Jorgensen Mix-SEC CRISPR

HI All,

I have been trying (without luck) the past few months to get an N-terminal insertion using the SEC method. My fellow lab mate has gotten it to work with C-terminal insertions, but for some reason we can’t get N-terminal insertions to work.
We seem to be getting stuck at isolating non-red rollers. After the injections we get red rollers then we do the Hygromysin treatment and we continue to get red rollers. We sometimes think we get non-red rollers, however there is always some faint red we can see from the Jorgensen mix that always seems to mark just one tissue.
Does anyone have any advice for something we’re doing wrong? I labeled this as Jorgensen Mix because of our problem with almost getting non-red rollers. The mix we use is: 10 ng/μL pGH8 , 5 ng/μL pCFJ104 , and 2.5 ng/μL pCFJ90 .

Thanks for the help.

Hi Nemo,

A couple of troubleshooting questions:

  • Have you tried multiple targets (for N-terminal insertion) or is it just one gene that’s causing problems?

  • Are you targeting an essential gene?

  • Compared to the C-terminal insertion that worked, when you try the N-terminal insertion do you see a comparable number of red rollers, or are there many more? Tons of red rollers can sometimes be an indication that there’s something wrong with the Cas9 plasmid.

HI Dan! Thanks for the help.

  1. Yes, we have tried multiple genes for an N-terminal insertion and have yet to get one to work. We have also tried different guides.
  2. 1 for sure is maternal effect lethal. The other 2 we’re not so sure about, as there exist no mutant. But RNAi for both genes are not lethal.
  3. Yes, for N- and C-terminal tags we get a comparable amount of F1 red rollers. They then give tons of F2 red rollers. For the C-terminal tags we always find non-red rollers.
    We are still using the ccdb based plasmids, but we always sequence, and digest to make sure we are getting the appropriately sized bands.

Thanks again!

As far as I know, both Cas9 and the repair machinery should be “blind” to which end of the gene you are targeting. So if you’ve had multiple C-terminal successes and multiple N-terminal failures, that’s really weird. The fact that you can get the C-terminal tags to work argues against the “usual suspects” (bad injections, dirty DNA etc). Also the fact that you’ve tried multiple sites and multiple guides argues against one construct having an undetected mutation or other issue. So I am honestly a bit stumped.

Have you looked at the F3s (~10 days post-injection, 1 week post hygro addition)? Sometimes we’ve seen lines come up if we wait an extra generation. This can be especially helpful if you have lots of array animals on the plates, since the insertions will tend to out-grow the arrays with time.

I’ve kept plates to look at F3s, but it’s a little tough because at that point there are so many red, rolling F3s.
I’m watching more careful this time around, and we have a few (~3) red, rolling F2s that are very faintly red. I’ve moved them to a new hygromycin plate and am hoping they give rise to non-red rollers that our grow the array.
Before we started using the Jorgensen mix (we only used on of the plasmids) I was able to detect some non-red rollers in the F2 generation but they turned out to be EX arrays instead of being CRISPR knock ins.
However with the C-terminal injections we only used one of the plasmids and were still able to get 100% rollers.

Thanks again!

The fact that you have tons of red rolling F3s worries me a little bit - in our hands, the array animals tend to be sick and slow-growing, so they are rapidly overtaken by dark worms on plates that have knock-ins. We might see one or two plates were there are just tons of red rollers, but nowhere close to every plate.

I’ve seen two exceptions to this: One was my attempt to tag RGA-4 (the one gene that didn’t work in my 2015 paper) and another was a similar case a couple years later. In both cases, we never got knock-ins, but there were tons of red rollers on every plate. It turned out that in those cases, we’d inadvertently introduced an inactivating point mutation in the Cas9 ORF (which I don’t routinely sequence because it’s so big) in the process of cloning the sgRNA.

Based on this and a couple of other observations, I’ve always suspected that active Cas9 is somewhat toxic, and thus acts as a (partial) negative selection against arrays. When Cas9 is inactive, you get tons of red animals but no insertions.

Considering what you’ve told me, I would recommend re-sequencing the Cas9 ORF in your sgRNA plasmids to rule out that you are experiencing a similar issue. I can’t guarantee that’s the problem, but it would at least be a good thing to rule out as part of your troubleshooting.

Interesting. I’ll for sure check out the Cas9 ORF.
From what I’ve seen the rolling red worms are very healthy and they propagate like crazy.

Hi Dan,

I just wanted to let you know you were right! The issue was with our guide plasmids. I recloned and verified my guides, and re-did the injections and was able to get a line from my first injection.
We did see what you were talking about some red-rollers were very healthy and gave tons of healthy red-rolling worms. But there were a few that were “sick” and we got our line from one of these worms. We did see some lethality before removal of the SEC, and we also got some self excision and saw TagRFP localisation.

Thanks again for the advice!