I have transfected cancer cell lines with pHAGE TRE dCas9-VP64 for overexpression. Now I need to select gene specific sequence for my guided-RNA. Any suggestion for specific and no off-target prone gRNA resource will be highly appreciated?
Hi Tushar Tomar,
I am using the following tools for gRNA design (also for CRISPRi/a):
https://crispr.med.harvard.edu/
http://crispr.cos.uni-heidelberg.de/
http://crispr.mit.edu/
I am cross-checking off-targets of the predicted guides by using the BLAT tool from UCSC genome browser.
https://genome.ucsc.edu/cgi-bin/hgBlat?command=start
Hope this helps.
You can try MIT gRNA designing tool and benchling as well. MIT only takes 250 bp DNA as input so you need to know where you want the cut in your GIO while benchling takes the whole gene and then designs the gRNA. Also, if your aim is to KO the gene then you should be aware of the region/ exon others have already used to KO. Personally, I use both the tools and then select the best gRNA after comparing.
crispr.mit.edu is best to design crispr gRNA.i design crispr gRNA for my gene with this and it works well.
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