{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T16:00:53Z","timestamp":1793030453288,"version":"4.5.0"},"reference-count":57,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2022,10,12]],"date-time":"2022-10-12T00:00:00Z","timestamp":1665532800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Genes to Cells"],"published-print":{"date-parts":[[2022,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Genome\u2010editing using the CRISPR\u2010Cas9 system has the potential to substantially accelerate crop breeding. Since off\u2010target editing is one of problems, a reliable method for comprehensively detecting off\u2010target sites is needed. A number of <jats:italic>in silico<\/jats:italic> methods based on homology to on\u2010target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE\u2010Seq analysis to predict potential off\u2010target sites. SITE\u2010Seq analysis is a comprehensive method that can detect double\u2010strand breaks <jats:italic>in vitro<\/jats:italic>. Furthermore, we developed a systematic method using SITE\u2010Seq in combination with web\u2010based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE\u2010Seq analysis of a rice genome targeted by <jats:italic>OsFH15<\/jats:italic> gRNA\u2010Cas9 as a model, and found 41 candidate off\u2010target sites in the annotated regions. Detailed amplicon\u2010sequencing revealed mutations at one off\u2010target site in actual genome\u2010edited rice. Since this off\u2010target site has an uncommon protospacer adjacent motif, it is difficult to predict using <jats:italic>in silico<\/jats:italic> methods alone. Therefore, we propose a novel off\u2010target assessment scheme for genome\u2010edited crops that combines the prediction of off\u2010target candidates by SITE\u2010Seq and <jats:italic>in silico<\/jats:italic> programs and the validation of off\u2010target sites by amplicon\u2010sequencing.<\/jats:p>","DOI":"10.1111\/gtc.12985","type":"journal-article","created":{"date-parts":[[2022,10,1]],"date-time":"2022-10-01T10:18:47Z","timestamp":1664619527000},"page":"706-718","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Unbiased prediction of off\u2010target sites in genome\u2010edited rice using <scp>SITE\u2010Seq<\/scp> analysis on a web\u2010based platform"],"prefix":"10.1111","volume":"27","author":[{"given":"Jumpei","family":"Narushima","sequence":"first","affiliation":[{"name":"Division of Biochemistry National Institute of Health Sciences  Kawasaki Kanagawa 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