scholarly journals Global detection of DNA repair outcomes induced by CRISPR-Cas9

2021 ◽  
Author(s):  
Mengzhu Liu ◽  
Weiwei Zhang ◽  
Changchang Xin ◽  
Jianhang Yin ◽  
Yafang Shang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Target Genes ◽  
Chromosomal Translocations ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Large Deletions ◽  
Mouse Cells ◽  
Repair Pathways ◽  
Cellular Dna

AbstractCRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, plasmid integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

scholarly journals The Chromatin Response to Double-Strand DNA Breaks and Their Repair

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1853
Author(s):  
Radoslav Aleksandrov ◽  
Rossitsa Hristova ◽  
Stoyno Stoynov ◽  
Anastas Gospodinov
Keyword(s):  
Dna Repair ◽  
Cancer Progression ◽  
Repair Process ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Double Strand Dna Breaks ◽  
Dna Repair Proteins ◽  
Dna Strands ◽  
Cellular Dna ◽  
Double Strand Dna

Cellular DNA is constantly being damaged by numerous internal and external mutagenic factors. Probably the most severe type of insults DNA could suffer are the double-strand DNA breaks (DSBs). They sever both DNA strands and compromise genomic stability, causing deleterious chromosomal aberrations that are implicated in numerous maladies, including cancer. Not surprisingly, cells have evolved several DSB repair pathways encompassing hundreds of different DNA repair proteins to cope with this challenge. In eukaryotic cells, DSB repair is fulfilled in the immensely complex environment of the chromatin. The chromatin is not just a passive background that accommodates the multitude of DNA repair proteins, but it is a highly dynamic and active participant in the repair process. Chromatin alterations, such as changing patterns of histone modifications shaped by numerous histone-modifying enzymes and chromatin remodeling, are pivotal for proficient DSB repair. Dynamic chromatin changes ensure accessibility to the damaged region, recruit DNA repair proteins, and regulate their association and activity, contributing to DSB repair pathway choice and coordination. Given the paramount importance of DSB repair in tumorigenesis and cancer progression, DSB repair has turned into an attractive target for the development of novel anticancer therapies, some of which have already entered the clinic.

Homozygosity for CHEK2 p.Gly167Arg leads to a unique cancer syndrome with multiple complex chromosomal translocations in peripheral blood karyotype

2019 ◽  
Vol 57 (7) ◽  
pp. 500-504 ◽  
Author(s):  
Tamar Paperna ◽  
Nitzan Sharon-Shwartzman ◽  
Alina Kurolap ◽  
Yael Goldberg ◽  
Nivin Moustafa ◽  
...  
Keyword(s):  
Dna Repair ◽  
Peripheral Blood ◽  
Early Onset ◽  
Genomic Analysis ◽  
Peripheral Blood Lymphocytes ◽  
Chromosomal Translocations ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Cancer Syndrome ◽  
Cancer Syndromes

BackgroundChromosomal instability, as reflected by structural or copy-number changes, is a known cancer characteristic but are rarely observed in healthy tissue. Mutations in DNA repair genes disrupt the maintenance of DNA integrity and predispose to hereditary cancer syndromes.ObjectiveTo clinically characterise and genetically diagnose two reportedly unrelated patients with unique cancer syndromes, including multiorgan tumourogenesis (patient 1) and early-onset acute myeloid leukaemia (patient 2), both displaying unique peripheral blood karyotypes.MethodsGenetic analysis in patient 1 included TruSight One panel and whole-exome sequencing, while patient 2 was diagnosed by FoundationOne Heme genomic analysis; Sanger sequencing was used for mutation confirmation in both patients. Karyotype analysis was performed on peripheral blood, bone marrow and other available tissues.ResultsBoth patients were found homozygous for CHEK2 c.499G>A; p.Gly167Arg and exhibited multiple different chromosomal translocations in 30%–60% peripheral blood lymphocytes. This karyotype phenotype was not observed in other tested tissues or in an ovarian cancer patient with a different homozygous missense mutation in CHEK2 (c.1283C>T; p.Ser428Phe).ConclusionsThe multiple chromosomal translocations in patient lymphocytes highlight the role of CHK2 in DNA repair. We suggest that homozygosity for p.Gly167Arg increases patients' susceptibility to non-accurate correction of DNA breaks and possibly explains their increased susceptibility to either multiple primary tumours during their lifetime or early-onset tumourigenesis.

