Targeted C-to-T conversion in human mitochondrial genome by CRISPR-base editor

Gene editing holds promise for potential treatments by correcting mutations in mtDNA in situ. Here we performed whole genome sequencing using randomly selected library to assess on- and off-target editing of mito-targeted CRISPR-CBE in the mitochondrial genome. Whole genomic DNA was extracted from unsorted 293T cells transfected with plasmids encoding MTSCBE and mito-gRNA targeting each of the eight mitochondrial genomic loci and pooled in equal proportions for high throughput DNA sequencing. Whole genomic DNA from untransfected 293T cells served as a control. Given that the samples were pooled equally from editing at eight different targets, on-target editing was identified, and the editing frequency was adjusted by a factor of 8. The adjusted frequencies ranged from 5.3% to 72.2% across 8 targets in three biologically independent samples. Off-target editing was detected in 56 loci in mtDNA with mean efficiency from n=3 ranged from 0.06% to 0.86%.