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Targeted Gene Editing Using CRISPR/Cas9 in a Wheat Protoplast System

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Targeted Gene Editing Using CRISPR/Cas9 in a Wheat Protoplast System Targeted Gene Editing Using CRISPR/Cas9 in a Wheat Protoplast System Xiucheng Cui Supervised by Dr. Thérèse Ouellet A thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the degree of Master of Science in Biology Department of Biology Faculty of Science University of Ottawa © Xiucheng Cui, Ottawa, Canada, 2017 1 Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a promising tool for targeted gene editing in a variety of organisms including plants. In this system, a 20 nt sequence on a single guide RNA (sgRNA) is the only gene-specific information required to modify a target gene. Fusarium head blight (FHB) is a devastating disease in wheat caused by the fungus Fusarium graminearum. The trichothecene it produces, deoxynivalenol (DON), is a major mycotoxin contaminant causing food production loss both in quality and yield. In this project, we used the CRISPR/Cas9 system to modify three wheat genes identified in previous experiments, including an ABC transporter (TaABCC6), and the Nuclear Transcription Factor X box-binding-Like 1 (TaNFXL1), both associated with FHB susceptibility, and a non-specific Lipid Transfer Protein (nsLTP) named TansLTP9.4 which correlates with FHB resistance. Two sgRNAs were designed to target each gene and were shown in an in vitro CRISPR/Cas9 assay to guide the sequence-specific cleavage with high efficiency. Another assay for CRISPR/Cas9 was established by the optimization of a wheat protoplast isolation and transformation system. Using a construct expressing a green fluorescent protein (GFP) as a positive control, estimated transformation efficiencies of about 60% were obtained with different batches of protoplasts. High-throughput sequencing of PCR amplicons from protoplasts transformed with editing constructs clearly showed that the three genes have been successfully edited with efficiencies of up to 42.2%. In addition, we also characterized by RT-qPCR the expression pattern of 10 genes in DON-treated protoplasts; seven of the genes were induced by DON in the protoplasts, consistent with their previously identified DON induction in treated wheat heads, while three genes expressed differentially between DON-treated wheat heads and ii protoplasts. Preliminary bioinformatics analyses showed that these differentially expressed genes are involved in different plant defense mechanisms. iii Abstrait Le système de répétitions palindromiques courtes groupées et régulièrement espacées (CRISPR) / Cas9 est devenu un outil prometteur pour l'édition ciblée de gènes dans divers organismes, y compris les plantes. Dans ce système, une séquence de 20 nt sur un guide unique d'ARN (sgRNA) est la seule information spécifique du gène nécessaire pour modifier un gène cible. La brûlure de l’épi ou fusariose (FHB) est une maladie dévastatrice du blé causée par le champignon Fusarium graminearum. Le trichothécène qu'il produit, le désoxynivalénol (DON), est une mycotoxine majeure qui cause une perte de production alimentaire à la fois en qualité et en rendement. Dans ce projet, nous avons utilisé le système CRISPR / Cas9 pour modifier trois gènes de blé identifiés dans des expériences antérieures, y compris un transporteur ABC (TaABCC6) et le facteur de transcription nucléaire X box-binding-Like 1 (TaNFXL1), tous deux associés à la susceptibilité à FHB, et une protéine de transfert de lipide non spécifique (nsLTP) nommée TansLTP9.4 qui corrélèle avec la résistance à FHB. Pour chaque gène, deux sgRNA spécifiques ont été conçus, et un essai in vitro CRISPR / Cas9 a été utilisé pour montrer qu’ils peuvent guider le clivage spécifique de séquence avec un rendement élevé. Un autre essai pour CRISPR / Cas9 a été établi par l'optimisation d'un système d'isolement et de transformation de protoplastes de blé. En utilisant une construction exprimant une protéine fluorescente verte (GFP) comme un contrôle positif, des efficacité de transformation d’environ 60% ont été obtenus avec différents lots de protoplastes. Le séquençage à haut débit des amplicons de PCR provenant de protoplastes transformés avec des constructions pour édition spécifique de gènes a clairement montré que les trois gènes ont été édités avec succès dans le système de protoplastes avec des rendements allant jusqu'à 42,2%. De plus, nous avons caractérisé par RT-qPCR le profil d'expression de 10 gènes dans des protoplastes traités avec DON; sept des gènes ont été induits par le DON dans les protoplastes, en cohérence avec leur induction de DON précédemment identifiée dans les têtes de iv blé traitées, tandis que trois gènes ont été exprimés différemment entre les têtes de blé traitées par DON et les protoplastes. Des analyses bioinformatiques préliminaires ont montré que ces gènes différentiellement exprimés sont associés avec différents mécanismes de défense dans les plantes. v Acknowledgement Two years of master study is coming to an end with the completion of this thesis. Continuing my study in plant molecular biology is a precious experience since it allows me to deepen the insights of the life secrets. Starting a new life abroad alone is always difficult and it wouldn’t be possible without the people who supported and journeyed with me. First, I express my grateful thanks to my supervisor, Dr. Thérèse Ouellet, for her impartial support and patience in guiding the experiments, as well as the enormous efforts in revising thesis drafts. I would like to thank our lab technicians, Margaret Balcerzak and Hélène Rocheleau, for solving my technical problems. The mentoring and encouragement from our lab members was especially valuable for the successful completion of my master study. Next, I would like to show gratitude to my thesis committee members, Dr. Douglas Johnson and Dr. Gopal Subramaniam, for reading my committee meeting reports and giving comments which were helpful to continue my experiments and complete them on time. I also thank Jhadeswar Murmu and Natalie Labbé, who helped me with protoplast isolation and transformation experiments. Lastly, my special and warmest thanks go to my parents, who supported me to pursue my dreams, and for their encouragement during tough times. vi Table of Contents Abstract ........................................................................................................................................... ii Abstrait ........................................................................................................................................... iv Acknowledgement ......................................................................................................................... vi Table of Contents .......................................................................................................................... vii List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiv List of Abbreviations .................................................................................................................... xv 1. Introduction ................................................................................................................................ 1 1.1 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ............................ 1 1.1.1 CRISPR/Cas9 genomic editing system in plants ......................................................... 5 1.2 Introduction to wheat (Triticum aestivum) .......................................................................... 8 1.3 Fusarium Head Blight (FHB) .............................................................................................. 9 1.4 Genes involved in responses to FHB disease in wheat ..................................................... 13 1.4.1 ABC transporter ......................................................................................................... 15 1.4.2 Nuclear Transcription Factor, X box-binding, like 1 (NFXL1) ................................. 19 1.4.3 Non-specific Lipid Transfer Protein (nsLTP) ............................................................ 21 1.5 Objectives .......................................................................................................................... 24 2. Materials and Methodology ..................................................................................................... 25 2.1. Plant materials ................................................................................................................... 25 vii 2.2. Design and test of sgRNAs in vitro .................................................................................. 25 2.2.1. Generation of sgRNA-encoding templates ................................................................ 26 2.2.1.1. Design of two sgRNAs for each gene ................................................................. 26 2.2.1.2. The amplification of sgRNA-encoding templates and in vitro transcription ...... 28 2.2.2. Design primers to amplify and sequence DNA cleavage template ............................ 30 2.2.3. Testing of sgRNA efficiency in vitro ......................................................................... 31 2.3. Testing of sgRNAs in a wheat protoplast system ............................................................. 32 2.3.1.
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