Puromycin selection problems with PX459 and PX462

[Fei Ann Ran]

Hello,

We have previously deposited the PX459 (Cbh::SpCas9-2A-Puro) and PX462 (SpCas9n-2A-Puro) plasmids at Addgene, which contain a puromycin cassette that some users have reported problems with. We found that a SNP in the puromycin gene may have reduced its efficacy and have an updated version of the plasmids. We are working with Addgene to make the V2.0 plasmids available, but in the mean time if you would like an aliquot, we are happy to send it if you fill out the form here:

https://docs.google.com/forms/d/1i9ESanzFan5-ta8FPAAwZLK8sq7mDw9YaSXNQ6DkELU/viewform

Please let me know if you have any questions,

Ann

[crispr.cas...@gmail.com]

Dear Ann,

Thanks a lot for your new plasmids. But which Cas9-Puro plasmid did you use in your Nature Protocol paper, the PX459-Cbh::SpCas9-2A-Puro or the updated version? Does it mean that the Cbh::SpCas9-2A-Puro with a SNP mutation can only work in your human ES cell lines rather than in other labs' human ES or iPS cell lines?

Best,

Ming

在 2015年2月6日星期五 UTC+1下午11:27:25，Fei Ann Ran写道：

[Lynn Quek]

Hi Ann
Do you know if the SNP is present in the lenti Guide Puro in GecKO version 2?

Thanks
Lynn

[Fei Ann Ran]

Hi Lynn,

I believe the SNP is not present in the GeCKO libraries, i.e. puro cassette should be fine.

Best,

Ann

[ZQ]

Dear Ann,

I used PX459 and PX462 last year, they were fine and I got my KO cell lines. But last week I did new transfections with PX459 and PX462 and all my transfected cells died 24 h after I added puromycin. I used same cell and same concentraction of puromycin as last year.  I am confused now.

May I ask what 's the problems the others reported? Maybe it helps me to find the reason why last time worked and this time doesn't.

Best,
Qing

[Fei Ann Ran]

Hi Qing,

The puromycin cassette in the previous PX459 and PX462 plasmids contained a SNP, which may have reduced its efficacy, as many users have reported cells dying with with puro selection. If you put in a request in the form above I'll be happy to send a replacement.

Best,

Ann

[Jason Scovell]

Ann - could you specify the snp so that I can compare to what we are using? Thanks for the plasmid and support.

Jason

[pasquale pensieri]

could you tell me how much purom I must to use for a good selection on Nih 3t3? Thanks

[Fei Ann Ran]

Hi Jason,

In the puro sequence, there's a R166H mutation in the old version (ETSAPHNLPFYE should be ETSAPRNLPFYE).

Best,

Ann

[Rami Masre]

Hi,

does the new version of the plasmid PX459 is already available in addgene?, 

if not, when it's supposed to be ?

regards,

Rami. 

[Mohsen Honarpisheh]

Dear Fei Ann Ran

I want to start my experiment for the new version of the plasmid.Can I just fill the form or I have to order from Addgene ? Please let me know.

Best Regards,

Mohsen

[Fei Ann Ran]

Hi All, 

The plasmids have been deposited and will be available from Addgene very soon. In the meantime, I can ship them out if you fill out the form linked above.

Best,

Ann

[Kyle]

Hi Ann-

I received the whatman paper with the new PX459 and PX462, but after trying to transfect PX459 twice, I am not getting any colonies.  I cut out the circle, put it in a tube and mashed it with 50 ul of DEPC water.  I took either 2 ul or 5 ul and did a chemical transfection into  competent cells.  After overnight amp selection on a LB plate, I get nothing.  Has anyone else had trouble with this?  Did I do something wrong in trying to tranfect it?  Nanodrop showed I should have 300 ng total.  Any help would be great!

Thanks-

Kyle

[Fei Ann Ran]

Hi Kyle,

Did you have trouble re-transforming PX462? That is strange given the nanodrop reading. I can re-send you another aliquot.

Ann

[Fei Ann Ran]

Hi all,

PX459 and PX462 V2.0 plasmids are now available from Addgene (Plasmid #62988 and Plasmid #62987). I will keep the request forms open until March 20, 2015 and fulfill any requests made before then. After that date, please contact Addgene directly.

Thank you for your patience and understanding!

Ann

[mohamad....@gmail.com]

hi ann about mosaicism in new px459 that sent out, could you explain it will affect anything or not? and how can we sole it if possible thanks

[Eduardo Rodenas]

Hi,

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

On Friday, February 6, 2015 at 5:27:25 PM UTC-5, Fei Ann Ran wrote:

[Fatwa Adikusuma]

Hi Ann,

I haven't received the plasmid from you. I don't know why, in fact I have fulfilled the request long time ago.

