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Aphidicolin: Increase HDR via Cell Cycle Synchronization
Isolated from the fungus C. aphidicola, aphidicolin is a potent (cell permeable) inhibitor of the B-family of DNA polymerases, (reversibly) arresting replication in eukaryotes, bacteria, and certain animal viruses (SV40, Herpes, Vaccinia) at the G1-S border. Used as a cell synchronization agent, it competes for the dCTP-binding site of eukaryotic DNA polymerase α, or viral-induced polymerases. Other DNA polymerases are also affected, but β and γ polymerases are not. [1,2,3]
|Target (in vitro)||IC50 (μM) |
|Mammalian (calf) DNA polymerase α||19.6 +/- 1.1|
Successful Applications in CRISPR
- Aphidicolin treatment coupled with timed delivery of pre-assembled Cas9 ribonucleoprotein (RNP) complex, targeting the EMX1 gene in HEK293T cells led to an increase in the rate of HDR from ∼9% in unsynchronized cells to ∼14%, with no detected off-target editing. Ss-donor templates were used. 
- Targeting the EMX1 gene in human primary neonatal fibroblasts (neoFB) led to a total editing (TE) increase of 17% and an HDR increase of 1.3%. TE in the unsynchronized condition was 5% but HDR was not detected in unsynchronized cells. 
- Aphidicolin treatment did not affect the frequency of HDR at the EMX1 locus in H9 human embryonic stem (hES) cells. [ 6 ]
- Drugs.ncats.io. 2020. NCATS Inxight: Drugs — APHIDICOLIN. [online] [Accessed 30 December 2020].
- Baranovskiy, A. G., Babayeva, N. D., Suwa, Y., Gu, J., Pavlov, Y. I., & Tahirov, T. H. (2014). Structural basis for inhibition of DNA replication by aphidicolin. Nucleic acids research, 42(22), 14013–14021.
- Longiaru, M., Ikeda, J. E., Jarkovsky, Z., Horwitz, S. B., & Horwitz, M. S. (1979). The effect of aphidicolin on adenovirus DNA synthesis. Nucleic acids research, 6(10), 3369–3386.
- Duy, L. H., Takenaka, Y., Mizushina, Y., Tanahashi, T. (2019). Evaluation of DNA polymerase inhibitory activities of depsidones, depsides and diaryl ethers from the Vietnamese lichen Rimelia clavulifera. Vietnam Journal of Chemistry, 57(1), 112-115.
- Chen, G., & Deng, X. (2018). Cell Synchronization by Double Thymidine Block. Bio-protocol, 8(17), e2994.
- Lin, S., Staahl, B. T., Alla, R. K., & Doudna, J. A. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife, 3, e04766.