Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases |
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Institution: | 1. Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany;2. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany |
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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. |
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