Design of a crispr cas9 system to overexpress osnramp7 in rice variety tbr225

VIETNAM AGRICULTURE ACADEMY FACULTY OF ECONOMY AND RURAL DEVELOPMENT GRADUATION THESIS DESIGN OF A CRISPR/CAS9 SYSTEM TO OVEREXPRESS OsNRAMP7 IN RICE VARIETY TBR225 Hanoi, July 2022 VIETNAM AGRICULTURE ACADEMY FACULTY OF ECONOMY AND RURAL DEVELOPMENT -  - GRADUATION THESIS TOPIC: DESIGN OF A CRISPR/CAS9 SYSTEM TO OVEREXPRESS OsNRAMP7 IN RICE VARIETY TBR225 Student : Ngo Thi Van Anh Class : K64CNSHE Major : Biotechnology Instructor : Dr Nguyen Duy Phuong : Assoc Prof Dr Dong Huy Gioi HANOI, 2022 GUARANTEE I hereby declare that this thesis is my research The collected data are true and have never been used or published in any documents, dissertations and scientific works ever before I hereby declare that the cited information in this thesis has been made of the source, ensuring cited as prescribed I bear full responsibility for these reassurances Hanoi, July 2022 Student Ngo Thi Van Anh i ACKNOWLEDGEMENTS In order to complete this graduation thesis, in addition to my constant efforts, I have received a lot of attention and help from groups and individuals inside and outside the school First and foremost, I would like to offer my deep gratitude to Assoc Prof Dr Dong Huy Gioi and teachers from Faculty of Biotechnology for imparting me critical and valuable knowledge during the study and training process at the Vietnam National University of Agriculture I would like to express my respect and appreciation to my supervisors Dr Nguyen Duy Phuong and the teachers at the Agricultural Genetics Institute (AGI) for having enthusiastically guided, advised and supported me throughout the process of learning and study; as well as Agricultural Genetics Institute (AGI) for creating favourable conditions to help me finished my thesis I would like to express my gratitude to Mr Nguyen Anh Minh has supported me in the lab works and my friends have helped and encouraged me during practice time Finally, I would like to send my sincerest thanks to my family and loved ones, who have supported and assisted me during practice time Due to limited time and knowledge, the thesis inevitably has certain limitations and shortcomings I would like to thank and appreciate the contributions from teachers, lecturers and students I sincerely thank! Hanoi, July 2022 Student Ngo Thi Van Anh ii LIST OF ABREVIATIONS A tumefaciens ABA Agrobacterium tumefaciens AP2 Bp Cas APETALA Base pair CRISPR associated protein CRISPR crRNA Clustered regularly interspaced short palindromic repeats CRISPR RNA dNTP Deoxynucleotide triphosphate DSB DNA double-strand break E coli EtBr Escherichia coli Ethidium Bromide gRNA HR HPT Guide RNA Homologous recombination Hygromycin phosphotransferase TBR225 Kb Rice cultivar TBR225 Kilobase NRAMP Natural Resistance Associated Macrophage Protein NHEJ Nu OsNRAMP7TBR225 PAM PCR sgRNA TAL Non-homologous end-joining Nucleotide The fragment of OsNRAMP7 gene was isolated from the TBR225 rice cultivar Protospacer adjacent motif Polymerase chain reaction single guide RNA Transcription activator-like TALEN tracrRNA ZFN TAL effector nuclease trans-encoded CRISPR RNA Zinc-finger nuclease Abscisic acid iii LIST OF TABLES Table 3.1 Sequence of oligonucleotides used in the research 37 Table 4.1 Characterization of TBR225 OsNRAMP7-editing sgRNAs 52 Table 4.2 Identification of TBR225 OsNRAMP7-editing sgRNAs 53 Table 4.3 Genomic DNA sequences homologous to designed OsNRAMP7-TBR225targeting crRNA 55 iv LIST OF FIGURES Figure 3.1 Diagram of the OsNRAMP7 structure 45 Figure 3.2 Diagram of designing pENTR4-V3/sgRNA-NRAMP7 47 Figure 3.3 Diagram of designing pENTR4-V3/sgRNA-NRAMP7-35S 48 Figure 3.4 Diagram of designing pCas9/sgRNA-NRAMP7-35S vector 50 Figure 4.1 Isolation of OsNRAMP7-TBR225 from genomic DNA 52 Figure 4.2 Sequencing