The CRISPR/Cpf1 system delivered into the mitochondria via the iron-sulfur protein Dosmit and causing changes in the mitochondrial DNA in Drosophila

Yu-Xuan Huang (黃宇瑄)1,2, Rei-Wen Liu (劉瑞⽂)1,2, Chih-Hsuan Chang (張芷瑄)1,3, Jian-Chiuan Li (李健全)1,

Chih-Fei Kao (⾼智⾶)2, Chun-Hong Chen (陳俊宏)1

1 Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan.

2 Institution of Biotechnology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan.

3 Institution of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

CRISPR/Cas9 system is a common gene editing engineering technology that has been widely used for years. Cpf1 nuclease is a new precision genomic editing tool for development and improved CRISPR/Cas system, Cpf1 unnecessary tracrRNA compared with Cas9, it only needs crRNA ,and the smaller size of Cpf1 combined with gRNAs makes it easier to transport and shuttle within cells. However, the mechanism of the CRISPR/Cpf1 system performs gene editing in organisms remains unclear. We used the GAL4-UAS system to drive target gene or protein in drosophila. A mitochondria-associated protein Dosmit, previous studies have shown that aging and mitochondrial enlargement are related to the transport of cytosolic proteins to mitochondria, and Dosmit may induce mitochondrial enlargement and the double-membrane vesicles formation. Therefore, in order to makes the Cpf1 delivered into the mitochondria more efficiently ,using the model organism overexpress protein Dosmit to observe the changes of mitochondrial in drosophila ,through the oxidative stress or mitochondrial DNA expression and that after adding T4-ligase to explore the regulatory mechanism of mitochondria.