Figure 4.

The schematic diagram of the CRISPR-based genome editing approach. The spacer and the donor DNA are cloned into pGE1 (blue rectangle) and transformed into S. islandicus LAL14/1 Δarrays cells. The cells are infected with the targeted virus. The homologous recombination (double-crossover) between the virus and the donor DNA generates the desired deletion allele in the viral genome. The crRNA produced from the plasmid-borne mini-CRISPR array is assembled with the endogenous Cas proteins into the CRISPR ribonucleoprotein (crRNP) complex that directs the cleavage of non-recombinant viruses, which are those still harboring the protospacer. Conversely, the recombinant viruses are not targeted and are able to propagate.