Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat

Haruyasu Hamada,Naoaki Taoka,Yuelin Liu,Yozo Nagira,Ryozo Imai,Ryuji Miki

doi:10.1038/s41598-018-32714-6

Abstract

The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T1 plants identified plants homozygous for the three homeologous genes, which were all derived from one T0 plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.

Highlights

Genome editing has been successfully applied in major crops, such as rice (Oryza sativa L.), maize (Zea mays L.) and wheat (Triticum aestivum L.), using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein[9] (Cas9) nuclease, which is a simple and versatile tool for inducing DNA double-stranded breaks at target DNA sites[1,2,3]
To achieve in planta genome-editing in wheat, we selected TaGASR7, which is involved in the control of grain length and weight and has been shown to be amenable to genome editing with CRISPR/Cas[9], as the target gene[12,13]
We mixed three plasmid constructs carrying expression cassettes for Staphylococcus pyogenes Cas[9], the single guide RNA (sgRNA) and a GFP reporter gene, respectively, coated gold particles with the mixture, and bombarded shoot apical meristems (SAMs) of mature embryos with the coated gold particles according to the standard in planta particle bombardment (iPB) delivery protocol[11]

Summary

Introduction

Genome editing has been successfully applied in major crops, such as rice (Oryza sativa L.), maize (Zea mays L.) and wheat (Triticum aestivum L.), using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein[9] (Cas9) nuclease, which is a simple and versatile tool for inducing DNA double-stranded breaks at target DNA sites[1,2,3]. In these cases, CRISPR/Cas[9] expression cassettes as well as a selectable marker gene were introduced into plant genomes using Agrobacterium tumefaciens-mediated or biolistic delivery. The method can be used to introduce DNA-free genome-editing in wheat

Methods

Results

Conclusion