Figure 3

Overview of the CRISPR-Cas9 system. Cas-9 is a nuclease that makes a double-strand break at a location defined by a guide RNA [108]. The latter comprises a scaffold (red) and a 20-nucleotide guide sequence (blue) that pairs with the DNA target immediately upstream of a 5'-NGG motif (this motif varies depending on the exact bacterial species of origin of the CRISPR used). There are two main approaches that can be followed. (Left) Repair of the double-strand break by nonhomologous end joining can be used to knock out gene function though incorporation of random indels at junction sites, where these occur within coding exons, leading to frameshift mutations and premature stop codons. (Right) Homology-directed repair can enable precise genome editing through the use of dsDNA-targeting constructs flanking insertion sequences or single-stranded DNA oligonucleotides to introduce single-nucleotide changes. Adapted with permission from [108].