CRISPR-Cas9 and Its Therapeutic Applications for Retinal Diseases

In 1987, Ishino et al1 made a fortuitous discovery in the 3′-end flanking region of the iap gene in Escherichia coli, finding 5 sequences of a partially palindromic, highly conserved 29-nucleotide repeats, with 32-nucleotide spacing intervals. These repeat sequences were subsequently found in other archaea and bacteria and thought to be associated with prokaryotic immunity against viruses via DNA repair and regulation.2–8 In 2002, Jansen et al9 named these sequences CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), and found adjacent genes, which were named Cas (CRISPR-associated) genes. Later work elucidated the mechanisms by which the CRISPR-Cas system mediates specific double-stranded DNA breaks (DSB) using single-guide RNAs (sgRNA), prompting investigation into its potential role in gene editing techniques.10,11 Initial trials of CRISPR-Cas technology included validation of the CRISPR-Cas system’s cleavage of bacteriophage and plasmid DNA in bacteria.12 Shortly afterwards, human cell gene editing was achieved, showing successful knock-out and introduction of reporter genes in HEK cells.13–15 Zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) technology are also prominent gene editing techniques utilizing site-specific nucleases. Briefly, ZFN consists of a fusion of the FokI nuclease domain and 2 zinc finger DNA binding modules, and TALEN consists of a fusion of the FokI nuclease domain and transcription activator-like DNA binding domains.16 Unlike CRISPR, which uses RNAguided DNA cleavage, both ZFN and TALEN involve a protein-DNA binding interaction to specify the site of cleavage. Thus, one major