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Suppression systems, on the other hand, are defined by an obligate link between an engineered transgene-expressing Cas9 and a gRNA-directing DNA cleavage element located within the drive cassette. In most cases, this element functions as an expression enhancer or is otherwise required for Cas9 activity and is referred to as a “promoter-less”-CRISPR-Cas9. The core design of an expression-enhanced CRISPR-Cas9 element (e-CRISPR) was first developed by the Gantz laboratory for the purposes of gene editing in human and mouse cells59. More recently, the same basic design was applied to mosquito gene drive systems61,63.
The e-CRISPR system relies on a promoterless Cas9 protein that can be split into two separate monomers, each fused to a functional guide RNA (gRNA) for cleavage of a specific site in the genome. The two protein monomers do not need to bind to the same DNA molecule to mediate genome cleavage but, rather, can bind independently and cooperatively to the target sequence. Of course, the monomers must bind to DNA in a sequence-specific manner and must be “spaced” appropriately in order to direct cleavage at the correct site. This approach is possible because Cas9 is not an obligate dimer, and monomeric versions of the protein can be constructed (for example, by truncating the N- or C-terminal amino acids of the Cas9 protein)61,62.
The first gene-drive system that was engineered to spread at high rates in the germline of A. aegypti was the As-drive80. It carries a Cas9 transgene, a transgene that expresses a gRNA targeting Cas9, and a second transgene expressing a copy of the As element that is inserted into the genome in place of the endogenous As sequence of A. aegypti85 (Figure 2). The As-drive system is based on the As element that had been previously developed to spread in D. melanogaster55. The As element is a mutagenic element that inactivates a selectable marker gene (GFP in the original version, LacZ in the current version) through induction of a DNA double-strand break at the 827ec27edc