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A Knock-in Reporter for a Novel AR-Targeted Therapy

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Technical Report,01 Mar 2015,29 Feb 2016

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Georgia Regents Research Institute, Inc Augusta United States

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Since castration resistance of prostate cancer is often caused by AR overexpression, down-regulation of AR expression using small molecules is a promising yet untested strategy to combat castration-resistant prostate cancer CRPC. The main objective of this research isto explore a possibility whether the CRISPR-Cas9 technology, an emerging genome-editing approach, could be applied to develop a reliable high-throughput drug screening platform for the identification of small-molecule AR transcriptional inhibitors to treat CRPC. We demonstrated in this report that the CRISPR-Cas9 system could indeed mediate high-efficient insertion of a selection gene into a site immediately downstream of the AR stop codon in castration-resistant C4-2 and 22Rv1 cells. Replacing the selection gene with a firefly luciferase FLuc gene led by an internal ribosomal entry site IRES through recombinase-mediated cassette exchange, we have successfully developed a C4-2-based recombinant cell line carrying FLuc in the AR gene locus and thereby bicistronically co-expressing the reporter gene with AR under the control of the endogenous AR promoter and enhancer. While the knock-in reporter is expected to faithfully reproduce chemical responses of the endogenous AR gene, we carried out a pilot screen using the NCI Diversity-set library and demonstrated that the genome-edited prostate cancer cells were useful in identifying small molecules inhibitory for AR expression.

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