A piggyBac-based toolkit for inducible genome editing in mammalian cells

Schertzer, Megan D.; Thulson, Eliza; Braceros, Keean C. A.; Lee, David M.; Hinkle, Emma R.; Murphy, Ryan M.; Kim, Susan O; Vitucci, Eva C.M.; Calabrese, J. Mauro

doi:10.1101/448803

Cited by 3 publications

(1 citation statement)

References 33 publications

(44 reference statements)

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“…8,9,44,[51][52][53][54][55] We generated a cell line stably expressing SNAPsense-Cdc42WT with biosensor expression under the control of a doxycycline-regulated promoter. 56 This proved substantially easier than using our previously published Cdc42 biosensor in which dye is incorporated through UAA labeling ; this requires co-expression of the UAA-specific tRNA synthetase and multiple copies its orthogonal tRNA. The biosensor was excited with a 561 nm laser and emission was split by a dichroic passing the wavelengths above and below 624 nm to different cameras (Semrock, FF624-Di01).…”

Section: Resultsmentioning

confidence: 99%

Using SNAP-tag for facile construction of dye-based biosensors in living cells

Pinkin

Liu

Pimenta

et al. 2020

Preprint

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Fluorescent biosensors based on environment-sensitive dyes have important advantages over alternative methodologies such as FRET, including the potential for enhanced brightness, elimination of bleaching artifacts, and more possibilities for multiplexing. However, such biosensors have been difficult to use because they required proteins to be covalently labeled and reintroduced into cells. Recent development of self-labeling enzymes that covalently react with membrane-permeable dyes (e.g. SNAP-tag) provide an opportunity to easily generate dye-based biosensors within cells. Here, we generate a new biosensor for Cdc42 activation by positioning SNAP-tag between Cdc42 and a peptide that binds selectively to active Cdc42. We generate a membrane-permeable Nile Red derivative that exhibits 50-fold fluorescence enhancement upon covalent labeling of the biosensor, then optimize the biosensor so the dye undergoes a 20 nm emission shift upon Cdc42 activation, enabling ratiometric imaging with a single dye. The biosensor, named SNAPsense Cdc42, is validated by examining its response to known regulatory proteins and studying Cdc42 activation during protrusion in living cells. Variants using other dyes are also presented.

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Section: Resultsmentioning

confidence: 99%

Using SNAP-tag for facile construction of dye-based biosensors in living cells

Pinkin

Liu

Pimenta

et al. 2020

Preprint

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A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells

Hazelbaker

Beccard

Angelini

et al. 2020

Sci Rep

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CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. collectively, our results provide proof-of-principle application of a stable, multiplexed pB gRnA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

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Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

Beccard

Mazzucato

et al. 2019

Preprint

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16CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) 17 approaches hold promise for functional genomic studies and genome-wide screens in 18 human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease 19 approaches, the efficiency of CRISPRi/a depends on continued expression of the dead 20 Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending 21 on transgene design and delivery. Here, we design new fluorescently labeled piggyBac 22 (PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, 23 we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9- 24KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of 25 cells that express both the dCas9 effectors and gRNAs. We then employ these systems 26 to target the TCF4 gene and conduct a rigorous assessment of expression levels of the 27 dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-28

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A piggyBac-based toolkit for inducible genome editing in mammalian cells

Cited by 3 publications

References 33 publications

Using SNAP-tag for facile construction of dye-based biosensors in living cells

Using SNAP-tag for facile construction of dye-based biosensors in living cells

A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells

Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

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