scholarly journals Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

2020 ◽  
Vol 6 (7) ◽  
pp. eaax2941 ◽  
Author(s):  
Boris V. Skryabin ◽  
Delf-Magnus Kummerfeld ◽  
Leonid Gubar ◽  
Birte Seeger ◽  
Helena Kaiser ◽  
...  
Keyword(s):  
Conditional Knockout ◽  
Nonhomologous End Joining ◽  
High Rate ◽  
Model Organisms ◽  
Pcr Analysis ◽  
End Joining ◽  
Dna Templates ◽  
Homology Directed Repair ◽  
Conditional Knockout Mouse ◽  
Wide Range

CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

scholarly journals Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events

2019 ◽  
Author(s):  
Boris V. Skryabin ◽  
Leonid Gubar ◽  
Birte Seeger ◽  
Helena Kaiser ◽  
Anja Stegemann ◽  
...  
Keyword(s):  
High Rate ◽  
Model Organisms ◽  
Pcr Analysis ◽  
End Joining ◽  
Dna Templates ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Wide Range ◽  
Non Homologous End Joining ◽  
Knock Out Mouse

AbstractCRISPR/Cas9 mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous end-joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis—in most cases—failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential, and offer practical solutions to correctly identify precisely edited chromosomes.

scholarly journals ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

2017 ◽  
Vol 114 (29) ◽  
pp. 7665-7670 ◽  
Author(s):  
Chun-Chin Chen ◽  
Elizabeth M. Kass ◽  
Wei-Feng Yen ◽  
Thomas Ludwig ◽  
Mary Ellen Moynahan ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Critical Role ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna Damage Signaling ◽  
Mutant Cells

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1–53BP1 antagonism and that its HDR function can become critical in certain contexts.

scholarly journals The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 550
Author(s):  
Matvey Mikhailovich Murashko ◽  
Ekaterina Mikhailovna Stasevich ◽  
Anton Markovich Schwartz ◽  
Dmitriy Vladimirovich Kuprash ◽  
Aksinya Nicolaevna Uvarova ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Gene Mutations ◽  
Nonhomologous End Joining ◽  
Published Data ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Chromosome Aberration Frequency

Incorrect reparation of DNA double-strand breaks (DSB) leading to chromosomal rearrangements is one of oncogenesis’s primary causes. Recently published data elucidate the key role of various types of RNA in DSB formation, recognition and repair. With growing interest in RNA biology, increasing RNAs are classified as crucial at the different stages of the main pathways of DSB repair in eukaryotic cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Gene mutations or variation in expression levels of such RNAs can lead to local DNA repair defects, increasing the chromosome aberration frequency. Moreover, it was demonstrated that some RNAs could stimulate long-range chromosomal rearrangements. In this review, we discuss recent evidence demonstrating the role of various RNAs in DSB formation and repair. We also consider how RNA may mediate certain chromosomal rearrangements in a sequence-specific manner.

scholarly journals MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

2016 ◽  
Vol 212 (4) ◽  
pp. 399-408 ◽  
Author(s):  
Tomas Aparicio ◽  
Richard Baer ◽  
Max Gottesman ◽  
Jean Gautier
Keyword(s):  
Dna Adducts ◽  
Topoisomerase Ii ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Egg Extracts ◽  
Additional Processing ◽  
Special Challenge

Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein–DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)–DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2–DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

scholarly journals Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration

2021 ◽  
Author(s):  
Chen-Tran Hsu ◽  
Yu-Hsuan Yuan ◽  
Yao-Cheng Lin ◽  
Steven Lin ◽  
Qiao-Wei Cheng ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Nonhomologous End Joining ◽  
Protoplast Regeneration ◽  
End Joining ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Dna Oligonucleotides ◽  
Vector Construction ◽  
Regenerated Plants ◽  
Low Efficiency

