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.

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…Cases where mutation could be detected in T2 but not in T1 have already been reported in Arabidopsis in previous studies (see, e.g. Feng et al ., 2014) especially when using specific promoters (Mao et al ., 2016). Two hypotheses could be proposed to explain the fact that mutations were not detected in T1 generation using sgRNA#1.…”

“…In Arabidopsis that was transformed using a method similar to that used for Camelina, the extensive analysis of several hundred lines across 12 different target genes demonstrated that the mutation rate in T1 was on average 71% (47%–92%) (Feng et al ., 2014). The mutagenesis rate observed in Camelina was in the range of multigene targeting in Arabidopsis.…”

“…Highly efficient, stable mutagenesis was subsequently demonstrated in Arabidopsis, as well as in several crops: rice, wheat, barley, Brassica oleracea , potato, maize and soybean (Brooks et al ., 2014; Butler et al ., 2015; Čermák et al ., 2015; Fauser et al ., 2014; Feng et al ., 2014; Ito et al ., 2015; Jiang et al ., 2014; Lawrenson et al ., 2015; Li et al ., 2015; Svitashev et al ., 2015; Wang et al ., 2014; Zhang et al ., 2014; Zhou et al ., 2014). The CRISPR‐Cas9 system can be used to achieve multiple mutagenesis by the expression of several sgRNAs targetting different genes (Lowder et al ., 2015; Ma et al ., 2015).…”

Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

“…Moreover, the mutations generated in the primary transgenic plants by the CRISPR/Cas9 system can be stably transmitted to the next generation (Brooks et al 2014;Feng et al 2014). Thus, the CRISPR/Cas9 system is becoming a powerful tool for genome editing in plants, whereas the reports of the usage and efficiency of the CRISPR/Cas9 system-mediated plant genome engineering are still limited.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…In plants, in case of stable transformation, the CRISPR-Cas system can complete genome editing in early developmental phases and generate T1 transgenic plants showing the expected mutant phenotypes, but it can also be active in later generations. It can thus be interesting to screen T2 and T3 progenies to detect newly formed edition events [26]. The sensitivity of the detection can also benefit from enrichment steps, such as the digestion of genomic DNA by restriction enzymes whose sites are located in the cleavage area.…”