scholarly journals Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

2020 ◽  
Vol 21 (4) ◽  
pp. 1380 ◽  
Author(s):  
Giovanni Pasquini ◽  
Virginia Cora ◽  
Anka Swiersy ◽  
Kevin Achberger ◽  
Lena Antkowiak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Time Course ◽  
Sequencing Analysis ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Gene ◽  
Repair Pathways

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

scholarly journals CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway

2017 ◽  
Author(s):  
Chris D Richardson ◽  
Katelynn R Kazane ◽  
Sharon J Feng ◽  
Nicholas L Bray ◽  
Axel J Schäfer ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Fanconi Anemia ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Dermal Fibroblasts ◽  
Human Cells ◽  
Individual Gene ◽  
Break Site ◽  
Repair Pathways

AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

scholarly journals Tissue Specific DNA Repair Outcomes Shape the Landscape of Genome Editing

2021 ◽  
Vol 12 ◽  
Author(s):  
Mathilde Meyenberg ◽  
Joana Ferreira da Silva ◽  
Joanna I. Loizou
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genetic Diseases ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Dna Damage And Repair ◽  
Tissue Specific ◽  
Precise Control ◽  
Repair Processes ◽  
Recent Developments

The use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 has moved from bench to bedside in less than 10years, realising the vision of correcting disease through genome editing. The accuracy and safety of this approach relies on the precise control of DNA damage and repair processes to achieve the desired editing outcomes. Strategies for modulating pathway choice for repairing CRISPR-mediated DNA double-strand breaks (DSBs) have advanced the genome editing field. However, the promise of correcting genetic diseases with CRISPR-Cas9 based therapies is restrained by a lack of insight into controlling desired editing outcomes in cells of different tissue origin. Here, we review recent developments and urge for a greater understanding of tissue specific DNA repair processes of CRISPR-induced DNA breaks. We propose that integrated mapping of tissue specific DNA repair processes will fundamentally empower the implementation of precise and safe genome editing therapies for a larger variety of diseases.

scholarly journals The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Lei Zhao ◽  
Chengyu Bao ◽  
Yuxuan Shang ◽  
Xinye He ◽  
Chiyuan Ma ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Ionising Radiation ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Repair Pathways

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

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 Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

2015 ◽  
Vol 35 (21) ◽  
pp. 3657-3668 ◽  
Author(s):  
Han Lin ◽  
Kyungsoo Ha ◽  
Guojun Lu ◽  
Xiao Fang ◽  
Ranran Cheng ◽  
...  
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Mitotic Exit ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Downstream Target ◽  
Damage Repair ◽  
Repair Pathways ◽  
Repair Defect

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.

scholarly journals NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Ekaterina Knyazhanskaya ◽  
Andrey Anisenko ◽  
Olga Shadrina ◽  
Anastasia Kalinina ◽  
Timofei Zatsepin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Wild Type Virus ◽  
Dna Breaks ◽  
End Joining ◽  
Double Stranded Dna ◽  
Repair Efficiency ◽  
Non Homologous End Joining ◽  
Cellular Dna ◽  
Successful Replication ◽  
Hiv 1

Abstract Background HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. Results We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Conclusions Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

scholarly journals Preserving genome integrity in human cells via DNA double-strand break repair

2020 ◽  
Vol 31 (9) ◽  
pp. 859-865 ◽  
Author(s):  
Ryan B. Jensen ◽  
Eli Rothenberg
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Human Cells ◽  
Cell Cycle Checkpoints ◽  
Human Diseases ◽  
Genome Integrity ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.

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