Fig. 3: Profiles of NHEJ events in single-generation crosses versus multigenerational cages.

Production of NHEJ events in single crosses (a–d) and cage trials (e–h). For single-cross data, target regions were amplified from single non-fluorescent F₂ individuals generated in Fig. 2, sequenced through Sanger sequencing and analyzed. A bar depicts the % of sGD⁺ (purple) and % of non-fluorescent (sGD⁻, gray) flies for each tested locus. Genotypic data is depicted in pie charts representing the prevalence of specific indel mutations in sGD⁻ individuals for a rab5, b spo11, c rab11, d prosalpha2 loci. Each section of the pie chart describes the kind of NHEJ that is formed and its percentage among the total tested sGD⁻ (NHEJ/WT) heterozygotes. The specific sequence of prominent NHEJ events, along with its corresponding prevalence, is reported under each pie chart. gRNA sequence of each sGD is depicted in blue with its PAM sequence shown in red. To generate the NHEJ cage trial data (e–h), non-fluorescent sGD⁻ individuals were pooled at every generation and used to amplify their target site region, which was deep sequenced to assess formation of NHEJ alleles. WT sequences (gray), in frame deletions (blue), frameshift deletions (red) and insertions (yellow) are shown in bars at each generation to represent the distribution of alleles in the sGD⁻ population (left) and among the total population (sGD⁺ and sGD⁻, right). Purple diagonally-dotted bars show the sGD⁺ population percentage.