Table 1.
Frequency of reads with GAATTC knockin based on second-generation sequencing
| Sample | Total no. of reads | No. of reads with GAATTC insertion | Knockin ratio |
|---|---|---|---|
| Crude mtDNA | |||
| ssODN1 | 5,028,392 | 343 | 0.00006821 |
| ssODN1 + RS-1 | 6,838,719 | 324 | 0.00004738 |
| mito-Cas9 + ssODN1 | 5,367,579 | 1608 | 0.00029958 |
| mito-Cas9 + ssODN1 + RS-1 | 6,208,098 | 8839 | 0.00142379 |
| Purified mtDNA | |||
| ssODN1 + RS-1 | 5,991,432 | 210 | 0.00003505 |
| mito-Cas9 + ssODN1 + RS-1 | 4,992,779 | 11,714 | 0.00234619 |
Counts for sequencing reads with the GAATTC insertion in a short PCR fragment (region m.11 600–11 820 in mtDNA) flanking the knockin site. Crude mtDNA and purified mtDNA were used as respective templates for PCR, and PCR products were sequenced by second-generation sequencing technology. Crude mtDNA, mtDNAs isolated from crude mitochondria in HEK293T cells transfected with ssODN1 alone, mito-Cas9 system (sgRNA1ND4-MTSCOX8A-Cas9-UTRSOD2 and ssODN1), with or without RS-1 treatment; purified mtDNA, mtDNAs isolated from crude mitochondria after DNase I digestion.