Coupling endonucleases with DNA end–processing enzymes to drive gene disruption

• Published in: Nature methods Vol. 9; no. 10; pp. 973 - 975
• Main Authors: Certo, Michael T, Gwiazda, Kamila S, Kuhar, Ryan, Sather, Blythe, Curinga, Gabrielle, Mandt, Tyler, Brault, Michelle, Lambert, Abigail R, Baxter, Sarah K, Jacoby, Kyle, Ryu, Byoung Y, Kiem, Hans-Peter, Gouble, Agnes, Paques, Frederic, Rawlings, David J, Scharenberg, Andrew M
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.10.2012; Nature Publishing Group
• Subjects: 631/1647/1513; 631/1647/338/552; 631/337/1427/2191; Animals; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; brief-communication; Cellular biology; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA End-Joining Repair; DNA Repair; Endonucleases - physiology; Enzymes; Exodeoxyribonucleases - physiology; Gene expression; HEK293 Cells; Humans; Life Sciences; Mice; Mutagenesis; Phosphoproteins - physiology; Proteomics; Receptors, CCR5 - physiology; Restriction enzymes, DNA; United States
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/nmeth.2177

Coexpression of DNA end–processing enzymes with targeted nucleases improves the efficiency of gene disruption in mammalian cells. Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end–processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.