1- Can you confirm you inject in the yolk? Is it not better to inject in the cell?
Yes, we have always injected in the yolk. We simply aim to inject in the centre of the yolk. Wu et al., 2018 (10.1016/j.devcel.2018.06.003) looked at yolk vs cell injections in great detail. Among others, they found that the RNPs are rapidly transferred to the cell. It is possible that injecting directly in the cell would create slightly more mutations. However, mutagenesis is not a limiting factor in our experience. We would actually be slightly more worried about generating too many double-strand breaks, especially when targeting multiple genes at once. Most importantly, yolk injections are much quicker, which means 1) all the eggs are injected rapidly just after they are laid; 2) higher Ns can be achieved, which is especially useful for experiments where a population of F0 knockouts need to be phenotyped. 2- My gene has a single/two exons, how should I proceed?
We would simply place all three target sites on the same exon (or two on one exon). In the publication, this was the case for mab21l2. You can read more about this in the Author response (question 7). Try to spread out the target sites as much as possible, especially avoiding overlap between each crRNA binding site/PAM sequence. This is to avoid as much as possible situations where one RNP mutates the binding site/PAM of another RNP.
3- Did any F0 knockout failed to replicate a stable mutant line phenotype?
At the time of writing this protocol, we have targeted 13 genes and replicated all expected phenotypes. All examples are presented in the publication.
4- In vitro transcribed sgRNA are more economic – is it necessary to use synthetic crRNA and tracrRNA?
Yes. It is highly likely that synthetic RNAs (i.e. not in vitro transcribed) are necessary for the success of the method. You can find an explanation and references in Author response (question 8).
5- It is more economic to use Cas9 mRNA – is it necessary to use Cas9 protein?
Yes. We think it is unlikely that the method would work as well with Cas9 mRNA. You can find an explanation and references in Author response (question 2).
6- How early should I inject?
We think it is crucial to inject very early, i.e. at the single-cell stage before the cell inflates. The cell should still be flat, wrapped around the yolk. I generally set everything up for injections (calibrate the needles, etc), then remove the divider of a few breeding boxes and wait for 7–8 minutes next to it, then collect the eggs, put the divider back, inject these eggs. All the eggs are generally injected within the first 20 minutes after they are laid. If for any reason some eggs have started inflating or dividing, I start over with the next clutch. This may be eased a little if the studied phenotype does not require maximum penetrance.