CRISPR: no idea what's happening

Model Organism: Worms Methods & Reagents
1

I’m using the co-CRISPR method (targeting dpy-10 with recCas9 : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566275/).
I get some F1 animals that are dumpy, but I also get lots of F1 that are dumpy AND roller at the same time. I have no idea how this could happen.
Has anyone seen this before?

2

Could they be mosaic?

3

From Arribere et al. Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224173/):

heteroallelic combinations mut/o confer a different phenotype from the desired mut/+ heterozygote … dpy-10(cn64)/dpy-10(o) are dumpy roller

4

::slight_smile: right! thanks

5

And in my hands I get this a lot. I tend to get more DpyRol than rollers. The recCas9 is REALLY efficient.

6

Talking about the recCas9, are you using purified protein or plasmid ?
Thanks

7

We’re moving towards using purified protein. A lot of UC folks are using it because Berkeley has a core that makes it and distributes it for a reasonable rate. I’ve made a large batch of my own and it’s pretty straightforward. I’ve tried two different Cas9 preps from two different protocols (Jinek and David Liu plasmids/preps) that a few colleagues generously gave me . I also tried commercial cr/trcRNA vs homemade sgRNA. The two Cas9s were comparable, and the cr/trcRNA seemed to work better, but the homemade sgRNA worked well. If one wants to save money, homemade sgRNAs work well (what I’m planning to do). For Cas9, the Jinek protocol will give you the cleanest prep, but it’s an extra day and a few extra steps, and needs an HPLC. The David Liu protocol is a day faster, and pretty easy. Both Jinek and Liu protocols use and FPLC. The method that Paix and Seydoux describe looks easiest (batch nickel purification, then flow over a Q ion exchange resin to bind DNA). The Cas9 RNPs really produce a ton of jackpot broods. I’m a reasonably decent injector and injecting 6-12 gives a few animals producing jackpots.

8

Thanks for your answer, I will see if our facility is producing this recCas9, otherwise, we might interested to get it.

I’ll contact you in case I need. :wink:
Cheers,

9

Yup! DM me if you need protocols/plasmids etc.