scholarly journals On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

2020 ◽  
Vol 21 (10) ◽  
pp. 3604 ◽  
Author(s):  
Alexey Korablev ◽  
Varvara Lukyanchikova ◽  
Irina Serova ◽  
Nariman Battulin
Keyword(s):  
Genome Engineering ◽  
Adaptive Immune System ◽  
Strand Break ◽  
Large Deletion ◽  
Dna Breaks ◽  
Adaptive Immune ◽  
Double Strand Dna Breaks ◽  
Mouse Zygotes ◽  
Genomic Editing ◽  
Cytoplasmic Injection

Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals The Schizosaccharomyces pombe rad60 Gene Is Essential for Repairing Double-Strand DNA Breaks Spontaneously Occurring during Replication and Induced by DNA-Damaging Agents

2002 ◽  
Vol 22 (10) ◽  
pp. 3537-3548 ◽  
Author(s):  
Takashi Morishita ◽  
Yasuhiro Tsutsui ◽  
Hiroshi Iwasaki ◽  
Hideo Shinagawa
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Strand Break ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Dna Breaks ◽  
Γ Irradiation ◽  
Synthetic Lethal ◽  
Double Strand Dna Breaks ◽  
Dna Double Strand Break

ABSTRACT To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and γ rays, epistatic to rhp51, and defective in the repair of DSBs caused by γ-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37°C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.

scholarly journals Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium

2020 ◽  
Vol 8 (5) ◽  
pp. 720 ◽  
Author(s):  
Meichen Pan ◽  
Matthew A. Nethery ◽  
Claudio Hidalgo-Cantabrana ◽  
Rodolphe Barrangou
Keyword(s):  
Genome Engineering ◽  
Rare Variants ◽  
Adaptive Immune System ◽  
Effector Proteins ◽  
Type I ◽  
Human Gut ◽  
Adaptive Immune ◽  
Naturally Occurring ◽  
History Of

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.

scholarly journals Modern methods of CRISPR/Cas genomic editing for modulation in plants aimed at viral diseases

2021 ◽  
Vol 136 (3) ◽  
pp. 64-85
Author(s):  
N. Iksat ◽  
◽  
Z. Stamgaliyeva ◽  
A. Madirov ◽  
S. Zhangazin ◽  
...  
Keyword(s):  
Abiotic Factors ◽  
Plant Biotechnology ◽  
Adaptive Immune System ◽  
Plant Interactions ◽  
Practical Applications ◽  
Biotic Stresses ◽  
Adaptive Immune ◽  
Defense Systems ◽  
World Agriculture ◽  
Genomic Editing

With the rapid growth of the world's population, food security has become a major concern, with more than 800 million people suffering from hunger and millions more at risk. World agriculture is constantly under an influence of various biotic and abiotic factors that limit productivity of agricultural crops. Pathogens, including fungi, bacteria, viruses, insects and parasitic plants, are severe biotic stresses that can cause severe crop losses. Molecular interactions between a virus and a plant are one of the main models in the understanding of antiviral defense systems and plant interference. The article discusses main classes of resistance genes, RNA interference and RNA-mediated adaptive immune system of bacteria and archaea - CRISPR/Cas. Recent studies indicate that the CRISPR/Cas system may play a significant role in conferring antiviral resistance to plants. The article aims to review recent advances in plant biotechnology that have potential practical applications in regulating virus-plant interactions.

scholarly journals CRISPR/Cas9 and cancer

2021 ◽  
Vol 53 (2) ◽  
Author(s):  
Nora Mimoune ◽  
Mohamed Wail Bahouh ◽  
Said Boukhechem ◽  
Djamel Khelef ◽  
Rachid Kaidi
Keyword(s):  
Cancer Research ◽  
Clinical Trials ◽  
Immune System ◽  
Genome Engineering ◽  
Adaptive Immune System ◽  
Powerful Method ◽  
Efficient Technology ◽  
Adaptive Immune ◽  
Mouse Models Of Cancer ◽  
The Impact

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found widespread use in genome engineering and in the activation or repression of gen expression. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review the current applications of the CRISPR/Cas9 technology for cancer research and therapy. We highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials applying CRISPR/Cas9 as a therapeutic approach against cancer.

