CRISPR/Cas9-mediated knock-in in ebony gene using a PCR product donor template in Drosophila

CRISPR/Cas9 technology has been a powerful tool for gene editing in Drosophila, particularly for knocking in base-pair mutations or a variety of gene cassettes into endogenous gene loci. Among the Drosophila community, there has been a concerted effort to establish CRISPR/Cas9-mediated knock-in protocols that decrease the amount of time spent on molecular cloning. Here, we report the CRISPR/Cas9-mediated insertion of a ∼50 base-pair sequence into the ebony gene locus, using a linear double-stranded DNA (PCR product) donor template. By circumventing the cloning step of the donor template, our approach suggests the PCR product as a useful, alternative knock-in donor format.