title: Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.
abstract: 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.
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors: Takeshi
authors: T
authors: Maruyama
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors: Stephanie K
authors: SK
authors: Dougan
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors: Matthias C
authors: MC
authors: Truttmann
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors: Angelina M
authors: AM
authors: Bilate
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors: Jessica R
authors: JR
authors: Ingram
authors: 1] Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Hidde L
authors: HL
authors: Ploegh
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D059766:DNA End-Joining Repair / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D059765:Homologous Recombination / Q000235:genetics; D051379:Mice; D011743:Pyrimidines / Q000008:administration & dosage; D012545:Schiff Bases / Q000008:administration & dosage; D016335:Zinc Fingers / Q000235:genetics
chemical_list: C578537:5,6-bis(benzylideneamino)-2-mercaptopyrimidin-4-ol; D011743:Pyrimidines; D012545:Schiff Bases; D011088:DNA Ligases; D000072481:DNA Ligase ATP