(I-344) Combined CRISPR-based technologies with artificial receptors for perturbing native gene expression programs in development

Introduction:: Various methods have been developed to molecularly trace developmental trajectories and record interaction histories of cells within a niche. Generally, these technologies involve artificial gene circuits that yield reporter protein expression (e.g., Brainbow) or archival of cellular responses to inputs via CRISPR-based scratchpads that can be read via single-cell sequencing and computationally mapped to reveal transaction logs of cells (e.g., MEMOIR, CAMERA, mSCRIBE). While powerful for unveiling the flow of information between cells and their niche, these methods do not enable facile, direct perturbation of cellular responses to selected inputs. Inspired by these and other advances in synthetic and developmental biology, we have the long-term goal of dissecting cell:niche interactions that enable reproducible organ and tissue morphogenesis by attempting to build signaling linkages de novo via artificial receptors. Here, we combine CRISPR-based transcriptional regulators with the synthetic Notch (synNotch) platform to tune mammalian cell behaviors to targeted disturbances in endogenous gene expression programs.

Materials and Methods:: We engineered GFP ligand in the PAX6 locus of 293T cells (293T-GFPL) using CRISPR/CAS9-mediated targeted integration. PAX6 is a transcription factor that serves as a marker of differentiation in several cell types, including neural progenitors. It is also expressed in HEK293 cells, which are known to express markers of not only renal progenitors and adrenal gland, but also of neuronal-specific genes. We tested the activation of synNotch in cell lines by this natively expressed GFP ligand. We also explored the induction of neuronal differentiation of pluripotent stem cells by the PAX6-driven GFP ligand expression. To test whether we can tune GFP-ligand expression by attenuating PAX6 profiles, we generated a CRISPRoff derivative of the PAX6-GFPLigand cell line. We then evaluated whether this targeted disturbance in synthetic ligand expression could relay corresponding level of signal transduction to synNotch receiver cells.


Results, Conclusions, and Discussions:: We successfully built a knock-in 293T-GFPL cell line, in which the expression of GFP ligand is controlled by native PAX6. The GFP ligand expressed in this way is sufficient to activate the synthetic Notch, yielding signal transduction in receiver cells. Perturbation of endogenous PAX6 expression is faithfully mirrored by the GFP ligand content, and this fine-tune signal can be transmitted to the activation level of synNotch and its downstream transgenic expression to regulate cell behaviors in a controllable manner. GFP ligand can induce the neural differentiation of pluripotent stem cells through Ngn2 transgenic expression.


Acknowledgements (Optional): : NSF CBET-2033800 to JMB


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