Mello lab CRISPR protocol (single stranded ends)

Model Organism: Worms Methods & Reagents
1

I’ve been using the new Mello lab method for generating GFP knock-ins using the annealed GFP products with overhanging single stranded ends. I’ve had some strange results with what looks like GFP expression that I’m expecting, but only in roller animals. Has anyone else experienced this? It is strange because it makes me think it must be expression from an array, but the cell specificity and bright expression would indicate that it is under the control of its endogenous promoter, which could only happen if it had been edited in properly. Has anyone else come across this? Also any tips to make this technique work optimally?

2

If you get a roller animal with the expected GFP expression, can you segregate GFP away from the Rol phenotype or are they linked?

3

If you read the paper carefully they suggest that the array carriers are much more likely to have the desired edit, so much so that they only screen for Rollers and then test via PCR for insertion.

That hasn’t been our experience but my guess is yours matches theirs.

We had to lower the amount of DNA by half to get better results, but we are trying to knock in much bigger fragments than just GFP, which might explain some differences in our hands.

Steve V

4

Have you done any PCR genotyping? I’ve gotten PCR false positives for insertion caused by array formation if I try to genotype using one primer in the locus and one in the repair template (PCR jumping or something), but if you use primers both external to the homology arms it should be easy to detect an insertion.

5

I haven’t done any PCR genotyping yet, but I know a lab member in my previous lab has done outcrossing and was able to segregate the roller from the GFP (which wasn’t happening by just selfing animals), and genotyping showed the insert in some cases, but was sometimes inconclusive. I’ll keep plugging away and see if I can segregate the roller phenotype from my lines, and also keep trying additional genes.

6

Is your knock in on chromosome II, near the dpy-10 co-CRISPR marker? Otherwise they should independently assort just fine.