Efficient genome editing of wild strawberry genes, vector development and validation.

Junhui Zhou,Guoming Wang,Zhongchi Liu

doi:10.1111/pbi.12922

Abstract

SummaryThe clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR–Cas9 to horticultural plants, especially with regard to germ‐line transmission of targeted mutations. Here, we report high‐efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6‐26 and the wild strawberry U6 promoter FveU6‐2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high‐efficiency genome editing in strawberry. To test germ‐line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild‐type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance.

Highlights

Fragaria vesca (F. vesca), the diploid wild strawberry, is an important member of the Rosaceae family and an emerging model system for the cultivated strawberry (Fragaria 9 ananassa) as well as other Rosaceae species
clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 employs the endonuclease activity of Cas9 to produce double-strand breaks (DSBs) at target genomic sites, with the specificity of the cutting site determined by an engineered single guide RNA
To construct a CRISPR genome editing vector that would work in strawberry with a high efficiency, we made a construct, where the single single guide RNA (sgRNA) was driven by the Fragaria vesca U6-2 promoter (Figure 1a)

Summary

The clustered regularly interspaced short palindromic repeats (CRISPR)–Cas system is an effective genome editing tool for plant and animal genomes. There are still few reports on the successful application of CRISPR–Cas to horticultural plants, especially with regard to germ-line transmission of targeted mutations. We report high-efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). To test germ-line transmission of the edited mutations and new mutations induced in the generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance

Introduction

Results

Discussion

Experimental procedures