The CRISPR-Cas9 system can be used for genome engineering in many organisms. PIWI-interacting RNAs (piRNAs) play a crucial role in repressing transposons to maintain genome integrity in Drosophila ovaries, and cultured ovarian somatic cells (OSCs) are widely used to elucidate the molecular mechanisms underlying the piRNA pathway. However, the germline-specific piRNA amplification system known as the ping-pong machinery does not occur in OSCs, making them unsuitable for elucidating the underlying mechanisms. Mutations in the lethal (3) malignant brain tumor gene (l(3)mbt) have been shown to cause ectopic expression of germline genes, including ping-pong factors. We therefore performed genome editing of Drosophila OSCs using the CRISPR-Cas9 system to achieve l(3)mbt knockout, resulting in successful induction of the piRNA amplification machinery. Here, we describe the detailed procedures for generating knockout and knockin OSC cells.
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