Optimizing CRISPR/Cas9 mutagenesis in Drosophila da neurons to avoid cytotoxicity
Duarte, V. N.; Najafi, V.; Thompson-Peer, K. L.
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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, but as with any new tool, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types. Here, we show that Cas9 expression alone causes cytotoxicity in the dendritic arborization (da) neurons that are widely used to study neuronal development and regeneration. We then systematically evaluate alternative Cas9 transgenes designed to lower total Cas9 expression, called uCas9 transgenes. We show that expression of these uCas9 transgenes results in little to no cytotoxicity to da neurons. Lastly, we demonstrate the ability of uCas9 transgenes to effectively and specifically gene edit in da neuro ns. Thus, we expand the toolkit of genetic perturbations available to researchers working with Drosophila da neurons or other cell types suceptible to cytotoxicity due to high expression of Cas9.
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