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

From the above facts, we will conduct the project "Design of a CRISPR/Cas9 system to overexpress OsNRAMP7 in rice variety TBR225" to create the research materials for the study on func

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

: Assoc Prof Dr Dong Huy Gioi

HANOI, 2022

i

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

ii

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

iii

LIST OF ABREVIATIONS

ABA Abscisic acid

Cas CRISPR associated protein

CRISPR Clustered regularly interspaced short palindromic repeats

dNTP Deoxynucleotide triphosphate

DSB DNA double-strand break

EtBr Ethidium Bromide

HR Homologous recombination

HPT Hygromycin phosphotransferase

TBR225 Rice cultivar TBR225

NRAMP Natural Resistance Associated Macrophage Protein

NHEJ Non-homologous end-joining

PCR Polymerase chain reaction

sgRNA single guide RNA

TAL Transcription activator-like

TALEN TAL effector nuclease

tracrRNA trans-encoded CRISPR RNA

ZFN Zinc-finger nuclease

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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-TBR225-

targeting crRNA 55

v

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

vi

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 8 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

1

CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF ABREVIATIONS iii

LIST OF TABLES iv

LIST OF FIGURES v

ABSTRACT vi

CHAPTER 1 INTRODUCTION 4

1.1 Introduction 4

1.2 Aim, objectives and meaning of research 5

1.1.1 Aim of the research 5

1.1.2 Objectives 6

1.1.3 Meaning of the topic 6

CHAPTER 2 OVERVIEWS OF LITERATURE 7

2.1 Plant metal transport proteins and the NRAMP family 7

2.1.1 Proteins involved in metal transport in plants 7

2.1.2 The roles of NRAMP protein in plants 9

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

2.3.3 Functional mechanism of CRISPR/Cas 9 24

2.3.4 Application of CRISPR/Cas9 in plant breeding 26

CHAPTER 3 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 OsNRAMP7-TBR225 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 4 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

3

OsNRAMP7 HDR-mediated editing 51

4.4.1 Insertion of NRAMP7-35S into pENTR4-V3/sgRNA 51

4.4.2 Confirmation of the recombinant pENTR4-V3/sgRNA-NRAMP7-35S 53

4.5 Design of plant transformation vector for editing TBR225 OsNRAMP7 55

4.6 Generation of A tumerfaciens strain containing the recombinant vector pCas9/sgRNA-NRAMP7-35S 58

CHAPTER 5 CONCLUSSIONS AND SUGGESTIONS 60

5.1 Conclussions 60

5.2 Suggestions 60

REFERENCES 61

4

CHAPTER 1 INTRODUCTION 1.1 Introduction

Rice (Oryza sativa L.) is one of the major food crops of the world in general

and Vietnam in particular, which affects the diet of at least 65% of the world's population Rice is a staple food, popular because it's a rich source of energy, is gluten-free, easy to digest, low in fat, and packed with vitamins, minerals, and other nutrients However, some key elements such as iron are often lost when the bran layer of unmilled brown rice is scrubbed away to produce white rice In developing countries, fortifying rice grains after milling may not be a viable option In addition, soils in which these crops are grown may lack some essential minerals, or minerals are not available for uptake by plant roots

There are concerns about micronutrient deficiencies in the diet, especially in countries where rice is the staple food The important role of rice has prompted scientists to research and develop new rice varieties, in order to contribute to improving the nutritional balance of rice, ensuring food quality and security The research to improve the nutritional value of rice was started nearly 20 years ago, initially using farming methods, traditional breeding and hybridization, now applying genetic technology, including genome editing

The NRAMP (natural resistance-associated macrophage protein) transporter protein family has seven members in rice, including OsNRAMP1, OsNRAMP2, OsNRAMP3, OsNRAMP4, OsNRAMP5, OsNRAMP6, and OsNRAMP7 NRAMP have been found to transport metal ions such as Zn2+, Mn2+, Fe2+, Cd2+, and others in

plants Expression studies in normal plant tissues indicate that while OsNRAMP1 is expressed primarily in roots, and OsNRAMP2 is primarily expressed in leaves,

OsNRAMP3 is expressed in both tissues Furthermore, OsNRAMP5 is the main

5

transpoter responsible for Fe, Mn and Cd, whereas OsNRAMP4 was identified as a transpoter for Al However, despite this, relatively little information about these

transporters is available, especially information regarding OsNRAMP7 The close

genetic relationship between OsNRAMP7 and the identified Zn transporter

AtNRAMP4 in Arabidopsis suggested the role of OsNRAMP7 in Zn accumulation

in rice

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 protein) system was discovered in bacteria and archaea and can degrade exogenous substrates It has garnered a lot of interest in recent years

as a effective gene-editing tool for gene mutation and transcriptional regulation in animals and crops This technique allow precise and efficient modification of genomic DNA in cells through non-homologous end joining (NHEJ) repair or homology-directed repair (HDR) mechanisms While NHEJ-mediated gene editing induces ramdom modification, HDR-mediated gene editing results in precise gene knock-in or replacement Therefore, it is considered as an ideal tool to replace the traditional gene transformation method to overexpression target genes

From the above facts, we will conduct the project "Design of a CRISPR/Cas9

system to overexpress OsNRAMP7 in rice variety TBR225" to create the research

materials for the study on functional identification of OsNRAMP7 and further high

nutritional rice breeding program in Vietnam

1.2 Aim, objectives and meaning of research

1.1.1 Aim of the research

Designing a CRISPR/Cas9 system targeting OsNRAMP7 for gene

overexpression in rice variety TBR225

6

1.1.2 Objectives

- Isolation and sequencing of the OsNRAMP7 gene/promoter involved in metal

accumulation in rice variety TBR225

- Design of a dual expression vector containing TBR225 OsNRAMP7-editing

sgRNA expression construct and DNA template cassette harboring the

promoter 35S and TBR225 OsNRAMP7-homologous sequences

- Design of a plant transformation CRISPR/Cas9 vector for overexpression of

TBR225 OsNRAMP7

1.1.3 Meaning of the topic

Developing research materials for the study on functional identification of

TBR225 OsNRAMP7 and further breeding program of rice variety TBR225 with

high micronutrient content

7

CHAPTER 2 OVERVIEWS OF LITERATURE 2.1 Plant metal transport proteins and the NRAMP family

