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 Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.
Highlights
The system of clustered, regularly interspaced, palindromic repeats (CRISPR)/Cas (CRISPR-associated) is the latest groundbreaking technology for genome editing and has become the dominant genome editing tool
Wu et al [56] analyzed the transcriptome of B. napus lines to investigate the defense responses to S. sclerotiorum using in-depth RNA sequencing (RNA-seq), results showed that BnWRKY11 and BnWRKY70 genes differentially expressed in resistant B. napus lines J964 after inoculated by S. sclerotiorum
The expression of six BnWRKY70 homologue genes were significantly down-regulated after inoculated by S. sclerotiorum and the expression level were getting lower and lower over inoculation time (Figure 1D)
Summary
The system of clustered, regularly interspaced, palindromic repeats (CRISPR)/Cas (CRISPR-associated) is the latest groundbreaking technology for genome editing and has become the dominant genome editing tool. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), due to the ease of sgRNA manipulation, the CRISPR/Cas system presents advantages in terms of simplicity, accessibility, cost and versatility [5,6,7]. This system has been used successfully in many organisms, including animals [8,9,10], plants [11,12], fungi [13] and bacteria [14]
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