scholarly journals A homology independent sequence replacement strategy in human cells using a CRISPR nuclease

Open Biology ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 200283
Author(s):  
Eric Danner ◽  
Mikhail Lebedin ◽  
Kathrin de la Rosa ◽  
Ralf Kühn
Keyword(s):  
Basic Research ◽  
Strand Break ◽  
Human Cells ◽  
End Joining ◽  
Linear Amplification ◽  
Promising Alternative ◽  
Independent Sequence ◽  
Homology Directed Repair ◽  
Dividing Cells ◽  
Non Homologous End Joining

Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing ( R ational e nd-joining p rotocol de l ivering a targeted sequen c e e xchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16–54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

scholarly journals A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

2020 ◽  
Author(s):  
Eric Danner ◽  
Mikhail Lebedin ◽  
Kathrin de la Rosa ◽  
Ralf Kühn
Keyword(s):  
Basic Research ◽  
Strand Break ◽  
End Joining ◽  
Linear Amplification ◽  
Promising Alternative ◽  
Independent Sequence ◽  
Replacement Strategy ◽  
Homology Directed Repair ◽  
Dividing Cells ◽  
Non Homologous End Joining

AbstractPrecision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

scholarly journals Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair

2020 ◽  
Vol 117 (44) ◽  
pp. 27566-27577
Author(s):  
Jacob V. Layer ◽  
Lydie Debaize ◽  
Alexandria Van Scoyk ◽  
Nealia C. House ◽  
Alexander J. Brown ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Double Strand Break ◽  
Human Cells ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Homology Directed Repair ◽  
Accessory Subunit ◽  
Radiation Induced ◽  
Translocation Frequency

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

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 (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

2022 ◽  
Vol 12 ◽  
Author(s):  
Alice Libri ◽  
Timea Marton ◽  
Ludovic Deriano
Keyword(s):  
Dna Repair ◽  
Gene Diversity ◽  
Receptor Gene ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pathway Choice ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

scholarly journals Canonical DNA non-homologous end-joining; capacity versus fidelity

2020 ◽  
Vol 93 (1115) ◽  
pp. 20190966 ◽  
Author(s):  
Atsushi Shibata ◽  
Penny A Jeggo
Keyword(s):  
High Efficiency ◽  
High Capacity ◽  
Active Regions ◽  
Strand Break ◽  
Human Cells ◽  
End Joining ◽  
Dsb Repair ◽  
First Choice ◽  
Radiation Induced ◽  
Non Homologous End Joining

The significance of canonical DNA non-homologous end-joining (c-NHEJ) for DNA double strand break (DSB) repair has increased from lower organisms to higher eukaryotes, and plays the predominant role in human cells. Ku, the c-NHEJ end-binding component, binds DSBs with high efficiency enabling c-NHEJ to be the first choice DSB repair pathway, although alternative pathways can ensue after regulated steps to remove Ku. Indeed, radiation-induced DSBs are repaired rapidly in human cells. However, an important question is the fidelity with which radiation-induced DSBs are repaired, which is essential for assessing any harmful impacts caused by radiation exposure. Indeed, is compromised fidelity a price we pay for high capacity repair. Two subpathways of c-NHEJ have been revealed; a fast process that does not require nucleases or significant chromatin changes and a slower process that necessitates resection factors, and potentially more significant chromatin changes at the DSB. Recent studies have also shown that DSBs within transcriptionally active regions are repaired by specialised mechanisms, and the response at such DSBs encompasses a process of transcriptional arrest. Here, we consider the limitations of c-NHEJ that might result in DSB misrepair. We consider the common IR-induced misrepair events and discuss how they might arise via the distinct subpathways of c-NHEJ.

scholarly journals A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

2020 ◽  
Vol 48 (21) ◽  
pp. e126-e126
Author(s):  
Rebeka Eki ◽  
Jane She ◽  
Mahmut Parlak ◽  
Mouadh Benamar ◽  
Kang-Ping Du ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
High Sensitivity ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Fluorescent Reporter ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Detection And Quantification

Abstract DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

scholarly journals A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

2019 ◽  
Author(s):  
Manuel M. Vicente ◽  
Afonso Mendes ◽  
Margarida Cruz ◽  
José R. Vicente ◽  
Vasco M. Barreto
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Relative Increase ◽  
End Joining ◽  
New Era ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Dna Lesion ◽  
Non Homologous End Joining ◽  
Cycle Dependent

ABSTRACTThe discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

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 Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

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