Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise [TM: inserted] genome editing

By T. Maruyama, S. Dougan, Matthias C. Truttmann, Angelina M. Bilate, J. Ingram, Hidde L. Ploegh. Nature Biotechnology, 2015

956 citations recorded by the source corpus.

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/f0d3a0d968c432c1f677a3820b0683e6c5938849

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In Molecular Biology

1. Min Wu
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4. Xia Zhao
5. Yang Yang
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Citations in the corpus

• Delivering CRISPR: a review of the challenges and approaches (2018, background): “Even in the presence of donor template DNA, NHEJ is the more frequent repair mechanism observed from CRISPR/Cas9 editing (Maruyama et al., 2015).”
• Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, neutral): “Although inhibiting the NHEJ pathway with gene silencing or chemical inhibitors can partially increase the efficiency of HDR pathway, the gene repair efficacy still needs to be further improved for disease therapy [82, 85].”
• Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, methodology): “Maruyama et al.(303) successfully employed SCR7 to inhibit NHEJ by targeting a key enzyme (DNA ligase IV) in the NHEJ pathway, thereby increasing the genome editing efficiency in cell lines and mice by up to 19-fold.”
• Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. (2016, neutral): “While making programmed cuts has become largely trivial, biasing DNA repair toward homology-directed repair rather than non-homologous end joining remains a challenge (Chu et al., 2015; Maruyama et al., 2015).”
• CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “Enhancement of HDR efficiency has been achieved via suppression of the NHEJ pathway through chemical inhibition of key NHEJ modulating enzymes such as Ku (77), DNA ligase IV (78), and DNAdependent protein kinases (DNA-PKcs) (79).”