title: Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases.
abstract: The development of customizable sequence-specific nucleases such as TALENs, ZFNs and the powerful CRISPR/Cas9 system has revolutionized the field of genome editing. The CRISPR/Cas9 system is particularly versatile and has been applied in numerous species representing all branches of life. Regardless of the target organism, all researchers using sequence-specific nucleases face similar challenges: confirmation of the desired on-target mutation and the detection of off-target events. Here, we evaluate the most widely-used methods for the detection of on-target and off-target mutations in terms of workflow, sensitivity, strengths and weaknesses.
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Zischewski
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany. Electronic address: luisa.bortesi@molbiotech.rwth-aachen.de.
authors.forename: Luisa
authors.initials: L
authors.lastname: Bortesi
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases
keywords: CRISPR; Cas9; Engineered nucleases; Genome editing; Indel detection; Mismatch detection assays; Unbiased off-target analysis