Fig. 3.

Different CRISPR approaches to mediate the dystrophin restoration in DMD patients. A The outcome of DMD exon 52 deletion and the two primary methods to restore dystrophin expression: (1) splice sites modifications: the donor site (gt) may be modified to ga or the acceptor site (ag) may be modified to gg. (2) The insertion in exon 51 of one nucleotide (A in red) to reframe exon 53 while conserving the same first amino acid codon for exon 53. B represents a strategy where a double-strand break can be made to generate an INDEL (micro-insertion or micro-deletion) in the exon preceding a stop codon. This stop codon was induced by a frameshift mutation which is compensated for by the engineered INDEL to restore the normal reading frame for the next exon. C The strategy where two single guide RNA can be used to completely delete one or several exons to restore the normal reading. D The strategy leading to the formation of a hybrid exon by inducing double-strand breaks. This hybrid exon not only restores the normal reading frame but also produces a dystrophin protein, which has a normal structure thus including a normal spectrin-like repeat structure at the junction site of the two exons. E The point mutation strategy where the abnormal nucleotide (G) in the figure forming a stop codon (TAG) is directly modified into another nucleotide (T in the figure) thus removing the stop codon