AbstractGenome editing requires insertion of DNA sequences into specific locations. Protocols involving clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins rely on homology-directed repair, require laborious vector construction, and have low efficiency. DNA oligonucleotides can be used as donors for targeted insertion via nonhomologous end joining. Our simple protocol eliminates the need for expensive equipment and vector construction by using polyethylene glycol to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. We achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated plants showed targeted insertions, and 1 contained a precise insertion of a 6-bp EcoRI site. Whole-genome sequencing showed that the DNA inserted only in the targeted positions, and genetic analysis showed that the inserted sequences transmitted to the next generation.

scholarly journals Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

2019 ◽  
Author(s):  
Tien Van Vu ◽  
Velu Sivankalyani ◽  
Eun-Jung Kim ◽  
Duong Thi Hai Doan ◽  
Mil Thi Tran ◽  
...  
Keyword(s):  
Genome Editing ◽  
De Novo ◽  
Phenotypic Selection ◽  
Culture Conditions ◽  
Nonhomologous End Joining ◽  
Physical Culture ◽  
End Joining ◽  
Dark Cycle ◽  
Homology Directed Repair ◽  
Error Prone Repair

ABSTRACTGenome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31°C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mM NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually as well as sexually reproducing plants.

scholarly journals A Homeostatic Shift Facilitates Endoplasmic Reticulum Proteostasis through Transcriptional Integration of Proteostatic Stress Response Pathways

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Liam Baird ◽  
Tadayuki Tsujita ◽  
Eri H. Kobayashi ◽  
Ryo Funayama ◽  
Takeshi Nagashima ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcriptional Regulation ◽  
Ubiquitin Proteasome System ◽  
Conditional Knockout ◽  
Protein Homeostasis ◽  
Knockout Mouse Model ◽  
Conditional Knockout Mouse ◽  
Wide Range ◽  
Ubiquitin Proteasome ◽  
Inducible Systems

ABSTRACT Eukaryotic cells maintain protein homeostasis through the activity of multiple basal and inducible systems, which function in concert to allow cells to adapt to a wide range of environmental conditions. Although the transcriptional programs regulating individual pathways have been studied in detail, it is not known how the different pathways are transcriptionally integrated such that a deficiency in one pathway can be compensated by a change in an auxiliary response. One such pathway that plays an essential role in many proteostasis responses is the ubiquitin-proteasome system, which functions to degrade damaged, unfolded, or short half-life proteins. Transcriptional regulation of the proteasome is mediated by the transcription factor Nrf1. Using a conditional knockout mouse model, we found that Nrf1 regulates protein homeostasis in the endoplasmic reticulum (ER) through transcriptional regulation of the ER stress sensor ATF6. In Nrf1 conditional-knockout mice, a reduction in proteasome activity is accompanied by an ATF6-dependent downregulation of the endoplasmic reticulum-associated degradation machinery, which reduces the substrate burden on the proteasome. This indicates that Nrf1 regulates a homeostatic shift through which proteostasis in the endoplasmic reticulum and cytoplasm are coregulated based on a cell's ability to degrade proteins.

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

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 Virulence and Karyotype Analyses of rad52Mutants of Candida albicans: Regeneration of a TruncatedChromosome of a Reintegrant Strain (rad52/RAD52) in the Host

2005 ◽  
Vol 73 (12) ◽  
pp. 8069-8078 ◽  
Author(s):  
Neeraj Chauhan ◽  
Toni Ciudad ◽  
Ane Rodríguez-Alejandre ◽  
Germán Larriba ◽  
Richard Calderone ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Large Deletion ◽  
Nonhomologous End Joining ◽  
Pcr Analysis ◽  
Wild Type ◽  
End Joining ◽  
Inoculum Concentration ◽  
A Cell ◽  
Karyotype Analyses ◽  
Cell Inoculum

ABSTRACT The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Δ mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 × 106, but there was a decrease in survival time at dosages of 1.5 × 106 and especially at 3 × 106 cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 × 106 and 1.5 × 106 cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.

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