Massively parallel saturation genome editing of an essential mitochondrial targeting sequence (TileSeq)

We employed saturation genome editing (SGE) to assess the functional consequences of synonymous, missense, and nonsense variants across KARS1 exon 2. Deep DNA sequencing of fixed-amplicon PCR products targeting the endogenous KARS1 Exon 2 locus was used to determine variant frequencies as part of a larger study to identify a set of reproducible enrichment scores indicating effects of variants on KARS1 function. Variant frequencies in homology-directed repair template libraries (2x replicates), and 4 biological replicates for two different sgRNAs were determined at the initial cell population stage (p0) and at three subsequent passages (p1, p2, p3).