ovoD 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^Dco-selection"), the only functional germ cells in injected females are those that have been edited at the ovo^D1locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovo^Dco-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.

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