Hi All,
I am planning to use 2gRNA & wild-type Cas9 to KO my target protein. I plan not to use HDR for my purpose but hope that by using 2gRNA I can create a specific deletion that help me successfully KO my gene + aid my following up selection.
I've gone through most of the Addgene posts about CRISPR-Cas9 system, but still can't decide which plasmids I should purchase from them.
I would be super grateful if anyone can answer these following questions for me:
1. Is there good vector that express 2gRNAs + Cas9 + GFP at the same time (all-in-one vector)?
2. Is it better to use "all-in-one" vectors or to separate into vectors that express gRNA, Cas9, and GFP independently?
3. If I need to send 2-3 vectors into mammalian cells, would the transfection efficiency become very low? If so, what would be the best strategies?
Thank in advance for any suggestions!!!
Best,
Lyra
I decided to use the vectors from Zhang's lab (https://www.addgene.org/crispr/zhang/)
- PX458
-PX459
-eSpCas9(1.1)
I have 4 gRNAs that I will colon into each of these vectors. Two of them target the 1st exon of my gene and two of them target the 2nd exon. I plan to try out if its better to use 2 gRNAs (screen for deletion) or 1gRNA (screen for Indel by Surveyor assay http://www.idtdna.com/pages/products/reagents/idt-surveyor-mutation-detection-kits).
I will let you guys know how it goes.
Best,
Lyra
I used NEB Builder to assemble 4 sgRNAs onto pX458. Each of them contains their own U6 promoter and they seem to work fine.
Wow! I don't know about the NEB builder before!!
Thanks many for sharing!!
Lyra
Hey,
Can any one tell me if there has been any study comparing the specificity of the Cas9 nickase & eSpCas9(1.1)? Or if you would know which is better between the two ? It would be great help!
Thank you.
Hi Ananya,
I have no idea whether they compared Cas9 nickase & eSpCas9(1.1) but I recently generated vectors with the PX backbone expressing Cas9 K848A, K1003A, & R1060A (the mutant Cas9 in eSpCas9(1.1) )fused with Puro or EGFP. This modification can make the eSpCas9(1.1) more selectable.
Will let you guys know whether the vectors work well or not :-)
Best,
Lyra
Also, I remember in the Zhang lab forum, they said something about the nickase have much lower efficiency than WT. Therefore you should use the WT if you are just making indel.
Hi Lyra,
Thanks for the information. I am sure it will be helpful to me.
And I would really appreciate you letting me know about the vectors you designed.
Best,
Ananya
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