ABSTRACT

Genome editing is a type of DNA engineering which allows precise modifications in the genomic DNA. Genome editing is mediated through programmable nucleases which recognize and bind specific sequences in the genome. 1 , 2 Once binding, the nucleases can be engineered to cut targeted genomic DNA, resulting in double-strand breaks (DSBs), which are efficiently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the presence of a donor DNA template. 1–5 NHEJ is error-prone, often introducing small or large insertions or deletions (indels) at the DNA cut site. 6 Depending on the types and positions, indels that shift the open reading frame (ORF) can lead to the mRNA degradation or production of abnormal and non-functional proteins. 7 Genome editing nucleases-mediated NHEJ is able to introduce long-term disruption of disease-prone genes. 8 It is also feasible to engineer nucleases-mediated NHEJ to restore ORF of a malfunctioning gene. 9–13 For example, mutations in dystrophin gene can lead to ORF shifting and severe disruption or deletion of dystrophin protein, to cause Duchenne muscular dystrophy. Disruption of the ORF-shifted exons to restore the ORF can rescue the function of dystrophin for treating Duchenne muscular dystrophy. 9–13 In contrast to error-prone NHEJ pathway, HDR-mediated genome editing enables precise modifications by incorporation of the donor DNA with homologous sequences. 14 Genome editing via HDR pathway could be used to precisely repair mutations or knock-in sequences at the desired loci. 15–17 Besides introducing DSBs, genome editing nucleases 390(e.g. Cas9) can be engineered to enable many different modifications, including epigenetic modification of targeted sequences, induction or suppression of gene expression, base editing, imaging of genomic locus, and many others. 8 , 18–25