High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal
Abstract
:1. Introduction
2. Results
2.1. Embryonic Cell Cycle Analysis by Nuclear Envelope Morphology
2.2. Mitosis-Specific High SSTR Efficiency
2.3. The Nuclear Envelope as A Major Barrier to the Nuclear Import of ssODNs
2.4. High SSTR Efficiency in Mitotic Synchronous Embryos and Cells
2.5. Minimal Impact of ssODN Size and Cdk1 on SSTR Efficiency
2.6. Improving SSTR Efficiency Using NLS-tagged ssODNs
3. Discussion
4. Materials and Methods
4.1. Animal and Embryo Preparation
4.2. Embryonic Whole-Mount Immunostaining for Cell Cycle Determination
4.3. In silico sgRNA Design and Homologous Template Preparation
4.4. Electroporation-Mediated Ribonucleoprotein Transfection into Embryos and Genotyping
4.5. Nuclear Localization Analysis for Cas9 Protein and ssODNs
4.6. Establishing SpCas9 Overexpression NIH3T3 Cells
4.7. Preparation of Modified ssODNs and Cdk1
4.8. Cell Cycle Synchronization and Transfection of Cells by Electroporation
4.9. Targeted Deep Sequencing
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Han, J.P.; Chang, Y.J.; Song, D.W.; Choi, B.S.; Koo, O.J.; Yi, S.Y.; Park, T.S.; Yeom, S.C. High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal. Int. J. Mol. Sci. 2020, 21, 3747. https://doi.org/10.3390/ijms21113747
Han JP, Chang YJ, Song DW, Choi BS, Koo OJ, Yi SY, Park TS, Yeom SC. High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal. International Journal of Molecular Sciences. 2020; 21(11):3747. https://doi.org/10.3390/ijms21113747
Chicago/Turabian StyleHan, Jeong Pil, Yoo Jin Chang, Dong Woo Song, Beom Seok Choi, Ok Jae Koo, Seung Youn Yi, Tae Sub Park, and Su Cheong Yeom. 2020. "High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal" International Journal of Molecular Sciences 21, no. 11: 3747. https://doi.org/10.3390/ijms21113747