Figure 2.

Efficient reframing by NHEJ

(A) sgRNAs for c.550delA. The purple squares mark the position of the mutation. (B) Schematic overview of the experiment. Patient-derived hiPSCs were transfected with vector containing either sgRNA no. 1 or no. 2 and Cas9:T2A:Venus. Venus-positive cells were enriched with FACS and cells were processed for bulk DNA analysis. (C) Representative Sanger sequencing result for edited (top) compared with unedited (bottom) samples. Binding site of sgRNA is indicated on top. The purple squares mark the position of the mutation, dotted line represents the cutting site of SpCas9. sgRNA no. 1 leads to a frameshift by insertion of one adenine (green box). (D) Predicted editing efficiency of three biological repeats. The editing efficiency was predicted using ICE tool. In gray the indel frequency is quoted, green shows the repair by +1 insertion. (E) As in (C), but for sgRNA no. 2. (F) As in (D), but for sgRNA no. 2. Green bar shows the efficiency of reframing by +1 insertion. (G) Potential mechanism of precise reframing. Top: DNA sequence of CAPN3 c.550delA; the purple square marks the position of the mutation, the dotted blue line indicates the SpCas9 staggered cutting model. Bottom: restored CAPN3 wild-type DNA sequence after repair of the DSB.