CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展

doi:10.11983/CBB18004

摘要/Abstract

摘要： 近年来, CRISPR定点编辑技术发展迅猛, 在动物、植物和微生物中均得到广泛应用。其中, 备受关注的脱靶现象也是研究的热点, 迄今已取得了重要进展。该文介绍了脱靶现象的产生原理及体内和体外检测脱靶现象的方法, 评价了通过改进sgRNA设计和优化CRISPR系统等来降低脱靶率的方法。在植物基因组定点编辑过程中, 应适时检测脱靶现象, 提高脱靶检测的精确度和准确度。

关键词: CRISPR/Cas9, 定点编辑, 脱靶

Abstract: CRISPR/Cas9 technology has rapidly developed in recent years. It is widely used for study of animals, plants and microorganisms. Among all developments, the use of the technology for the off-target issue has progressed, with important breakthroughs for this hot topic. This paper elucidates the principle of off-target generation and the method for detecting off target both in vitro and in vivo. Also, how to reduce off-targets by improving single-guide RNA design and optimizing the CRISPR system is evaluated. Further more, detecting off-targets at the right time and improving the precision and accuracy of off-target detection are proposed for plant site-specific genome editing.

Key words: CRISPR/Cas9, site specific editing, off-target

王影, 李相敢, 邱丽娟. CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展. 植物学报, 2018, 53(4): 528-541.

Wang Ying, Li Xianggan, Qiu Lijuan. Research Progress in Off-target in CRISPR/Cas9 Genome Editing. Chinese Bulletin of Botany, 2018, 53(4): 528-541.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18004

https://www.chinbullbotany.com/CN/Y2018/V53/I4/528

图/表 6

参考文献 69

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检测方法	优点	缺点
软件预测+测序	简单, 易操作	无法覆盖全基因组, 只检测软件预测的序列
深度测序	简单, 易操作	成本高, 数据分析复杂
T7E1检测	成本低, 速度快	需辅助软件预测脱靶位点, 无法覆盖全基因组
GUIDE-seq	精确度高(0.1%), 全基因组检测	受细胞转染的限制, dsODNs的整合效率影响结果
HTGTS	全基因组覆盖	只能检测到与断裂片段结合的脱靶位点, 存在遗漏
IDLV	全基因组覆盖, 无偏见检测脱靶位点	精确度较低(1%)
BLESS	摆脱了特定核酸酶的限制, 可检测任何酶所产生的DSB 中的突变	操作复杂, 只能检测特定时期所产生的突变
ChIP-seq	全基因组检测	体外检测, 未考虑切割频率, 准确度低
Digenome-seq	高效率, 高灵敏度(0.1%), 全基因组检测	体外检测, 成本高, 分析难度大
Circle-seq	全基因组检测, 灵敏度高, 提供分析平台	体外检测, 准确度不高
FIND-seq	全基因组检测, 灵敏度高	体外检测, 存在个别遗漏
Site-seq	全基因组检测, 灵敏度高	体外检测, 测序结果分析需要特定的算法

检测方法	优点	缺点
软件预测+测序	简单, 易操作	无法覆盖全基因组, 只检测软件预测的序列
深度测序	简单, 易操作	成本高, 数据分析复杂
T7E1检测	成本低, 速度快	需辅助软件预测脱靶位点, 无法覆盖全基因组
GUIDE-seq	精确度高(0.1%), 全基因组检测	受细胞转染的限制, dsODNs的整合效率影响结果
HTGTS	全基因组覆盖	只能检测到与断裂片段结合的脱靶位点, 存在遗漏
IDLV	全基因组覆盖, 无偏见检测脱靶位点	精确度较低(1%)
BLESS	摆脱了特定核酸酶的限制, 可检测任何酶所产生的DSB 中的突变	操作复杂, 只能检测特定时期所产生的突变
ChIP-seq	全基因组检测	体外检测, 未考虑切割频率, 准确度低
Digenome-seq	高效率, 高灵敏度(0.1%), 全基因组检测	体外检测, 成本高, 分析难度大
Circle-seq	全基因组检测, 灵敏度高, 提供分析平台	体外检测, 准确度不高
FIND-seq	全基因组检测, 灵敏度高	体外检测, 存在个别遗漏
Site-seq	全基因组检测, 灵敏度高	体外检测, 测序结果分析需要特定的算法

