Massively parallel saturation genome editing of an essential mitochondrial targeting sequence (Tn5)

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. Tagmented libraries were prepared for homology-directed repair template pDNA and genomic DNA collected from HAP1 cells (4 biological replicates for two different sgRNAs) transfected with sgRNA only or sgRNA + HDR template at two passages post-puromycin selection (p1, p2) to assess overall sgRNA efficacy and HDR knock-in rates for saturation genome editing.