1) Is this new Cas9 expression system developed to to eliminate random integration of the construct? If so, this is certainly important and it should be explained more clearly in the text.
2) My understanding is that both the lox-p sites and the TK gene, are serving the same purpose: to have a way of deactivating Cas9 in case in sticks around after genome editing. I see this a little redundant. Maybe authors should briefly explain different situations in which each of them are more favorable.
3) Along the same line, it seems like GFP and puromycin can both be used for positive selection of transfected cells. It would be nice if authors explain this redundancy as well. Perhaps one would be used for FACS sorting of the cells, and one would be for drug selection. I emphasize on redundancy because large size of these types of plasmids make them hard to transfect in many cell types.
4) "Kleinstiver et. al. 2016" should be cited along with "Slaymaker et. al. 2015 " as these two came out around the same time. Although, none of these two improve the "mutagenesis" of Cas9, but rather its specificity.