2.1.1 Proteins involved in metal transport in plants

Plants require a variety of essential transition metals as micronutrients for normal growth and development These metals are required for most redox reactions, which are fundamental to cell function Fe is an important component of haem proteins (such as cytochrome, catalase and Fe-S-containing proteins such as ferredoxin) and various enzymes Cu is an integral part of several electron transport proteins in photosynthesis (such as plastocyanins) and respiration (such as cytochrome c oxidase) and participates in liquefaction (laccase), although Mn has low redox activity, also participates in photosynthesis (evolved O2, etc.) Zinc is not involved in redox activity, but is an important structure and/or catalyst involved in many proteins and enzymes Other transition metals such as Ni and Mo are also micronutrients essential for plant function Loss of one of these metals can lead to a

variety of deficiency symptoms and impaired growth (Marschner et al., 1995)

However, although these metals are essential, they can be toxic if present in excess, and the production of reactive oxygen species and the harmful effects of oxidation

are of particular importance (Schützend et al., 2002) Therefore, their intracellular

concentrations need to be carefully controlled, and thus plants and other organisms have many potential mechanisms for metal ion homeostasis and tolerance, including

transmembrane transport (Clemens et al., 2001; Hall et al., 2002) Therefore, for

healthy plant growth and development, a wide range of transition metals must be absorbed from the soil, transported within the plant, and distributed and divided in various tissues and cells It is clear that membrane transport systems are likely to play an important role in this situation

8

During the last ten years or more, much research has been carried out, especially

with regard to Saccharomycer cereviriae, and to the applications of this knowledge

to the transport processes from yeast to organisms other eukaryotes (Eide et al., 1998; Nelson et al., 1999; Van Ho et al., 2002) With the use of genetic and molecular

engineering such as sequence comparison for transporter identification, functional complementation of mutant yeasts and plant transformation to regulate gene activities, a wide range of Gene families have now been identified in plants potentially involved in transition (or heavy) metal transport These include heavy metal ATPases (or CPx-types), natural resistance-associated macrophage proteins

(Nramps) and cation diffusion aid (CDF) (Williams et al., 2000), the ZIP gene family (Guerinot et al., 2000), and counter-carrier cations (Gaxiola et al., 2002)

Although there are some assumptions that the movement of metal cations across the membrane by some systems is accomplished through proton repulsions generated

by an ATP-powered H+ pump, direct evidence for co-transport in plants is still incomplete For most of these transporters, the gene family is quite large For

example, in Arabidoprir, there are 8 heavy metal ATPases (Mills et al., 2003), 6 Nramps (Williams et al., 2000) and 15 ZIPs (Maser et al., 2001) This diversity can

be formed for a variety of reasons: to provide the high and low affinity systems needed to accommodate the metal's presence in the soil; to provide specific requirements for transport in different cell membranes and organs in the plant; and

to respond to a variety of stressful conditions

Of course, some of these gene family members may be functionally redundant Techniques such as functional analysis in yeasts, studies of expression in plants under different concentrations of metal presence, and analysis of phenotypic variation will provide answers to several questions of these questions More interest

9

in these transporter gene families includes their potential for gene editing of certain species, or for improving the metal content of crops increasing nutrition for humans and animals or for plant treatment purposes, or the technology of using plants to remove toxins from the soil

2.1.2 The roles of NRAMP protein in plants

2.1.2.1 Functional characteristics of NRAMP

The NRAMP family has been shown to be functionally involved in metal transport Starting with SMF1, this protein was first identified as part of the Mn

uptake system in yeast (Supek et al., 1996) Next, Gunshin et al identified the

DCT1/DMT1/NRAMP2 protein as the major absorbed Fe transporter in the mammalian duodenum and has been shown to be capable of transporting a wide

range of heavy metals (Gunshin et al., 1997) A new concept that is slowly gaining popularity is that NRAMP encodes transition metal transporters with a broad

spectrum of specificity The metal transport function of plant NRAMPs has been demonstrated by adding them to yeast strains with a metal uptake impairment mutant

OsNRAMP1, AtNRAMP1, 3, 4 and 5 can complement Fe uptake in yeast strains with

inactivation of two fet3fet4 genes (Dix et al., 1994; De Silva et al., 1995) leading to defects in both high and low affinity iron absorption systems (Belouchi et al., 1997;; Curie et al., 2000;; Thomine et al., 2000) In contrast, OsNRAMP2 and AtNRAMP2 could not supply Fe to this strain (Curie et al., 2000;) The ability of some plant NRAMP homologues to transport other metals has also been examined AtNRAMP1,

3, 4 and 5 complement the phenotype of smf1, which has a defective ability to absorb

manganese (Thomine et al., 2000) In addition, AtNRAMP4, but not AtNRAMP1 or

3, compensated the growth of yeast zrt1zrt2 (Zhao and Eide, 1996a; Zhao and Eide,

1996b) with disrupted low-and high-affinity Zn transporters The addition of Fe, Mn,

10

and Zn uptake systems on the yeast plasma membrane requires robust constitutive

expression of plant NRAMP genes, suggesting that plant NRAMP-expressing yeast

cells at plasma membrane with low efficiency Furthermore, the expression of plant

