Hi,
I am planning to use RNP nucleofection to insert a DNA fragment by HDR-mediated editing by CRISPR-Cas9. I have always used dual promoter plasmids for this, so I don't have experience with the different synthetic gRNA in the market.
In your hands, which gRNAs (from which supplier) give the best results?
Regardless of cost, do you prefer crRNA:tcrRNA or one-system sgRNA?
Thank you
Cris
https://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/crispr-cas9-gene-editing/sygrnafaq.html
Hope you can find your answer by following this link
https://www.thermofisher.com/bd/en/home/life-science/genome-editing/geneart-crispr/crispr-libraries/trueguide-grnas.html
I clone my own multiple promoter'ed (up to 3) guides on a single Cas9 expression plasmid. Several such are now available on the AddGene website.
Thank you Randolph,
That is what I have always used. However, now I want to directly transfect RNPs (gRNA-CAS9 ribonucleoprotein).
Cristina Gutierrez Caballero
I clone the guides and the Cas 9 protein all on one single plasmid. I do not transfect with multiple plasmids, just one plasmid, with 0, 1 or more linearized HR inserts.
Hi,
I used gRNA-Cas9 online tool to designation and ordered to Merck group to terrifically synthesis gRNA in the primer caset for dual plasmid.
Amy Johnson
ok, and the Merc group is 8 EURs per nucleotide and seems cheaper than kit.https://www.sigmaaldrich.com/catalog/product/sigma/crispr?lang=it®ion=IT
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