title: Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.
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.
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
authors.forename: Zhengyan
authors.initials: Z
authors.lastname: Feng
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Mao
authors.forename: Nanfei
authors.initials: N
authors.lastname: Xu
authors.forename: Botao
authors.initials: B
authors.lastname: Zhang
authors.forename: Pengliang
authors.initials: P
authors.lastname: Wei
authors.forename: Dong-Lei
authors.initials: DL
authors.lastname: Yang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: Zhengjing
authors.initials: Z
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Zheng
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.forename: Liang
authors.initials: L
authors.lastname: Zeng
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Liu
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D017343:Genes, Plant; D059765:Homologous Recombination; D009154:Mutation; D020641:Polymorphism, Single Nucleotide
keywords: genome engineering; targeted gene modification