FIG. 1.

Base editing in Drosophila. (A) Schematic of Drosophila BE2. A catalytically inactive dCas9m4 is fused through flexible linkers to CD and uracil DNA glycosylase inhibitor domains. (B) Summary of mechanisms inducing point mutations. A gRNA directs the base editor to a 20 nucleotide target. A cytidine residue may be deaminated, resulting in conversion of the cytidine (C) to a uridine (U). During DNA repair or replication, the uridine has base-paring properties of thymine (T). This results in a C-to-T change on the top strand, or a guanosine-to-adenosine (G-to-A) change if the gRNA targeted the bottom strand. (C) Summary of fly crosses used to induce base editing. In this example, males containing one or more gRNAs (inserted on the second chromosome using the pCFD4 construct)²⁰ are crossed to the base editor (inserted on the X chromosome) that uses the Actin5C promoter to express in all cells. Base editing occurs in male F₁ progeny. Editing can be examined directly in F₁ males, or mutant lines obtained by crosses to balancers to isolate the mutagenized chromosome. BE2, base editor 2; CD, cytidine deaminase; gRNA, guide RNA; dCas9m4, dead Cas9 containing four mutations. Color images are available online.