Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Cellular Response ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Precise Integration ◽  
Guide Rna ◽  
Double Strand Dna Breaks ◽  
Human Therapy ◽  
P53 Activation ◽  
Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

scholarly journals p53 drives a transcriptional program that elicits a non-cell-autonomous response and alters cell state in vivo

2020 ◽  
Vol 117 (38) ◽  
pp. 23663-23673 ◽  
Author(s):  
Sydney M. Moyer ◽  
Amanda R. Wasylishen ◽  
Yuan Qi ◽  
Natalie Fowlkes ◽  
Xiaoping Su ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cellular Response ◽  
Target Genes ◽  
Transcriptional Program ◽  
Sequencing Data ◽  
Tissue Specific ◽  
Chip Sequencing ◽  
Cell State ◽  
P53 Activation

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion ofMdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which includedMdm2but notp21. Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

scholarly journals RNA-programmed genome editing in human cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Martin Jinek ◽  
Alexandra East ◽  
Aaron Cheng ◽  
Steven Lin ◽  
Enbo Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Human Cells ◽  
Limiting Factor ◽  
Dna Breaks ◽  
Genetic Changes ◽  
Rna Sequence ◽  
Guide Rna ◽  
Double Strand Dna Breaks

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Role played by BRCA1 in regulating the cellular response to stress

2003 ◽  
Vol 31 (1) ◽  
pp. 257-262 ◽  
Author(s):  
P.M. Gilmore ◽  
J.E. Quinn ◽  
P.B. Mullan ◽  
H.N. Andrews ◽  
N. McCabe ◽  
...  
Keyword(s):  
Cellular Response ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Breast Cancer Susceptibility ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Breast Cancer Susceptibility Gene ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Response To Stress

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer. In this review, we examine the role played by BRCA1 in mediating the cellular response to stress. We review the role played by BRCA1 in detecting and signalling the presence of DNA damage, particularly double-strand DNA breaks, and look at the evidence to support a role for BRCA1 in regulating stress response pathways such as the c-Jun N-terminal kinase/stress-activated protein kinase pathway. In addition, we examine the role played by BRCA1 in mediating both cell-cycle arrest and apoptosis following different types of cellular insult, and how this may be modulated by the presence or absence of associated proteins such as p53. Finally, we explore the possibility that many of the functions associated with BRCA1 may be based on transcriptional regulation of key downstream genes that have been implicated in the regulation of these specific cellular pathways.

scholarly journals A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP

2020 ◽  
Author(s):  
Natalia Felipe-Medina ◽  
Sandrine Caburet ◽  
Fernando Sánchez-Sáez ◽  
Yazmine B. Condezo ◽  
Dirk de Rooij ◽  
...  
Keyword(s):  
Missense Variant ◽  
Primary Ovarian Insufficiency ◽  
Dna Breaks ◽  
Ovarian Insufficiency ◽  
Knock Out ◽  
Recombination Nodules ◽  
Meiotic Gene ◽  
Double Strand Dna Breaks ◽  
Gene Functional Analysis

AbstractPrimary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a new HSF2BP knock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency.SummaryFelipe-Medina et al. describe a missense variant in the meiotic gene HSF2BP in a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, that in vivo impairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.

scholarly journals Prevalence and Characterization of Some Colibactin Genes in Clinical Enterobacteriaceae isolates from Iraqi Patients

2020 ◽  
Vol 17 (3(Suppl.)) ◽  
pp. 1113
Author(s):  
Mohammed T. Hussein ◽  
Safaa ahmed S. Al-Qaysi ◽  
Munther H. Rathi ◽  
Qasim I. Hussein ◽  
Tarek A.A. Moussa
Keyword(s):  
Hela Cells ◽  
Cellular Response ◽  
Polyketide Synthase ◽  
Giant Cells ◽  
Nuclear Chromatin ◽  
Cell Nuclei ◽  
Dna Breaks ◽  
Cytotoxic Effects ◽  
E Coli ◽  
Double Strand Dna Breaks

The members of the family of Eentrobacteriaceae harbour a gene cluster called polyketide synthase (pks) island. This cluster is responsible for the synthesis of the genotoxin colibactin that might have an important role in the induction of double-strand DNA breaks, leading to promote human colorectal cancer (CRC). Eleven out of the eighty eight isolates (12.5%) were pks+, distributed as 7 (8%) isolates of E. coli, 2 (2.25%) of K. pneumoniae and 2 (2.25%) of E. aerogenes. The cytotoxic effects of selected pks+ isolates (E. coli and E. aerogenes) on HeLa cells were represented by decreasing cell numbers and enlarged cell nuclei in comparison to the untreated cells. Cytological changes were observed when the infected HeLa cells cultures were stained with AO/EBr and visualized under fluorescent microscope. Some changes that happened in the color of the nuclear chromatin were accompanied by DNA condensation and degradation and fragmentation of nuclei. HeLa cells with green unchanged nuclear chromatin were alive while those with orange-dark and bright red nuclei were dead. It was concluded that a proportion of the Entreobacteriaceae isolates from Iraqi patients was pks+, which exerted cytotoxic effects upon using them to kill HeLa cells. In this study the microscopic observation of the cell morphology reveals the cellular response to the genotoxic insult, with reduced numbers, striking giant cells phenotype (megalocytosis) and fragmentation of nuclei due to the cell cycle arrest and cellular senescence

scholarly journals In vivo evolution of tumour cells after the generation of double-strand DNA breaks

