Hi RG community,
I'm about to order some single guide RNAs (gRNAs) for CRISPR/Cas9 site-directed knockouts following Bassett and Liu (2014) protocol (paper below). Briefly, two partially overlapping primers (an ~ 64 nt forward target-specific and an 80 nt universal reverse) are used in PCR amplification to generate a DNA template, which will then be used for T7 in vitro transcription (gRNA synthesis).
Now, I've always ordered these primers PAGE-purified, reasoning that more full-length oligos (non-truncated) would generate more full-length functional gRNAs, thus getting more mutagenesis efficiency. However, some colleagues have been using standard de-salted oligos instead, and they have the impression that works just fine. Considering that they're way cheaper than PAGE-purified oligos, it might worth a try, but I'm very skeptical about it.
So, that's the question: are PAGE purified oligos really necessary for CRISPR/Cas9 mutagenesis?
Any and all comments are very appreciated!
Dani.
Bassett and Liu (2014):
Article CRISPR/Cas9 mediated genome engineering in Drosophila
Currently, I am using standard de-salted oligos which I ordered from eurofin genomics. They work perfectly, the precision of my gRNA is yet to be fully tested after my current experiment is over however the current preliminary protein expression results shows that my Gene of interest has been knockout using the CRISPR/cas9 with the ordered standard oligos. Thank you, Daniel, your question have questioned my procedure and I will certainly want to know if there exist any significant difference too.
Joseph Ayariga, thanks for your input. I've also been able to generate mosaic mutants using standard de-salted oligos, but in a very low frequency. Among other parameters, it could be just the gRNA efficiency, which might be improved using PAGE-purified versions. Testing some next week. Good luck with your experiments.
Thank you very much Dr. Daniel, I am glad to hear your feedback, and thanks too for your good wishes. Wishing you the best too.
Daniel Fenando Paulo standard de-salted oligos finally working well? I need to do primers with 55-59 nt to synthesize gRNA and I not also sure that desalting quality is enought
Hi Alexandra. Yes, standard desalted primers are good enough. The sgRNA sequence itself is way more important than this. Best luck, - Dani.
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