Development of a Bioassay for Continuous Monitoring of TWIST1 Activity

Czapiński, J; Kałafut, Joanna; Przybyszewska-Podstawka, A; Pawlicka, M.; Roszkowska, A; Kiełbus, M; Borkiewicz, L; Dudziak, Karolina; Czerwonka, Arkadiusz; Stepulak, A; Rivero-Müller, Adolfo

doi:10.20944/preprints202103.0265.v1

Cited by 2 publications

(3 citation statements)

References 64 publications

(88 reference statements)

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“…TWIST1 activity is closely associated with EMT, and correlates with tumor growth, metastasis and drug resistance, thus diminishing the survival of cancer patients 47 . Using an artificial promoter containing TWIST1 binding domains ( TWIST1-BD ) 35 , and the other halve under the p hCEA , only cells undergoing EMT will express both Cas4.5 halves. To trigger EMT, we exposed cells to TGFβ.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid #62988 32 ), pU6-sgGFP-NT1 was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid #46914; http://n2t.net/addgene:46914; RRID:Addgene_46914) 8 ), pCAG-EGxxFP- Cetn1 was a gift from Masahito Ikawa (Addgene plasmid #50717; http://n2t.net/addgene:50717; RRID:Addgene_50717 33 ), AmCyan-P2A-mCherry was previously created by us 34 (Addgene plasmid #45350; http://n2t.net/addgene:45350; RRID:Addgene_45350 34 ). The TWIST1 binding domain ( TBD ) promoter was created previously by us 35 , DnaE intein plasmids were a gift from Hideo Iwai (Addgene plasmid #34549; http://n2t.net/addgene:34549; RRID:Addgene_34549 36 and #15335; http://n2t.net/addgene:15335; RRID:Addgene_15335 37 ). Cell lines HEK-293, HSF, H1299, H2170, SW480, A375 and HeLa, were obtained from ATCC; H2170 stable cell line with vimentin reporter (VRCs) was created by us 38 .…”

Section: Methodsmentioning

confidence: 99%

“…). The TWIST1 binding domain (TBD) promoter was created previously by us35 , DnaE intein plasmids were a gift from Hideo Iwai (Addgene plasmid #34549; http://n2t.net/addgene:34549; RRID:Addgene_34549 36 and #15335; http://n2t.net/addgene:15335; RRID:Addgene_1533537 …”

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confidence: 99%

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Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviours by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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Synthetic circuits based on split Cas9 to detect cellular events

Przybyszewska-Podstawka,

Czapiński,

Kałafut

et al. 2023

Sci Rep

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Synthetic biology involves the engineering of logic circuit gates that process different inputs to produce specific outputs, enabling the creation or control of biological functions. While CRISPR has become the tool of choice in molecular biology due to its RNA-guided targetability to other nucleic acids, it has not been frequently applied to logic gates beyond those controlling the guide RNA (gRNA). In this study, we present an adaptation of split Cas9 to generate logic gates capable of sensing biological events, leveraging a Cas9 reporter (EGxxFP) to detect occurrences such as cancer cell origin, epithelial to mesenchymal transition (EMT), and cell–cell fusion. First, we positioned the complementing halves of split Cas9 under different promoters—one specific to cancer cells of epithelial origin (phCEA) and the other a universal promoter. The use of self-assembling inteins facilitated the reconstitution of the Cas9 halves. Consequently, only cancer cells with an epithelial origin activated the reporter, exhibiting green fluorescence. Subsequently, we explored whether this system could detect biological processes such as epithelial to mesenchymal transition (EMT). To achieve this, we designed a logic gate where one half of Cas9 is expressed under the phCEA, while the other is activated by TWIST1. The results showed that cells undergoing EMT effectively activated the reporter. Next, we combined the two inputs (epithelial origin and EMT) to create a new logic gate, where only cancer epithelial cells undergoing EMT activated the reporter. Lastly, we applied the split-Cas9 logic gate as a sensor of cell–cell fusion, both in induced and naturally occurring scenarios. Each cell type expressed one half of split Cas9, and the induction of fusion resulted in the appearance of multinucleated syncytia and the fluorescent reporter. The simplicity of the split Cas9 system presented here allows for its integration into various cellular processes, not only as a sensor but also as an actuator.

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Development of a Bioassay for Continuous Monitoring of TWIST1 Activity

Cited by 2 publications

References 64 publications

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Synthetic circuits based on split Cas9 to detect cellular events

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