AtNRAMP1, 3 and 4 genes in wild-type yeasts increases their sensitivity to the toxic

metal cadmium (Thomine et al., 2000) This supports the hypothesis that in addition

to essential metals, plant NRAMP transport proteins can also transport toxic metals Different tested NRAMP proteins do not transport the same metal For example,

although AtNRAMP1, 3, and 4 all induce the same Cd susceptibility, AtNRAMP3 and

4 complement fet3fet4 with greater efficiency than AtNRAMP1, and together with AtNRAMP4 is the only gene among the tried NRAMP genes Experiments could

complement the growth of zrt1zrt2 in low-zinc media (Thomine et al., 2000) The

transport selectivity of plant NRAMP proteins does not depend on whether they

belong to group I or group II in the phylogenetic tree Although other plant NRAMP

genes have been shown to encode metal transporters, no metal transport function could account for EIN2 It has been proposed that EIN2 can act as a metal sensor in plant cells (Hirayama and Alonso, 2000) In the future, the determination of the

complete substrate spectrum for each NRAMP transporter and the identification of

structural features underlying the differences in selectivity will hold great promise

The transport mechanism by which plants utilize NRAMP remains unclear Based on the strong pH sensitivity of the phenotypic complementation of AtNRAMP3 and 4 with the yeast fet3fet4 (Thomine et al., 2000), it was hypothesized that the metals

could be co-transported with protons, as demonstrated for mammalian

NRAMP2/DCT1/DMT1 and yeast SMF1 (Gunshin et al., 1997; Chen et al., 1999) Attempts to express AtNRAMP3 and 4 in Xenopus oocytes for a more detailed

transport mechanism analysis did not yield any significant metal flux

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2.1.2.2 Regulation of NRAMP gene expression in plants

Expression of plant NRAMP genes was investigated using Northern blot

techniques and transient gene expression In contrast to the metal transport genes

from the ZIP family, IRT1 and IRT2, which are completely root-specific, the

NRAMPs of some plants are expressed in both roots and shoots Some members of

the family are expressed preferentially in roots (AtNRAMP1 and 2, LeNRAMP1 and

3 and OsNRAMP1) and others in shoots (AtNRAMP3 and 4, OsNRAMP2 and 3) The

preferential expression of plant NRAMPs in roots or shoots is independent of whether they belong to group I or group II of the phylogenetic tree For example,

OsNRAMP3 and AtNRAMP1 and LeNRAMP1 both belong to group I OsNRAMP3

expression is stronger in shoots while AtNRAMP1 and LeNRAMP1 are more strongly expressed in roots (Curie et al., 2000; Belouchi et al., 1997; Bereczky et al., 2003) Expression of AtNRAMP5 is limited to reproductive organs in Arabidopsis Consistent with its expression pattern restricted to root epidermal cells, IRT1 encodes

a major metal-absorption transporter that promotes efflux of iron from the soil into

root cells (Vert et al., 2002) In contrast, the expression of NRAMPs in both roots

and shoots suggests that they are involved in metal homeostasis in all plant tissues NRAMP2/DMT1/DCT1 plays an important role in iron uptake and recycling in

mammalian cells Expression of some NRAMPs from Arabidopsis and rice could rescue the growth on low-iron media of mutant yeast fet3fet4, which is defective in iron absorption To investigate the possible function of NRAMP in iron homeostasis

in plants, several studies have tested the upregulation of NRAMP expression under iron-deficient conditions AtNRAMP1, AtNRAMP3 and AtNRAMP4 may support the

Fe uptake mutant in yeast by enhancing root system activity under iron deficiency

(Belouchi et al., 1997; Thomine et al., 2000) This is a demonstration that they

12

function in iron homeostasis in plants LeNRAMP1, the copper protein of AtNRAMP1

in tomato plants, is also expressed in iron deficiency (Bereczky et al., 2003) However, the expression of LeNRAMP3, a quasi-homologous protein of AtNRAMP3

in tomato, was insensitive to the iron nutritional status of tomato

The activation of LeNRAMP1 under iron-deficient conditions is controlled by

fer (Bereczky et al., 2003) Tomato fer mutant was unable to activate iron uptake

responses under Fe deficiency conditions Recently, fer has been cloned and encodes

a transcription factor with a basal Helix Loop Helix (bHLH) DNA-binding domain

(Ling et al., 2002) Transcription of LeNRAMP1 may also be one of the direct target actions of fer In addition, LeNRAMP1 is optimally regulated in tomato, acting when the plant is iron deficient Further analysis of the modes of regulation of NRAMP genes under Fe deficiency is currently underway (Bereczky et al., 2003; Ling et al., 2002) This can be done by monitoring NRAMP gene expression of known iron- responsive mutants or by selecting novel Arabidopsis mutants that are defective during NRAMP induction under Fe-starved conditions It is also possible that some plant NRAMP genes are regulated by an excess or starvation of other metal-

converting cations such as Zn, Mn or Cu

Studies using a transcriptional combination between the AtNRAMP promoters and geneβ-glucuronidase (GUS) have shown that AtNRAMP3 and AtNRAMP4 are expressed in the vascular tissues of roots and shoots (Thomine et al., 2003) This

suggests that they may be involved in metal translocation between different plant

organs These studies also revealed that the up-regulation of AtNRAMP gene

expression in iron deficiency occurs at the transcriptional level Indeed, the GUS

marker gene under the control of the AtNRAMP3 or AtNRAMP4 promoter is activated

in the absence of Fe This is in contrast to the control of iron-regulatory genes in

13

mammals, which occurs only at the post-transcriptional level and is implicated in

mRNA stability through regulation of IRE (iron response elements) (Casey et al., 1988; Klausner et al., 1993) However, AtNRAMP upregulation is consistent with the upregulation of other iron-responsive genes such as IRT1, IRT2 and ferritin, which

have been shown to be transcriptionally controlled in response to iron deficiency

(Vert et al., 2002; Petit et al., 2001; Vert et al., 2001)

2.2 Research on breeding rice varieties with high zinc content

2.2.1 Selection of rice varieties with high Zn content

Zinc plays an important role in the catalytic function of most enzymes necessary for the structural stability and activity of more than 3000 proteins, helps maintain membrane stability and protects tissues and cells from oxidative damage (Cakmak

