I am naive to this CRISPR/Cas9 system for gene editing. As a part of my research project, I want to establish this protocol in my lab to knockout the E6 and E7 oncogene of HPV16 and HPV18 virus. I have designed the gRNA using the ZiFit software and now I have to clone the gRNA fragments. Can anyone suggest me a good and easy protocol for cloning gRNA fragments? Also the software has suggested nearly 7 gRNA, do I have to clone them all and screen one by one to check the efficiency of gene knockout?
We currently use the linked protocol from the Zhang lab. We use the updated one step protocol on page 4 and it works very efficiently. As for screening different guides, you may get lucky and the first one would work or you may have to go through all of them, there isn't really a way to know until you try.
http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf
Hi Shahid,
For your cloning, you must check if your gRNA plasmid is compatible with the protocol from Zhang lab, the digestion enzymes can change and the approach also, check if you use the good promoteur for your plasmid on zifit design web-page. Finaly, if everything is ok, for your 7 gRNA , try to choose the best one, I mean the one with less off-target and the best location in your ooncogene.
In one word: Don't. You can have the entire expression cassette with U6 and scaffold synthesized in your gene synthesis company's default vector for under $100. That simply does not justify the trouble under any circumstances I can imagine. Then, co-transfect each synthetic vector with your Cas9 plasmid.
Dear Shahid
You can do in vitro digestion experiment to screen the efficiency of 7 guide sequences. If you have more research funds then you can go for this experiment because you need to buy in vitro transcription kit, cas9 enzyme and order your guide sequence flanking with T7 promoter and gRNA scaffold sequences at both 5' and 3' ends respectively. I will send you further details if you are ready to do this experiment. All the best.
Hello Shahid!
I'm into something similar research. I see this question was asked 4 years ago. Did you get any results of it? Just curious as I'm in the process of constructing a Cas9 plasmid targeting E6/E7.
Thank you!
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