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The CRISPR system has been widely used for genome manipulation in various cells, tissues and whole organisms. Although an increasing variety of inducible CRISPR systems have been exploited for a variety of applications, such as chemical switch (Zetsche et al., 2015), photo switch(Shao et al., 2018) and solution ligand switch (Baeumler et al., 2017; Kipniss et al., 2017; Schwarz et al., 2017) systems, a cell-cell interaction inducible system is absent. The synthetic Notch (synNotch) receptor is a recently developed cell-cell contact sensing platform, which contains a customized extracellular sensor module, a transmembrane core domain of native Notch, and a customized intracellular responder module (Morsut et al., 2016). Because of its extraordinary flexibility in terms of customizable sensing/response behaviors, the synNotch receptor serves as a powerful tool for cell engineering (Roybal et al., 2016a; Roybal et al., 2016b; He et al., 2017). In the current study, we combined the synNotch receptor with the CRISPR/Cas9 system to develop a cell-cell interaction inducible gene regulated tool. Since the previously reported synNotch receptor was based on mouse Notch1 (M1) (Morsut et al., 2016), we tried to develop other synNotch receptors using different Notch family members from several species, including human (H), mouse (M), drosophila (Fly) and zebrafish (Z), with antiCD19-ScFv/mCherry as a sensor/responder module (Fig. 1A). We found that the new synNotch receptors demonstrated better activation than the M1 synNotch (especially for Z3, 78.0% ± 9.8% of cells were activated with a 158.4 ± 19.7-fold change), while showing a background ranging from 0.5% to 45% (Figs. 1B, 1C and S1A). M4 system was not activated when treated with CD19+ Cells (Figs. 1B and S1A), which was consistent with the previous report that Notch4 does not signal in response to ligand but inhibits signaling from the Notch1 receptor (James et al., 2014). To decrease the background noise, P2A-Gal4KRAB or P2A-Gal4 was added downstream of Gal4-VP64 (Fig. S1B). Gal4KRAB completely blocked activation (Fig. S1C), and Gal4 dramatically decreased the background noise (M2, ∼10%; all the others, <5%) but simultaneously attenuated activation significantly (all, <15%) (Fig. S1D). It has been reported that EGF (epidermal growth factor) repeats can prevent the constitutive activation of Notch (Sakamoto et al., 2005). Therefore, we included an extra EGF repeat on the extracellular domain between the antiCD19 ScFv and the Notch core domain (Fig. S2A). By including an extra EGF, the background in the Z1, Z2, and Z3 systems was decreased, and nearly eliminated in the Fly system (Fig. S2B). However, it also affected activation, as represented by the Z3 system with an efficiency of decreasing to approximately 41.2% when stimulated (Fig. S2B and S2C). To balance background and efficiency, we shortened the EGF repeat by half (eZ3), which remarkably increased the stimulation efficiency to 62.8% while maintaining a tolerable background (8.5%) (Fig. S2B and S2C). Next, we tested the ability of the synNotch receptors to respond to different sender cells and the response flexibility of the receiver cells. In addition to K562-CD19 cells, CD19transduced B16 melanoma cells (B16-CD19) with either high (B16-10) or low (B16-3) CD19 expression could activate mCherry expression in the M1 and H1 systems (Fig. S3A and S3B). However, mouse splenocytes expressing mouse CD19 failed to activate the M1 and H1 receptors, demonstrating the specificity of the synNotch systems (Fig. S3C and S3D). To test the flexibility of the response modules in the synNotch systems, we replaced the mCherry reporter with EGFP and SIRPɑ-Fc in the H1, Z3 and eZ3 systems (Fig. 1A). FACS and fluorescence microscopy analyses showed that EGFP was potently activated by the CD19+ cells (Figs. 1D, 1E and S4). High-affinity SIRPɑ-Fc (CV1hIgG4) holds great therapeutic potential and has entered a phase I clinical trial for solid tumor treatment (Weiskopf et al., 2013). SIRPɑ-Fc also could be secreted by the receiver cells triggered by CD19+ sender cells (Fig. 1F). To develop a cell-cell contact-induced gene editing system, we swapped the Gal4-VP64 domain into Cas9 (SpCas9). In HEK293T-dEGFP cells that stably express a fast-degradable EGFP variant, transfected synNotch-Cas9 and EGFP-targeting sgRNA could down-regulate EGFP even without stimulation by CD19+ cells, suggesting a

Cell-cell contact-induced gene editing/activation in mammalian cells using a synNotch-CRISPR/Cas9 system