基于基因组编辑技术的水稻靶向突变特征及遗传分析

唐丽, 李曜魁, 张丹, 毛毕刚, 吕启明, 胡远艺, 韶也, 彭彦, 赵炳然, 夏石头

doi:10.16288/j.yczz.16-052

遗传 >

2016 , Vol. 38 >Issue 8: 746 - 755

DOI: https://doi.org/10.16288/j.yczz.16-052

研究报告

基于基因组编辑技术的水稻靶向突变特征及遗传分析

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1. 湖南农业大学生物科学技术学院,植物激素与生长发育湖南省重点实验室,长沙 410128;
2. 湖南杂交水稻研究中心,杂交水稻国家重点实验室,长沙 410125

唐丽,在读博士研究生,研究方向：生物物理学。E-mail: tangli@hhrrc.ac.cn

收稿日期: 2016-02-03

修回日期: 2016-06-29

网络出版日期: 2016-07-14

基金资助

湖南省自然科学基金项目(编号：14JJ7084)和湖南省重大科研项目(编号：湘财农指[2014]180)资助

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Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice

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1. Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China;
2. State Key Laboratory of Hybird Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China

Received date: 2016-02-03

Revised date: 2016-06-29

Online published: 2016-07-14

Supported by

Supported by the Natural Science Foundation of Hunan Province (No; 14JJ7084) and the Major Scientific Research Project of Hunan Province (No; XCNZ[2014180)

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摘要

TALEN(Transcription activator-like effector nucleases)系统和CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)系统是当前广泛应用的两大基因组编辑技术。本文比较分析了水稻(Oryza sativa L.)中这两大系统诱导突变的突变率、突变类型、突变位置、突变时间和遗传模式,发现TALEN系统和CRISPR/Cas9系统均可在T₀代水稻中有效诱发位点特异性突变,且CRISPR/Cas9的突变率更高。两大系统都以诱发10 bp以内的InDel突变为主,TALEN系统易诱导10 bp以内的缺失突变,而CRISPR/Cas9系统易诱导1 bp的插入突变,且CRISPR/Cas9系统诱发的DNA双链断裂(Double-strand breaks,DSBs)位置更加精确。此外,DSBs在修复过程中能以低频同源重组(Homologous recombination,HR)途径修复,产生DNA片段重复突变。对于相邻双靶点CRISPR/Cas9系统而言,双靶点间的DNA片段可发生缺失或倒位,这种双靶点间DNA片段突变的发生频率与双靶点各自的突变率无正相关性。两大系统诱导的突变最早发生在已转化的愈伤组织中,少量发生在水稻的体细胞中,导致了纯合突变、杂合突变、双等位突变和嵌合突变4种遗传模式,其中双等位突变比例最高,嵌合突变比例最低。除嵌合突变外,纯合突变、杂合突变、双等位突变的突变序列均可稳定遗传给下一代。

关键词： TALEN; CRISPR/Cas9; 水稻; 靶向突变; 遗传特性

本文引用格式

唐丽, 李曜魁, 张丹, 毛毕刚, 吕启明, 胡远艺, 韶也, 彭彦, 赵炳然, 夏石头 . 基于基因组编辑技术的水稻靶向突变特征及遗传分析[J]. 遗传, 2016 , 38(8) : 746 -755 . DOI: 10.16288/j.yczz.16-052

Abstract

The transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are two current genome editing technologies. Here, we compare and analyze the characteristics of the targeted mutations mediated by these two systems, such as efficiency, type, position, time, and genetic patterns. Both the TALEN and CRISPR/Cas9 systems can induce site-specific mutations in T₀ rice plants effectively, but CRISPR/Cas9 is more effective. The major mutation type in both systems is the short insertion/deletion(InDel) mutation within 10 base pairs: deletions ranging from 1 to 10 bps are more often in TALEN, and 1bp insertions are more often in CRISPR/Cas9. Moreover, double-strand breaks (DSBs) generated by CRISPR/Cas9 are more precise than TALEN. In addition, DSBs could be repaired by the homologous recombination at a low frequency, causing DNA fragment duplication mutations. In some cases, the DNA fragments between the two close targets are deleted or inverted, and the mutation efficiency does not positively correlatewith the mutation efficiency of each target. Mutagenesis mediated by the TALEN or CRISPR/Cas9 system can occur as early as in transformed callus cells, and less frequently in somatic cells. Consequently, four different mutation types are formed, including homozygous, heterozygous, bi-allelic and chimeric mutations, with bi-allelic mutations having the highest rate and chimeric mutations having the lowest rate. All, except chimeric mutations, can descend stably into the next generation.

Key words： TALEN; CRISPR/Cas9; rice; site-targeted mutagenesis; inheritance character

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