Hello Mishael.

That's due to the presence of non-target guide RNAs even if target guide RNAs are present.

Please read the article (listed) through and Nature paper to understand this evolving field.

https://greenfluorescentblog.wordpress.com/2017/10/09/new-cripsr-based-rna-imaging-tool/

Best wishes.

Thank you so much Sang lee. That was so helpful.

I forgot to mention one thing. I used gRNA that target DNAJA1 with high specificity. The sequence of the gRNA was obtained from a library that predicates highly effective single guide RNAs with minimal off targets.

Dear Mishael,

Not all expressed dCas9 will go into the nucleus even if it have multiple NLS. Generally, shuttling the protein to the nucleus follows an active balance involving nuclear pore import receptors. Therefore if you overexpress a lot of the NLS-tagged protein some will remain cytosplasmic and this pool may increase further with the increased overexpression.

How long did your knockdown experiment last? Are you sure that the turnover of the protein you want to knockdown is short enough to match the time you gave to the experiment to allow seeing the protein decrease?

Perhaps your cells need more time achieve an effective knockdown or it also may happen that the gRNAs you use are more effective in some cell types than in others. To test this last point, try the same approach in different cell types.

Hope it may help you.

Sebastián Dupraz

First of all thank you so much for your answer. It was so helpful.

To answer your question:

The experiment lasted for almost 8 days. And I don't know the turnover of my protein(it is a heat shock protein though).

I agree with you regarding that my cells perhaps needed more time to achieve an effective knockdown.

Thank you again.

Hi again Mishael,

Heat shock protein, makes you issue more interesting. Could it be that your protein somehow is getting into aggregates or is forming complexes into aggregosomes and therefore being protected/non-accessible for degradation? That may increase the half life of the protein and mask the knockdown effectiveness.

Hello Sebastián Dupraz ,

As far as I know HSPs are proteins that don't denature, therefore, they don't aggregate because of their physicochemical features, like : 1-Better hydrogen bonding 2-Better hydrophobic internal packing 3-enhanced secondary structure propensity and so many other features. Many studies have shown that HSPs can be degraded, so I don't think that they are non-accessible for degradation.

You're totally right. I wasn't thinking of your HSP being aggregate itself, what I meant is whether your protein may be involved in chaperon-like interaction in aggregates thus changing its soluble ratio, which can indeed affect its targeting for degradation.

I think you should test the knockdown in other human-derived cell lines to have an idea if this issue is cell type specific.

Best,

Sebastián Dupraz I got what you're trying to say.

Yes actually I am planing to try the knockdown using another cell line.

Thank you so much for your suggestions.

Hi,

I have the same problem. There is supposed to be only dCas9-BFP, no any sgRNAs (targeting nor non-targeting) in the cells. I see the BFP signal is localized mainly in cytoplasm (99%). Does this indicate there is contamination of sgRNAs in the cells? Thanks!