crispr/cas9jishuzuoweidisandaijiyinzubianjijishu，pingjieqicaozuojiandan、jiagedilianhebianjixiaolvgaodengyoudianyijingguangfandeyingyongyushengwugongcheng、yiliao、nongyehehuanjingdenglingyudeyanjiu，bingqiecanyutuidongyaowukaifa、mianyizhiliaohejiyinzhiliaolingyudetupo。hetalenhezfnsjishuyiyang，crispr/cas9jishuzaiyingyongshiyecunzaiyidinggailvdetuobawenti——cas9hesuanmeizaifeimubiaoweidianfashengqiege，yinrufeiyuqidejiyintubian。tuobaqiegeyinrudetubiankenenghuizhijiepohuaixibaodejibengongneng，dailaibukeyukongdeyanzhonghouguo。yinci，weiletigaocrisprjishuzailinchuangyanjiushangdeanquanxing，tongguogaolingmindudequanjiyinzutuobaxiaoyingjianceyouxuantuobaxiaoyingdideshiyanfanganshituijinjiyinbianjijishuzouxianglinchuangyingyongdeguanjian。
anbiqishengwujianlilegaoxiaochengshudejiyinzubianji、gaotongliangcexuheshengwuxinxixuefenxipingtai，weikehutigongquanmianjingquedecrispr/cas9tuobaxiaoyingjiancehefenxifuwu。womendejiancefangfabaokuoquanjiyinzucexu、guide-seqhecircle-seq。
biao1.changyongdetuobajiancefangfa
| 检测方法 | 方法原理 | 特点 |
| 脱靶预测结合PCR检测法 | 利用脱靶预测软件预测潜在的脱靶位点，PCR扩增预测的脱靶位点，利用直接测序或酶切法检测脱靶情况。 |
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| 全基因组测序 | 一种通过高通量测序检测脱靶突变的方法，需要选择适当的参考基因组过滤背景突变，数据比对分析获得含有PAM基序的潜在修饰位点，进一步基因扩增验证其突变情况。 |
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| BLESS | 利用生物素标签对DSBs进行原位标记，后经PCR扩增实现对于生物素标记片段的富集，并通过二代测序实现脱靶位点检测。 |
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| LAM-HTGTS | 片段化的gDNA经过LAM-PCR引入接头，然后进行全基因组易位测序。 |
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| GUIDE-Seq | 将dsODNs标签整合到DSBs位点，通过二代测序检测这些标签所在的基因组区域，从而确定脱靶突变的位置。 |
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| Digenome-Seq | 片段化的gDNA与CRISPR/RNP混合孵育，进行全基因组测序检测脱靶。 |
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| CIRCLE-Seq | 将gDNA打断并环化，环化的DNA与CRISPR/RNP孵育，NGS测序检测线性化的DNA片段，确定脱靶位点。 |
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anbiqishengwutigongsgrnatuobaxiaoyingpinggufuwu，bangzhunintiaoxuangaoduteyi，tuobalvdidesgrnajinxinghouxushiyan。tongshi，womenyexiezhuninjiancefenxijiyinbianjihouxibaoyangpindetuobaqingkuang，tongguogezhonggaotongliangcexujiancejishu，womennengzhunquedefenxiquanjiyinzufanweineidetuobaweidian，tuidongcrispr/cas9jishuzaizhiliaoyaowukaifahelinchuangyanjiuzhongdeyingyong。
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参考文献：
zhang xh, et al. (2015) 'off-target effects in crispr/cas9-mediated genome engineering', mol ther nucleic acids 4, e264.
zischewski j, et al. (2016) 'detection of on-target and off-target mutation generated by crispr/cas9 and other sequence-specific nucleases', biotechnology advances, 35(1): 95-104.
tsai sq, et al. (2015) 'guide-seq enables genome-wide profiling of off-target cleavage by crispr-cas nucleases', nature biotechnology, 33(2): 187-97.
kim d, et al. (2015) 'digenome-seq: genome-wide profiling of crispr-cas9 off-target effects in human cells', nature methods, 12(3): 237-43.
tsai sq, et al. (2017) 'circle-seq: a highly sensitive in vitro screen for genome-wide crispr-cas9 nuclease off-targets', nature methods, 14(6).
