Ectopic Expression of RAD52 and Dn53BP1 Improves Homology-directed Repair During CRISPR-Cas9 Genome Editing

Overview

Journal Nat Biomed Eng

Publisher Springer Nature

Specialty Biomedical Engineering

Date 2019 Apr 25

PMID 31015609

Citations 59

Authors

Bruna S Paulsen

Pankaj K Mandal

Richard L Frock

Baris Boyraz

Rachita Yadav

Srigokul Upadhyayula

Paula Gutierrez-Martinez

Wataru Ebina

Anders Fasth

Tomas Kirchhausen

Michael E Talkowski

Suneet Agarwal

Frederick W Alt

Derrick J Rossi

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

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