Generation of a null allele and humanized containing human disease-variants.

Clinical variants of are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and other degenerative diseases. The predicted ortholog of is encoded by , but functional orthology has not been demonstrated We undertook CRISPR/Cas9-based genome editing of the locus to create a complete loss of function allele; all exons and introns were deleted, creating , which resulted in neurodegeneration after oxidative stress. Next, we undertook CRISPR-based genome editing to replace exons with human TARDBP coding sequences, creating humanized ( ) expressing TDP-43 Based on the efficiency of this genome editing, we suggest that iterative genome editing of the target locus using linked coCRISPR markers, like , would be a more efficient strategy for sequential assembly of the large engineered transgenes. decreased the neurodegeneration defect of , demonstrating functional cross-species orthology. To develop models of FTD and ALS, we inserted five different patient variants in the locus. Only one clinical variant increased stress-induced neurodegeneration; other variants caused inconsistent or negligible defects under these conditions. Combined, this work yielded an unambiguous null allele for , a validated, humanized and multiple ALS/FTD patient-associated variant models that can be used for future studies.