CRISPR-Cas9 repair complexity in Drosophila melanogaster: NHEJ-induced deletions and HDR variability in the bantam microRNA gene

CRISPR-Cas9-mediated gene editing was used to generate specific mutants of the bantam gene in Drosophila melanogaster. To drive non-homologous end joining (NHEJ) and achieve a precise deletion of most of the bantam locus, two guide RNAs targeting sites 90 base pairs apart were expressed in the germline using the UAS/GAL4 system. Thirty lethal and eight viable lines were established and analyzed. One lethal line exhibited the expected 90 bp deletion, while the others carried diverse indels at one or both cleavage sites. Among the viable lines, seven harbored a single-nucleotide deletion that did not disrupt bantam function. Notably, one viable line, band1-44, carried a hypomorphic allele that reduced organismal size without affecting viability. To generate precisely edited bantam variants, CRISPR-Cas9-mediated homology-directed repair (HDR) was used using donor plasmids containing engineered mutations in the miRNA seed region, along with a scarless dsRED fluorescent marker. Approximately 40% of the resulting fluorescent lines were correctly edited, demonstrating the efficiency of this strategy for producing specific bantam variants. The remaining lines exhibited unexpected outcomes, among which partial HDR events, where the dsRED marker was integrated but not the bantam mutations, and full donor plasmid integrations, which led to duplication of the bantam locus. These findings reveal the complexity of CRISPR-Cas9 outcomes, emphasizing the need for thorough screening and characterization of individual candidates in gene-editing experiments. They also provide valuable insights for optimizing genome editing strategies. Article summaryThe authors used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing to mutate the bantam microRNA in Drosophila melanogaster. They guided Non-Homologous End Joining to induce a precise 90 bp deletion. It was the rarest occurrence, while small inactivating indels occurred frequently. They employed Homology-Directed Repair using a fluorescent marker to specifically target the bantam seed region. This efficiently produced the intended mutations but also led to unexpected outcomes, including partial sequence replacements and full donor plasmid integrations. These results reveal the complexity of gene editing outcomes and highlight the importance of thorough molecular characterization in genome engineering experiments.