CRISPR/Cas9-Mediated Mutagenesis of RCO in Cardamine hirsuta

Abstract

The small crucifer Cardamine hirsuta bears complex leaves divided into leaflets. This is in contrast to its relative, the reference plant Arabidopsis thaliana, which has simple leaves. Comparative studies between these species provide attractive opportunities to study the diversification of form. Here, we report on the implementation of the CRISPR/Cas9 genome editing methodology in C. hirsuta and with it the generation of novel alleles in the RCO gene, which was previously shown to play a major role in the diversification of form between the two species. Thus, genome editing can now be deployed in C. hirsuta, thereby increasing its versatility as a model system to study gene function and evolution.

Keywords: CRISPR/Cas9; Cardamine hirsuta; RCO; leaf development.

Figure 1

Schematic representation of the ChRCO locus targeted for mutagenesis. The two sgRNA complementary sequences are shown in red, and the PAM site is shown in blue. The yellow asterisk in exon 2 marks the position of the premature stop codon causing the first described rco mutant allele.

Figure 2

TIDE analysis of T1 Cardamine plants to estimate CRISPR heritable mutations in the sgRNA 1 target site. Plants with 50% or more wild-type sequence traces are classified either as carrying targeted mutations with ‘low heredity probability’, or as ‘wild type’ if indels are present with less than 15%.

Figure 3

Leaf shape comparison between wild-type and rco mutant lines. (A) Representative silhouettes of the fifth rosette leaf of the C. hirsuta wild type, the original rco mutant [2], and of two independently CRISPR-derived rco mutant lines. Scale bar: 1 cm; (B) Dissection index of the fifth rosette leaf. A red dot indicates the mean and error bars ± 1SD. Significance groups are determined based on ANOVA and Tukey’s HSD test for multiple pairwise comparisons. Only pairwise comparisons involving different groups, labeled a-b, are significantly different below the 5% level; (C) a schematic representation of the RCO locus. Mutations due to single nucleotide insertions in three independently CRISPR-derived alleles are indicated in lower case magenta. The previously known ‘non-CRISPR’ mutation in the second exon is shown in lower case green.

Figure 4

Simple flow diagram of the CRISPR/Cas9-mediated mutagenesis process, as followed in C. hirsuta in order to generate novel rco mutant alleles.