2000; Broadley et al., 2007; Andreini et al., 2009; Maret and Li 2009) Zinc deficiency is one of the major causes of death in children worldwide (Black et al.,

2008), estimated to affect more than 178 countries (WHO 2003) Zn-deficient children are susceptible to diarrhea, respiratory disease, poor cognitive function, and

stunting (Brooks et al., 2004; Sazawal et al., 2007; Tielsch et al., 2007; Young et al.,

2014) Zn deficiency in the first 1000 days after birth causes irreversible damage leading to less chance of survival, poor immune system and cognitive ability, and stunting (UNICEF 2013) Therefore, a steady daily supply of Zn is essential, but this

is rarely achieved for most of the resource poor Thus, adequate nutrition of Zn is essential for good health, especially for children and pregnant women for growth and development (IZiNCG 2009) Individuals' daily Zn requirements vary from 9 to 11 ppm depending on age, sex, and health status, but preschool children and pregnant and lactating women require more Zn (IOM 2001; Welch and Graham 2004; Iqbal

et al., 2020; Alqabbani and AlBadr 2020)

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Rice is the most important source of energy and nutrition for more than half of the world's population (Gross and Zhao 2014) It is a staple crop in more than 40 countries and provides at least 20% of the daily calories of more than 4523.5 billion people (FAO 2014) Asia, with 60% of the global population, consumes more than 90% of the total rice produced annually (Milovanovic and Smutka 2017) Annual per capita rice consumption exceeds 100 kg in some Asian countries (FAO 2016) However, ground rice is less nutritious; therefore, most of the poor who mostly depend on rice without access to a varied diet rich in minerals suffer from hunger and cold, including Zn deficiency deficiency, especially when multiple deficiency

conditions occur (Torheim et al., 2010; Szymlek-Gay et al., 2009) Some studies

report that the consumption of a varied diet and crops enriched with mineral elements

provide more nutrients (Brown et al., 2002; WHO 1998) Recently, plant

microbiology has become a popular method to tackle malnutrition It is the process

of increasing the density of bioavailable mineral elements by breeding or

biotechnological approaches (Garg et al., 2018) for major food crops such as rice,

which has been has received increasing attention from breeders and policy makers in recent times Biofortification has the lowest per capita cost compared to other interventions, and it is particularly accessible and affordable for rural populations

(Ma et al., 2008) Therefore, increasing the Zn content of cereals will have a

significant impact on human health One estimate is that an addition of 8 μg/g Zn in coarse milled rice compared with baseline Zn (16 μg/g) in cultivars could result in

an amount equivalent to 30% of the Estimated Average Requirements per days (HarvestPlus 2012)

2.2.2 Molecular basis of Zn accumulation in rice grain

Increasing the amount of bioavailable Zn in the rice endosperm is a major goal

of rice microbiology There is a variation in the pattern of Zn distribution in rice

15

grains with the aleurone layer having 25–30% of the total Zn, and this layer being lost during processing, while the endosperm layer has 60–75% Zn, this layer is

retained even after polishing (Hansen et al., 2009) The genetic basis of high-grain

Zn in brown rice/milled rice is very complex and a better understanding of the genetic basis of high-grain Zn in rice is essential for the systematic use of rice germplasm in future chapters process to improve the content of microelements Zn Zn seeds have

a moderate to high wide sensory heritability and can be improved by crossbreeding

(Norton et al., 2010; Zhang et al., 2014), whereas heritability is reported narrow

sensory transmission clearly indicates significant additive and dominant genetic effects In addition, Zn particles are also significantly affected by environmental

factors (Gregorio 2002; Chandel et al., 2010; Anuradha et al., 2012a) Genetic

characterization of Zn seeds in several recombinant Inbred Lines (RILs) and also in rice seed collections have shown a significant Phenotypic Effect of Variation (PCV), Genotyping efficiency of variation (GCV), heritability, and genomic advancement (GA) In 12 of 14 studies, parental mapping populations were used, and in two studies, germ cell collection was used for genetic characterization of Zn

concentrations A population descended from the wild ancestor O rufipogon Among

different studies, PCV and GCV for grain Zn concentrations varied from 9.3% to 40% and from 9.2% to 36%, respectively, while heritability varied from 41 % to 99.4% and GA varies from 18.6% to 66.6% The highest PCV and GCV values were reported in the Azucena × Moromutant population, while the lowest in the TRY(R)

2 × Mapillaisamba population Heritability and genetic progress were highest in the BPT5204 × HPR14 and Azucena x Moromutant populations, respectively All of these results suggest that there is an adequate variability or concentration of Zn particles with moderate to high heritability and genetically progressive Therefore, it

is possible to improve the Zn concentration of popular rice varieties by exploiting high Zn genes in breeding programs

16

2.2.3 Application of marker-assisted selection in high Zn rice breeding

2.2.3.1 Genes/QTLs associated with high Zn content trait

The identification of the key quantitative trait genes/Locus (QTLs) and capture the molecular background of Zn seeds in rice will facilitate the breeding of rice varieties with high Zn content through Selection Based on molecular markers (MAS) Several genes/gene families involved in Zn homeostasis have been well characterized in rice Root exudates or phytosiderophores help to efficiently release

and absorb metals from the soil (Bashir et al., 2006; Widodo et al., 2010; Nozoye et

al., 2011) Several gene families such as OsNAS, OsTOM1, OsDMAS, OsSAMS and OsNAAT are involved in the biosynthesis, transport and secretion of

phytosiderophore in the rhizosphere and thus increase the uptake of metals by the

roots (Inoue et al., 2003, 2008; Bashir et al., 2006; Widodo et al., 2010; Nozoye et

al., 2011; Johnson et al., 2011) ZIP family genes are important metal transporters

found to be involved in Zn transport within and between different parts of rice, and

their expression varied under different Zn conditions to each other (Ramesh et al., 2003; Ishimaru et al., 2007,2011) The OsZIP1 gene is upregulated under Zn- deficient conditions, while OsZIP3 is up-regulated both under control and Zn- deficient conditions in rice (Ramesh et al., 2003) Overexpression of OsIRT and

