Optimizing CRISPR/Cas9 mutagenesis in Drosophila dendritic arborization neurons to avoid cytotoxicity

Genetic perturbations are one of the great strengths of the model organism Drosophila melanogaster, with approaches such as classical mutagenesis and RNA interference enabling a wealth of biological discoveries. A more recent approach for altering gene expression is CRISPR/Cas9-based mutagenesis. As with any new tool, however, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types, including class IV dendritic arborization (da) neurons. In this study, we provide evidence that Cas9 expression causes cytotoxicity in class I da neurons, in addition to class IV da neurons, both of which are widely used to study neuronal development and regeneration. We then systematically evaluated available Cas9 transgenes designed to titrate Cas9 expression, called uCas9 transgenes. We show that the expression of these uCas9 transgenes results in little to no cytotoxicity in various classes of da neurons. Immunostaining revealed drastic reductions in Cas9 protein levels for da neurons expressing the uCas9(L) transgene. Lastly, we demonstrate that the uCas9(L) transgene effectively and specifically gene edits in both class I and class IV da neurons, lowering the expression of GFP-tagged proteins and producing loss-of-function morphological phenotypes when targeting endogenous loci. Thus, we refine the use of CRISPR mutagenesis in Drosophila da neurons through titration of Gal4/UAS-mediated Cas9 expression using existing uCas9 transgenes, a feasible and flexible approach that may be useful for other labs encountering Cas9 cytotoxicity in their own model systems.