CRISPR: Stressed about p53?

Foronda, Miguel; Dow, Lukas E.

doi:10.1016/j.molmed.2018.06.010

Cited by 6 publications

(11 citation statements)

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“…PFT-µ probably improve the knock-in efficiency of BFF cells by decreasing the apoptosis of cells with DSB, not by changing the cell cycle. Considering of the severe chromatin damage brought by p53 inhibition (84,85), a short-term use of p53 inhibitors not only provide a window for genome editing temporarily, but also the expression level of p53 can restoration (59).…”

Section: Discussionmentioning

confidence: 99%

“…Severe deleterious consequences of Cas9-induced DNA damage, mainly DNA double-strand breaks (DSBs), have been observed in some studies (57,58). What's more, some researchers have found that a single targeted DNA break is enough to cause cell cycle arrest or apoptosis in some cell types (59,60). Further experimental evidence in human pluripotent stem cells showed that introduction of DSBs at a single locus is sufficient to cause a P53-dependent molecular response and that transient suppression of the activity of p53 can improve the efficiency of precise genome engineering (60).…”

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confidence: 99%

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Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

et al. 2020

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Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-µ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 µg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6-8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

et al. 2020

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“…The double-strand dna breaks created by crispr/Cas9 as part of its mechanism of action activate a gene called p53, which is known as the "guardian of the genome"-involved in the repair of dna damage and, if that damage is sufficiently significant, in apoptosis, or the destruction of the cell containing the damaged dna. It is because of these functions that p53-a tumor suppressor gene-is known to be mutated in more than half of all human cancers (Hollstein et al, 1991;Foronda and Dow, 2018); if p53 cannot carry out its normal activities, damaged cells may go on to become tumorous (Ferrarelli, 2018). This is an issue because, as one might expect, p53 blocks crispr/Cas9 activity; and it therefore follows that cells that are experimentally modified by crispr, must, thus, tolerate dna damage, and so must have deficient p53.…”

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confidence: 99%

“…It has been suggested that such cells could be identified and eliminated by in vitro screening (Foronda and Dow, 2018), but various problems remain. Just one, single dna break seems to be sufficient to prime p53 activity, and lead to cell arrest or death (Foronda and Dow, 2018;Ihry et al, 2018), so the problem may be greater than first thought. Some have inferred or implied that this is a new discovery, but it is not: almost quarter of a century ago, this was demonstrated in human fibroblasts (Di Leonardo et al, 1994).…”

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confidence: 99%

“…Because break-induced toxicity has not been detected in all cell types, but also due to it not being seen in some cell types that do have functional p53, it means that "the induced arrest phenomenon will have to be tested and addressed for each type of target cell" as "that pathway is not the whole story" (Carroll, 2018). Finally, while selection is possible in vitro, it is not an option for in vivo somatic gene correction, in which this would have serious consequences for animals and humans (Foronda and Dow, 2018).…”

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Genetic Modification of Animals: Scientific and Ethical Issues

Bailey¹

2019

Animal Experimentation: Working Towards a Paradigm Change

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Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss

et al. 2021

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Hearing loss (HL) is a major global health problem of pandemic proportions. The most common type of HL is sensorineural hearing loss (SNHL) which typically occurs when cells within the inner ear are damaged. Human induced pluripotent stem cells (hiPSCs) can be generated from any individual including those who suffer from different types of HL. The development of new differentiation protocols to obtain cells of the inner ear including hair cells (HCs) and spiral ganglion neurons (SGNs) promises to expedite cell-based therapy and screening of potential pharmacologic and genetic therapies using human models. Considering age-related, acoustic, ototoxic, and genetic insults which are the most frequent causes of irreversible damage of HCs and SGNs, new methods of genome editing (GE), especially the CRISPR/Cas9 technology, could bring additional opportunities to understand the pathogenesis of human SNHL and identify novel therapies. However, important challenges associated with both hiPSCs and GE need to be overcome before scientific discoveries are correctly translated to effective and patient-safe applications. The purpose of the present review is (a) to summarize the findings from published reports utilizing hiPSCs for studies of SNHL, hence complementing recent reviews focused on animal studies, and (b) to outline promising future directions for deciphering SNHL using disruptive molecular and genomic technologies.

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CRISPR: Stressed about p53?

Cited by 6 publications

References 10 publications

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

Genetic Modification of Animals: Scientific and Ethical Issues

Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss

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