ABSTRACT
Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage, marker-assisted strategy to facilitate precise, scarless edits in Drosophila with little need for molecular screening. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a novel transformation marker. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Added new experimental data on HDR mediated CRISPR and building of new Drosophila balancers.