MxIRT genes in rice increases Fe and Zn concentrations in rice grains

Similarly, OsOZT1, OsVIT1 and OsVIT2 are important vacuolar metal

transporters that participate in the transport of Zn across the vacuolar membrane and

also help sequester Zn within the cell (Lan et al., 2013; Zhang et al., 2012) While,

OsYSL family proteins play an important role in sieve circuit transport and metal

transport over long distances (Inoue et al., 2009; Aoyama et al., 2009; Lee et al., 2009; Ishimaru et al., 2010; Sasaki et al., 2011; Kakei et al., 2012) The OsYSL2

17

gene increased Fe content in rice fourfold (Ishimaru et al., 2010; Masuda et al., 2013) OsHMA3 overexpression enhanced Zn uptake by upregulating ZIP family genes in roots (Sasaki et al., 2014) Meanwhile, the OsHMA2 gene is involved in Zn loading into the growing tissues of rice (Yamaji et al., 2013) Several studies have shown that overexpression of OsNAS genes improved the grain Fe and Zn concentrations severalfold, OsNAS2 and OsNAS3 over the expression showed increased Fe and Zn accumulation (Lee et al., 2011; Johnson et al., 2011) OsIRO2 increases Fe content in rice plants grown on limestone soils (Ogo et al., 2011) The ferritin gene OsFer2 overexpressed in basmati rice (Pusasugandh II) accumulated higher levels of Fe and Zn (Paul et al., 2012) Several transcription factors such as

OsNAC, NAM-B1, OsIDEF1, OsIDEF2, and OsIRO2 also play an important role in

the regulation of genes involved in metal homeostasis (Ogo et al., 2006, 2007, 2008; Waters et al., 2009; Banerjee et al., 2010; Ogo et al., 2011; Gande et al., 2014) In

an expression analysis study with 25 metal-related genes, it was found that 9 genes

such as OsYSL6, OsYSL8, OsYSL14, OsNRAMP1, OsNRAMP7, OsNRAMP8,

OsNAS1, OsFRO1 and OsNAC5 were specifically expressed on the leaves and gave

found a significant correlation with Fe and/or Zn concentration in the seeds (Sperotto

et al., 2010) Similarly, transcriptome analysis of 25 metal homeostasis genes in

different tissues of the 12 rice genotypes revealed the expression of the highest

number of genes (24) in fenugreek, while genes such as OsZIP4, OsZIP11,

OsNRAMP5, OsNRAMP7, OsYSL2, OsYSL4, OsYSL6, OsYSL9, OsNAAT1, OsNAC, OsFER1, OsVIT1, OsFRO2, OsIRT1, OsFER2, OsZIP7, OsZIP8, OsZIP9, OsNRAMP4, OsNRAMP6 and OsYSL12 are expressed at the root The expression of OsNAC, OsYSL2, OsYSL9, OsZIP4, OsVIT1, OsNAAT1 and OsNRAMP7 genes in

coriander leaves was highly correlated with high grain Zn content (Banerjee and Chandel 2011) The Zn-deficient line RIL46 was found to produce higher levels of

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mugineic acid and low-molecular-weight organic acids than the Zn-deficient line

IR74 under Zn-deficient conditions (Widodo et al., 2010) In another study with RIL

by Madhkar x Swarna, OsNAS and OsHMA were expressed in leaves (Priya et al., 2015), in the same group of subjects under Fe-deficient conditions NAS2, IRT2,

DMAS1 and YSL15 were expressed in shoots, while NAS2, IRT1, IRT2, YSL2 and ZIP8 were in roots (Agarwal et al., 2014) Similarly, Chadha-Mohanty et al (2015)

reported that OsZIP5 and OsFRO1 were upregulated in the roots and leaves of rice

lines with high Zn content It is therefore clear that several genes and gene networks involved in metal uptake, translocation, uptake and loading, and their coordinated action, play an important role in homeostasis metallic lips in rice

2.2.3.2 Achievements in breeding rice varieties with high Zn content

Zn-fortified rice has great potential to combat malnutrition in the consuming poor countries of Asia, Africa and Latin America HarvestPlus, in collaboration with the International Rice Research Institute (IRRI) and the International Center for Tropical Agriculture (CIAT) and National Agricultural Extension Systems and Research (NARES) partners in several countries, is

rice-implementing program to develop rice varieties with high Zn content (Bouis et al.,

2013) The International Rice Research Institute (IRRI) is also working to include high seed micronutrient characterization as an integral part of all mainstream breeding projects The main target countries of the Zn biomicrofiltration program are India, Bangladesh, Indonesia and the Philippines The Microbiological Breeding Research Group at IRRI has identified a number of rice sprouts with high Zn content

as donors, early generation and advanced high Zn material based on popular rice varieties such as IR64, Swarna, Swarna Sub1, NSICRc222, PSBRc82, BR28, BR29, BR11, and Ciherang were produced and shared with national partners Overall, IRRI

is coordinating the crossbreeding activities of its domestic partners and is also

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encouraging them to generate high-Zn material in the genetic background of locally applied popular rice varieties using using the high-Zn donors provided by IRRI

(Swamy et al., 2015) The first batch of high Zn materials with an additional 6–8 mg

kg -1 Zn (18–20 mg kg -1 versus 12–14 mg kg -1 Zn of the common grades) is ready for release in the partner country, and a second cohort of high Zn lines with 8–10 mg

kg -1 Zn is in development Two rice varieties with high Zn content, BRRI dhan 62 and BRRI dhan 64, were introduced into cultivation during the Aman and Boro crops

in Bangladesh These two varieties have 19 mg kg -1 and 25 mg kg -1 Zn with yield potentials of 4.2 tons/ha and 6 tons/ha, respectively (HarvestPlus 2014), and also many high Zn strains in different stages advanced evaluation for seed release in Bangladesh IRRI also shared with the family of early generation materials combining high Zn and submergence tolerance, for further evaluation and selection

in the Bangladesh environment

In the Philippines, high-Zn propagation materials shared by IRRI are in the National Pre-NCT Collaborative Trial (Pre-NCT) and the National Collaborative Trial (NCT) for release There are many high-Zn strains being evaluated in trials at

the Philippine Rice Research Institute research station (Inabangan-Asilo et al., 2015)

