基于CRISPR编辑系统的DNA片段删除技术

doi:10.11983/CBB20203

植物学报 ›› 2021, Vol. 56 ›› Issue (1): 44-49.DOI: 10.11983/CBB20203 cstr: 32102.14.CBB20203

基于CRISPR编辑系统的DNA片段删除技术

谢先荣, 曾栋昌, 谭健韬, 祝钦泷, 刘耀光^*()

华南农业大学生命科学学院, 亚热带农业生物资源保护与利用国家重点实验室, 广州 510642

收稿日期:2020-12-11 接受日期:2021-02-25 出版日期:2021-01-01 发布日期:2021-02-25
通讯作者: 刘耀光
作者简介:*E-mail: ygliu@scau.edu.cn
基金资助:
广东省基础与应用基础研究重大项目(2019B030302006)

CRISPR-based DNA Fragment Deletion in Plants

Xianrong Xie, Dongchang Zeng, Jiantao Tan, Qinlong Zhu, Yaoguang Liu^*()

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China

Received:2020-12-11 Accepted:2021-02-25 Online:2021-01-01 Published:2021-02-25
Contact: Yaoguang Liu

摘要/Abstract

摘要： 基于CRISPR/Cas9系统的基因组编辑技术已成为基因功能研究和遗传修饰的重要工具。在引导RNA的引导下, Cas9蛋白对基因组靶位点进行精准切割产生DNA双链断裂(DSB), 借助细胞内的DSB修复机制, 可实现基因组靶位点碱基的缺失、插入或者替换, 甚至发生片段删除。该文介绍了基于CRISPR/Cas9基因组编辑系统的DSB微同源末端连接修复方式(MMEJ)提高基因组DNA片段删除效率的方法, 主要从靶点设计和突变检测方面展开详细描述。

关键词: 基因组编辑, CRISPR/Cas9, MMEJ, 微同源, 片段删除

Abstract: CRISPR/Cas9-based genome editing technology has been an important tool to study the gene function and genomic modification. Directed by a guide RNA, Cas9 protein can cleavage the genomic DNA at the target site, and produce mutations, including deletion, insertion, substitution and fragment deletion, by DNA double strand break (DSB) repair mechanism. In this protocol, we introduce the method to use CRISPR/Cas9 system to increase the efficiency of genomic DNA fragment deletion with microhomology-mediated end joining, especially the details in target design and detection of mutant plants.

Key words: genome editing, CRISPR/Cas9, MMEJ, microhomologous, fragment deletion

谢先荣, 曾栋昌, 谭健韬, 祝钦泷, 刘耀光. 基于CRISPR编辑系统的DNA片段删除技术. 植物学报, 2021, 56(1): 44-49.

Xianrong Xie, Dongchang Zeng, Jiantao Tan, Qinlong Zhu, Yaoguang Liu. CRISPR-based DNA Fragment Deletion in Plants. Chinese Bulletin of Botany, 2021, 56(1): 44-49.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20203

https://www.chinbullbotany.com/CN/Y2021/V56/I1/44

图/表 3

图1 利用微同源介导的末端连接(MMEJ)修复进行DNA片段删除的原理(改自Xie et al., 2020) MHS: 微同源序列

Figure 1 The principle for microhomology-mediated end joining (MMEJ)-based DNA fragment deletion(modified from Xie et al., 2020) MHS: Microhomologous sequence

图2 基于微同源介导的末端连接(MMEJ)介导DNA片段删除的实验流程

Figure 2 The experimental workflow for microhomology-mediated end joining (MMEJ)-mediated DNA fragment deletion

图3 再生植株突变体突变类型的鉴定策略 (A) 如果删除片段小于或约等于800 bp, 在靶点1 (T1)的上游和靶点2 (T2)的下游设计正向引物(T1-F)和反向引物(T2-R)进行PCR扩增; (B) 如果删除片段大于800 bp, 合成4条引物(使以下各扩增产物长度相互有约70-100 bp的差别), 设3组PCR反应(T1-F/T1-R、T2-F/T2-R和T1-F/T2-R), 也可以在同一个PCR反应使用4条引物(如果只扩增出T1-F/T1-R片段和T2-F/T2-R片段而扩增不出T1-F/T2-R小片段, 表明没有发生片段删除; 如果只扩增出T1-F/T2-R小片段而没有扩增出T1-F/T1-R片段和T2-F/T2-R片段, 表明2个等位序列都产生了片段删除; 如果3个片段都被扩增出, 表明一个等位序列产生了片段删除而另一等位序列没有片段删除); (C) 按策略(A)的引物设计的PCR扩增电泳检测图。WT: 未编辑的野生型; 1-9为T0编辑植株, 其中植株1-4的2个等位序列都发生了片段删除; 植株5、6、8、9有1个等位序列发生了片段删除, 植株7未发生片段删除。

Figure 3 The strategy for identification of the mutant types in transgenic plants (A) Amplification of the target region using the forward primer (T1-F) upstream the T1 and reverse primer (T2-R) downstream T2 if the length of desirable deleted fragment is less than 800 bp; (B) Four primers (with the length of the following amplification products is about 70-100 bp different from each other) with three combinations (T1-F/T1-R, T2-F/T2-R, and T1-F/T2-R) or pooled in an amplification are suggested if the length of desirable deleted fragment is more than 800 bp (there is no deletion of the target region if the resulting bands are produced with primer groups (T1-F/T1-R and T2-F/T2-R) but not T1-F/T2-R; both alleles had fragment deletion if the resulting bands are produced with primer group T1-F/T2-R but not T1-F/T1-R and T2-F/T2-R; one allele had fragment deletion and the other allele had no fragment deletion if all three fragments were produced; (C) The example of gel electrophoresis according to the detection method of (A). WT: Wild type; 1-9 indicate edited T0 plants in which 1-4 with biallelic fragment deletion, 5, 6, 8 and 9 with heterozygous deletion and 7 with no deletion.

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基于CRISPR编辑系统的DNA片段删除技术

CRISPR-based DNA Fragment Deletion in Plants

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