the amplicon of OsNRAMP7-TBR225 52 Figure 4.3 Alignment of the OsNRAMP7 53 Figure 4.4 Location of crRNA on OsNRAMP7-TBR225 58 Figure 4.5 Secondary structure of sgRNA targeting OsNRAMP7-TBR225 59 Figure 4.6 Insertion of crRNA12-OsNRAMP7 into vector pENTR4-V3 60 Figure 4.7 Validation of the plasmid pENTR4-V3/sgRNA-NRAMP7 61 Figure 4.8 Sequencing the recombinant vector pENTR4/sgRNA-NRAMP7 62 Figure 4.9 Insertion of NRAMP7-35S into pENTR4-V3/sgRNA-NRAMP7 64 Figure 4.10 Validation of the plasmid pENTR4-V3/sgRNA-NRAMP7-35S 65 Figure 4.11 Sequencing the vector pENTR4/sgRNA-NRAMP7-35S 66 Figure 4.12 Insertion of sgRNA-NRAMP7-35S into pCas9 67 Figure 4.13 Validation of the plasmid pCAS9/sgRNA-NRAMP7-35S 68 Figure 4.14 Transformation of pCAS9/sgRNA-NRAMP7-35s 69 v ABSTRACT Micronutrients are not only necessary for plant growth and development, but also for human and animal health Natural resistance-associated macrophage protein (NRAMP) family, that have been found to be related to the accumulation of micronutrients in plants In rice, there were gene belong to OsNRAMP family of which OsNRAMP1, OsNRAMP4, OsNRAMP5, OsNRAMP6 and OsNRAMP8 were reported to transport metals such as iron, zinc, etc Recent, AtNRAMP5 which was closely related to OsNRAMP7 was identified that function in Zn uptake in Arabidopsis In order to overexpress OsNRAMP7 of rice variety TBR225, in this study, a part of the promoter and the Exon I of the gene was isolated with an approximate 800 bp in size for structural design of a gene editing construct The isolated DNA fragment is 99.8 % similar to the homologous OsNRAMP7 sequence published on the GenBank (code CP012620) Based on the sequencing analysis, the TBR225 OsNRAMP7-targeting sgRNA and TBR225 OsNRAMP7-homologous DNA template fragment containing CaMV35S promoter sequence were designed by the bioinformatic software These fragments were, respectively, inserted into LguI and BamHI sites on pENTR4-V3 carrying the sgRNA expression construct which then subcloned into plant transformation vector pCas9 This research is the basis for functional identification of OsNRAMP7 and further creating new rice varieties with high micronutrient content by genetic engineering in Vietnam vi CONTENTS ACKNOWLEDGEMENTS ii LIST OF ABREVIATIONS iii LIST OF TABLES iv LIST OF FIGURES v ABSTRACT vi CHAPTER INTRODUCTION 1.1 Introduction 1.2 Aim, objectives and meaning of research 1.1.1 Aim of the research 1.1.2 Objectives 1.1.3 Meaning of the topic CHAPTER OVERVIEWS OF LITERATURE 2.1 Plant metal transport proteins and the NRAMP family 2.1.1 Proteins involved in metal transport in plants 2.1.2 The roles of NRAMP protein in plants 2.2 Research on breeding rice varieties with high zinc content 13 2.2.1 Selection of rice varieties with high Zn content 13 2.2.2 Molecular basis of Zn accumulation in rice grain 14 2.2.3 Application of marker-assisted selection in high Zn rice breeding 16 2.3 CRISPR/Cas9 gene editing system 20 2.3.1 An overview of gene editing systems 20 2.3.2 An overview of CRISPR/Cas9 22 2.3.3 Functional mechanism of CRISPR/Cas 24 2.3.4 Application of CRISPR/Cas9 in plant breeding 26 CHAPTER RESEARCH MATERIALS AND METHODS 29 3.1 Time and place of study 29 3.2 Research subject 29 3.3 Research materials 29 3.4 Equipments and chemicals 29 3.5 Research methods 30 3.5.1 General molecular biology techniques 30 3.5.2 Isolation of TBR225 OsNRAMP7 by PCR 35 3.5.3 Design of the sgRNA expression construct for targeting OsNRAMP7TBR225 36 3.5.4 Design of dual-sgRNA expression vector carrying DNA template for TBR225 OsNRAMP7 HDR-mediated editing 38 3.5.5 Design of plant transformation vector for editing TBR225 OsNRAMP7 39 CHAPTER RESULTS AND DISCUSSIONS 41 4.1 Isolation and sequencing analysis of the TBR225 OsNRAMP7 41 4.1.1 Isolation of TBR225 OsNRAMP7 41 4.1.2 Sequencing analysis