In Indonesia and India, rice lines with high Zn content are in the advanced stage of evaluation in multisite and station trials These first and second highest Zn lines had 18–22 mg kg -1 Zn with acceptable yield potential, grain quality and agronomic traits

(Swamy et al., 2015) These strains can meet 16–20% of the estimated average need

for Zn, which is enough to overcome the serious health problems caused by Zn deficiency In the coming years, we hope to release several high Zn rice lines in the target countries and see them deployed on a large scale The initial success of high

Zn rice varieties and other high Zn rice varieties developed and published provided further impetus to expand the program to other poorer countries in Asia

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2.3 CRISPR/Cas9 gene editing system

2.3.1 An overview of gene editing systems

Genome editing technology enables precise modification of DNA sequences in vivo and holds great promise for the exploitation of plant genes in crop improvement

(Shan et al., 2013, Tian et al., 2021) Gene editing is a technique to precisely alter an

organism's genomic DNA with the aim of altering the function of a target gene This method induces small changes in the target DNA sequence by taking advantage of the cell's DNA repair system to induce small changes in the target DNA sequence, through the use of synthetic nucleases to generate produces double-stranded breaks (DSB) mutations at specific locations in the genome Precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases to initiate non-homologous end-joining (NHEJ)-based DNA repair reactions or homologous directed recombination (HDR) NHEJ is an efficient but imprecise common cellular repair mechanism by inducing nucleotide addition or loss mutations that lead to off-targeting errors, while HDR has the potential to high accuracy through the use of a template with a homologous sequence at the coupling, thus preserving the DNA sequence but less efficiently The outcome of DBS repair can be either exact repair or the emergence of mutations (gene substitution, loss or

insertion), depending on whether the pathway used is HDR or NHEJ (Liu et al.,

2017)

Recently, scientists have been advantageous in gene editing studies thanks to the development of nucleases The Zinc-finger nuclease (ZFN) gene editing system belongs to the first generation SDN group, which is one of the quite popular gene editing techniques in the past In the field of plant biotechnology, the zinc-finger protein family (ZFP) is used in two ways: ZFN for double-stranded DNA cutting (DBS) and ZFN-TF (Zinc-finger nuclease – Transcription factor) for regulation gene

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expression Because ZFN has a flexible natural conformation (modular form), it is not too difficult to redesign the ZFN molecular structure with specific functional regions (domains) for DSB generation and gene editing The nature of ZFN is a fusion protein consisting of a domain with DNA cutting activity and a domain with specific DNA binding activity ZFN has been successfully used in genome editing of many different plants including tobacco, maize, soybean, etc However, ZFN has some disadvantages such as the construction of target enzymes which is time consuming and expensive, low specificity, and high off-target mutations Later, ZFN gave way to a new technology – TALEN (transcription activator–like effector nucleases) TALEN is a DNA-cutting enzyme that can be easily reprogrammed to recognize and cut specific DNA sequences This system has been widely used to generate non-homologous mutants with greater efficiency in a wide range of

organisms (Shan et al., 2013; Arora et al., 2017) However, this technique is still

time-consuming and requires a long process as well as the need to modify proteins when designing the system Therefore, ZFN and TALEN have very few applications

in plants Until the advent of technology CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) with many outstanding advantages such as easier to design, lower cost, and has more robust applications has changed replace both ZFN and TALEN for widespread use as a novel approach to genome editing in eukaryotic host cells Thanks to continuous advances in efficiency, the CRISPR/Cas9 tool has been increasingly used in the field

of plant science and has become an extremely promising gene editing tool (Arora et

al., 2017)

The field of molecular biology and DNA editing techniques has benefited from recent developments and advances in biotechnology Many research projects using clinical biotechnology, such as gene editing, are currently being finalized, offering hope for technology research and development These technologies have the

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potential to be the future in developing new varieties of plants that will benefit our lives in the future (Sikandar, 2019)

2.3.2 An overview of CRISPR/Cas9

CRISPR/Cas is an immune mechanism that has been found in prokaryotes such

as bacteria and archaea since the late 1980s CRISPR/Cas has been detected in algae

(90%), bacteria (50%) and archaea (90%) (Rouseau et al., 2009; Grissa et al., 2007; Makarova et al., 2015) The distinguishing feature of these systems is the CRISPR

array This genomic location consists of alternating identical repeats and unique

spacers (Ishino et al., 1987; Jansen et al., 2002) which first suggested that Cas may function as a prokaryotic defense mechanism (Bolotin et al., 2005; Mojica

CRISPR-et al., 2005; Pourcel CRISPR-et al., 2005) After decoding the role of the CRISPR/Cas system

in bacteria and archaea, the system was modified for use as a genome editing tool

(Jun et al., 2019)

CRISPR has previously been classified into three types (I, II, and III) based on the presence of the Cas3, Cas9 (or Csn1) and Cas10 proteins CRISPR/Cas type I is present in both algae and bacteria, while CRISPR/Cas type II (CRISPR/Cas9) is only found in bacteria and CRISPR/Cas III is found mainly in algae and some bacterial species determined The three CRISPR/Cas types all share the CRISPR locus and the Cas1 and Cas2 proteins, but the crRNA binding domain (CRISPR RNA) and cleavage initiation domain are different The CRISPR locus includes information on previously exposed pathogens (foreign genetic material); Cas genes encode proteins that mediate CRISPR-based immunity CRISPR loci are made up of Palindromic symmetric sequences approximately 30 nucleotides long and constitute sequencers (Spacers) generated from foreign genetic material For the CRISPR/Cas system to function as an immune mediator against pathogens, it requires the CRISPR loci, the

