Generation of Human CRY1 and CRY2 Knockout Cells Using Duplex CRISPR/Cas9 Technology

Börding, Teresa; Abdo, Ashraf N; Maier, Bert; Gabriel, Christian; Kramer, Achim

doi:10.3389/fphys.2019.00577

Cited by 23 publications

(24 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S2E). Knocking out the CRY1 in this cell line resulted in a shorter period compared to wild-type controls as in agreement with previously published data 27 . Notably, when U2OS CRY1 -/-Bmal1-dLuc cells were treated with the M47, no change was observed in the period length of the circadian rhythm (Fig.…”

Section: Characterization Of M47supporting

confidence: 93%

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice

Gül

Akyel

Gul

et al. 2021

Preprint

View full text Add to dashboard Cite

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous level of the CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis is unknown. Here, we discovered a molecule that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The small molecule, called M47, selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and the period of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 enhanced degradation rate of the CRY1 level in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced cisplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Finally, systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a very promising molecule to treat forms of cancer depending on the p53 mutation.

show abstract

Section: Characterization Of M47supporting

confidence: 93%

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice

Gül

Akyel

Gul

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…The resulting fluorescent knock-in cell lines enabled us to visualize endogenously expressed PER2 and CRY1 proteins -two canonical circadian repressors -for the first time in single human live cells. We are confident that the fusion proteins are functional, because cells with a bi-allelic knock-in of PER2or CRY1-fusion proteins display normal circadian rhythms, in contrast to Per2 or Cry1 knockout cells which are hardly rhythmic (34,35). This is further supported by reports that homozygous Per2-Venus and Per2-Luc knock-in mice display normal circadian rhythms, and rhythmically expressed CRY1-EGFP can rescue rhythmic behavior in otherwise arrhythmic Cry1/2 double knock-out mice (16,36).…”

Section: Discussionmentioning

confidence: 97%

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

Gabriel

Olmo

Zehtabian

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator.Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.Recent biochemical studies with mouse liver lysates suggest that during the repressive phase, essentially all nuclear PER and CRY proteins are coordinated together in one large repressive complex, with only a minor amount of CRY1 monomers detectable (17). Again, double knock-out of either Per1/2 or Cry1/2 completely prevented the formation of this repressive complex.Most of the current knowledge of PER and CRY protein dynamics resulted either from biochemical data with mixed lysates of many thousand cells, or from single-cell imaging of over-expressed fluorescent tagged fusion proteins (12,13,17,18). Both approaches have clear limitations: Population samplinge.g. cell fractionation followed by Western Blot, chromatography or immunoprecipitation -not only conceals spatial information but also suffers from much reduced temporal resolution. Most importantly, however, population sampling averages signals from thousands of cells thereby masking individual cell properties (e.g. regarding circadian period, phase and amplitude) and degree of noise.While fluorescent tagged proteins constitute an outstanding tool to monitor protein expression and localization in individual living cells, overexpression of PER-and CRY-proteins in most cases disrupts the circadian oscillator and data from such experiments have to be conceived with caution (19,20).Such limitations can be overcome by incorporating a fluorescent tag directly into the proteins' genomic locus. In this case, expression dynamics and level of the resulting fusion protein often remain similar to the wild-type protein and the clock stays intact. Indeed, the PER2-Luciferase and the PER2-Venus knock-in mice -in which PER2 is tagged at the genomic level with ...

show abstract

“…In doing so, they transfected U2-OS cells stably expressing a Bmal1-Luciferase reporter with a lentivirus expressing Cas9 and two sgRNA targeting different sites in the www.nature.com/scientificreports/ Fbxl3 gene and reported that CRISPR-Cas9-based Fbxl3 KO dampened Bmal1 promoter-driven luminescence oscillation. The same group then utilized a CRISPR-Cas9 system to generate CRY1 and CRY2 double KO cells 36 . Furthermore, Clock targeting has been efficient in mouse embryonic stem cells when investigating the non-clock functions of the Clock gene 25 , while Lee and colleagues expanded the repertoire of direct CRISPR-Cas9 targets by designing an adenoviral vector system for efficiently generating null mutant cell lines 28 .…”

Section: Discussionmentioning

confidence: 99%

Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Kim

Chun

et al. 2021

Sci Rep

View full text Add to dashboard Cite

The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.

show abstract

Generation of Human CRY1 and CRY2 Knockout Cells Using Duplex CRISPR/Cas9 Technology

Cited by 23 publications

References 20 publications

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Contact Info

Product

Resources

About