TY - JOUR
T1 - Use of the CRISPR-Cas9 system for genome editing in cultured Drosophila ovarian somatic cells
AU - Ishizu, Hirotsugu
AU - Sumiyoshi, Tetsutaro
AU - Siomi, Mikiko C.
N1 - Funding Information:
Current studies in the authors’ laboratory are supported in part by JSPS KAKENHI Grant Numbers 17H03632 , 16H01214 to H.I. and 26113004 , 25221101 to M.C.S.
Publisher Copyright:
© 2017
PY - 2017/8/15
Y1 - 2017/8/15
N2 - 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.
AB - 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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U2 - 10.1016/j.ymeth.2017.05.021
DO - 10.1016/j.ymeth.2017.05.021
M3 - Article
C2 - 28552546
AN - SCOPUS:85020011078
SN - 1046-2023
VL - 126
SP - 186
EP - 192
JO - Methods
JF - Methods
ER -