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

Huang, Hongxin; Zhang, Xin; Lv, Jie; Yang, H.; Wang, Xinlong; Ma, Shufeng; Shao, Ruoyang; Peng, Xin; Lin, Ying; Rong, Zhili

doi:10.1007/s13238-020-00690-1

Cited by 18 publications

(22 citation statements)

References 15 publications

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“…Another recent study using the Cas9:p300 fusion protein showed that the expression of a given gene could be either activated or suppressed after synNotch receptor activation [7]. Indeed, the authors of this study could demonstrate synNotch-mediated indel formation but observed a high unspecific background activity or LIA when the Cas9:p300 expression was triggered via a Gal4-UAS-system.…”

Section: Discussionmentioning

confidence: 72%

“…Even if synNotch receptors are reported to have a given amount of ligand-independent activation (LIA) [3,6,7,9], one could assume that this drawback may be reduced by different means. As reported by Yang et al, an additional hydrophobic sequence (named as RAM7) which is present in the native notch receptor significantly reduces the LIA [8].…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, synNotch receptors also contain a cytosolic domain that is cleaved upon receptor stimulation. This γ-secretase-dependent cleavage can cause ligand-independent activation [3,6,7] of the SynNotch receptor, but enhanced synthetic notch receptors (esNotch) are reported to significantly reduce the ligand-independent activation [8]. This intracellular domain can be substituted with various proteins that interact with compounds of the cytoplasm or the nucleus [3].…”

Section: Introductionmentioning

confidence: 99%

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

Sgodda

Alfken

Schambach

et al. 2020

Cells

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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.

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Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Sgodda

Alfken

Schambach

et al. 2020

Cells

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“…Biomedicine and biotherapeutics will benefit further from controlling CCIs, particularly in modifying disease courses, as is currently done with immune checkpoint inhibitors 159 , 160 . As a proof of concept, a tool that induces gene activation when specific cells interact or are directly in contact was recently built by combining a synthetic Notch receptor and the CRISPR–Cas9 system 161 . This biological device enabled customization of cell behaviours as observed through the expression of reporter genes.…”

Section: Challenges and Future Directionsmentioning

confidence: 99%

Deciphering cell–cell interactions and communication from gene expression

et al. 2020

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“…To construct transcriptional activators, we mutated the two key amino acid residues, D8A and H559A ( 20 ), within the nuclease domain of Cj Cas9 to generate d Cj Cas9 ( Supplementary Figure S1A ). T7E1 and PAGE assays demonstrated loss of DNase catalytic activity of d Cj Cas9 at an endogenous site (AAVS1 site) in HEK293T cells and an exogenous site (EGFP site) in a dEGFP HEK293T reporter cell line (a short half-life EGFP variant knock-in line ( 37 )) ( Supplementary Figure S1B, C ). Moreover, FACS assay showed that d Cj Cas9 failed to disrupt EGFP protein expression in the reporter cells, further demonstrating the inability of d Cj Cas9 to induce double-strand DNA breaks ( Supplementary Figure S1D ).…”

Section: Resultsmentioning

confidence: 99%

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

Zhang

Luo

et al. 2021

Nucleic Acids Research

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CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

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

Cited by 18 publications

References 15 publications

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

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

Deciphering cell–cell interactions and communication from gene expression

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

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