设计软件	网址	软件功能
Cas-OFFinder	http://www.rgenome.net/	针对CRISPR/Cas9系统, 通过使用者提供的目标位点序列, 推测所选择的目标基因组中潜在的脱靶位点。可选择不同的PAM、错配数量和是否允许错位配对(Bae et al., 2014)
CHOPCHOP	https://chopchop.rc.fas.harvard. edu/	针对CRISPR/Cas9和TALEN系统, 根据用户给出的目标基因序列, 在目标基因组或染色体中查找潜在的脱靶位点。可查找2个以内碱基错配的脱靶序列(Montague et al., 2014)
CRISPR Design	http://crispr.mit.edu/	在所给基因序列中设计sgRNA, 能够预测该sgRNA在基因组中的脱靶情况, 并标出最特异的sgRNA
CRISPR/Cas9 gRNA Finder	http://spot.colorado.edu/~slin/cas9.html	在所给出的基因序列中, 查找可能的目标切割位点, 给出合适的sgRNA序列, 并推测其二级结构(Mali et al., 2013)
CRISPRfinder Christine	http://crispr.u-psud.fr/Server/	在公开的微生物基因组中定位CRISPR重复序列位置, 并能报告间隔序列(Pourcel and Drevet, 2013)
E-CRISP	http://www.e-crisp.org/E-CRISP/	设计并评估CRISPR目标位点, 输入基因ID、FASTA序列进行搜索。可针对不同的CRISPR系统进行设计(Hsu et al., 2014)
CRISPR-Plant	http://www.genome.arizona.edu/crispr/	此软件针对一系列植物基因组设计CRISPR目标位点, 在所给出的基因序列或染色体序列中查找合适的目标位点(Lozano-Juste and Cutler, 2014)
CRISPR MultiTargeter	http://www.multicrispr.net	可用于设计同时靶向几个基因或1个基因中的多个位点的sgRNA (Prykhozhij et al., 2015)
sgRNA Designer	http://www.broadinstitute.org/rnai/public/	适用于人类和小鼠基因组中sgRNA的设计。能够推荐最高特异性的sgRNA, 但不会给出可能的脱靶位点(Doench et al., 2016)
sgRNA Scorer	https://crispr.med.harvard.edu/sgRNAScorer/	可以设计sgRNA并评估sgRNA的体内切割活性(Chari et al., 2015)

设计软件	网址	软件功能
Cas-OFFinder	http://www.rgenome.net/	针对CRISPR/Cas9系统, 通过使用者提供的目标位点序列, 推测所选择的目标基因组中潜在的脱靶位点。可选择不同的PAM、错配数量和是否允许错位配对(Bae et al., 2014)
CHOPCHOP	https://chopchop.rc.fas.harvard. edu/	针对CRISPR/Cas9和TALEN系统, 根据用户给出的目标基因序列, 在目标基因组或染色体中查找潜在的脱靶位点。可查找2个以内碱基错配的脱靶序列(Montague et al., 2014)
CRISPR Design	http://crispr.mit.edu/	在所给基因序列中设计sgRNA, 能够预测该sgRNA在基因组中的脱靶情况, 并标出最特异的sgRNA
CRISPR/Cas9 gRNA Finder	http://spot.colorado.edu/~slin/cas9.html	在所给出的基因序列中, 查找可能的目标切割位点, 给出合适的sgRNA序列, 并推测其二级结构(Mali et al., 2013)
CRISPRfinder Christine	http://crispr.u-psud.fr/Server/	在公开的微生物基因组中定位CRISPR重复序列位置, 并能报告间隔序列(Pourcel and Drevet, 2013)
E-CRISP	http://www.e-crisp.org/E-CRISP/	设计并评估CRISPR目标位点, 输入基因ID、FASTA序列进行搜索。可针对不同的CRISPR系统进行设计(Hsu et al., 2014)
CRISPR-Plant	http://www.genome.arizona.edu/crispr/	此软件针对一系列植物基因组设计CRISPR目标位点, 在所给出的基因序列或染色体序列中查找合适的目标位点(Lozano-Juste and Cutler, 2014)
CRISPR MultiTargeter	http://www.multicrispr.net	可用于设计同时靶向几个基因或1个基因中的多个位点的sgRNA (Prykhozhij et al., 2015)
sgRNA Designer	http://www.broadinstitute.org/rnai/public/	适用于人类和小鼠基因组中sgRNA的设计。能够推荐最高特异性的sgRNA, 但不会给出可能的脱靶位点(Doench et al., 2016)
sgRNA Scorer	https://crispr.med.harvard.edu/sgRNAScorer/	可以设计sgRNA并评估sgRNA的体内切割活性(Chari et al., 2015)

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