Molecular engineering and synthetic biology approaches have been applied (1) to replace the intracellular domain (NICD) of Notch with synthetic transcription factors1 that bind to genes other than natural Notch targets; (2) to replace human Notch extracellular domain (NECD) with recognition motifs against novel targets (e.g. cognate receptor ligands such as Delta versus single-chain variable fragments raised against a specific antigen). The project aims to interrogate and engineer the fate of a receiver cell to surface-receptor levels of a sender cell by harnessing contact-mediated Notch-Delta signaling and RNA-guided CRISPR genome engineering. We have developed Notch1-Cas9 chimeric receptors that, upon binding Delta ligand to the extra-cellular Notch domain, cleave and release Cas9 to manipulate gene expression of transgenic reporter constructs - and - of endogenous targets in mammalian cells. First, we describe the challenges and solutions in the construction of Notch-Cas9, which led to the discovery of a naturally occurring sequence in bacterial Cas9 that is being co-opted as a nuclear localization signal (NLS) when expressed in mammalian cells. To our knowledge, this is the first reported evidence of a cognate NLS in bacterial Cas9. Second, we expand the toolkit with not only Delta-dependent gene activation, but also with gene editing in two Cas9 orthologs (Streptococcus pyogenes and Staphylococcus aureus Cas9). The toolkit performs well in reporter-gene activation and editing assays. Finally, we demonstrate activation of and phenotypic changes associated with endogenous genes encoding for regulators of the cell-division cycle, CDKN1B and CASP8; and editing of CXCR4 and CD47, receptors relevant to immune trafficking and tolerance. This Notch-Cas9 fusion technology opens up novel biological behaviors in response to cell-cell contact and can be utilized to interrogate or manipulate existing signaling pathways.
Citation Format: P C Dave P. Dingal, Nathan Kipniss, Yuchen Gao, Lei S. Qi. Conversion of extracellular signals to programmable genome manipulation via CRISPRouter [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B075.
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