Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

被引:503
作者
Feng, Zhengyan [1 ,2 ,3 ]
Mao, Yanfei [1 ]
Xu, Nanfei [1 ]
Zhang, Botao [1 ]
Wei, Pengliang [1 ,3 ]
Yang, Dong-Lei [1 ]
Wang, Zhen [1 ,3 ]
Zhang, Zhengjing [1 ,3 ]
Zheng, Rui [1 ,3 ]
Yang, Lan [1 ]
Zeng, Liang [1 ]
Liu, Xiaodong [1 ]
Zhu, Jian-Kang [1 ,4 ]
机构
[1] Chinese Acad Sci, Shanghai Ctr Plant Stress Biol, Shanghai 200032, Peoples R China
[2] Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Plant Physiol & Ecol, Shanghai 200032, Peoples R China
[3] Univ Chinese Acad Sci, Shanghai 200032, Peoples R China
[4] Purdue Univ, Dept Hort & Landscape Architecture, W Lafayette, IN 47907 USA
关键词
targeted gene modification; genome engineering; ZINC-FINGER NUCLEASES; ONE-STEP GENERATION; TARGETED MUTAGENESIS; GENOME; RNA; MULTIPLEX;
D O I
10.1073/pnas.1400822111
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, similar to 22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.
引用
收藏
页码:4632 / 4637
页数:6
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