期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 533, 期 3, 页码 449-457出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2020.09.053
关键词
CRISPR/Cas9; DNA ligase IV; Non-homologous end joining; Homology directed repair; RAD51; In utero electroporation
资金
- JSPS KAKENHI [25282242, 19H03544, 19H03846, 15H04917, 16K14592, 24650183, 15K15730]
- Takeda Science Foundation
- Uehara Memorial Foundation
- Ichiro Kanehara Foundation
- Grants-in-Aid for Scientific Research [15K15730, 24650183, 15H04917, 25282242, 19H03846, 19H03544, 16K14592] Funding Source: KAKEN
CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the beta-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the beta-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons. (C) 2020 Elsevier Inc. All rights reserved.
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