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

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, Dieter Egli and Alberto Ciccia ()
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Tarun S. Nambiar: Columbia University Irving Medical Center
Pierre Billon: Columbia University Irving Medical Center
Giacomo Diedenhofen: Columbia University Irving Medical Center
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

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-11105-z

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