Trouble with Puro resistance using pSpCas9-(BB)-2A-Puro

[Nate Young]

Hi,

I have been unable to use the Puro-resistance gene in the pSpCas9-(BB)-2A-Puro vector to successfully select for transiently transfected cells across multiple cell types (mESC, 293T, U2OS), and I was wondering if anyone else has had similar problems. Although control transfection wells (with empty Cas9-BB-2A-GFP) always show that the transfection is relatively efficient, when I treat the pSpCas9-(BB)-2A-Puro-transfected cells for 1-2 days with Puro, all the cells die. Control experiments using other plasmids with Puro-resistance cassettes work fine, and I have sequenced verified the PuroR gene in the original vector. Is it possible that the expression of of PuroR downstream of the 2A is  diminished to the point it's only functional for very low doses of puro? Does anyone have any suggestions (besides cloning all my sgRNAs into the pSpCas9-BB-2A-GFP vector and using FACS)? Thanks for any advice.

Nate

[JP]

You can co-transfect with a GFP plasmid if you don't want to do any additional cloning and don't mind making the assumption that GFP+ means pSpCas9+. I make a similar assumption when transfecting with pX330 and GFP and then sorting. It works well for me.

[lcsbcel...@gmail.com]

Hi, I'm also trying this plasmid recently. Unfortunately, it doesn't work. Can you contact me through my email: qingxi...@gmail.com? 

Best,

Xiaobing

[Jens Refluxx]

Hey guys,

I am planning to use this plasmid as well. Has anyone had success with it yet? Also Nate, what concentration of puro did you use and what concentration is recommended considering the promoter? Would be great if someone can post a protocol for successful clonal selection with the puro or GFP vector.

Cheers, Jens

[Yangfei Xiang]

Hi,

I had similar results. After 48 h treatment with 1ug/ml puro, I could hardly find any difference between the px459 group and the cell control, with nearly all the cells died in both groups... Cell would survive if I use px330 and a puro plasmid.

Yangfei 

[Kazu]

Hi all

I'm new to CRISPR and planing to knock out some genes with mouse ES cells. I've already got pX459 (pSpCas9(BB)-2A-Puro) from Addgene, but now I'm a little bit worried about this plasmid after hearing you guys had problems with it. Did anyone successfully use it? Thanks in advance.

Kazu

[Fatwa Adikusuma]

Hi All,

I did nucleofect PX459 in mouse ES cells, change to Puro media (2 ug/ml) the next day for 48 hours and they grew well. I got plenty of clones.

Cheers,

Fatwa

[lcsbcel...@gmail.com]

Hi, did you try it in human iPS cells?

[Nat]

Hi Fatwa,

Would you mind telling me conditions of your pX459 nucleofection assay?

really appreciate it!

Best,

Nat

[Jonathan Geisinger]

Looking through all the responses, three things come to mind:

1) Amount of plasmid transfected.  This one is important because if everyone is using different amounts, then the same puromycin concentration will have different selection results.

2) Targeted gene.  This is really important because if you're getting really efficient targeting and the gene is essential for the cell to stay alive, you're going to have a bad time getting resistant cells.

3) Cell type.  Different types of cells respond to puromycin selection in different ways.  I've observed for a different vector (unrelated to CRISPR/Cas9)  a massive difference in response using the same concentration of puromycin and the same amount of transfected vector for HEK293 versus H9 hESCs.

These are important things to keep in mind.

[Andrei Ry]

Same here, px462 do not protect my C2C12 vs 2ug puromycin

[Patrick David Hsu]

I would recommend doing a kill curve for every cell line with puro (titrate puro concentration without virus and see what you need to get 100% death in 4-5 days). oftentimes you will find you are using way too much puro

best

Patrick

zlab

[Jonathan Geisinger]

It's also going to depend on your lot and manufacturer of puromycin.  

For example, my puromycin from Life Technologies may give me a different kill curve than someone's puromycin from Invivogen on the same cells.

[Fatwa Adikusuma]

I used Neon nucleofector to double knock-out genes. One million mouse ES cells nucleofected with 4ug PX330 and 2ug PX459, 1400 V, 10ms, 3 pulses . Spread them all to 1 or 2 10cm dishes. I made 5 replicates (in total I used 5 million cells and spread into more than 5 10cm dishes). Grow in normal ES media, and change with Puro media (2ug/ml) the next day for another 2 days. After 2 days of puro selection, change with normal media, collect some pool cells for preliminary screening using surveyor/T7E assay, while let the rest of clones to grow. If surveyor assay shows positive result you can continue growing the clones and pick individually. If not, abort the experiment and change your gRNA.

Cheers.

Fatwa

[Raphael Bendriem]

Hi all,

I'm having similar problems as the rest of the members of this thread. I'm using HEK293 cells and transfecting pX459 containing my sgRNA of interest. After a few days of puromycin treatment (1-2 ug/mL), all cells in the transfected wells have died and most cells in the control (untransfected) well have survived. Any insight or advice will be most appreciated.

Best,

R

[Daniel Rosen]

I am having the same issues with px459. Even at low concentrations of puro, all cells die. I checked this via hem0cytometer and flow. Has anyone had better luck with px260?

