Extended Data Fig. 2: A two-step genome editing approach using CRISPR–Cas9 and HDR to generate knock-in Atpα lines of Drosophila melanogaster.

In the first step, the region encoding the H1–H2 extracellular domain was replaced with a 3×P3::GFP marker through CRISPR–Cas9-mediated HDR. This generated a common parent line with the deletion allele Atpα^{Deletion (GFP+)}. In the second step, the 3×P3::GFP marker was replaced with each of the synonymous and non-synonymous point mutation alleles through an additional round of CRISPR–Cas9-mediated HDR to generate the knock-in lines. The crossing schemes to establish the deletion line and the knock-in lines following the first and second rounds of HDR, respectively, are also shown. See also Methods and Supplementary Tables 4–7 for further details on the genome engineering strategy and crosses behind the establishment of the knock-in lines.