The use of CRISPR to generate a whole-gene humanized and the examination of P301L and G272V clinical variants, along with the creation of deletion null alleles of and loci.

To study important genes involved in Frontotemporal Dementia ( , and ), we created deletion alleles in the three orthologous genes ( , , and ). Simultaneously, we replaced the gene with the predicted orthologous human gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an mutation rescue technique. Clinical ALS/FTD missense variants G272V and P301L were successfully inserted in . Neither loss or clinical variants caused neuronal defects in young adult or aged , based on examination of glutamatergic phasmid neurons. Yet, we noted decreased survival to day 9 in the P301L strain, compared to control strains. Based on these results, we comment on strategies for humanization, including the importance of confirming gene predictions and identifying loss of function defects for each gene before embarking on humanization, and we report the creation of strains and a new gene-editing selection strategy that will be useful for future studies.