Thank you,

Dan

[Benjamin Schmid]

Hi all,
We are having a similar problem with puro selection. We are using the plasmid pSpCas9(BB)-2A-Puro (Addgene plasmid ID: 48139) and we are working in human iPS cells. We made a kill curve and found that the ideal concentration for our cell line was 0.125 µg/mL. This concentration kills all the cells after 3 days (lower concentrations don't kill our cells even after a week). We are using the Amaxa 4D nucleofector (1 million cells, 5 µg DNA, program CA 137) and we are getting 50% GFP positive cells when we use the plasmid pSpCas9(BB)-2A-GFP (Addgene plasmid ID:48138) with a mean fluorescence intensity of 100,000. However, when we use the puro-plasmid, they all die after 3 days of puro selection like the non-nucleofected. We start the selection always the day after nucleofection – that’s when the GFP nucleofected cells are already green. Is that too early? However, when we use HEK 293 T cells, they survive very well – here we don’t have any problems with the selection. For the HEK cells, we use Lipofectamine 3000 and 2 µg/mL of puromycin. So, the puromycin plasmid is working in HEK cells but not in our hiPS cells. Our thinking was that the promoter may be very ineffective in iPS cells. We were using Endo-free plasmids for our experiments.Is the puromycin-plasmid working for somebody in human iPS cells without problems?

Best,

Benjamin

[Andrei Ry]

Hi Benjamin,

Stability of GFP and Puro variant, might be different in different cells, plus plasmid dissolving due to cell divisions (GFP is pale after 4 days, right?). I have found that additional transfection and shorter time of selection may help (at least in my C2C12 cells). I am hitting cells by two transfections (24 hrs apart from first) with px462, and adding puro (4mg/ml) only 6 hrs afterwards (after second transfection), and my selection period only 48hrs. Concentration of puromycin is high enough to kill 99.9% of control cells during this period.     

[Benjamin Schmid]

Thx Andrej for your help - I think I will try a double nucleofection then and only a 6h selection with puromycin. Will come back to this forum with the results. If somebody else has already experience with iPS cells and this puro plasmid I'm very open to your suggestions :-)
Best,
Benjamin

[Tessa]

Hi,

I have also been trying to select cells (H460) with puromycin after transfection of the pSpCas9-(BB)-2A-Puro plasmid. However, it's not working well.

We were not able to have more surviving cells in the transfected cell plates vs the control plates.

After 2 days we remove the puromycin and grow the cells for about a week. 

Now the control plates (that seemed to be all dead) suddenly also had a lot of colonies (even less then the transfected ones).

My concern now is that we're not really selecting for Cas9 positive cells since the puromycin selection was not different from the control plates.

Does anyone have any suggestions to overcome this selection problem?

We did titre the concentration on the wt cells for a 2 day exposure to the antibiotic in order to have all your wt cells killed.

Did anyone try to do a longer puromycin selection, around 5 days for instance, and did this improve the selection?

Thanks,

Tessa

[贾伟彦]

Hi Tessa,

I met the same situation in H460 as you!

Have you tried the GFP selection marker of CRISPR plasmid, such as px461 and px 458,  in H460 cells? Besides, the transfection efficiency is very lower in H460 and how you overcame this problem?

Weiyan

在 2015年1月8日星期四 UTC-6上午8:55:48，Tessa写道：

[amrendra ajay]

I used pX459 and used 2-5 ug/ml Puro. Sounds transfected cells survived. I am still in the process of selecting clones.

Ajay

[Xiaobing Qing]

Hi Benjamin,

Any news about your result? We start to use a dsDNA vector with puromycin-resistant cassette as donor.

And also we are waiting for the px459 V2.0. Hope it will work.

Best,

Xiaobing

在 2015年1月8日星期四 UTC+1下午2:32:11，Benjamin Schmid写道：

[Eduardo Rodenas]

Hi Xiaobing,

I tried px459 V2.0 last week and it seems to be working well. I transfected U2OS cells (around 70% confluent) with 2 microg px459 V2.0 (6 well plate) and 24 hrs after transfection I pass the cells to a 10 cm plate and add puromycyn (3microg/mL). After 24 hrs of puromycin selection I could see how in my control plate all the cells had died and around 40-50 % of the cells transfected with px459 V2.0 were alive. I hope this is helpful,

Eduardo

[Xiaobing Qing]

Hi Eduardo,

Thanks for your good news. Did you try the px456 v1.0 before, and how was it compared to v2.0?

Best,

Xiaobing

在 2015年3月17日星期二 UTC+1下午9:33:50，Eduardo Rodenas写道：

[Eduardo Rodenas]

I tried before px459 V1.0 and after adding puromycin all my cells died. Using px459 V2.0 40-50 % of the cells survived after putomycin addition.

Eduardo

[Ashutosh Dhingra]

Dear All, 

I recently started nucleofection of human iPS cells. I have 4D nucleofector system. Using P3 kit and CB-150, yielded less than 10 % transfection efficiency while with mirus Lt-1, the efficiency is in the range of 30-40 % (puromycin selection in iPS cells at 0.2 ug/ml is killing transfected iPS cells but not HEK at 2 ug/ml). I will try CA-Mirus137 program. Cell viability is not of major concern at the moment. I will try 137 program on Amaxa 4Dx unit. 

Can someone please provide a cell number and plasmid quantity? I used 2 x 10 ^6 cells with 5 ug of plasmid in 100 uL rxn CB-150, P3 kit, amaxa 4D unit.

  

Thanks a lot!

Best regards

Ashutosh

[slava berger]

Hi Eduardo,

We want to use the U2OS cells for inducing a knock-in mutation using CRSPR with px461 plasmid with Cas9 mutant variant. What reagent did you use to transfect the cells? In which growth medium they were grown?

Thanks,

Slava

[SALAH ADLAT]

Would you please share your protocol?