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Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant

Author

Listed:
  • Tarun S. Nambiar

    (Columbia University Irving Medical Center)

  • Pierre Billon

    (Columbia University Irving Medical Center)

  • Giacomo Diedenhofen

    (Columbia University Irving Medical Center
    University of Rome Tor Vergata)

  • Samuel B. Hayward

    (Columbia University Irving Medical Center)

  • Angelo Taglialatela

    (Columbia University Irving Medical Center)

  • Kunheng Cai

    (Columbia University Irving Medical Center)

  • Jen-Wei Huang

    (Columbia University Irving Medical Center)

  • Giuseppe Leuzzi

    (Columbia University Irving Medical Center)

  • Raquel Cuella-Martin

    (Columbia University Irving Medical Center)

  • Andrew Palacios

    (Columbia University Irving Medical Center)

  • Anuj Gupta

    (Columbia University Irving Medical Center)

  • Dieter Egli

    (Columbia University Irving Medical Center)

  • Alberto Ciccia

    (Columbia University Irving Medical Center)

Abstract

Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.

Suggested Citation

  • Tarun S. Nambiar & Pierre Billon & Giacomo Diedenhofen & Samuel B. Hayward & Angelo Taglialatela & Kunheng Cai & Jen-Wei Huang & Giuseppe Leuzzi & Raquel Cuella-Martin & Andrew Palacios & Anuj Gupta &, 2019. "Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11105-z
    DOI: 10.1038/s41467-019-11105-z
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    Cited by:

    1. Ron Baik & M. Kyle Cromer & Steve E. Glenn & Christopher A. Vakulskas & Kay O. Chmielewski & Amanda M. Dudek & William N. Feist & Julia Klermund & Suzette Shipp & Toni Cathomen & Daniel P. Dever & Mat, 2024. "Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    2. Gaofeng Cui & Maria Victoria Botuyan & Pascal Drané & Qi Hu & Benoît Bragantini & James R. Thompson & David J. Schuller & Alexandre Detappe & Michael T. Perfetti & Lindsey I. James & Stephen V. Frye &, 2023. "An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    3. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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