences with CRISPRed vs non-CRISPRed cells that is measurable via AFM.

Perhaps you should instead target genes known to contribute to the structure of the cell wall, which I would expect to be the dominant material contributing to the Young's modulus. Since you mentioned GFP as a tool for selecting for "successful" cuts, it might be worthwhile looking into generating a multiplex plasmid that allows you to target several loci with a single construct. Such kits are available on Addgene and contain associated publications and manuals on how they can be used.

So to summarize, no, I don't think there is anything physically different about cells that have been CRISPR treated compared to a control group. If you did want to use CRISPR to effect a knockout of the fluorescence in the competent cells you described and alter the cell mechanics, you would likely have to generate a multiplex CRISPR plasmid with multiple gRNA cassettes for both GFP and perhaps some gene involved in cell wall formation. Keep in mind that your efficiency percentage is compounded with each new target in the same multiplex – instead of a single cut being deemed successful, you will have raised the bar to 2 cuts being deemed successful without doing anything to improve the efficiency of a single cutting event.

Hope this helps, and I'd be happy to discuss this more.

| improve this answer | |

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.