Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory.

Guillaume Bernard,Harmony Alves Dos Santos,David Gagneul,Caroline Rambaud,Audrey Etienne,Jean-Louis Hilbert

doi:10.3390/ijms20051155

Abstract

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

Highlights

Genome editing, which consists of targeting and digesting DNA at a specific site in the genome, is an important tool for gene function analysis and crop improvement [1]
clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology was already successfully used to knock out genes in other plants of the Asteraceae family such as salsify [40], lettuce [41] and dandelion [30]
Many successes in genome editing have been achieved with plants belonging to the Poaceae family, not because of their great ability to regenerate but because they are certainly more studied

Summary

Introduction

Genome editing, which consists of targeting and digesting DNA at a specific site in the genome, is an important tool for gene function analysis and crop improvement [1]. Three specific genome-editing technologies have been developed: zinc finger nucleases (ZFNs) [2], transcription activator-like effector nucleases (TALENs) [3], and clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (CAS) system. These methods induce double strand-breaks (DSBs) in the targeted DNA. In eukaryotic cells, these breaks can be repaired in two different pathways: the non-homologous end-joining (NHEJ) and homologous recombination (HR). The CAS9 endonuclease forms a ribonucleoprotein complex with the crRNA, which guides the endonuclease to a specific target DNA. Low cost, versatility, and high efficiency, the CRISPR/Cas system has become the most widely used technology for genome editing in many organisms such as bacteria, yeasts, animals, and plants

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