Investigating expression of a human optimized cas9 transgene in Neurospora crassa

The CRISPR-associated Cas9 enzyme is used in molecular biology to engineer the genomes of a wide range of organisms. While Cas9 can be injected or transfected into a target cell to achieve the desired goal, there are situations where stable expression of Cas9 within a target organism is preferable. Here, we show that the model filamentous fungus Neurospora crassa is recalcitrant to heterologous expression of a human-optimized version of Streptococcus pyogenes cas9 . Furthermore, partial optimization of cas9 by synonymous codon exchange failed to improve its expression in the fungus. Finally, we show that transgene expression can be detected when cas9Hs sequences are placed in the 3’ UTR regions of transgene-derived mRNAs, but not when the same sequences are in the translated part of the transgene-derived mRNA. This finding suggests that the primary obstacle to high cas9Hs expression levels in N. crassa is translational in nature. ### Competing Interest Statement The authors have declared no competing interest.