My understanding is that you wouldn't really get anything - maybe a couple of base pairs before the U6 termination sequence (TTTTT). But someone more familiar with polIII promoters might have a better answer.

I agree with Jill, although I've never used this specific plasmid.

Yes, it is probably transcribed but you wouldn't really get anything besides the stretch of bases before the termination sequence. You can see the sequence on the addgene website or if you want a plasmid that just expresses the SpCas9 those are also available.

http://www.addgene.org/crispr/zhang/

Dear Jill and Caitlin. Thank you for your comments. In my case, I want to have a stable Cas9-expressing cell line, but without sgRNA expression; I will apply the sgRNA-coding sequence separately, as a PCR amplicon. As Caitlin noted, there is a plasmid that only expresses SpCas9, but that plasmid does not have a selection marker (puromycin), hence, I cannot make my cells stably transfected with that vector. Consequently, I need the vector that has the selection antibiotic resistance gene but not the sgRNA-coding sequence. I am now planning to use the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector, but before the stable transfection, I will delete the TATA box of the U6 promoter, in order to prevent any unwanted transcription that might, somehow, bind to an unexpected region within the genome... Would you have any comments on this strategy?

As this plasmid contains NLS on the Puro sequence, the resistance conferred may be compromised because of that. I observed that in my experiments, the resistance was really poor. I suggest to consider other approach.

Dear Mutlu

In this type of vectors you only insert crRNA's sequence (20bp) exactly tracrRNA upstream. The crRNA+ tracrRNA together= sgRNA which is a fusion form. In fact, tracrRNA's coding sequence is next to Bbs1 and under U6 promoter in pSpCas9(BB)-2A-Puro (PX459) V2.0 vector. Real order of the sequences is: U6 promoter+ Bbs1 restriction site+ tracrRNA + U6 termination sequence (TTTTT). You have to insert you ordered oligo at Bbs1 site. Without inserting your 20bp oligo, you will gain tracrRNA's transcription which is NOT functional alone. If you want to delete sgRNA completely, I suggest you make a digestion with AflIII & Xba1 or Kpn1 restriction enzymes.

I may be wrong, but I would assume that the non-functional tracrRNA will compete with sgRNA for Cas9 binding site. If true, this might compromise the efficiency.

Dear Lech

There is no competition at all! sgRNA is a combination of crRNA (20bp ordered oligo) + tracrRNA. By omitting each component, Cas9 won't be able to recognize the structure, so will be non-functional.

Hadi, do you have any reference or data that make you so sure?

Dear Hadi,

I see your point, but when I look at the sequence of the pSpCas9(BB), I see that there is a 26bp region immediately after the U6 promoter. You can see the photo of the sequence in the link that I attached. That 26bp sequence has been highlighted as blue in between the U6 promoter and the gRNA scaffold. Additionally, I could not locate any BbsI restriction site, moreover,  XbaI and KpnI restriction sites seem to be at irrelevant positions to delete sgRNA completely as you suggested. I prepared the linked image by copying the full sequence from the Addgene website and pasting it to SnapGene Viewer, then compiled that sequence. The Viewer just recognised the motifs automatically.

And dear Lech, NLS sequence seems to be fused to Cas9 coding sequence, not to PuromycinR coding sequence. Between the NLS and PuromycinR gene, there seems to be a T2A peptide coding region, which leads to separate synthesis of the NLS and PuroR.

http://i.imgur.com/l3WCYVr.png

Dear Mutlu

Yes, you are right. SnapGene Viewer or others can't recognize Bbs1 restriction sites. In fact, there are 2 restriction sites for Bbs1 in that  26bp under U6. Kindly see attached pic which is introduced in Ran et al. (2013) "Genome engineering using the CRISPR-Cas9 system". We are currently working with pSpCas9(BB) and successfully cut it with Bbs1.

Dear Mutlu,

Then the scheme (figure 4c) in the Ran et al. paper is wrong, as it depicts NLS on the Puro. In any case, the conferred Puro resistance in ES cells was poor. But maybe it's better in other cell types. Good luck!

The sequences order is correct U6+Bbs1+ sgRNA scaffold. Yes, dear Lech you are right. NLS is located between cas9n & T2A.

Hello Lech, you are right, that figure is incorrect. The map in the figure must have been configured as Cas9-NLS-T2A-Puro. Indeed, there is no meaning of fusing Puro with an NLS (Nuclear Localization Signal), the molecule that must be directed to nucleus is Cas9.

Hello Hadi, ok, Bbs1 can be used. BTW, I still could not understand what that 26bp does actually encode for, however, I blasted the sequence but it practically corresponds to nothing.

Thank you all for the answers. I will write here if any "funny" result comes up...

Dear Mutlu

Hello,

In fact, the 26bp doesn't encode for anything!!! it's just a place for inserting crRNA after cutting your vector with Bbs1. Actually, gRNA scaffold encodes for tracrRNA. After inserting crRNA's oligo into the digested 26bp, the crRNA and gRNA scaffold together express sgRNA.

Good luck with your experiment.