of the TBR225 OsNRAMP7 42 4.2 Design of sgRNA for OsNRAMP7-TBR225-targeting CRISPR/Cas9 complex 43 4.3 Construction of OsNRAMP7-TBR225-targeting sgRNA expression cassette 48 4.3.1 Insertion of OsNRAMP7-TBR225-targeting crRNA into pENTR4-V3 48 4.3.2 Confirmation of the recombinant pENTR4-V3/sgRNA 50 4.4 Design of dual sgRNA expression vector carrying DNA template for TBR225 Figure 4.11 Sequencing the vector pENTR4/sgRNA-NRAMP7-35S Note: A part of sequencing results of pENTR4/sgRNA-NRAMP7-35S with attL-R primer The arrow (→) represents a part of OsNRAMP7-homologous fragment sequence; the stars (*) show promoter 35S sequence From the above results, we conclude that we have successfully generated the recombinant vector pENTR4-V3/sgRNA-NRAMP7-35S The vector product was then stored for further experiments 4.5 Design of plant transformation vector for editing TBR225 OsNRAMP7 To generate plant transformation vector for expression of the TBR225 OsNRAMP7-overexpressing CRISPR/Cas9 complex, the sgRNA expression cassette and the donor DNA template sequence (refferred as sgRNA-NRAMP7-35S) on pENTR4-V3/sgRNA-NRAMP7-35S were recombined into the T-DNA expressing Cas9 by using LR clonase Two vectors pENTR4-V3/sgRNA-NRAMP7-35S and pCas9 were used as raw materials for Gateway reaction (subsection 3.5.5) which was then transformed into E coli cells Transformants appearing on selective medium supplemented with spectinomycin (Figure 4.12A) suggest that colonies carry the recombinant plasmid pCas9 Colonies were screened by PCR with the primers sgRNA-F/sgRNA-R The obtained results showed that the PCR products from colonies give a single DNA band with size approximately 56 kb (corresponding to the size of the region between 54 the sites of sgRNA-F and sgRNA-R primers) (Figure 4.12B, lane 1-6) on 1.0% agarose gel electrophoresis In contrast, the product electrophoresis of the negative control PCR from non-DNA-transformed colony showed no DNA band (Figure 4.12B, lane 7) From the above results, we initially concluded that colonies carrying recombinant plasmids containing sgRNA-NRAMP7-35S fragment were obtained One colony was selected for culture, plasmid DNA extraction and PCR checked with primers sgRNA-F/sgRNA-R (specific for sgRNA expression construct), UbiF/Cas9-t- R (specific for Cas9 expression construct), HPT-F/HPT-R (specific for the selective gene HPT) and NRAMP7.1-F/NRAMP7.2-R (specific for the donor DNA template NRAMP7-35S) (Figure 4.13) Figure 4.12 Insertion of sgRNA-NRAMP7-35S into pCas9 Note: (A) Growth of transformed E coli cells on selective medium containing 50 µg/ml spectinomycin (B) Screening the transformants by colony-directed PCR with the primers sgRNAF/sgRNA-R; PCR products were electrophoresed on 1% agarose gel; lane M: 1.0 kb DNA ladder; lanes 1-6: templates were transformed colonies; lane 7: negative control (template was nontransformed colony) The PCR products from purified plasmid when electrophoresed on a 1.0% agarose gel gave DNA bands of approximately 0.56 (Figure 4.13, well 2), 0.36 kb 55 (Figure 4.13, well 4), 0.8 kb (Figure 4.13, well 6) and 1.2 kb (Figure 4.13, well 8), respectively The size of the obtained DNA bands is completely consistent with the theoretical calculation of the amplified DNA fragments These results indicated that the recombinant pCas9/sgRNA-NRAMP7-35S plasmid carries the Cas9 gene expression construct (controlled by the Ubiquitin promoter), the HPT gene expression structure and the gene-edited sgRNA expression construct OsNRAMP7TBR225 (controlled by U6 promoter) Figure 4.13 Validation of the recombinant plasmid pCAS9/sgRNA-NRAMP7-35S Note: Validation of the pCAS9/sgRNA-NRAMP7-35S by PCR; lane M: kb DNA Ladder; lane 1,2: with primers Ubi-F/Cas9-t-R; lane 3,4: with primers HPT-F/HPT-R; lane 5,6: with primers sgRNA-F/sgRNA-R, lane 7,8: with