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buffer, and the Cas gene The Cas gene is commonly associated with the CRISPR

locus, although it may also be located elsewhere in the genome The target of the CRISPR/Cas system is foreign DNA or RNA that enters the cell Many Cas proteins have RNase and DNase activities, which play an important role in the functioning of

the CRISPR/Cas system (Shan et al., 2013; Arora et al., 2017)

Conventional CRISPR-Cas tools have significantly facilitated the generation of targeted genetic variants in plants by generating random indels via an incompatible

terminal linkage repair pathway (NHEJ) (Xie and Yang, 2013; Molla et al., 2020a)

Base editing, an emerging technology, can precisely install four switch points and

two switch point mutants (Molla and Yang, 2019; Molla and Yang, 2020ª; Molla et

al., 2020b; Molla et al., 2020c) However, neither CRISPR-Cas nor base editing were

able to produce precise indicators, which are also important for improving crop traits

To generate the correct indels, we mainly depended on using the HDR pathway Unfortunately, HDR is stereotype-oriented and ineffective, limiting its application in crop improvement Cas9-induced double-strand breaks (DSBs) in DNA are repaired mainly through the NHEJ pathway in higher plants Thus, unlike HDR, NHEJ-mediated mutagenesis is highly efficient in plants If we can predict the outcome of DSB repair, it will facilitate the generation of accurate indicators

HDR is valuable in precise gene replacement, addition and installation of complex modifications However, achieving good results in higher plants is a major obstacle to the frequent use of HDR in crop improvement For HDR to be successful, suitable donor repair samples should be available near the DSB The temporal and spatial co-ordination between DSB generation and adequate feeding of the donor's

samples are considered to be major bottlenecks in HDR (Li et al., 2018; Huang and Puchta, 2019) Some strategies include the use of geminivirus clones (Čermák et al.,

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2015), chimeric guide RNA (cgRNA) (Butt et al., 2017), chemically modified donor DNA, HDR tandem repeats (TR-HDR) (Lu et al., 2020) and transcriptionally patterned HDR (TT-HDR) (Li et al., 2019) were used to overcome these bottlenecks

To make donor samples available in situ of the DSB, one attractive approach is to combine them with guide RNA (gRNA) sequencing For example, the use of chimeric guide RNA (cgRNA), which contains a gRNA coupled to a donor template,

has been shown to induce HDR in rice (Butt et al., 2017) The cgRNA strategy is

based on template fusion of the 3'-end donor of the sgRNA and RNA-templated DNA

repair (Butt et al., 2017)

2.3.3 Functional mechanism of CRISPR/Cas 9

CRISPR/Cas9 has been used to efficiently alter genomes in many plant species,

including Arabidopsis, rice, tobacco, maize, and tomato, and has been tested in

Arabidopsis thaliana, Nicotinana benthamiana, rice, wheat, tomato, grape and maize

(Li J et al., 2013) CRISPR/Cas9 has now become a technique used in plant gene

editing, not only to knock out genes, but also to insert or delete genes via disruption using CRISPR/Cas9-mediated DNA breaks to increase the level of homozygous recombination

CRISPR/Cas 9 (Class II) is the simplest and most efficient system, with four proteins (Cas1, Cas2, Cas9 and Cas4/Csn2) Cas9 is a large protein with two domains: the RuvC nuclease domain and the HNH nuclease domain, which can catalyze double-stranded cleavage of target DNA at specific sites and process prerRNA molecules to produce full crRNAs A tracrRNA (conversion encoded CRISPR RNA) molecule is also present in this system, which controls DNA cleavage Adaptation, expression, and intervention are the three stages that all CRISPR/Cas systems go through

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The procedure involves two steps: (1) foreign DNA recognition and (2) integration of the foreign DNA sequence (buffer) into the CRISPR locus The sequence PAM-protospacer adjacent motif, which is a conserved DNA fragment of 2-5 Nu length that acts as a recognition motif during foreign DNA acquisition, is commonly found in front of buffer (protospacer) One of the two copies of the spacer will be placed at the top of the CRISPR sequence after it has been duplicated twice The immune response to infections can be impeded by mutations in the PAM sequence site Expression is the second stage in the action sequence of the CRISPR system, in which a pre-crRNA molecule is transcribed With the help of Cas proteins (Cas1, Cas2, Cas9 and Cas4/Csn2) and the tracrRNA molecule, the encoding from

the CRISPR locus is converted to the full crRNA In Streptococcus pyogenes,

tracrRNA has been found to participate in pre-crRNA processing The tracrRNA molecule contributes to the conversion of pre-crRNA to full crRNA by pairing with

a repeat region on the crRNA according to the principle of complementarity In CRISPR, the entire crRNA molecule participates in and aids in the recognition of a specific target DNA site The crRNA molecule directs the Cas protein complex to a specific target region on the foreign DNA strand during the interference phase DNA disruption is catalyzed by enzymes that support a cell's ability to resist infection with

pathogens (Arora et al., 2017; Ma et al., 2016)

Using the Cas9 enzyme based on the Class II-CRISPR system, and tracrRNA: crRNA was produced into sgRNA (single guide RNA) The method by which the endonuclease-regulated sgRNA Cas9 selectively disrupts DNA, thereby activating the cellular double-strand break (DSB) repair machinery, which is believed to be the

functioning mechanism of CRISPR/Cas9 (Bortesi et al., 2015) The HDR or NHEJ pathways can repair flat-ended DNA strands (Razzaq et al., 2019; Chen et al., 2019)

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The NHEJ repair process is prone to errors, leading to insertion or loss of nucleotides

on the target sequence Cells utilize this repair process during most phases of the cell cycle and do not require homologous template sequences for repair Therefore, inducing small changes (Nu insertion/deletion) at specific sites on target genes has become a popular method for gene inactivation The NHEJ mechanism can cause significant chromosomal deletions when a pair of sgRNAs is expressed

