Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts

Li, Guoling; Liu, Dewu; Zhang, Xianwei; Quan, Rong; Zhong, Cuili; Mo, Jiawei; Huang, Yaoqiang; Wang, Haoqiang; Ruan, Xiaofang; Xu, Zheng; Zheng, Enqin; Gu, Tao; Hong, Linjun; Li, Zicong; Wu, Zhenfang; Yang, Huaqiang

doi:10.1016/j.biocel.2018.04.011

Cited by 36 publications

(29 citation statements)

References 28 publications

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“…In murine cells the lack of Ku70 could lead to depleted expression of Ku80 [ 26 ]. In human cells, however, silencing of Ku70 may not result in depletion of Ku80 [ 25 , 27 ]. In the current study we showed that Ku70 silencing (by targeted shRNA) did not alter Ku80 mRNA and protein expression in THP-1 cells.…”

Section: Discussionmentioning

confidence: 99%

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes

Sun

Yin

et al. 2020

aging

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In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.

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Section: Discussionmentioning

confidence: 99%

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes

Sun

Yin

et al. 2020

aging

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“…KU is the first protein to bind to DSBs in c-NHEJ regardless of cell cycle stage, and cells will first attempt to repair DSBs by c-NHEJ if the ends are compatible. Owing to the role of KU in promoting NHEJ and suppressing HDR, Li et al used KU-specific siRNA to downregulate the expression of Ku70 and Ku80, which resulted in a significant increase in the frequency of CRISPR/Cas9-mediated HDR in pig fibroblasts [94]. Further upstream of NHEJ repair, treating DNA-PK with two chemical inhibitors, NU7441 and KU-0060648, led to a twofold increase in the rate of HDR at DSBs induced by Cas9 in HEK-293T cells [95].…”

Section: Suppression Of Key Nhej Factorsmentioning

confidence: 99%

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

Yang

Ren

et al. 2020

IJMS

145

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Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

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“…HDR pathway can make this desired edit easy but it's lower efficiency renders it's use of precise Gene editing for highly limiting clinical intervention and NHEJ can be used as default pathway for human cells repairing. Because of chemical inhibition ofNHEJ modulating enzymes like DNA ligase IV [48,52], DNA dependent protein kinases [48,53] and Ku [48,54] it causes suppression of NHEJ pathway through which enhancement in HDR efficiency has been achieved [48].…”

Section: Challenges Using Crispr -Cas9mentioning

confidence: 99%

Crispr - Cas9 Gene Editing: A Review

Desai¹,

ijar²,

Panchal³

et al. 2020

IJAR

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CRISPR is an RNA guided genome editing technique of genetic engineering which works like genetic scissors. Based on simplified version of bacterial CRISPR-Cas9 antiviral defense system. It is more accurate, faster and cost efficient than other genome editing methods. There are two components in this system: First component includes a single guide RNA (sgRNA) of system which will identify target sequence in genome and Second component will include Cas9 nuclease of system which will act as a pair of scissors to spilt the double strands of DNA. CRISPR has promising therapeutic applications. This current review focuses on mechanism, therapeutic applications, delivery systems, limitations and different approaches used for gene editing using CRISPR.

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Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts

Cited by 36 publications

References 28 publications

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

Crispr - Cas9 Gene Editing: A Review

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