Optimizing genome editing strategy by primer-extension-mediated sequencing

Published on Dec 1, 2019in Cell discovery
路 DOI :10.1038/s41421-019-0088-8
Jianhang Yin1
Estimated H-index: 1
(PKU: Peking University),
Mengzhu Liu1
Estimated H-index: 1
(PKU: Peking University)
+ 6 AuthorsJiazhi Hu12
Estimated H-index: 12
(PKU: Peking University)
Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
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