A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.

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Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea

International Journal of Molecular Sciences ◽

10.3390/ijms222413296 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13296

Author(s):

Mariarosaria De Falco ◽

Mariarita De Felice

Keyword(s):

Cell Cycle ◽

Dna Repair ◽

Genome Stability ◽

Developmental Disorders ◽

Strand Break ◽

Human Diseases ◽

Dna Damages ◽

Dna Repair Mechanisms ◽

Repair Mechanisms ◽

Repair Pathways

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

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Control of gene editing by manipulation of DNA repair mechanisms

Mammalian Genome ◽

10.1007/s00335-017-9688-5 ◽

2017 ◽

Vol 28 (7-8) ◽

pp. 262-274 ◽

Cited By ~ 25

Author(s):

Eric Danner ◽

Sanum Bashir ◽

Saniye Yumlu ◽

Wolfgang Wurst ◽

Benedikt Wefers ◽

...

Keyword(s):

Dna Repair ◽

Gene Editing ◽

Dna Repair Mechanisms ◽

Repair Mechanisms

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Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.601541 ◽

2020 ◽

Vol 2 ◽

Author(s):

Elizabeth K. Benitez ◽

Anastasia Lomova Kaufman ◽

Lilibeth Cervantes ◽

Danielle N. Clark ◽

Paul G. Ayoub ◽

...

Keyword(s):

Dna Repair ◽

Gene Editing ◽

Ex Vivo ◽

Specific Gene ◽

Gene Correction ◽

End Joining ◽

Therapeutic Success ◽

Hematopoietic Stem ◽

Dna Repair Mechanisms ◽

Repair Mechanisms

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

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Faculty Opinions recommendation of Quantitative live cell imaging reveals a gradual shift between DNA repair mechanisms and a maximal use of HR in mid S phase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717952551.793492296 ◽

2014 ◽

Author(s):

Wade Clapp ◽

Grzegorz Nalepa

Keyword(s):

Dna Repair ◽

Live Cell Imaging ◽

Cell Imaging ◽

S Phase ◽

Live Cell ◽

Dna Repair Mechanisms ◽

Gradual Shift ◽

Repair Mechanisms

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Faculty Opinions recommendation of Quantitative live cell imaging reveals a gradual shift between DNA repair mechanisms and a maximal use of HR in mid S phase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717952551.793458053 ◽

2012 ◽

Author(s):

Junjie Chen

Keyword(s):

Dna Repair ◽

Live Cell Imaging ◽

Cell Imaging ◽

S Phase ◽

Live Cell ◽

Dna Repair Mechanisms ◽

Gradual Shift ◽

Repair Mechanisms

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DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽

10.3390/cancers13030504 ◽

2021 ◽

Vol 13 (3) ◽

pp. 504

Author(s):

Takayuki Saitoh ◽

Tsukasa Oda

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Tumor Microenvironment ◽

Dna Damage Response ◽

Genetic Instability ◽

Dna Repair Mechanisms ◽

Damage Response ◽

Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

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Ancient Sturgeons Possess Effective DNA Repair Mechanisms: Influence of Model Genotoxicants on Embryo Development of Sterlet, Acipenser ruthenus

International Journal of Molecular Sciences ◽

10.3390/ijms22010006 ◽

2020 ◽

Vol 22 (1) ◽

pp. 6

Author(s):

Ievgeniia Gazo ◽

Roman Franěk ◽

Radek Šindelka ◽

Ievgen Lebeda ◽

Sahana Shivaramu ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Embryo Development ◽

Hatching Rate ◽

Embryo Survival ◽

Dna Repair Mechanisms ◽

Acipenser Ruthenus ◽

Repair Mechanisms ◽

Sterlet Acipenser Ruthenus

DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 µM benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 µM etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.

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