scholarly journals CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing

2020 ◽  
Author(s):  
Gavin Kurgan ◽  
Rolf Turk ◽  
Heng Li ◽  
Nathan Roberts ◽  
Garrett R. Rettig ◽  
...  
Keyword(s):  
Genome Editing ◽  
Amplicon Sequencing ◽  
Strand Break ◽  
Analysis Tool ◽  
Sequencing Data ◽  
Dna Breaks ◽  
End Joining ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Security Standards

AbstractCRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair footprints that result from non-homologous end joining (NHEJ) after double stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building our understanding into a novel analysis tool (CRISPAltRations) improved results’ quality. We validated our software using simulated rhAmpSeq™ amplicon sequencing data (11 gRNAs and 603 on- and off-target locations) and demonstrate that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user-interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a UI we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.

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.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

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 ATR inhibition enhances 5-fluorouracil sensitivity independent of non-homologous end-joining and homologous recombination repair pathway

2020 ◽  
Author(s):  
Soichiro S. Ito ◽  
Yosuke Nakagawa ◽  
Masaya Matsubayashi ◽  
Yoshihiko M. Sakaguchi ◽  
Shinko Kobashigawa ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Homologous Recombination Repair ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Drug Induced ◽  
Nucleotide Synthesis ◽  
Double Strand Dna Breaks ◽  
Recombination Repair ◽  
Non Homologous End Joining

ABSTRACTThe anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly, BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.

scholarly journals A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

2016 ◽  
Author(s):  
Marella D. Canny ◽  
Leo C.K. Wan ◽  
Amélie Fradet-Turcotte ◽  
Alexandre Orthwein ◽  
Nathalie Moatti ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Cell Types ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Non Homologous End Joining ◽  
G1 Cells ◽  
Programmable Nucleases ◽  
End Resection

AbstractThe expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1–3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

scholarly journals Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Jes Alexander ◽  
Gregory M. Findlay ◽  
Martin Kircher ◽  
Jay Shendure
Keyword(s):  
Genome Editing ◽  
Cpg Island ◽  
Strand Break ◽  
End Joining ◽  
Exogenous Dna ◽  
Cpg Dinucleotides ◽  
Epigenome Editing ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

scholarly journals The Impact of Single- and Double-Strand DNA Breaks in Human Spermatozoa on Assisted Reproduction

2020 ◽  
Vol 21 (11) ◽  
pp. 3882 ◽  
Author(s):  
Ashok Agarwal ◽  
Cătălina Barbăroșie ◽  
Rafael Ambar ◽  
Renata Finelli
Keyword(s):  
Negative Impact ◽  
Sperm Dna Fragmentation ◽  
Reproductive Outcomes ◽  
Dna Breaks ◽  
End Joining ◽  
Double Strand Dna Breaks ◽  
Sperm Dna ◽  
Dna Strands ◽  
Non Homologous End Joining ◽  
The Impact

Several cellular insults can result in sperm DNA fragmentation either on one or both DNA strands. Oxidative damage, premature interruption of the apoptotic process and defects in DNA compaction during spermatogenesis are the main mechanisms that cause DNA breaks in sperm. The two-tailed Comet assay is the only technique that can differentiate single- (SSBs) from double- (DSBs) strand DNA breaks. Increased levels of the phosphorylated isoform of the H2AX histone are directly correlated with DSBs and proposed as a molecular biomarker of DSBs. We have carried out a narrative review on the etiologies associated with SSBs and DSBs in sperm DNA, their association with reproductive outcomes and the mechanisms involved in their repair. Evidence suggests a stronger negative impact of DSBs on reproductive outcomes (fertilization, implantation, miscarriage, pregnancy, and live birth rates) than SSBs, which can be partially overcome by using intracytoplasmic sperm injection (ICSI). In sperm, SSBs are irreversible, whereas DSBs can be repaired by homologous recombination, non-homologous end joining (NHEJ) and alternative NHEJ pathways. Although few studies have been published, further research is warranted to provide a better understanding of the differential effects of sperm SSBs and DSBs on reproductive outcomes as well as the prognostic relevance of DNA breaks discrimination in clinical practice.

