title: Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.
abstract: DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair.
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Mu-Dan
authors: MD
authors: He
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Feng-Hua
authors: FH
authors: Zhang
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Hua-Lin
authors: HL
authors: Wang
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors: Hou-Peng
authors: HP
authors: Wang
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors: Zuo-Yan
authors: ZY
authors: Zhu
authors: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Electronic address: yhsun@ihb.ac.cn.
authors: Yong-Hua
authors: YH
authors: Sun
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000502:physiology*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D029961:Zebrafish Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus
keywords: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9; Double-strand breaks repair; Ligase 3; Ligase 4; Microhomology-mediated end joining (MMEJ); Transcription activator-like effector nuclease (TALEN)