Cheers,

Fatwa

[Anoeska]

Hi Ann,

I received the plasmids (thank you!) but am having problems to get colonies as well. 

For the pX462 I managed to get a few so I'll test them and hopefully it's OK, but nothing for the pX459. 

Could you maybe send them again? Preferably via regular mail, since for FedEx I need to ask permission at the institute again. 

Thanks,

Anoeska

Op vrijdag 6 februari 2015 23:27:25 UTC+1 schreef Fei Ann Ran:

[gkavakl...@ku.edu.tr]

Hi, 

I have received PX459 and PX462 in a filter paper, denoted as 1 and 2. Could you inform me which one corresponds to 1 and 2?

Thanks

Gulnihal

On Saturday, February 7, 2015 at 12:27:25 AM UTC+2, Fei Ann Ran wrote:

[Winston Yan]

Hi Gulnihal, 1 means px459 , and 2 is px462. Good luck!

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[Gülnihal Kavaklıoğlu]

Hi, 

 

I tried transformation 2 times for both PX459 and PX462. In the first transformation I got very few for colonies for both and tested them with diagnostic digestion. The expected bands did not show up for PX462 and i could not see any band for PX459. I repeated transformation and this time I got few colonies for PX462 and nothing for PX459.

What could be the reason for this?

Thanks,

Gulnihal

On Saturday, February 7, 2015 at 12:27:25 AM UTC+2, Fei Ann Ran wrote:

[Stuart Blakemore]

Hi Ann,

I have also been using the first version of this plasmid, and if it is okay would like an aliquot of V2.0, however the online form is no longer functioning. What's the best way to get hold of some PX459 V2.0 please?

Many thanks,

Stuart

On Friday, February 6, 2015 at 10:27:25 PM UTC, Fei Ann Ran wrote:

[Tim Vierbuchen]

--> http://www.addgene.org/62988/

[v.co...@sheffield.ac.uk]

Hi,

I've transfected PX462 V2.0 into my MDA-MB-231 cells but the cells are dying at 1.0ug/ml Puromycin, which was determined to be the optimal concentration following a kill curve. The transfection efficiency is good as I get >50% when using pMax-GFP as a control. Could anyone enlighten me as to why this is happening? I always transfect empty plasmid without sgRNA so surely some of these cells should survive?

Thank you,

Vicky

[Rocamar]

I transfert this plasmide in u2os..and it works nicely (I am selecting single clone)

48h selection with puromycin then I remove puro and plate for single cells

 Didier 

--

[Kashif Khan]

Even some users of pX459 V2.0 have reported a mosaic clone with a frame-shifted EcoRI site upstream of Puro in one of the batches. The mosaicism is about 50% defective EcoRI site and 50% intact. We tested our construct and it was fine. 

I would recommend performing a EcoRI digest and PX459 drops out a ~670bp Puro fragment. 

You might want to do another plasmid prep as some time it is only a bad prep.

Hope it helps

Kashif

[Jorn Lakowski]

Hi,

I am using the pX459 v2.0 in H9 hESC and I have observed pretty much loss of all colonies after 3 days of puromycin selection at 0.2ug/ml. The puro fragment drops out after EcoRI digest so if I understand your post correctly my plasmid is fine?

I have also seen some post implying that the Chb promoter in the pX459 2.0 is not driving Cas9/puro expression efficiently in hESC and hiPSC which could also account for the loss of the colonies. 

Any ideas?

bw,

Jorn

[Aidan]

Hi Jorn,

Did you ever get an answer to your question?

I am also using pX459 V2.0 and after selection everything dies. I also saw the 670 bp fragment after digestion (XbaI and EcoRI 2x digest). I was assuming that was the correct as that fits with the sequence on addgene.

I will shortly do PCR + T7 assay to check if I can see edits with any of my sgRNAs in cells without selection. If I see some edits I guess that might answer my question. I am curious if you worked anything out or if you ended up with a work around like pX458 etc.

My cell type is hard to grow at low density so it could be that selection is just wiping everything out, or there are some other posts in this forum that suggest the promoter might not express in my cells....

Aidan

[Explorer]

PX459 V2.0 works fine in our hands using at least 3 different cell lines. Usually we collect cells before starting selection to blot for Flag signal as indicator of good Cas9 expression. 

You might want to do a kill curve for your cell line as one should not put too much puro.

Hope it helps

[Aidan]

Thanks,

Yeah a kill curve would be worth it if I see edits. I did try 1 micro g/ mL and could see cells that weren't stained with trypan blue. again though, they are transformed B lymphoblast cells which don't transfect well and don't do well at low culture density so its hard to say where my problem is exactly.

It could be transfection is low, it could be Cas9/Puro expression is low it could be a problem with the puro selection (too much or not resistant).

Thanks again for the feedback.

