Has anyone used recombinant cas9 protein for injection? Any success? And how much to use?
The Genetics paper (Genetics November 1, 2013 vol. 195 no. 3 1177-1180; https://doi.org/10.1534/genetics.113.155853) did injection with protein but it was mixed with
in vitro transcribed sgRNA. I want to just inject with a Pu6 sgRNA plasmid with the protein and compare the efficiency.
Thanks for your attention.
Please see this thread:
http://forums.wormbase.org/index.php?topic=3052.0
The protocol in question was from Paix and Seydoux, but be warned that the concentrations therein are off. Re-calculate them yourself! And I suppose be careful about the steps discussed in the thread.
Another protocol we’ve used is from Abby Dernburg’s and Simone Kohler’s blog post on the IDT website (with link to more detailed protocol at the end of the blog): https://www.idtdna.com/pages/education/decoded/article/genome-editing-using-the-alt-r-crispr-system-in-em-c.-elegans-em. This protocol is similar to the Paix protocol.
One other point to keep in mind is that I don’t know of anyone who has used Cas9 protein with an sgRNA expressed from a plasmid. All protocols that I’ve seen use either in vitro transcribed sgRNA or trcRNA+crRNA. You can try the approach you outlined, but there might be some limitations. Your window of editing will be defined by Cas9 protein half-life and the time it takes for the U6 promoter to express the sgRNA. So there will be a period of time where Cas9 is not complexed with an sgRNA and therefore inactive. I’m not sure how long Cas9 is active in the germline. I know from old labmates working in mammalian cells that the half life of Cas9 could vary strikingly between cell lines (gone in under 6 hours in some cells, detectable after more than a day in others). The advantage to using Cas9 protein and RNA assembled in vitro is that you can use equimolar amounts (or a slight excess of sgRNA) so that in theory most Cas9 is complexed with sgRNA. It’s not clear what the molar ratios would be with Cas9 protein and sgRNA expressed from a plasmid. Cas9 protein and a U6 driven sgRNA plasmid will likely produce edits, the only question is whether you’ll take a hit in efficiency and also risk losing the longer window of editing that comes with using all plasmid components.