primers NRAMP7.1-F/NRAMP7.2-R; lane 2,4,6,8: the recombinant pCAS9/sgRNA-NRAMP7-35S; lane 1,3,5,7: negative control (no DNA template) From the above results, we can confirm that the recombinant pCas9/sgRNANRAMP7-35S vector was successfully generated The vector product was stored at -20⁰C for use in subsequent transgenic studies on TBR255 56 4.6 Generation of A tumerfaciens strain containing the recombinant vector pCas9/sgRNA-NRAMP7-35S For the purpose of producing materials for further studies, the pCas9/sgRNA-NRAMP7-35S vector was transformed into A tumerfaciens EHA105 bacterial cells The transformation experiment obtained the colonies which could grow on the medium supplemented with the spectinomycin and rifampicin antibiotics (Figure 4.14A) Figure 4.14 Transformation of pCAS9/sgRNA-NRAMP7-35s into A tumefaciens Note: (A) Growth of transformed A tumefaciens cells on selective medium containing 50 µg/ml spectinomycin and 50 µg/ml rifampicin (B) Screening the transformants by colony-directed PCR with the primers NRAMP7.1-F/NRAMP7.2R; PCR products were electrophoresed on 1% agarose gel; lane M: 1.0 kb DNA ladder; lanes 1-3: templates were transformed colonies; lane 4: positive control (template was pCas9/sgRNA-NRAMP7-35S); lane 5: negative control (template was nontransformed colony) The A tumefaciens transformants bearing pCas9/sgRNA-NRAMP7-35S plasmid were selected based on the results of colony PCR with primers NRAMP7.1-F/NRAMP7.2-R Specifically, the PCR products from the transformed A.tumefaciens colonies gave a single DNA band approximately 1.2 kb on the agarose gel (Figure 4.14B, lane 1-3) which was similar to the product size of positive control PCR using pCas9/sgRNA-NRAMP7-35S as the template (Figure 4.14B, lane 4) In contrast, PCR products from non-transformed A 57 tumefaciens were not obtained (Figure 4.14B, lane 5) Based on this result, it can be concluded that the vector pCas9/sgRNA-NRAMP7-35S was successfully transformed into A tumefaciens bacterial cells 58 CHAPTER CONCLUSSIONS AND SUGGESTIONS 5.1 Conclussions The findings of this study prove and give conclusions as following: - Successfully isolated and sequenced the OsNRAMP7-TBR225 gene fragment the rice cultivar TBR225 The isolated 806-bp sequence were 99.8% and 96% similarity with OsNRAMP7 of Oryza saltiva var Indica (accession number: CP012620) and Oryza sativa var Japonica (accession number: AP014959), respectively - Successfully identifed a sgRNA sequence for CRISPR/Cas9 complex targeting TBR225 OsNRAMP7 - Successfully designed the sgRNA expresssion construct for editing TBR225 OsNRAMP7, which was confirmed by PCR, digestion and sequencing analysis - Successfully constructed the dual OsNRAMP7 sgRNA expression vector carrying DNA template with 35S promoter, which was confirmed by PCR, digestion and sequencing analysis - Successfully created T-DNA vector expressing CRISPR/Cas9 complex to overexpress TBR225 OsNRAMP7 via HDR-mediated editing The construct was successfully transformed to A tumefaciens EHA105 5.2 Suggestions Based on the obtained conclusions, we make some recommendations as follows: - Continue to transfer the OsNRAMP7-TBR225 gene editing system into the rice variety TBR225 - Transgenic rice lines will be further analyzed for genotype and phenotypic characteristics to demonstrate the fucntional ability of the designed construct 59 REFERENCES ENGLISH REFERENCES: Alqabbani H.M & Albadr N.A (2020) Zinc status (intake and level) of healthy elderly individuals in Riyadh and its relationship to physical health and cognitive impairment Clinical Nutrition Experimental, 29: 10-17 Andreini C., Bertini I., & Rosato A (2009) Metalloproteomes: a bioinformatic approach Accounts of chemical research, 42(10): 14711479 Arora L., & Narula A (2017) 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