Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis - PubMed (original) (raw)

. 2014 Mar 25;111(12):4632-7.

doi: 10.1073/pnas.1400822111. Epub 2014 Feb 18.

Yanfei Mao, Nanfei Xu, Botao Zhang, Pengliang Wei, Dong-Lei Yang, Zhen Wang, Zhengjing Zhang, Rui Zheng, Lan Yang, Liang Zeng, Xiaodong Liu, Jian-Kang Zhu

Affiliations

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

Zhengyan Feng et al. Proc Natl Acad Sci U S A. 2014.

Abstract

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, ∼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.

Keywords: genome engineering; targeted gene modification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Mutations and segregation of the GAI gene modified by CRISPR/Cas.

Fig. 2.

Fig. 2.

Modifications by HR and NHEJ of a transformed nonfunctional GUUS reporter gene triggered by CRISPR/Cas in Arabidopsis. (A) Schematics to show vector construction and possible outcomes after a DSB in the GUUS reporter gene. (B) A T2 plant with mosaic GUS staining indicating that parts of the plant had correct HR events of the GUUS genes. (C) A T2 plant with uniform GUS staining suggesting that all cells on the plant contain a functional copy of the GUS gene as a result of correct HR. (D) Ratios of various GUS staining patterns and genotypes in progenies from five different T1 transgenic lines.

Fig. 3.

Fig. 3.

CRISPR/Cas-induced NHEJ mutation types and mutation lengths. Graph and Inset statistics are from combined data of 12 different target sites at T2 generations. (Left Inset) Occurrence of deletion (d), insertion (i), replacement (r), and combined (c) mutation types. (Right Inset) Frequency of different mutation lengths. In x axis: d#, number of bases deleted from a target site; i#, number of bases inserted at a target site; c#, combined mutations; x, mutation occurred but the number of mutated bases could not be determined; t, deletion between two nearby target sites.

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