Kinetics model of DNA double-strand break repair in eukaryotes

Shen, Wangtao; Ma, Yun; Qi, Huizhou; Wang, Wuzhou; He, Junyan; Xiao, Fangzhu; Zhu, Hui; He, Shuya

doi:10.1016/j.dnarep.2020.103035

Cited by 7 publications

(5 citation statements)

References 37 publications

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“…The donor DNA integrates into the DSB locus when the donor DNA is used as the HA template. However, the HR efficiency is much lower than NHEJ in cells [ 47 , 48 ], which explains the lower KI rate compared to the KO rate [ 49 ]. Therefore, understanding the NHEJ/HR switch mechanism is the foundation for improving HR efficiency.…”

Section: Discussionmentioning

confidence: 99%

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Ouyang

Yang

Chen

et al. 2022

IJMS

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Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.

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Section: Discussionmentioning

confidence: 99%

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Ouyang

Yang

Chen

et al. 2022

IJMS

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“…The recruitment of pH2AX was described as being rapid. The phosphorylation of serine 139 on H2AX is completed within minutes after the occurrence of double-strand breaks [41]. In the context of ROS induction, this process happens within 20 min, e.g., after neocarzinostatin treatment [42].…”

Section: Dna Double Strand Break Repair Is Accelerated Upon Addition ...mentioning

confidence: 99%

“…Antioxidants 2023, 12, x FOR PEER REVIEW 11 of 20 completed within minutes after the occurrence of double-strand breaks [41]. In the context of ROS induction, this process happens within 20 min, e.g., after neocarzinostatin treatment [42].…”

Section: Dna Double Strand Break Repair Is Accelerated Upon Addition ...mentioning

confidence: 99%

Lactobacillus sp. Facilitate the Repair of DNA Damage Caused by Bile-Induced Reactive Oxygen Species in Experimental Models of Gastroesophageal Reflux Disease

et al. 2023

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Gastroesophageal reflux disease (GERD) leads to the accumulation of bile-induced reactive oxygen species and oxidative stress in esophageal tissues, causing inflammation and DNA damage. The progression sequence from healthy esophagus to GERD and eventually cancer is associated with a microbiome shift. Lactobacillus species are commensal organisms known for their probiotic and antioxidant characteristics in the healthy esophagus. This prompted us to investigate how Lactobacilli survive in a bile-rich environment during GERD, and to identify their interaction with the bile-injured esophageal cells. To model human reflux conditions, we exposed three Lactobacillus species (L. acidophilus, L. plantarum, and L. fermentum) to bile. All species were tolerant to bile possibly enabling them to colonize the esophageal epithelium under GERD conditions. Next, we assessed the antioxidant potential of Lactobacilli and role in bile injury repair: we measured bile-induced DNA damage using the ROS marker 8-oxo guanine and COMET assay. Lactobacillus addition after bile injury accelerated repair of bile-induced DNA damage through recruitment of pH2AX/RAD51 and reduced NFκB-associated inflammation in esophageal cells. This study demonstrated anti-genotoxic and anti-inflammatory effects of Lactobacilli, making them of significant interest in the prevention of Barrett’s esophagus and esophageal adenocarcinoma in patients with GERD.

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“…Studies of DSBs induced by CRISPR/Cas9 in endogenous chromatin have primarily focused on mutagenic repair, revealing many aspects affecting editing outcome and repair-pathway choice 41,42 . In addition, kinetic studies have provided insight into the dynamics of the process of induction and error-prone repair [43][44][45][46] .…”

Section: Introductionmentioning

confidence: 99%

Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks

Ben‐Tov

Mafessoni

Cucuy

et al. 2023

Preprint

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CRISPR/Cas9-mediated genome editing relies on error-prone repair of targeted DNA double-strand breaks (DSBs). Understanding CRISPR/Cas9-mediated DSB induction and subsequent repair dynamics requires measuring the rate of cutting and that of precise repair, a hidden-variable of the repair machinery. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterized the dynamics of DSB induction, processing and repair at endogenous loci along a 72-hour time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is not an accurate reflection of DSB induction efficiency due to prominent precise re-ligation, accounting for 40-70% of all repair events. Altogether, this system exposes previously unseen flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high fidelity repair in limiting CRISPR editing efficiency in somatic cells.

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Kinetics model of DNA double-strand break repair in eukaryotes

Cited by 7 publications

References 37 publications

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Lactobacillus sp. Facilitate the Repair of DNA Damage Caused by Bile-Induced Reactive Oxygen Species in Experimental Models of Gastroesophageal Reflux Disease

Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks

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