Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing

Abstract

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%–45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.

SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo

“…When analysing transgene expression within single samples, we found that mCherry distribution follows a bimodal on/off response, indicative of the presence of receiver cells which do not interact with sender cells at low sender:receiver cell ratios. This bimodal pattern of transgene induction is also evident in data from Cantz and colleagues (Sgodda et al, 2020). The ability of individual sender cells to induce mCherry induction in STC receiver cells (as seen in Movies 1 and 3) implies that this system can be effectively employed to study the effect of interactions between receiver cells and individual and/or rare sender cells in relevant model systems.…”

“…We generated sender cell lines expressing high and uniform levels of extracellular membrane-tethered EGFP. This transgenic construct was previously used for SynNotch sender cells (Morsut et al, 2016; Sgodda et al, 2020), and comprises EGFP fused to an N-terminal mouse IgGK signal sequence and a C-terminal human PDGFRB transmembrane domain. The addition of HA and Myc tags at the N- and C-termini of EGFP did not affect the ability of sender cells to induce mCherry expression in STC receiver cells (Fig.…”

“…Previous studies have co-cultured senders and receiver cells at different ratios (ranging from 1:50 to 5:1), and for varying times (ranging from 10 minutes to several days) (Cho et al, 2018; Choe et al, 2021; He et al, 2017; Huang et al, 2020; Luo et al, 2019; Matsunaga et al, 2020; Morsut et al, 2016; Roybal et al, 2016; Sgodda et al, 2020; Srivastava et al, 2019; Toda et al, 2018; Toda et al, 2020; Wang et al, 2021; Yang et al, 2020). We analysed transgene induction in STC receiver cells at 11 different sender:receiver cell ratios (ranging from 1:19 to 9:1), and observed that higher proportions of sender cells in culture result in a higher proportion of receiver cells inducing mCherry.…”

“…We observed low levels of mCherry induction in STC receiver cells following 2 hours of co-culture with sender cells, provided we allowed time for subsequent protein maturation, and observed maximum mCherry induction following 48 hours of co-culture. Previous studies making use of lentiviral-delivered transgenes suggest that 10 minutes may be sufficient for transgene activation in HEK293 receiver cells, and that 1 hour may be sufficient for transgene induction in L929 receiver cells, as long as protein maturation time is allowed (Morsut et al, 2016; Sgodda et al, 2020). This is significantly faster than what we observed in this study, and may be ascribable to lentiviral transduction leading to higher levels of SynNotch receptor and/or integration of multiple transgene copies compared to our clonal mouse ESC lines.…”

“…SynNotch technology has been used for monitoring cell-cell interactions, generating synthetic patterns, generating synthetic morphogen gradients, inducing contact-mediated gene editing, and generating custom antigen receptor T-cells (Cho et al, 2018; Choe et al, 2021; He et al, 2017; Huang et al, 2020; Roybal et al, 2016; Sgodda et al, 2020; Toda et al, 2018; Toda et al, 2020; Wang et al, 2021). SynNotch technology has been established in Drosophila (He et al, 2017) as well as in immortalised cell lines and differentiated cell types, but its potential in the study of mammalian developmental events remains largely untapped.…”