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.
Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant / Nambiar, T. S.; Billon, P.; Diedenhofen, G.; Hayward, S. B.; Taglialatela, A.; Cai, K.; Huang, J. -W.; Leuzzi, G.; Cuella-Martin, R.; Palacios, A.; Gupta, A.; Egli, D.; Ciccia, A.. - In: NATURE COMMUNICATIONS. - ISSN 2041-1723. - 10:1(2019), p. 3395. [10.1038/s41467-019-11105-z]
Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant
Diedenhofen G.Membro del Collaboration Group
;
2019
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
