dc.contributor.author	Bao, X. Robert
dc.contributor.author	Pan, Yidan
dc.contributor.author	Lee, Ciaran M.
dc.contributor.author	Davis, Timothy H.
dc.contributor.author	Bao, Gang
dc.contributor.funder	National Institutes of Health	en
dc.contributor.funder	Cancer Prevention and Research Institute of Texas	en
dc.date.accessioned	2021-01-13T09:11:32Z
dc.date.available	2021-01-13T09:11:32Z
dc.date.issued	2020-12-07
dc.date.updated	2021-01-12T12:47:53Z
dc.description.abstract	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.	en
dc.description.sponsorship	National Institutes of Health (UG3HL151545; R01HL152314; OT2HL154977); Cancer Prevention and Research Institute of Texas (RR140081)	en
dc.description.status	Peer reviewed	en
dc.description.version	Accepted Version	en
dc.format.mimetype	application/pdf	en
dc.identifier.citation	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	en
dc.identifier.doi	10.1038/s41596-020-00431-y	en
dc.identifier.eissn	1750-2799
dc.identifier.endpage	26	en
dc.identifier.issn	1754-2189
dc.identifier.journaltitle	Nature Protocols	en
dc.identifier.startpage	10	en
dc.identifier.uri	https://hdl.handle.net/10468/10901
dc.identifier.volume	16	en
dc.language.iso	en	en
dc.publisher	Nature Research	en
dc.rights	© 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	en
dc.subject	Double-stranded breaks	en
dc.subject	Zinc-finger nucleases	en
dc.subject	Genome-wide analysis	en
dc.subject	In-vitro	en
dc.subject	Unbiased detection	en
dc.subject	High-effeciency	en
dc.subject	DNA cleavage	en
dc.subject	Stem-cells	en
dc.subject	Web tool	en
dc.subject	CAS9	en
dc.title	Tools for experimental and computational analyses of off-target editing by programmable nucleases	en
dc.type	Article (peer-reviewed)	en

Tools for experimental and computational analyses of off-target editing by programmable nucleases

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