Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma^-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma^-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is trun-cated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma^-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J Gzma^S211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteo-lytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.