ovo(D) Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila

Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovo(D1). In this system (“ovo(D) co-selection”), the only functional germ cells in injected females are those that have been edited at the ovo(D1) locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovo(D) co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.