Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure.

Anthrax infection is caused by, a bacterium that once established within the host releases lethal toxin (LeTx). Anthrax LeTx is internalized by the capillary morphogenesis protein 2/anthrax toxin receptor 2 (CMG2/ANTXR2) cell surface receptor on mammalian cells. Once inside the cell, LeTx cleaves mitogen-activated protein kinases (MAPKs), ultimately leading to cell death. Previous reports have shown that decreased expression of reduces cell susceptibility to LeTx. By ablating the gene in cells in vitro, we observed complete resistance to LeTx-induced cell death. Here, we directed CRISPR/dCas9-based tools to the promoter to modulate expression without altering the underlying gene sequence in human cell lines that express the receptor at high levels. We hypothesized that downregulating the expression of the gene at the genomic level would mitigate the impact of toxin exposure. In one epigenetic editing approach, we employed the fusion of DNMT3A DNA methyltransferase and dCas9 (dCas9-DNMT3A) to methylate CpGs within the CpG island of the promoter and found this repressed gene expression resulting in significant resistance to LeTx-induced cell death. Furthermore, by multiplexing gRNAs to direct dCas9-DNMT3A to multiple sites in the promoter, we applied a broader distribution of CpG methylation along the gene promoter resulting in enhanced repression and resistance to LeTx. In parallel, we directed the dCas9-KRAB-MeCP2 transcriptional repressor to the promoter to quickly and robustly repress expression. With this approach, in as little as two weeks, we created resistance to LeTx at a similar level to gene-ablated cells. Overall, we present a transcriptional tuning approach to inhibit the effects of LeTx and provide a framework to repress toxin-binding cell surface receptors.