Co-conversion CRISPR

Hi All,

I was hoping to get some advice for my co-conversion CRISPR. I have tried 3 different guides (with scores 97, 97, and 94), I am using a ssODN that has the desired change, a mutation in the pam, and has 50 bp homology arms. I have done 8 separate injections and usually all my injections worms give rolling progeny, and I get jackpot broods that have 40 or more rollers. I screen all the rolling progeny and about 60 non-rolling progeny, and I have yet to get a successful edit (verified by sequencing).
Does anyone have any suggestions/ideas why I’m not getting any editing? I can believe that one guide might not be efficient, but all 3? Also, my ssODN is the strand opposite the guide. Should I have the ssODN be the same strand as the guide?

Thanks in advance for any suggestions.

Try to design some new ssODN maybe useful. Check this paper. https://www.nature.com/articles/nbt.3481

A few questions/ideas:

-there’s no reason that the desired mutation would cause lethality is there? Just want to make sure that we can rule out the possibility that you’re getting the edits, but unable to recover the edited animal.

-how far from the DSB is your desired edit? Less than 10 bp? If more, than the efficiency can drop off. How big is your insert?

-where is the PAM relative to the insertion site? This orientation is the key determinant of choosing strand polarity. The paper that shzhhua693 pointed out discusses that Cas9 dissociation is asymmetrical and it remains bound to one strand. I remember Bobby Farboud talking about this point at the CRISPR workshop at the last worm meeting. Alex Paix and Geraldine Seydoux had a recent paper discussing editing in human cells and mouse embryos where they discuss donor DNA design rules. From these studies, if you are trying to knock sequences in 5’ to the PAM, use the protospacer strand (this would be an ssDNA that would be the same strand as the guide, often called a sense ssODN). If your edits are 3’ to the PAM, use the spacer strand (this would be the strand opposite to the guide, often called and antisense ODN). If your insertion is right as the DSB site, then strand doesn’t seem to be a big efficiency determinant
http://www.pnas.org/content/early/2017/11/27/1711979114

-you could try adjusting Cas9 concentration; the Mello lab had a nice paper showing that Cas9 had some toxicity and that reducing the amount of Cas9 could boost editing efficiency. Given that you’re getting jackpot rollers, this probably won’t help with your oligo knock-in issue, but is probably a useful practice. They also describe hybrid dsDNA-ssDNA repair templates that worked better to knock-in GFP sized edits
https://www.biorxiv.org/content/early/2018/06/20/352260

-if the oligo polarity doesn’t sort things out, you might consider trying a selection-based approach. Matt Schwartz and Erik Jorgensen has a nice Micropublication describing modified SapTrap vectors for introducing point mutations with unc-119 selection. You could also us this approach with the SapTrap SEC selection plasmids that Dan Dickinson recently published. The power of this approach would be that if you position the point mutation between your DSB and selection cassette, then if you get the selection cassette inserted, then the point mutation will be incorporated. You could put the selection cassette in a native intron, or flank the cassette with splice-site sequences
https://www.micropublication.org/schwartz_2018_saptrapvectors.html
https://www.micropublication.org/dickinson_2018_saptrap_sec.html

Good luck!