Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing

• DOI: 10.1038/s41551-017-0145-2
• Publication Date: 2017-10-09T09:13:20Z
• Authors: Bruna S. Paulsen; Pankaj K. Mandal; Richard L. Frock; Baris Boyraz; Rachita Yadav; Srigokul Upadhyayula; Paula Gutierrez-M...; Wataru Ebina; Anders Fasth; Tomas Kirchhausen; Michael E. Talkowski; Suneet Agarwal; Frederick W. Alt; Derrick J. Rossi
• Keywords: Gene Editing; 0301 basic medicine; DNA End-Joining Repair; DNA Repair; Induced Pluripotent Stem Cells; Recombinational DNA Repair; Ectopic Gene Expression; Rad52 DNA Repair and Recombination Protein; 03 medical and health sciences; HEK293 Cells; Humans; DNA Breaks, Double-Stranded; CRISPR-Cas Systems; Tumor Suppressor p53-Binding Protein 1

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.