scholarly journals The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions

2019 ◽  
Vol 20 (13) ◽  
pp. 3371 ◽  
Author(s):  
Serge M. Rozov ◽  
Natalya V. Permyakova ◽  
Elena V. Deineko
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Transcription Activator ◽  
End Joining ◽  
Knock Out ◽  
Genome Region ◽  
Higher Eukaryotes ◽  
Non Homologous End Joining ◽  
Recombination Pathway ◽  
Donor Template

The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e., the insertion of them into a target genome region, are between one and two orders of magnitude less frequent. First and foremost, this is associated with the specific features of the repair systems of higher eukaryotes and the availability of the donor template in accessible proximity during double-strand break (DSB) repair. This review briefs the main repair pathways in plants according to the aspect of their involvement in genome editing. The main methods for increasing the frequency of knock-ins are summarized both along the homologous recombination pathway and non-homologous end joining, which can be used for plant objects.

scholarly journals EPCO-15. TUMOR TREATMENT WITH IONIZING RADIATION IS ASSOCIATED WITH A CLINICALLY RELEVANT DELETION SIGNATURE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii72-ii72
Author(s):  
Emre Kocakavuk ◽  
Kevin Anderson ◽  
Kevin Johnson ◽  
Frederick Varn ◽  
Samirkumar Amin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Radiation Therapy ◽  
Ionizing Radiation ◽  
Cox Regression ◽  
Strand Break ◽  
Rank Test ◽  
Sequencing Data ◽  
End Joining ◽  
Signed Rank Test ◽  
Non Homologous End Joining

Abstract Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 2249 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified a significant increase in the frequency of small deletions following radiation therapy that was independent of other factors. These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P = 1.2e-02 and P = 8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P < 2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P = 3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P = 4.7e-02, HR = 2.59 [95% CI: 1.01, 6.60] and P = 2.5e-02, HR = 1.43 [95% CI: 1.05, 1.94], respectively; multivariable Cox regression), suggesting that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments.

scholarly journals EccDNA formation is dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA double-strand break

2020 ◽  
Author(s):  
Teressa Paulsen ◽  
Pumoli Malapati ◽  
Rebeka Eki ◽  
Tarek Abbas ◽  
Anindya Dutta
Keyword(s):  
Strand Break ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Complex Interactions ◽  
Double Strand Dna Breaks ◽  
Dna Double Strand Break ◽  
Circular Dnas ◽  
In Cells ◽  
Double Strand Dna

ABSTRACTExtrachromosomal circular DNAs (eccDNA) are widespread in normal and cancer cells and are known to amplify oncogenic genes. However, the mechanisms that form eccDNA have never been fully elucidated due to the complex interactions of DNA repair pathways and lack of a method to quantify eccDNA abundance. Through the development of a sensitive and quantitative assay for eccDNA we show that the formation of eccDNA is through resection dependent repair of double-strand DNA breaks, especially micro-homology mediated end joining, and through mismatch repair. The most significant decreases in eccDNA levels occurred in cells lacking PARP1, POLQ, NBS1, RAD54, and FAN1. Further, a significant increase in eccDNA occurred in cells lacking c-NHEJ proteins DNA-PKcs, XRCC4, XLF, LIG4 and 53BP1. This suggests that when alt-NHEJ pathways are utilized to repair DNA breaks by necessity, the formation of eccDNA is increased. Induced and site-directed double-strand DNA breaks increase eccDNA formation, even from a single break. Additionally, we find that eccDNA levels accumulate as cells undergo replication in S-phase and that levels of eccDNA are decreased if DNA synthesis is prevented. Together, these results show that the bulk of eccDNA form by resection based alt-NHEJ pathways, especially during DNA replication and the repair of double-strand breaks.

Sign in / Sign up

Export Citation Format

Share Document