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

Significance The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been used to generate targeted gene editing in plants. However, it is not known whether CRISPR/Cas-induced gene modifications in plants occur in somatic cells only or whether some or all of the modifications can enter the germ line to become heritable. Through systematic and multigenerational analysis, this study demonstrates that although the majority of gene modifications detected in the first generation CRISPR/Cas transgenic Arabidopsis plants were somatic mutations only, heritable mutations could be found in subsequent generations. In addition, deep sequencing of CRISPR/Cas-modified Arabidopsis genomes did not detect any off-targets. The work demonstrates that the CRISPR/Cas method can effectively create specific gene modifications in planta that are stably transmitted through the germ line to future generations. 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.