Biological Activity

Tamoxifen is a selective estrogen receptor modulator (SERM) that evokes tissue-dependent effects. As an antagonist of estrogen receptor (ER) activity in breast tissue and breast cancer cells, it is commonly used in the prevention and treatment of (ER)-positive breast cancer patients. In cells, it is converted to active metabolites including (Z)-4-hydroxytamoxifen (4-HT) by cytochrome P450 (CYP) enzymes. 4-HT exhibits an approximately 100-fold higher ER affinity than the parent drug and the ability to inhibit cell proliferation. ^[1] 4-HT has also been used to control Cas9 function post-translationally, which reduced off-target effects in CRISPR-mediated gene editing. [2]

Successful Applications in CRISPR

To optimize the ratio of on-target:off-target DNA cleavage, researchers engineered two conditionally active Cas9 nucleases [intein-Cas9(S219) and intein-Cas9(C574)] to be active only in the presence of 4-HT. Along with wild-type, they were expressed in HEK293-GFP cells and treated with or without 1 µM 4-HT (for 12 hours) and then assayed for their ability to modify three on-target loci and 11 known off-target genomic sites. It was found that the on-target:off-target indel modification ratios for both intein-Cas9 variants in the presence of 4-HT were on average 6-fold to 25-fold higher than wild-type Cas9 due to similar on-target frequencies as wild-type (slightly less active), but lower off-target frequencies. ^[2]

Reference:

1. Brauch, H., Mürdter, T. E., Eichelbaum, M., & Schwab, M. (2009). Pharmacogenomics of tamoxifen therapy. Clinical chemistry, 55(10), 1770–1782. https://doi.org/10.1373/clinchem.2008.121756
2. Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., & Liu, D. R. (2015). Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nature chemical biology, 11(5), 316–318. https://doi.org/10.1038/nchembio.1793

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