CRISPR/Cas-Systems: characteristics and possibilities of use for editing bacterial genomes

Bacteriology ◽  
2020 ◽  
Vol 5 (2) ◽  
pp. 38-48
Author(s):  
I.A. Blatov ◽  
◽  
A.S. Shchurova ◽  
D.Yu. Guschin ◽  
S.D. Zvereva ◽  
...  
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Bacterial Genomes ◽  
Modern Biology ◽  
Adaptive Immune ◽  
History Of ◽  
Genomic Editing ◽  
Classification Structure

CRISPR-Cas is the adaptive immune system of bacteria and archaea. Since 2012, when the first opportunity to use the CRISPR/Cas system for genome editing was realized, the number of studies in this area has been growing rapidly. Today, genomic editing to modify specific regions of the genomes of various organisms is considered one of the key methodologies of modern biology. This review is devoted to the history of discovery, classification, structure, operational mechanisms of CRISPRCas systems and strategies for editing the genomes of various bacterial species using this technology. Key words: genome editing, genome, CRISPR-Cas system, bacteria

scholarly journals In VitroExpansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Ian Hare ◽  
Marieta Gencheva ◽  
Rebecca Evans ◽  
James Fortney ◽  
Debbie Piktel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Strand Break ◽  
Dna Lesions ◽  
Cellular Damage ◽  
Human Mscs ◽  
Dna Breaks ◽  
Double Strand Dna Breaks

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies.Ex vivoexpansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs within vitropassage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passagein vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passagedin vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate thatex vivoexpansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

scholarly journals Genetic instability in the RAD51 and BRCA1 regions in breast cancer

2007 ◽  
Vol 12 (2) ◽  
Author(s):  
Maria Nowacka-Zawisza ◽  
Magdalena Bryś ◽  
Hanna Romanowicz-Makowska ◽  
Andrzej Kulig ◽  
Wanda Krajewska
Keyword(s):  
Breast Cancer ◽  
Genetic Instability ◽  
Strand Break ◽  
The Other ◽  
Cancer Development ◽  
Allelic Loss ◽  
Cancer Type ◽  
Polymorphic Markers ◽  
Dna Breaks ◽  
Double Strand Dna Breaks

AbstractBreast cancer is the most prevalent cancer type in women. Accumulating evidence indicates that the fidelity of double-strand break repair in response to DNA damage is an important step in mammary neoplasias. The RAD51 and BRCA1 proteins are involved in the repair of double-strand DNA breaks by homologous recombination. In this study, we evaluated loss of heterozygosity (LOH) in the RAD51 and BRCA1 regions, and their association with breast cancer. The polymorphic markers D15S118, D15S214 and D15S1006 were the focus for RAD51, and D17S855 and D17S1323 for BRCA1. Genomic deletion detected by allelic loss varied according to the regions tested, and ranged from 29 to 46% of informative cases for the RAD51 region and from 38 to 42% of informative cases for the BRCA1 region. 25% of breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 31% of breast cancer cases manifested LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA1 regions. LOH in the RAD51 region, similarly as in the BRCA1 region, appeared to correlate with steroid receptor status. The obtained results indicate that alteration in the RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and BRCA1 and thus enhance the risk of breast cancer development.

scholarly journals Telomeric chromosome ends are highly mobile and behave like free double-strand DNA breaks

2019 ◽  
Author(s):  
Mathias Toulouze ◽  
Assaf Amitai ◽  
Ofir Shukron ◽  
David Holcman ◽  
Karine Dubrana
Keyword(s):  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Strand Break ◽  
Spindle Pole ◽  
Dna Breaks ◽  
Computational Approaches ◽  
Pole Body ◽  
Double Strand Dna Breaks ◽  
Chromosome Arm ◽  
Approaches To Study

AbstractChromosome organization and dynamics are critical for DNA transactions, including gene expression, replication, and DNA repair. In yeast, the chromosomes are anchored through their centromeres to the spindle pole body, and their telomeres are grouped into clusters at the nuclear periphery, constraining chromosome mobility. Here, we have used experimental and computational approaches to study the effects of chromosome-nuclear envelope (NE) attachments on the dynamics of S. cerevisiae chromosomes. We found that although centromere proximal loci were, as predicted, more dynamically constrained than distal loci, telomeres were highly mobile, even when positioned at the nuclear periphery. Polymer modeling indicated that polymer ends are intrinsically more mobile than internal sites. We tested this model by measuring the mobility of a double strand break (DSB) end within a chromosome arm. Upon separation of the DSB ends, their mobility significantly increased. Altogether, our results reveal that telomeres behave as highly mobile polymer ends, despite interactions with the nuclear membrane.

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