Aidan

[niksasi]

hey, Aidan,

I am trying to transfect EBV transformed B lymphoblast cells for CRISPR. I was planning on using Nucleofection, do you have any advice on what would be the best transfection method?  Thank you.

Best,
Nikhil

[Aidan]

Hey Nikhil,

I did my T7 assays for 4 different guide RNAs and I did not observe any editing. This was in cells transfected with pX459 but without puromycin selection. My conclusion is either that the CBh promoter does not express in this cell type or the vectors are not getting into the cells in the transfection.

I get decent (>50%) pMaxGFP transfection with good viability using a variety of the best nucleofection kit + program combinations but that is 3.5 kb in size while pX459 is 9.1 kb so not a good transfection control. I should note that a homemade GFP vector ~6kb in size when transfected with similar nucleofection protocols results in <1% GFP positive cells so...thinking its just the transfection problems with these cells.

Currently I am trying to try in a different cell type that is easier to transfect and I may reclone my guides into pX330 in case there is a problem with the pX459 I got from addgene.

As for different transfection methods there are papers out there which talk about different nucleofection programs and which are the most efficient.

Let me know if you have any success!

Aidan

[Diego León]

Hi Nikhil

I've been trying to nuclefect 459 or 458 plasmids into LCLs withouth any success. I got decent nucleofection (>60%) using pmax , EW-113 program and the 4d nucleofector but just 1% of GFP+ cells when using 458 v2.o plasmid (up to 1.5 ug). Have you had any success? I heard from an ex-colleague that he observed cutting in EBV transformed B lymphoblast cells using 5 ug of the px330 plasmid (although that seems to me as a huge amount of DNA). I will do an experiment next week with these conditions. I will let you know if I have any success.

BW,
Diego

[Milena Lesseva]

Hi,

Is it possible to swap out the Puro resistance gene in PX459 with a gene for resistance to another antibiotic? Alternatively, do you have the PX459 with alternative backbone and another antibiotic resistance?

I am trying to deactivate a Puro resistance gene that is inserted in the Dnmt3b knockout mESC. I need to knockdown another gene and observe the effect in the Dnmt3b knockout background. My shLenti confers Puro resistance, so obviously even my non-infected cells are not dying off. For this purpose, I was thinking of using CRISPR to disrupt the PuroR in the original Dnmt3b KO..

Now, it turns out the PX459 vector also confers PuroR. Any idea how to solve such a conundrum?

Many thanks!

Milena

On Saturday, February 7, 2015 at 6:27:25 AM UTC+8, F Ann Ran wrote:

Hello,

We have previously deposited the PX459 (Cbh::SpCas9-2A-Puro) and PX462 (SpCas9n-2A-Puro) plasmids at Addgene, which contain a puromycin cassette that some users have reported problems with. We found that a SNP in the puromycin gene may have reduced its efficacy and have an updated version of the plasmids. We are working with Addgene to make the V2.0 plasmids available, but in the mean time if you would like an aliquot, we are happy to send it if you fill out the form here:

https://docs.google.com/forms/d/1i9ESanzFan5-ta8FPAAwZLK8sq7mDw9YaSXNQ6DkELU/viewform

Please let me know if you have any questions,

Ann

[F Ann Ran]

Hi Milena,

The 2A-Puro region is flanked by EcoRI, so you can cut it out and put another selection marker in place of Puro. 

Please see sequence here:

https://www.addgene.org/browse/sequence/106324/

Best,

Ann

[Dadi Jiang]

Hi Ann,

To replace the Puro, can I just insert a coding sequence starting with ATG after the first EcoRI site? Does the original Puro have it's own promoter etc. between the two EcoRI sites?

Thanks!

Dadi

[vittorio calderone]

Hi Aidan,

I also have problems using the v2.0 of PX459 and MCF10A cells. Transfection efficiency is OK, but 48 hours after puro treatment (2ug/ml), all the cells die. Could you figure out the problem at the end. I sequenced the pure gene of the plasmid and it was also OK...

Thanks in advance,

Vittorio

[Aidan]

For me I think the vectors don't drive expression of Cas9/Puro in my cell type. You can check for Cas9 expression (and Puro). Also my cells seem to be very sensitive to puro naturally so one way or another they always died.

[Cheng Xu]

Hi Vittorio,

How's your experiment going? Have you solve your problem yet?

I've just began doing CRISPR-Cas9 in MCF-7 cells a few weeks ago and I'm using the V2.0 of PX459 plasmid. I met the same problem with you. After 2.5ug/mL of Puromycin treatment, all the transfected cells die just like the control group. I'm wondering if you have figured out this problem and this might be very helpful to me.

Thank you!

Cheng




在 2016年7月18日星期一 UTC-4上午4:04:16，vittorio calderone写道：