We have tried doing CRISPR/Cas9 using the Ribonucleoprotien method. We got Cas9, crRNA (dpy-10 and my gene), and tracrRNA from IDT. We are trying to do co-conversion with the dpy-10 ssODN. My repair template is 1kb (GFP) with 35nt homology arms generated via PCR, and I inject 3.15ug .
I’ve tried injecting a few times (~15 worms an injection) and I haven’t been able to see any rollers at all for any of the injections. Does anyone have any idea on what I may be doing wrong? We are following the Paix et al 2015 paper.
Make sure you incubate the injection mix at 37C before injection, as directed in Alex’s protocol. I think this is necessary for assembly of the Cas9 RNP - I forgot to do it once, and the efficiency was really low.
Check your injections - it’s possible you’re just not hitting the germline. You can add a fluorescent co-injection marker to your injection mix as a positive control for successful injection.
Staging is really important in my experience, because the dpy-10 worms only roll as adults. Checking 3 or 4 days after injection is optimal. If you look too early or too late, you can fool yourself into thinking it didn’t work.
If you struggle with co-CRISPR, drug selection is a very robust alternative method for GFP tagging of endogenous genes.
In terms of incubating the Cas9 RNP, I read of some people hybridizing the RNAs at 95C. Would incubating only at 37C still be okay?
I’m pretty sure I’m hitting the gonads, I’ve made a few worm lines with extra chromosomal arrays before. I’ve even had a few other more experienced lab mates try the RNP injections and they haven’t gotten anything.
Thanks for the tip on staging, usually if I don’t see L4 rollers I give up. How many P0s would you suggest I inject?
95C?? I’d be surprised if that works - it would denature the Cas9. We’ve done incubation at 37C which is sufficient for successful CRISPR in our hands.
In terms of number to inject, I usually do 20 - it’s overkill if you are a skilled injector, but better to have too many than not enough. Out of 20 injected worms, at least 15 should give Rollers if your injections are good.
One other thought - are you making your own Cas9, or are you using commercial stuff? If it’s a homemade prep, it might be worth checking activity in vitro and/or buying a batch of commercial stuff just to get it working.
With the 95ºC, are you just meaning the RNA? The protocol that I got involved heating the sgRNA then cooling to refold, then to do as Dan indicated and add Cas9 and incubate at 37ºC. One additional question is whether you are seeing dumpy worms? Sometimes I got low numbers of rollers, but usually see at least a few dumpy animals.
Sorry for the confusion. What some people have told me is to incubate the RNAs (tracr, and crRNAs) at 95C then cool it down, and then add it to the Cas9. We are currently using commercial Cas9 (and RNA) from IDT.
When I do an injection (~15 worms) I do see a few dmpy F1 worms (~4), but no rollers. The dmpy does not get passed on to the F2s, the F2s look WT.
Does the concentration of repair template play a role? So for my gene repair I’m using a PCR product of ~1kb (GFP). Is there a minimum amount of repair DNA I should be adding to my mix? As I said below I add 3.15ug total repair DNA (157.5ng/uL).
Again, I agree with Dan that injecting 20 is overkill and you should be getting tonnes of dpys and rollers from that amount. Are you using a ratio of dpy:target? If so, what ratio? I’d try a straight-up 100% dpy crRNA+50 ng/µl oligo+Cas9 injecton. IF you don’t get dpys and rollers in that experiment, either there is something wrong with your Cas9 or crRNA, or you’ve got injection issues. For repair template, I don’t think that I’ve gone that high. So if the experiment that I suggested worked, then it could be too much DNA causing an issue and you could try less.
Jordan’s suggestion to try a few injections with dpy-10 + Cas9 alone is a good one. That being said, I’ve never heard anyone report having problems as a result of too much repair template - unless it’s toxic, but that seems unlikely for a GFP PCR product.
Even if the GFP integration doesn’t work, you should get Rollers out no matter what (they’ll just be GFP-negative if the co-conversion isn’t working). Overall I’d agree with Jordan that this sounds like a case of either dead Cas9 or a problem with the injections.
I have found that PCR product purified through a standard zymo-kit can be quite toxic when injected into worms. You may want to make sure the product is as clean as possible.