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Fig. 1 Generation of YARS2 knockout HeLa cell lines using CRISPR/Cas9 system. A, schematic representation of CRISPR/Cas9 target site at exon 1 as used in this study. An allele, YARS2del14bp was produced by a 14 bp delete in the exon 1 and a truncated nonfunctional protein with 24 amino acids. B, western blot analysis of YARS2 in various cells. Twenty micrograms of total cellular proteins of each cell line was electrophoresed through and hybridized with antibodies specific for YARS2 (1:1000 dilution) and with TOM20 (1:2000 dilution) as a loading control. WT, wild-type cells; KO, YARS2KO; KO+YARS2, exogenous YARS2 expression in YARS2KO; KO+vector, vector transfected in YARS2KO. Three independent experiments were performed. C, in vivo aminoacylation of mitochondrial tRNA assays. Ten micrograms of total cellular RNAs purified from various cell lines under acid conditions was electrophoresed through an acid (pH 5.2) 10% polyacrylamide-7 M urea gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probe specific for the tRNATyr, tRNALeu(UUR), and tRNAThr, respectively. Samples for WT cells were deacylated (DA) by heating for 10 min at 60 °C at pH 8.3, electrophoresed, and hybridized with DIG-labeled oligonucleotide probes as described above. Three independent experiments were performed.