2003 ◽  
Vol 88 (11) ◽  
pp. 1763-1771 ◽  
Author(s):  
H Mekid ◽  
O Tounekti ◽  
A Spatz ◽  
M Cemazar ◽  
F Z El Kebir ◽  
...  
Keyword(s):  
Tumour Cells ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

scholarly journals Mre11-Rad50-Nbs1 complex is activated by hypertonicity

2006 ◽  
Vol 291 (5) ◽  
pp. F1014-F1020 ◽  
Author(s):  
Mee Rie Sheen ◽  
Seung Whan Kim ◽  
Ju-Young Jung ◽  
Joon Young Ahn ◽  
Juong G. Rhee ◽  
...  
Keyword(s):  
Cellular Response ◽  
Control Level ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Damages ◽  
Mrn Complex ◽  
Double Strand Dna Breaks ◽  
Functionally Impaired ◽  
Mutant Cells ◽  
Dose Dependent

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including γH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and γH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.

scholarly journals p53 pathway is involved in cell competition during mouse embryogenesis

2017 ◽  
Vol 114 (3) ◽  
pp. 498-503 ◽  
Author(s):  
Guoxin Zhang ◽  
Yinyin Xie ◽  
Ying Zhou ◽  
Cong Xiang ◽  
Lai Chen ◽  
...  
Keyword(s):  
Cellular Response ◽  
Physiological Role ◽  
Tumor Suppressor P53 ◽  
Cell Competition ◽  
Cycle Arrest ◽  
Genetic Mosaic ◽  
P53 Activation ◽  
Murine Double Minute ◽  
High Level

The function of tumor suppressor p53 has been under intense investigation. Acute stresses such as DNA damage are able to trigger a high level of p53 activity, leading to cell cycle arrest or apoptosis. In contrast, the cellular response of mild p53 activity induced by low-level stress in vivo remains largely unexplored. Murine double minute (MDM)2 and MDM4 are two major negative regulators of p53. Here, we used the strategy of haploinsufficiency of Mdm2 and Mdm4 to induce mild p53 activation in vivo and found that Mdm2+/−Mdm4+/− double-heterozygous mice exhibited normal embryogenesis. However, closer examination demonstrated that the Mdm2+/−Mdm4+/− cells exhibited a growth disadvantage and were outcompeted during development in genetic mosaic embryos that contained wild-type cells. Further study indicated the out-competition phenotype was dependent on the levels of p53. These observations revealed that cells with mild p53 activation were less fit and exhibited altered fates in a heterotypic environment, resembling the cell competition phenomenon first uncovered in Drosophila. By marking unfit cells for elimination, p53 may exert its physiological role to ensure organ and animal fitness.

scholarly journals ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

2020 ◽  
Author(s):  
Georgia Chatzinikolaou ◽  
Kalliopi Stratigi ◽  
Kyriacos Agathangelou ◽  
Maria Tsekrekou ◽  
Evi Goulielmaki ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genotoxic Stress ◽  
Gene Promoters ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Genome Wide ◽  
Human Disorders ◽  
Or Gene ◽  
Type Ii Dna Topoisomerases

AbstractType II DNA Topoisomerases (TOP II) generate transient double-strand DNA breaks (DSBs) to resolve topological constraints during transcription. Using genome-wide mapping of DSBs and functional genomics approaches, we show that, in the absence of exogenous genotoxic stress, transcription leads to DSB accumulation and to the recruitment of the structure-specific ERCC1-XPF endonuclease on active gene promoters. Instead, we find that the complex is released from regulatory or gene body elements in UV-irradiated cells. Abrogation of ERCC1 or re-ligation blockage of TOP II-mediated DSBs aggravates the accumulation of transcription-associated γH2Ax and 53BP1 foci, which dissolve when TOP II-mediated DNA cleavage is inhibited. An in vivo biotinylation tagging strategy coupled to a high-throughput proteomics approach reveals that ERCC1-XPF interacts with TOP IIβ and the CTCF/cohesin complex, which co-localize with the heterodimer on DSBs. Together; our findings provide a rational explanation for the remarkable clinical heterogeneity seen in human disorders with ERCC1-XPF defects.

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