Chinese Bulletin of Botany ›› 2018, Vol. 53 ›› Issue (4): 528-541.doi: 10.11983/CBB18004

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Research Progress in Off-target in CRISPR/Cas9 Genome Editing

Wang Ying1,2,*(), Li Xianggan2, Qiu Lijuan1,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2Syngenta Biotechnology China Co, LTD, Beijing 102206, China
  • Received:2018-01-03 Accepted:2018-03-29 Online:2018-09-11 Published:2018-07-01
  • Contact: Wang Ying,Qiu Lijuan E-mail:ying.wang@syngenta.com;qiulijuan@caas.cn
  • About author:† These authors contributed equally to this paper

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

Figure 1

Schematic diagram of genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and integrase-defective lentiviral vectors (IDLV)"

Figure 2

Simplified process of high-throughput genomewide translocation sequencing (HTGTS) (modified from Frock et al., 2015)"

Figure 3

Schematic diagram of chromatin immunoprecipitation sequencing (ChIP-seq)"

Figure 4

Process of FIND-seq and Circle-seq"

Table 1

Advantage and disadvantage of in vivo and in vitro off-target detection methods"

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

Table 2

Summary of sgRNA design tools"

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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