CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response

Here, we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.

[1]  Sruthi Mantri,et al.  CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells , 2016, Nature.

[2]  Jacob E Corn,et al.  Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA , 2016, Nature Biotechnology.

[3]  You-xin Chen,et al.  Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future. , 2018, International journal of ophthalmology.

[4]  Hao Yin,et al.  Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype , 2014, Nature Biotechnology.

[5]  S. Hohmann,et al.  Structural analysis of the 5′ regions of yeast SUC genes revealed analogous palindromes in SUC, MAL and GAL , 1988, Molecular and General Genetics MGG.

[6]  Dana Carroll,et al.  Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells , 2016, Science Translational Medicine.

[7]  T. Kivioja,et al.  Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion , 2016, bioRxiv.

[8]  T. Kivioja,et al.  CRISPR/Cas9 screening using unique molecular identifiers , 2017, bioRxiv.

[9]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[10]  Jing Wang,et al.  WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit , 2017, Nucleic Acids Res..

[11]  H. Lockstone,et al.  53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms , 2016, Molecular cell.

[12]  P. Sicinski,et al.  Cell cycle proteins as promising targets in cancer therapy , 2017, Nature Reviews Cancer.

[13]  D. Durocher,et al.  Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency , 2017, Nature Biotechnology.

[14]  E. Lander,et al.  Identification and characterization of essential genes in the human genome , 2015, Science.

[15]  Łukasz M. Boryń,et al.  Resolving systematic errors in widely-used enhancer activity assays in human cells , 2017, Nature Methods.

[16]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[17]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[18]  Hidde L Ploegh,et al.  Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise [TM: inserted] genome editing , 2015, Nature Biotechnology.

[19]  Daniel Durocher,et al.  The control of DNA repair by the cell cycle , 2016, Nature Cell Biology.