Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis
Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis
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DOI：
10.1073/pnas.1400822111
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发表时间：
2014-03-25
影响因子：
11.1
通讯作者：
Zhu, Jian-Kang
中科院分区：
文献类型：
--
作者：
Feng, Zhengyan;Mao, Yanfei;Zhu, Jian-Kang
CRISPR（clustered regularly interspaced short palindromic repeat）/Cas（CRISPR associated）系统已成为许多生物（包括植物）中靶向基因编辑的强大工具。然而，所有报道的植物研究都集中在瞬时系统或CRISPR/Cas系统稳定转化到植物中后的第一代。在这项研究中，我们检查了在12个不同靶位点上具有7个基因的几代植物，以确定拟南芥中CRISPR/Cas诱导的基因突变或校正的模式，效率，特异性和遗传性。携带任何突变（嵌合、杂合、双等位基因或纯合）的植物比例在T1、T2和T3代分别为71.2%、58.3%和79.4%。CRISPR/Cas诱导的突变主要是1 bp插入和短缺失。在T1植物中检测到的基因修饰主要发生在体细胞中，因此没有基因修饰事件纯合的T1植物。相比之下，发现类似于22%的T2植物对于修饰的基因是纯合的。所有纯合子对下一代都是稳定的，在靶位点没有任何新的修饰。通过检查靶位点和与靶位点高度同源的序列以及通过深入的全基因组测序，没有任何脱靶突变的迹象。总之，我们的研究结果表明，CRISPR/Cas系统是一种有用的工具，可以在植物的靶基因上特异性地产生多功能和可遗传的修饰。
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.