CRISPR questions

Hello All,

I was wondering how people are faring with the plasmids from Friedland et al. in Nature Methods. I got the constructs from Addgene (Peft-3::cas9-SV40_NLS::tbb-2 3’UTR and PU6::unc-119_sgRNA) and verified them by control digest. I injected 13 animalsaccording to the mix in the paper: Cas9 (50ng/ul), sgRNA (45ng/ul), pCFJ104 (5ng/ul)–100ng/ul total.

I scored the F2 progeny of 125 mCherry positive F1s, and I unfortunately had no Unc-119 worms. I had a few funny looking Unc worms, but they were definitely not Unc-119. I also had a lot of dead eggs in F2. A few other labs I’ve communicated with have similar results, but no CRISPR effect.

Are there any tricks to this that we’re missing? Is anyone else having problems or have people been successful on the whole?


It’s not a directly helpful reply to your question - we’ve done CRISPR-Cas by a fairly different approach, and I can’t speak from experience to the specific problems you’re having - but there are six papers available in advance online publication at Genetics describing a range of methods to use CRISPR-Cas to get mutants in C. elegans; one warns of possible toxicity with Peft-3::Cas9 expression constructs.

(edited to add: I have just been made aware of a seventh paper available as an early online publication.)

Thank you!

I will definitely explore all available options. I just thought that this one should be pretty reliable, so I’m wondering about others’ experiences with the Frieldland et al. system.

rescue of unc-119 …

Also, please see this:

I have had similar results with the Hsp16.46 that is been used in the Boxem lab ( I also tried the Peft-3::cas-9, but in my hands the worms don’t produce any mCherry+ F1’s. Only dead eggs are sometimes seen.