Aphidicolin: Increase HDR via Cell Cycle Synchronization 

Biological Activity

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]

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. ^[5]
• 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. ^[6]
• Aphidicolin treatment did not affect the frequency of HDR at the EMX1 locus in H9 human embryonic stem (hES) cells. ^{[ 6 ]}

Reference:

1. Drugs.ncats.io. 2020. NCATS Inxight: Drugs — APHIDICOLIN. [online] [Accessed 30 December 2020].
2. 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.
3. 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. 
4. 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. 
5. Chen, G., & Deng, X. (2018). Cell Synchronization by Double Thymidine Block. Bio-protocol, 8(17), e2994. 
6. 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. 

Related Product:

190303 Aphidicolin, 98%, from Nigrospora oryzae [CAS: 38966-21-1]