TY - JOUR
T1 - Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining
AU - Maruyama, Takeshi
AU - Dougan, Stephanie K.
AU - Truttmann, Matthias C.
AU - Bilate, Angelina M.
AU - Ingram, Jessica R.
AU - Ploegh, Hidde L.
N1 - Funding Information:
We thank members of the Ploegh laboratory, especially N. Pishesha and F. Tafesse, for critical reading of the manuscript; P.A. Koenig, L.K. Swee, C.S. Shivalila, H. Wang, H. Yang and R. Jaenisch for discussions; T. Wang and D.M. Sabatini for pCW-Cas9; P. Thiru and G. Bell of BARC (WIBR) for assistance with statistical analysis; and F. Zhang (Addgene) for pX330. This work was supported by the National Institutes of Health (RO1 grant AI087879-01 to H.L.P.), Japan Society for the Promotion of Science (to T.M.), Japan Heart Foundation (to T.M.), AACR-Pancreatic Cancer Action Network (to S.K.D. and H.L.P.) and an SNSF Early Postdoc Mobility fellowship (to M.C.T.).
Publisher Copyright:
© 2015 Nature America, Inc. All rights reserved.
PY - 2015/5/12
Y1 - 2015/5/12
N2 - Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.
AB - Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.
UR - http://www.scopus.com/inward/record.url?scp=84929166074&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84929166074&partnerID=8YFLogxK
U2 - 10.1038/nbt.3190
DO - 10.1038/nbt.3190
M3 - Article
C2 - 25798939
AN - SCOPUS:84929166074
SN - 1087-0156
VL - 33
SP - 538
EP - 542
JO - Nature biotechnology
JF - Nature biotechnology
IS - 5
ER -