I am currently trying to generate stable cell lines using pSpCas9(BB)-2A-Puro (Addgene plasmid ID: 48139). The sgRNAs that were designed are successful in knocking down the protein of interest, however the knockdown achieved is only 10%. I am working with AR42J cells, which exhibits a poor viability and are relatively slow growing.
My question was whether I need to keep on selecting the cells under puromycin for several weeks or if the cells could be exposed to the drug for a shorter period of time (4-5 days) and that should be sufficient enough to kill untransfected cells/cells that have not undergone genome editing? Since the crispr/cas systems permanently edits the genome, I was thinking that the cells don't need to be cultured under the antibiotic selection.
Also is there any way I can increase the transfection efficiency so that a higher knockdown could be achieved with my transient transfections? I have tried lipofectamine 3000 which claims to provide a better transfection efficiency than others.
I don't know specifically for AR42J, but I would not leave puromycin in your culture for more than a couple of days. You are just transfecting a plasmid which is going to remain episomal and will be degraded/diluted with time, so you will inevitably loose the puromycin resistance. With hESC I usually start selection after 24h and leave puro for just 24h more. I am afraid that if you leave it for longer you will have a very poor survival.
About the transfection protocol, one easy thing to do is to increase the amount of DNA that you transfect... You can try other protocols such electroporation/nucleofection, which work way better than lipofection in some cell types.
Another strategy would be to clone your sgRNA into the lentiCRISPR plasmid (addgene #49535 - this is for Joanne! :P), produce the lentiviral vector and transduce your cells with it. This will mediate a stable integration of your sgRNA and Cas9, which will theoretically increase (to almost 100%) the cleaving efficiency. See Shalem and Sanjana et al, Science, 2014 for more details.
Hope this helps.
Thanks for referencing the Addgene number! That very popular plasmid was deposited by the lab of Feng Zhang and his website has a lot of information to help with protocols http://crispr.genome-engineering.org/ Also the Addgene genome engineering pages have useful links!
I don't know specifically for AR42J, but I would not leave puromycin in your culture for more than a couple of days. You are just transfecting a plasmid which is going to remain episomal and will be degraded/diluted with time, so you will inevitably loose the puromycin resistance. With hESC I usually start selection after 24h and leave puro for just 24h more. I am afraid that if you leave it for longer you will have a very poor survival.
About the transfection protocol, one easy thing to do is to increase the amount of DNA that you transfect... You can try other protocols such electroporation/nucleofection, which work way better than lipofection in some cell types.
Another strategy would be to clone your sgRNA into the lentiCRISPR plasmid (addgene #49535 - this is for Joanne! :P), produce the lentiviral vector and transduce your cells with it. This will mediate a stable integration of your sgRNA and Cas9, which will theoretically increase (to almost 100%) the cleaving efficiency. See Shalem and Sanjana et al, Science, 2014 for more details.
Hope this helps.
Addgenies get so excited when someone references one of our plasmids in a Research Gate suggestion..you made our day Albert. Good luck with your experiments Dhruvit...call Addgene if we can help.
Joanne: would you know when is Addgene planning on releasing the new lentiCRISPR plasmid. I am planning to perform the lentiCRISPR transduction as recommended by Albert, but I was hoping to get the newer version of that plasmid.
I think the new ones are out or about to come out...you can set up an alert for it on the researchers page. Or check the CRISPR pages too. Addgene alerts are an easy way to know when a plasmid comes out (no spam..only an alert on the researcher's plasmid being available). You can also sign up for gene alerts which is kind of handy.
Hi all,
I'm also using the pSpCas9-(BB)-2A-Puro and am trying to do a transient transfection, becasue I don't want a permanent integration of my plasmid into the genome. We were actually trying to work with the Nature protocol that is referred to by Mohammad Ameen Al-Aghbar above. However, after 2 days of puromycin selection we changed the medium to antibiotic-freemedium and grew the cells for about a week. What we see is that there are actually a lot of cells growing in the control plate and even a little less in the transfected plates (we did titre the concentration for a 2 day puro selection and it seemed like all cells had died).
Anyone had similar problems? Or maybe someone has some additional advice? Do we need a longer puromycin selection for selectively keeping the Cas9 positive cells (like 5 days or even longer)? Also, does anyone know how long it will take after transfection voor indel events?
Thanks.
Tessa
Joe, with the info that you give in your question, I am not sure if you remove Puro, or you keep it in the media for 2 weeks. Make sure that you only keep puromycin for 2days at most. Otherwise you will be selecting for a random integration of the px459 plasmid, which I doubt is what you want.
Tessa, it does not make much sense, to have surviving cells in the control plate. Are you sure your "parental" cells do not have puromycin resistance already? Or maybe you changed the lot number of puromycin? You may want to repeat your kill curve anyway. I usually use it at 0.5-1 ug/ml and untransfected cells die within 24h. I would not go over 2 days for transient selection.
Hi All,
I am trying to generate a stable cell line using CRISPR cas9 in SKBR3 and T47D for the second attempt. I added the puromycin selection at 2 ug/ml and still seeing a lot of living cells in the control. I repeated the puromycin twice again and then all the transfected and control died and become very sticky to the wells and difficult to remove. Now I repeated again the protocol and add the puromycin at 0.5 and 1 ug/ml still alot of living cells in the control.Please does anyone have experience with CRISPR selection, should I repeat the puromycin selection again? is it usual that is in effective with control ?
I suggest that you should transiently expose your cells to the CRISPR CAS9. After the transfection, you should select your cells with deletion immediately, such as PCR of the single colony, or fluorescent selection etc. The reason you should not keep your cells with the plasmids for long period of time is that you will lead the plasmid integrated into your cell's genome, and in that case you will artificially introduce a insertion mutation, even though you have successfully deleted your target gene...
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