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Tools for experimental and computational analyses of off-target editing by programmable nucleases
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Bao, X. Robert
Lee, Ciaran M.
Davis, Timothy H.
Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.Off-target effects of programmable nucleases remain a critical issue for therapeutic applications of genome editing. This review compares experimental and computational tools for off-target analysis and provides recommendations for better assessments of off-target effects.
Double-stranded breaks , Zinc-finger nucleases , Genome-wide analysis , In-vitro , Unbiased detection , High-effeciency , DNA cleavage , Stem-cells , Web tool , CAS9
Bao, X. R., Pan, Y., Lee, C. M., Davis, T. H. and Bao, G. (2020) 'Tools for experimental and computational analyses of off-target editing by programmable nucleases', Nature Protocols, 16, pp. 10-26. doi: 10.1038/s41596-020-00431-y
© 2020, Springer Nature Limited. This is a post-peer-review, pre-copyedit version of an article published in Nature Protocols on 7 December 2020. The final authenticated version is available online at: https://doi.org/10.1038/s41596-020-00431-y