Highly frequent undesired insertional mutagenesis during Drosophila genome editing

CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.