This method was outlined in the Zhang lab paper. I previously tried transient transfection of lenticrispr v2; which expresses both cas9 and 1 sgRNA. Actually, I took a deletion approach(2 sgRNAs targeting opposite alleles of an exon to create a deletion-transfecting 2 lenticrispr plasmids). The reason i chose this approach is because it is easy to detect the deletion with conventional pcr. I got a clone with homozygous deletion but it doesn't seem very efficient(maybe it is also a transfection efficiency problem); most of the deletion were heterozygous and some were no deletion. Maybe that's how crispr is. I was looking for a way to make the process easier and more efficient. People said Cas9 stable expression is going to give non-specific cutting but if you don't have guide RNA, will it still be cutting? I was thinking that the sgRNA pcr amplicons will only express transiently so it may not have the long term off target effects that people are talking about. There is additional flexibility for multiplexing or targeting different genes without having to clone it again and again into a plasmid. But another thing is that this approach requires long primers(around 130bp); which seem costly. People have also published with stable expression of both cas9 and sgRNA using lentivirus. Actually, the stable expression of both cas9 and sgRNA would be my second choice but ,if possible, i would like to do the cas9 stable and sgRNA pcr amplicon approach. I can also try the transient transfection again but i feel the cells don't have good transfection efficiency with plasmids.
I would just like to get some idea.
Thank you,
Santosh
Hi Santosh,
I strongly recommend the google groups moderated by the Zhang lab:
https://groups.google.com/forum/#!forum/crispr
It all really depends on what you want to do in the end with your CRIPR experiment. You can do single cell dilutions and find the clone that has your desired deletion. You can create a stable Cas9 expressing line - and yes even without sgRNA high Cas9 expression may cause DSBs in some cells. long oligos are a bit costly indeed, you might wwant to clone the short guides first (e.g. into lentiCRISPR) and then just use a U6 fwd and sgRNA rev primer to obtain an expression cassette that can be transfected. Of course you can also package lentiCRISPR v2 and get stable expression. It all really depends on what you want to do wiith the cells in the end. Short term stuff -then off-target DSBs may be less important. Long term stuff you want to be as specific as possible.
Maybe you also want to optimize your transfection method - are you using electroporation? That's usually very efficent.
Good luck!
Dear Santosh,
You may try lentiviral constructs allowing doxyciclin-inducible expression of Cas9 like for example pCW-Cas9 (Addgene Plasmid #50661). Thus your cells will stably contain Cas9 expression cassette circumventing the problem of low-efficient transient transfection BUT protein Cas9 is not permanently expressed to limit off-target effects. You induce Cas9 expression when you want by treating your cells with doxycyclin.
Another way to take advantage of lentiviral infection efficiency without having a permanent expression of Cas9 is to use IDLV (Integrase deficient lentivirus). IDLV infect cells with high efficiency but are progressively eliminated as the cells divide (like a transiently transfected plasmid).
Thank you very much Thomas and David. You helped me to get some good idea on what to do.
Based on your input, I will most likely use the inducible cas9 plasmid above to make stable cells. Then I will use the u6 forward (probably can use the same primer outlined in Zhang lab paper)and sgRNA reverse primer to make the pcr amplicons to use for making the deletions(I already cloned the sgRNA into a plasmid so I think I can use it as a template). Since the plasmid already has the sgRNA, I guess I can just take the beginning part of the reverse primer given in the Zhang paper(so same f and r primers can be used for all the sgrnas as long as I keep track which plasmid has what sgRNA).Since the pCW-Cas9 system is inducible, will I be adding doxycycline at the time of transfection or couple of hours after transfection? Is there any protocol for that or is it pretty straight forward? Some people say that inducible systems are leaky(fbs has some dox it seems). But I guess if the expression isn't too high, there won't be too much non-specific effects.
To me this plan sounds ok but I was still interested to know whether the pcr amplicons approach works well? But it should probably work fine since its the same thing as what's on the plasmid; just taken out of the plasmid. I hope that the transfection efficiency will be much better with the pcr amplicons and help to increase the efficiency of genomic deletions.
Even with this in mind, it may be easier for me just to do lenticrispr transduction(cas9+sgRNA). I saw a paper doing transduction, selection then using the bulk selected cells for their experiments. They saw good kd/ko. But as you said, it probably depends on whether it is for short term or long term use. I would do some functional assays and maybe animal work; so it may count as long term.
So all in all, the inducible system with pcr amplicons sounds right for me. My mind wasn't going back and forth but I was just trying to make a clear decision.
Thank you once again,
Santosh
Santosh Timilsina ,
Could you please send me a link or some information about the paper you were talking about?
"I saw a paper doing transduction, selection then using the bulk selected cells for their experiments. They saw good kd/ko."
Thanks in advance.
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