CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

Abstract

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In <i>Arabidopsis</i>, <i>AtWRKY11</i> and <i>AtWRKY70</i> genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (<i>Brassica napus</i> L.), <i>BnWRKY11</i> and <i>BnWRKY70</i> genes were found to be differently expressed after inoculated with the pathogenic fungus, <i>Sclerotinia sclerotiorum</i> (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of <i>BnWRKY11</i> and four copies of <i>BnWRKY70</i> were designed to generate <i>BnWRKY11</i> and <i>BnWRKY70</i> mutants respectively. As a result, twenty-two <i>BnWRKY11</i> and eight <i>BnWRKY70</i> independent transformants (T₀) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in <i>BnWRKY11</i> and <i>BnWRKY70</i> transformants respectively. Eight and two plants with two copies of mutated <i>BnWRKY11</i> and <i>BnWRKY70</i> were obtained respectively. In T₁ generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of <i>BnWRKY70</i> mutants showed editing in three copies of <i>BnWRKY70</i> in examined T₁ plants. <i>BnWRKY70</i> mutants exhibited enhanced resistance to <i>Sclerotinia</i>, while <i>BnWRKY11</i> mutants showed no significant difference in <i>Sclerotinia</i> resistance when compared to non-transgenic plants. In addition, plants that overexpressed <i>BnWRKY70</i> showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that <i>BnWRKY70</i> may function as a regulating factor to negatively control the <i>Sclerotinia</i> resistance and CRISPR/Cas9 system could be used to generate germplasm in <i>B. napus</i> with high resistance against <i>Sclerotinia</i>.