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Abstract
Introduction: Different cancer cells, including those from breast cancer, show increased APOBEC3 activity [Citation1,Citation2], a single strand DNA cytidine deaminase, probably responsible for mutation clusters in tumors. The working hypothesis is that inhibition of APOBEC3 prevents development of mutagenesis-dependent breast cancer cells resistance to chemotherapy. To test this hypothesis, we used breast cancer cell lines that overexpress APOBEC3G and performed a knock-out of APOBEC3G by CRISPR-Cas9 system.
Materials and methods: Selected guides against the APOBEC3G gene were cloned into the pX459 plasmid expressing both single guide RNA (sgRNA) and Cas9 protein and verified by Sanger sequencing. The sgRNA constructs were introduced in MCF10A and HCC1806 breast cancer cell lines by lipofection. 24h after transfection puromycin selection was applied for 48h. Genomic DNA was extracted for the detection of the indels caused by double strand break DSB.
Results: We have chosen four different pairs of guides to inactivate APOBEC3G gene, using the online tool (http://crispor.tefor.net/). After performing transfection of MCF10A and HCC1806 cells on small scale, the best candidate guides A3Gguide285 and A3Gguide265 () were chosen for further transfection on large scale with the aim of individual clones’ isolation. Several clones were obtained and are presently being subjected to genomic DNA analysis for the presence of indels in APOBEC3G gene.
Figure 1. Representation of the two selected guides that target the APOBEC3G gene.
Discussion and conclusions: We have obtained several clones to screen for the successful knock-out of both alleles of the APOBEC3G gene. These cell lines will be used as control, against stably transfected cells expressing both Vif and APOBEC3G proteins to check the effect of Vif/APOBEC3 interaction on chemotherapy resistance.
Acknowledgements
The authors acknowledge funding from FCT PTDC/BIM-MEC/6631/2014 and Egas Moniz, CRL.