Fig 1.
Scheme of guide RNA and PAM sequence targeting exon 1 in the bovine TFAM gene.
Note the complete sequence of Exon 1. The horizontal red underline represents the PAM sequence. The horizontal black underlined region represents the guide sequence and the cut site is the vertical black dotted line.
Fig 2.
Test of Amaxa Nucleofector program by flow cytometry analysis.
Control pCAG (0.0%), Cell viability (65.2%), A-24 program (84.8%), T-016 program (96.3%), U-012 (98.4%), U-013 (99.5%) and V-013 (98.4%).
Fig 3.
Agarose gel (2.5%) used for the T7EI cleavage assay.
Note the different gRNA tested (1 to 4) and controls (1 to 4). The gRNA 1 and 2 with no mutation rate, gRNA 3 with 7.2% mutation rate and gRNA 4 with 10% mutation rate. It’s possible to see 2 brighter bands on gRNA 3 and gRNA 4.
Table 1.
Primers sets used for PCR and the T7EI assay.
Table 2.
Primers used for relative quantification of the target gene (mtDNA) and endogenous control (ACTB).
Fig 4.
Photomicrographs of fibroblasts after transfection with CRISPR Cas9.
In A, C and E note culture of fibroblasts (control). In C note the cluster formed (arrow) and in E adherent cells with 80% confluence. In B, D and F observe the cells positively stained.
Fig 5.
Clones after 7-day growth in 6-well plates. Note adherence to plastic and high cell confluence. In A-B, cells in 96-well plates at 80% confluence; C-D confluent cells in 6-well plates.
Fig 6.
Mutation detection of TFAM gene by T7EI cleavage assay and sequence analysis.
In the T7EI assay note mutations in numbers 1 to 3. It’s possible to see 2 bands in the center of the gel and the numbers 4, 5, and 6 are controls of clones 1–3. In the PCR sequencing analysis note A, B and C (nucleotides 163–165) showing the PAM region of the sequence. The cleavage site is 3bp prior to PAM (160–162). In A, note the insertion of two A residues at the site that should be GC. At nucleotide 155, there also occurred an insertion of G, normally an A. In B, note the insertion of an A substitution for G. In C the insertion of AT nucleotides involving positions 156 and 157. This site is normally GC. Also insertion of a T at the A site of nucleotide 151, and insertion of C at the G site at nucleotide 144.
Fig 7.
Sequences of alleles identified by Sanger sequencing. The sequence of gRNA is shown in horizontal black underlined region, the PAM site is the horizontal red underline. In 1A, 1B, 2A, 2B sequenced colonies mutations with 39bp deletion (dotted); 1C, 2C, 2D, 3B sequenced colonies wild-type alleles. In 3A sequenced colonies mutations with 9bp deletion (dotted).
Fig 8.
Number of copies of mtDNA per cell, non-edited clones (control) and edited clones.
Note the 100% percentage of copy number to non-edited clones (used as reference) with 2.912 mitochondrial number copy and 56.8% to edited-clones with 1.655 mitochondrial number copies. Significative difference (P ≤ 0.01).