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.
Keywords: CRISPR; Cas9; Engineered nucleases; Genome editing; Indel detection; Mismatch detection assays; Unbiased off-target analysis.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.