the eggs of CRISPR worm all dead

Hi all, I am doing CRISPR knock in to get a fusion GFP with my target protein. Luckily I got one worm seems like the GFP worm I wanted. While the eggs of this GFP+ worm all dead.
The knockout strain of my target gene can survive. Is that mean the design of guide RNA have unspecific cutting that causes lethal? The normal and the dead eggs were attached.

Your GFP+ picture doesn’t look like dead eggs - they look like unfertilized oocytes to me. Is it possible your GFP+ worm was just old?

When you say you “got one worm,” do you mean you have one line, or really just one worm? If your CRISPR is working, you should never have just one worm, you should have lots of worms (even if they are descended from a single founder).

I’d discount the possibility that off-target effects are to blame for what you are seeing, since (to my knowledge) no one has ever reported a bona fide off-target CRISPR mutation in C. elegans (though we and several other groups have looked for them).

Yes, I agree. Your GFP+ animal could have just been an old P0 or F1 that accumulated gut autofluorescence which can resemble GFP expression.

When you say “the knockout of your target gene can survive” - I take it you mean that a previously existing null mutant is homozygous-viable, not that you knocked it out using CRISPR? Because if you haven’t previously gotten CRISPR to work there, it seems likely that your CRISPR at this target just isn’t working very well so far.

Also: are you screening for knock-in only by GFP fluorescence, or also by PCR? Are you using a co-CRISPR/co-transformation marker to identify promising F1s?

Thanks for your information :). I really got only one. The offspring of this worm seems very little. I am waiting for them to grow then I’ll check again.
Just pick the GFP+ worm for single worm lysis today, I will do the PCR tomorrow.
Before that, I took images of the worm with the one express my co-injection marker (myo-2::gfp) as control.
I haven’t seen autofluorescence looks like this before. Would you mind give me some suggestion?

I don’t know how much more I can say without knowing what gene you tagged, what protocol you are using, and what you hope to see. The intestinal fluorescence in that worm definitely looks like autofluorescence to me, so what is the GFP signal you think you are seeing? Those bright blobs near the posterior? I’m not sure I’ve seen anything that looks exactly like that either, but I’d be skeptical unless you get progeny that all show the same pattern.

For the purpose of increase the CRISPR insertion frequency, I choose a gRNA target gives score only 72 (the specific ones are not within 100bp of cutting site) in, which means it have several off target cutting sites in prediction.
I think I can backcross with wild type after. That’s one reason I think I might get off target which causes mutation.

I am agree with dan_dickinson, the “dead egg” is unfertilized oocytes. Since you mentioned the progeny is small, I guess your CRISPR may mutated the energy metabolism genes, which cause defects in both fertility and development.

Thanks for your reply. I tried by single worm PCR. The GFP+ worm was heterozygote for the insertion. However, it seems that all the offsprings do not have the GFP anymore. I will do single worm PCR to all the offspring to confirm. I think I’d better change the gRNA mutation site.