2.3.4 Application of CRISPR/Cas9 in plant breeding

Although the CRISPR/Cas9 gene editing system has only been around for a few years, there are already plenty of studies using it CRISPR has made genetic engineering significantly easier since its introduction, and it can now be used in virtually any model organism, from zebrafish to rice to large mammals like monkeys CRISPR/Cas9 technology has also been used to alter the genomes of many plant species Many obstacles to the classification of GMO crops are expected to be solved using CRISPR/Cas9 modified crops Tomato plants modified with CRISPR/Cas9 are expected to have better taste, sugar content and aroma than current commercial varieties; maize is drought tolerant and gives high yield per hectare; wheat was engineered to resist powdery mildew, and the fungus was engineered to reduce

melanin (Arora et al., 2017; Ma et al., 2016)

Single gene inactivation such as DSB mutations induced by CRISPR/Cas9 can lead to Indel mutations at the site of disruption of the NHEJ DNA repair mechanism Indels appearing on the open reading frame of a gene often cause a frameshift mutation that alters the expression of the gene, leading to a gene inactivation

mutation (Jun et al., 2019) For example, Arabidopsis’s ab1 mutated gene using the CRISPR/Cas9 tool (Razzaq et al., 2019) has a 5 bp deletion mutation at the target

site, so the open reading frame of the target gene is altered, leading to the appearance

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of the trinity of cessations This demonstrates that the abp1 mutant carries a recessive

allele and that ABP1 is not an important component of the auxin signaling pathway

and does not affect Arabidopsis growth In plants, homologous genes in gene families

are often used for the purpose of fine-tuning cellular biological processes Inactivation of a single gene in a family may not induce a phenotypic change due to compensatory activity of other genes in the same family Therefore, a molecular tool for simultaneous editing of many genes will be a very useful solution to support genomic functional studies and applied research Scientists can easily combine multiple sgRNA expression constructs on the same plasmid, thereby editing multiple

genes using the CRISPR/Cas9 system (Jun et al., 2019) For example, in plants, two homologous magnesium-chelatase I (CHLI) subunit genes are involved in photosynthesis The scientists engineered two sgRNAs, each affecting a CHLI gene and transforming into Arabidopsis Plant lines carrying mutations in both genes

expressed the albino phenotype The results of this study elucidated the function of two genes on chlorophyll biosynthesis By using one or more sgRNAs simultaneously, the polygene editing system is a powerful support tool for studies that analyze the function of gene family members or study the genetic relationships

between genes gene by different evolutionary pathways (Jun et al., 2019) In

addition, when using the CRISPR/Cas9 gene editing system in combination with a double-ended template DNA sequence similar to the DSB region, scientists can replace and/or move the gene into the desired location through the organism's HDR repair mechanism, for plant biology research and plant genetic improvement research

Elimination of negative factors is one of the effective methods for genetic improvement of plants Thus, inactivation of genes that control undesirable traits is

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an application of CRISPR/Cas9 Plant traits that have been improved by CRISPR/Cas9 in this way include yield, quality, and tolerance to biotic and abiotic stressors Hybrid breeding techniques and many other important issues related to

crop yield have also been improved using CRISPR/Cas9 (Chen et al., 2019) For

example, inactivation of all three copies of the GASR7 gene in the wheat genome increased grain weight significantly Another example is the successfully obtained bi-allelic rice mutants by transforming a Cas9 expression construct, gRNA, and a gene targeting (GT) vector containing an HDR template into the calli of Oryza sativa

to target the acetolactate synthase (ALS) gene (Endo et al., 2016) Furthermore, the

HDR-mediated CRISPR/Cas9 system has been used successfully to create precise

and heritable changes in tomato, maize, and soybean (Cermak et al., 2015; Svitashev

et al., 2015; Li et al., 2015)

The OsNRAMP7 gene is involved in the transport and accumulation of Fe

microelements in the rice variety TBR225

3.3 Research materials

The vector systems: Vector pENTR4-V3 (carrying the sgRNA expression construct), and pCas9 (carrying the Cas9 protein expression construct driven by the

Ubiquitin promoter), were provided by Dr Sebastien Cunnac (Research Center for

Developmental Events, Montpellier, France)

Bacterial strains: E coli strain DH5α and A tumerfaciens strain EHA105 were

purchased from Thermo Fisher Scientific

Rice DNA samples: TBR225 rice varieties are supplied by ThaiBinh Seed Group Joint Stock Company

3.4 Equipments and chemicals

Chemicals: Restriction enzymes, T4 DNA ligase, Taq DNA polymerase, Pfu

DNA polymerase, 1.0 kb DNA standard scale, GeneJETTM Plasmid Miniprep plasmid DNA purification kit, GeneJETTM Gel Extraction agarose DNA purification kit, were ordered from Thermo Fisher Scientific and Gateway kit purchased from Invitrogen The basic chemicals used for molecular biology were ordered from Sigma Company and Merk Company (Germany)

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Equipments: Perkin Elmer 9700 PCR machine, Biorad DNA electrophoresis

system, Hettich Universal 30RF centrifuge, Heraus Biofuge 28RS, and Firereader fluorescence imager UVITEC…

The oligonucleotides (Table 3.1) were designed based on the sequences

published on GenBank and ordered from Invitrogen (USA)

Table 3.1 Sequence of oligonucleotides used in the research

Primers Primer sequence (5' - 3') Gene /vector

3.5.1 General molecular biology techniques

3.5.1.1 Isolation of plasmid DNA from bacterial cells

DNA plasmid was purified from E coli cells using the GeneJET Plasmid

Miniprep kit (Thermo Fisher Scientific) A single colony was cultured overnight in 10.0 ml of liquid LB broth added a suitable antibiotic at 37°C with shaking at 220