scholarly journals Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

2017 ◽  
Author(s):  
Yuping Huang ◽  
Caitlin McCann ◽  
Andrey Samsonov ◽  
Dmitry Malkov ◽  
Gregory D Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Cell Cycle Control ◽  
Strand Break ◽  
End Joining ◽  
Genetic Changes ◽  
A Cell ◽  
Non Homologous End Joining ◽  
Green Fluorescent ◽  
Chromosomal Sequences

ABSTRACTTargeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

scholarly journals Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

2021 ◽  
Author(s):  
Anie Day D C Asa ◽  
Rujira Wanotayan ◽  
Mukesh Kumar Sharma ◽  
Kaima Tsukada ◽  
Mikio Shimada ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Strand Break ◽  
Growth Defect ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Stable Transfectants ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Green Fluorescent

Abstract Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.

DNA double-strand break repair within heterochromatic regions

2012 ◽  
Vol 40 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Johanne M. Murray ◽  
Tom Stiff ◽  
Penny A. Jeggo
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Strand Break ◽  
Higher Order ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Dna Dsbs ◽  
A Cell ◽  
Non Homologous End Joining

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases

Cytometry ◽  
2001 ◽  
Vol 45 (3) ◽  
pp. 225-234 ◽  
Author(s):  
Christopher J. Donahue ◽  
Maxine Santoro ◽  
Donald Hupe ◽  
Jay M. Jones ◽  
Brian Pollok ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Protein Substrate ◽  
A Cell ◽  
Group Ii ◽  
Green Fluorescent

scholarly journals Regulatory networks integrating cell cycle control with DNA damage checkpoints and double-strand break repair

2011 ◽  
Vol 366 (1584) ◽  
pp. 3562-3571 ◽  
Author(s):  
Petra Langerak ◽  
Paul Russell
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Regulatory Networks ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Strand Break ◽  
Dsb Repair ◽  
Cycle Progression ◽  
Non Homologous End Joining

Double-strand breaks (DSBs), arising from exposure to exogenous clastogens or as a by-product of endogenous cellular metabolism, pose grave threats to genome integrity. DSBs can sever whole chromosomes, leading to chromosomal instability, a hallmark of cancer. Healing broken DNA takes time, and it is therefore essential to temporarily halt cell division while DSB repair is underway. The seminal discovery of cyclin-dependent kinases as master regulators of the cell cycle unleashed a series of studies aimed at defining how the DNA damage response network delays cell division. These efforts culminated with the identification of Cdc25, the protein phosphatase that activates Cdc2/Cdk1, as a critical target of the checkpoint kinase Chk1. However, regulation works both ways, as recent studies have revealed that Cdc2 activity and cell cycle position determine whether DSBs are repaired by non-homologous end-joining or homologous recombination (HR). Central to this regulation are the proteins that initiate the processing of DNA ends for HR repair, Mre11–Rad50–Nbs1 protein complex and Ctp1/Sae2/CtIP, and the checkpoint kinases Tel1/ATM and Rad3/ATR. Here, we review recent findings and provide insight on how proteins that regulate cell cycle progression affect DSB repair, and, conversely how proteins that repair DSBs affect cell cycle progression.

scholarly journals Apalutamide Sensitizes Prostate Cancer to Ionizing Radiation via Inhibition of Non-Homologous End-Joining DNA Repair

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1593 ◽  
Author(s):  
Wenhao Zhang ◽  
Chen-Yi Liao ◽  
Hajar Chtatou ◽  
Luca Incrocci ◽  
Dik C. van Gent ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Locally Advanced ◽  
External Beam Radiation ◽  
Combination Regimen ◽  
End Joining ◽  
Beam Radiation ◽  
Non Homologous End Joining

Androgen-deprivation therapy was shown to improve treatment outcome of external beam radiation therapy (EBRT) for locally advanced prostate cancer (PCa). DNA damage response (DDR) was suggested to play a role in the underlying mechanism, but conflicting results were reported. This study aims to reveal the role of the androgen receptor (AR) in EBRT-induced DDR and to investigate whether next-generation AR inhibitor apalutamide can radiosensitize PCa. PCa cell lines and tissue slices were treated with anti-androgen alone or combined with EBRT. The effect of treatments on cell growth, tissue viability, DDR, and cell cycle were investigated. RAD51 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) levels were determined by Western blotting. Homologous recombination (HR) capacity was measured with the directed repeats-green fluorescent protein (DR-GFP) assay. We report the radiosensitizing effect of anti-androgens, which showed synergism in combination with EBRT in AR-expressing tumor slices and cell lines. Moreover, a compromised DDR was observed in AR-expressing cells upon AR suppression. We found that AR inhibition downregulated DNA-PKcs expression, resulting in reduced non-homologous end-joining repair. DDR through HR was a secondary effect due to cell-cycle change. These data provide a mechanistic explanation for the combination regimen and support the clinical use of apalutamide together with EBRT for localized PCa patients.

scholarly journals Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin

2001 ◽  
Vol 12 (4) ◽  
pp. 971-980 ◽  
Author(s):  
Nasser M. Rusan ◽  
Carey J. Fagerstrom ◽  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
A Cell ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
Quantitative Immunoblotting

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

scholarly journals A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

2019 ◽  
Author(s):  
Manuel M. Vicente ◽  
Afonso Mendes ◽  
Margarida Cruz ◽  
José R. Vicente ◽  
Vasco M. Barreto
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Relative Increase ◽  
End Joining ◽  
New Era ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Dna Lesion ◽  
Non Homologous End Joining ◽  
Cycle Dependent

ABSTRACTThe discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

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