In this protocol there is every detail you need regarding CRISPR/Cas homologous recombination. There are plasmids in Addgene from the Feng Zhang lab that would suit your purpose- those details are also found in this paper.

Additionally, it may be useful to check out http://www.addgene.org/browse/pi/227/articles/ (Rudolf Jaenisch Lab Plasmids) and http://www.addgene.org/Philippe_Soriano/ both of these PIs have deposited many plasmids designed for use in genome engineering and homologous recombination templates.

Hi everyone!! Thanks a lot for your answers. We found this addgene plasmid that we think it could work. Addgene #11072. Thanks a lot for your suggestions!

Ari

Hi Ariel,

Recently our lab ordered the pSpCas9(BB)-2A-GFP (PX458) plasmid from Addgene (Plasmid# 48138), I designed the gRNAs by using both CRISPR design tool (http://crispr.mit.edu/ ) as well as sample gRNAs which were already listed in the GeCKO version 2 library (http://genome-engineering.org/gecko/?page_id=15).

Yet as I'm new in the field I was unsure about appropriate overhangs which need to be designed at the 5' of oligos for insertion of gRNA sequence into the pSpCas9(BB)-2A-GFP plasmid. I found an example in the FAQs of Addgene website:

Forward: 5'-CACCGN...N-3'

Reverse: 5'-AAACN...NC-3'

I appreciate if you can kindly confirm my understanding of the overhang design for this specific plasmid.

Many Thanks,

Sara

Hi Sara,

I'm starting also with the same CRISPR system and i'm also a beginner in this field. I designed my oligos as you mentioned  above.  For me , they look good. Please tell me if it works in your hands.

Thanks

Hi Sara! Yes, I designed them exactly like that too. :D

Good luck!

Ariel

Dear Chaveroux and Ariel,

Thank you very much for your respond and confirming the gRNA overhang design. We just received the plasmid from Addgene and I'm going to sequence its Cas9 first. Following confirmation of the plasmid, I'll insert our gRNAs.

Sure Chaveroux, I'll update you with the outcome.

Thanks again!

Sara

Hi guys,

I come baack to you, since we tried to generate a pLentiCRISPR from Zhang lab by following their protocol. We got no clones at all and we tried with 8 different oligos. Obviously, we have a technical problem. The plasmid restriction was OK. So many a problem with the oligos annealing or ligation. Does anyone could help me to succeed ? thank a lot in advance

Hello Everyone,

The topic of discussion seems pretty interesting to me. I have used the same vector; designed and inserted my gRNA for my target gene and seems to works wonderful.

I have a question: Does any one has the vector NTI file (.gb) for the pSpCas9(BB)-2A-GFP vector??

I am desperately looking for it as I am looking forward to introduce RFP instead of GFP.

Any suggestions/comments will be highly appreciated.

Thanks. 

Here is the VNTI version of the vector

hi.. I recently used pSpCas9(BB)-2A-GFP (PX458) for knocking out one of the targets in T-cells. I get very good transfection effeciency monitored by an unrelated GFP. When i transfect pSpCas9(BB)-2A-GFP (PX458) or pSpCas9(BB)-2A-GFP (PX458) with the guide cloned ( sequence verified), I see GFP signal in vector alone ( without gRNA) but no GFP signal in the ones with the guide. Can anyone please help sugggest if this has been observed before?

Thanks so much

Interesting, I also am seeing GFP signal in vector (PX458) alone (without gRNA). But my PX458 target clones (with gRNA) also show GFP expression. Did you ever figure out why your empty PX458 (without gRNA) expressed GFP and no GFP in your ones with guide?

Hi Tessa,

Have you found the reason for your finding? I have the same issue?

thanks

I have a tip about getting plasmid maps for VectorNTI from Addgene. You just save the .gbk file, rename it to .gb and open in your Vector NTI program.

Hello Everyone,

I am also using the same vector - PX458_pSpCAs9-2A-GFP. The Plasmid restriction is fine but i am having trouble in cloning the guides ( Oligo annealing and Ligation ). Could any of you help me to succeed?.

I've seen a few suggesting here a problem with annealing/ligation. My trainees sometimes commit the mistake of not ordering the oligos in 5' -> 3' orientation. In fact, if you were to simply follow the original Fang Zhang lab protocol, replace your guide with their guide, and order those. Your top guide (forward guide) would be fine, but your bottom guide (reverse guide) would be 3' -> 5' instead of 5' -> 3', and direction matters. I know it seems simple, but it happens to the best of us.