Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not both

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Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not both

Postby Dean22April » Nov 27 2017 9:49 am

Hi a CRISPR/Cas9 question if anyone can help (please see attached photo for better clarity):

We have 36 transformed wheat lines with CRISPR/Cas9 and tested in those lines which had been successfully transformed with the Cas9 RNA and the sgRNA (the two things needed) but (as in the picture) we had several plants that expressed Cas9 but not guide RNA and vice versa, even though they were transformed in equal proportions.

Is there a reason why some plants have Cas9 mRNA (including those that were the highest expressers- starred) but not guide RNAs (e.g. no.3), or why did some plants have almost all the guide RNA's expressed but no mRNA (e.g.14, 4)? Or is this chance?

Thank you so so much for any help!
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby r.rosati » Nov 28 2017 9:26 am

Hi,
what plasmids and system are you using? Specifically, how many plasmids/DNAs are you trying to transfect at once? And what transfection protocol are you using?
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby Dean22April » Nov 28 2017 10:37 am

r.rosati wrote:Hi,
what plasmids and system are you using? Specifically, how many plasmids/DNAs are you trying to transfect at once? And what transfection protocol are you using?


Hi thanks for your reply! We used biolistic transformation with tungsten rings coated in DNA that were then fired into the cells. We had separate plasmids of sgRNA and CRISPR that were fired in equal quantities at the same time.

Any help is hugely appreciated thank you!
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby 29yrsExperience » Nov 28 2017 11:17 am

My first question was, are the Cas9 gene and the guide RNAs encoded on the same plasmid or separate plasmids? you have answered that they are separate. Therefore, I am not surprised you are getting some transfectants with one (or some) but not the other(s). In a transfection of multiple plasmid constructs, only a proportion (according to the laws of probability) will pick up more than one. Which is why you are screening the clones in the first place. Every experiment produces “duds.” Are you doing stable transfections? Do your plasmid(s) have different eukaryotic selection markers like neo, hygro, or whatever is appropriate for plant cells, and are you getting the resistance(s) but not the linked Cas9 or sgRNA? Is that the issue?
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby Dean22April » Nov 28 2017 11:33 am

29yrsExperience wrote:My first question was, are the Cas9 gene and the guide RNAs encoded on the same plasmid or separate plasmids? you have answered that they are separate. Therefore, I am not surprised you are getting some transfectants with one (or some) but not the other(s). In a transfection of multiple plasmid constructs, only a proportion (according to the laws of probability) will pick up more than one. Which is why you are screening the clones in the first place. Every experiment produces “duds.” Are you doing stable transfections? Do your plasmid(s) have different eukaryotic selection markers like neo, hygro, or whatever is appropriate for plant cells, and are you getting the resistance(s) but not the linked Cas9 or sgRNA? Is that the issue?


Hi, thanks for the reply again! Yes, they were stable transfectants and no plasmids did not have selection markers on, so the issue (which you have now cleared up) I believe is to do with the random probability that certain plasmids won't be transformed
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby 29yrsExperience » Nov 28 2017 3:45 pm

I think you've got it now. By the way, if your goal is to find one clone with all 7 sgRNAs and the Cas9, you may have a lot of screening to do! I'm not sure if this would work, but perhaps you could pick a clone expressing most of your RNAs and then re-transfect it with just the plasmids that are missing. I don't know if you would lose any genes that are already in there (more screening would be needed of course). But it's a thought. Let us know how it turns out, and good luck!
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Re: Bizarre CRISPR/Cas9 expression of Cas9 RNA or sgRNA not

Postby Dean22April » Nov 29 2017 1:00 pm

29yrsExperience wrote:I think you've got it now. By the way, if your goal is to find one clone with all 7 sgRNAs and the Cas9, you may have a lot of screening to do! I'm not sure if this would work, but perhaps you could pick a clone expressing most of your RNAs and then re-transfect it with just the plasmids that are missing. I don't know if you would lose any genes that are already in there (more screening would be needed of course). But it's a thought. Let us know how it turns out, and good luck!



Thanks very much for all your help! We only really need at least one sgRNA and Cas9 to be expressed so multiple plasmids with both a single sgRNA and Cas9 would maybe work for future direction (could you think of any issues why this wouldn't work? I can't off the top of my head).

We have another 100 or so plants to test yet aha! I'll let you know the results! We are finding out results for whether we have editing (after doing NGS) on Friday hopefully we'll have some edits in the few plants that had the Cas9 and then at least one sgRNA!
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