scholarly journals Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 112 ◽  
Author(s):  
Oliver Patrick March ◽  
Thomas Kocher ◽  
Ulrich Koller
Keyword(s):  
Genome Editing ◽  
Epidermolysis Bullosa ◽  
External Environment ◽  
Strand Break ◽  
Clinical Applications ◽  
Structural Proteins ◽  
Wound Management ◽  
Genes Encoding ◽  
Repair Pathways ◽  
Genetic Context

The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes encoding structural proteins. Therapeutic options for this debilitating group of diseases, including epidermolysis bullosa, primarily consist of wound management. Genome editing approaches co-opt double-strand break repair pathways to introduce desired sequence alterations at specific loci. Rapid advances in genome editing technologies have the potential to propel novel genetic therapies into the clinic. However, the associated phenotypes of many mutations may be treated via several genome editing strategies. Therefore, for potential clinical applications, implementation of efficient approaches based upon mutation, gene and disease context is necessary. Here, we describe current genome editing approaches for the treatment of genodermatoses, along with a discussion of the optimal strategy for each genetic context, in order to achieve enhanced genome editing approaches.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

2020 ◽  
Author(s):  
Ruben Schep ◽  
Eva K. Brinkman ◽  
Christ Leemans ◽  
Xabier Vergara ◽  
Ben Morris ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

scholarly journals Multiple mechanisms contribute to double-strand break repair at rereplication forks inDrosophilafollicle cells

2016 ◽  
Vol 113 (48) ◽  
pp. 13809-13814 ◽  
Author(s):  
Jessica L. Alexander ◽  
Kelly Beagan ◽  
Terry L. Orr-Weaver ◽  
Mitch McVey
Keyword(s):  
Follicle Cell ◽  
Strand Break ◽  
Follicle Cells ◽  
End Joining ◽  
Genomic Context ◽  
Dsb Repair ◽  
Origin Firing ◽  
Experimental Systems ◽  
Genes Encoding ◽  
Repair Pathways

Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. InDrosophilafollicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants,mus308displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of bothDrosophilaRad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.

scholarly journals Different DNA repair pathways are required following excision and integration of the DNA cut & paste transposon piggyBat in Saccharomyces cerevisiae.

2015 ◽  
Author(s):  
Weifeng She ◽  
Courtney Busch Cambouris ◽  
Nancy L. Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Single Gene ◽  
Donor Site ◽  
Insertion Site ◽  
Strand Break ◽  
Host Factors ◽  
A Genome ◽  
Genes Encoding ◽  
Repair Pathways

The movement of transposable elements from place to place in a genome requires both element-encoded and host-encoded factors. In DNA cut & paste transposition, the element-encoded transposase performs the DNA breakage and joining reactions that excise the element from the donor site and integrate it into the new insertion site. Host factors can influence many aspects of transposition. Notably, host DNA repair factors mediate the regeneration of intact duplex DNA necessary after transposase action by repairing the double strand break in the broken donor backbone, from which the transposon has excised, and repairing the single strand gaps that flank the newly inserted transposon. We have exploited the ability of the mammalian transposon piggyBat, a member of the piggyBac superfamily, to transpose in Saccharomyces cerevisiae and used the yeast single gene deletion collection to screen for genes encoding host factors involved in piggyBat transposition. piggyBac transposition is distinguished by the fact that piggyBac elements insert into TTAA target sites and also that the donor backbone is restored to its pre-transposon sequence after transposon excision, that is, excision is precise. We have found that repair of the broken donor backbone requires the non-homologous end-joining repair pathway (NHEJ). By contrast, NHEJ is not required for DNA repair at the new insertion site. Thus multiple DNA repair pathways are required for piggyBac transposition.

scholarly journals Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1506
Author(s):  
Angelos Papaspyropoulos ◽  
Nefeli Lagopati ◽  
Ioanna Mourkioti ◽  
Andriani Angelopoulou ◽  
Spyridon Kyriazis ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Mechanisms ◽  
Non Coding Rnas ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Living Organisms

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

scholarly journals Phosphorylation: The Molecular Switch of Double-Strand Break Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
K. C. Summers ◽  
F. Shen ◽  
E. A. Sierra Potchanant ◽  
E. A. Phipps ◽  
R. J. Hickey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genomic Stability ◽  
Molecular Switches ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Brca1 And Brca2 ◽  
Damage Response ◽  
Repair Mechanisms ◽  
Repair Pathways

Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

scholarly journals Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

2010 ◽  
Vol 79 (2) ◽  
pp. 316-330 ◽  
Author(s):  
Richa Gupta ◽  
Daniel Barkan ◽  
Gil Redelman-Sidi ◽  
Stewart Shuman ◽  
Michael S. Glickman
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Break Repair
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