Hi all,
I'm currently designing constructs for CRISPR- and HR-mediated epitope tagging of ORFs in their genomic context, and could do with a little guidance.
From what I've read, it sounds like the best bet is to use two opposite-sense, closely-juxtaposed gRNAs and the D10A nickase version of Cas9 (Cas9n) to enhance specificity and reduce off-target effects; however, to minimise cloning and enhance the efficiency of my targeting, I'd like to express both gRNAs and the Cas9n from the one plasmid - combine the whole targeting system onto one vector.
My question is, does anyone know of such a plasmid that contains two gRNA target sites and also encodes a Cas9n nickase? I've done a bunch of reading and have been unable to find this exact permutation of the CRISPR system. The closest I've found is the pX333 from the Ventura lab which expresses wt Cas9 and two gRNAs, though from what I can see they use these gRNAs to target two separate genomic locations (hence the wt not nickase Cas9).
If such a plasmid does exist, I'd be hugely grateful if someone could let me know - and, if possible, send a small aliquot (private message me for information). If not, suggestions on cloning strategies are always welcome - I'm looking at either sourcing pX333 and doing a SDM for D10A mutant, or taking e.g. pX461 and cloning a second U6-gRNA-tracrRNA unit into the XbaI site using PCR amplification and XbaI/NheI digestion (followed by mutagenesis to convert the BbsI sites to e.g. BsaI).
Thanks very much for any/all help available!
Regards,
Dan
Hi
Hopefully, this article will be helpful for you.
http://www.cell.com/cell-reports/abstract/S2211-1247(15)01493-X
.................
Hassanur
Hi Daniel Dehany Scott ,
at the end, did you find this plamid?
best,
Antonino
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