{"refrec":{"BRefID":355509,"RR":"<b>Malinin, N.L.; Lee, G.; Lazzarotto, C.R.; Li, Y.; Zheng, Z.; Nguyen, N.T.; Liebers, M.; Topkar, V.V.; Iafrate, A.J.; Le, L.P.; Aryee, M.J.; Joung, J.K.; Tsai, S.Q.</b> (2021). Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq. <i>Nature Protocols 16(12)</i>: 5592-5615. <a href=\"https://dx.doi.org/10.1038/s41596-021-00626-x\" target=\"_blank\">https://dx.doi.org/10.1038/s41596-021-00626-x</a>","BEntID":353222,"PublicFlag":1,"CheckedFlag":0,"wosflag":1,"vabbflag":1,"RefStringPartII":". <i>Nature Protocols 16(12)</i>: 5592-5615. <a href=\"https://dx.doi.org/10.1038/s41596-021-00626-x\" target=\"_blank\">https://dx.doi.org/10.1038/s41596-021-00626-x</a>","DocTypID":8,"DocType":"Journal article","MarineFlag":1,"FreshFlag":0,"BrackishFlag":0,"TerrestrialFlag":0,"Authorstring":"Malinin, N.L.; Lee, G.; Lazzarotto, C.R.; Li, Y.; Zheng, Z.; Nguyen, N.T.; Liebers, M.; Topkar, V.V.; Iafrate, A.J.; Le, L.P.; Aryee, M.J.; Joung, J.K.; Tsai, S.Q.","OrigTitleTranslFlag":0,"Authorstringtrunc":"Malinin, N.L. <i>et al.</i>","Englishabstract":"Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.","AbstractOtherLang":null,"BibLvlCode":"AS","StandardTitle":"Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq","OrigTitleLangCode":"en","OrigTitleLangCodeExtended":"eng","OrigTitleLangID":15,"DateLastModified":{"date":"2024-12-10 01:33:17.368041","timezone_type":1,"timezone":"+01:00"},"UserAccessRight":null,"UserAccID":null,"AuthorKeywords":null,"OtherDescriptors":null,"Notes":null,"AnaPub":2021,"MonPub":null,"DateUpdate":"2022-09-16","DateCreate":"2022-09-16","SecASFANote":null,"ConfID":null,"PeerRev":1,"VlizCoreFlag":1,"WoScode":"WOS:000717898500001","VABBcode":null,"OpenAcc":0,"DOI":"10.1038/s41596-021-00626-x"},"refs":null,"anarec":{"AnaID":355509,"PubliDate":2021,"Pagination":"5592-5615","XtraPublOfAnaID":null,"ISBN":null,"Volume":"16","Issue":"12","BRefMon":null,"BRefMonRR":null,"BRefXtra":null,"BRefXtraRR":null,"SerBRefID":275198,"SerRR":"Nature Protocols. 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