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CRISPR/Cas9 By Transient Expression

Mohamed E. M. Mosalam

Master’s Thesis

Department of Agriculture Science

University of Helsinki

May 2020

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Abstract

HELSINGIN YLIOPISTO ¾ HELSINGFORS UNIVERSITET ¾ UNIVERSITY OF HELSINKI

Tiedekunta/Osasto ¾ Fakultet/Sektion ¾ Faculty

Faculty of Agriculture and Forestry

Osasto ¾ Sektion ¾ Department

Department of Agricultural Sciences

Tekijä ¾ Författare ¾ Author

Mohamed Mosalam Työn nimi ¾ Arbetets titel ¾ Title

CRISPR/Cas9 By Transient Expression Oppiaine ¾Läroämne ¾ Subject

Erasmus Mundus Programe in Plant Breeding (rmPLANT)

Työn laji ¾ Arbetets art ¾ Level

M.Sc. Thesis

Aika ¾ Datum ¾ Month and year

May 2021

Sivumäärä ¾ Sidoantal ¾ Number of pages

40

Tiivistelmä ¾ Referat ¾ Abstract

The objectives of the study were to assess the efficiency of transient expression of

sgRNA/Cas9 construct in Petunia V26, where sgRNA targeted a cytosine deaminase gene (CodA)

that converts 5-fluorocytosine into the toxic compound 5-fluorouracil. Disrupting CodA by transient

expression of sgRNA/Cas9 introduced a conditional negative selection system that allowed plants

with mutated CodA to regenerate on media containing 5-fluorocytosine.

The single transcriptional unit vector pMOH2 was designed to carry two amplified sgRNAs

guiding Cas9 targeting at HinfI cutting sites. The expression vector was transformed into Petunia

V26 using Agrobacterium tumefaciens (pGV2260). Successful mutations were detected on 62.5

mg/L 5-fluorocytosine. Large numbers of in vitro shoots were regenerated from the transformed

leaves on a modified MS-media containing 1 mg/L zeatin.

The study revealed that transient expression of the sgRNA/Cas9 construct is efficient and can

be used to target other genes in Petunia V26. pMOH2 targeted its sites successfully, and proved that

CodA can be used as a conditional negative selection marker to detect cells with an edited genome. Avainsanat ¾ Nyckelord ¾ Keywords

CRISPR/Cas9, Transient Expression, CodA, STU, Agroinfiltration, Petunia V26, Tissue Culture. Säilytyspaikka ¾ Förvaringsställe ¾ Where deposited

Master's Programme in Agricultural Sciences, Department of Agricultural Sciences Muita tietoja ¾ Övriga uppgifter ¾ Further information

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Dedication

”I would like to dedicate this work to the memory of my grandfather and to my parents and my brother who always believe in me"

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Acknowledgment This thesis resulted from collective valuable efforts of brilliant trustworthy people and lovely

friends, who supported me through my research endeavors. I take this opportunity to express

my gratitude to Professor Teemu Teeri, I am extremely thankful to him for sharing sincere

guidance and valuable expertise and for his patience and support that made me overcome the

many obstacles that I have been facing through my research. I will always carry positive

memories with him.

I wish to express my truthful thanks and respects to Professor Essam El-Gendy. I was lucky to

joint his lab for seventeen months to carry out my graduation project. I used to discuss and

argue with him and he had a unique teaching style and enthusiasm for his topic that made a

strong impression on me. He is no longer with us, but he lives in the heart of his sincerely

student.

It has been a pleasure to be a member of gerbera group, I will miss all the moments that I have

spent in the lab together with my colleagues and friends. I would like to thank Feras Hadid,

Feng Wang, Mamunur Rashid, Minhazur Rahman, and Steven Taniwan, it has been a fantastic

opportunity to work alongside them. I would like to gift Petunia flowers to Anu Rokkanen,

Katrin Artola, and Marja Huovila.

Many thanks to my dear colleagues; AB Siddique, Ana Paula, Arletys Mogena, Bikila Gelana,

Eyerusalem Sija, Gauranvi, Hafiz Umar Farooq, Kaltra Xhelilaj, Mark Melchior, Paul

Adunola, Romain Dlm, and the talented chef Umama Hani.

I would like to give a special acknowledgment to my teachers and colleagues in Cairo University, National Gene Bank of Egypt, and Swedish University of Agriculture Sciences. Also, to the unique country of Finland: Kiitos Suomi ja Suomalaiset.

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Table of Contents

Abstract ...................................................................................................................................... 2 Dedication .................................................................................................................................. 3

Acknowledgment ....................................................................................................................... 4 Table of Contents ....................................................................................................................... 5

List of Abbreviations ................................................................................................................. 7 1. Introduction ............................................................................................................................ 8

2. Literature Review ................................................................................................................. 10 2.1 Early Events of Gene Editing Before CRISPR (B.C). ................................................... 10

2.2 A Brief History of CRISPR ........................................................................................... 11 2.3 CRISPR/Cas9 Mechanism of Action ............................................................................. 13

2.4 Petunia V26 .................................................................................................................... 14 2.5 Transient Expression of CRISPR/Cas9 by Agrobacterium tumefaciens ....................... 15

2.6 CodA – A Conditional Negative Selection Marker ....................................................... 16 3. Research Objectives ............................................................................................................. 17

4. Materials and Methods ......................................................................................................... 18 4.1 Plant material ................................................................................................................. 18

4.2 Optimizing plant growth medias .................................................................................... 18 4.3 Cas9 construction design ............................................................................................... 18

4.4 Transformation ............................................................................................................... 18 4.4.1 For Escherichia coli DH5a ..................................................................................... 18 4.4.2 For Agrobacterium tumefaciens GV2260 ............................................................... 19

4.5 Construct verification ..................................................................................................... 19 4.5.1 Boiling prep ............................................................................................................ 19 4.5.2 Miniprep .................................................................................................................. 20 4.5.3 Restriction enzyme digestion .................................................................................. 20 4.5.4 Sequencing .............................................................................................................. 20

4.6 Agroinfiltration of plant leaves ...................................................................................... 20

4.7 Surface sterilization ....................................................................................................... 21 4.8 DNA extraction .............................................................................................................. 21

4.9 DNA concentration and purity ....................................................................................... 21 4.10 HinfI assay ................................................................................................................... 21

4.11 PCR .............................................................................................................................. 22 4.12 Agarose gel electrophoresis (AGE) ............................................................................. 22

4.13 Gel documentation ....................................................................................................... 22 4.14 Mutation detection ....................................................................................................... 22

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5. Results .................................................................................................................................. 23 5.1 Optimizing plant growth media ..................................................................................... 23

5.2 Construct Verification .................................................................................................... 24 5.3 HinfI Assay ................................................................................................................... 26

5.4 Mutation Detection ........................................................................................................ 28 5.5 CodA selection assay ..................................................................................................... 29

6. Discussion ............................................................................................................................ 31 7. Conclusion ........................................................................................................................... 33

8. References ............................................................................................................................ 34 9. Appendix .............................................................................................................................. 38

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List of Abbreviations BAP 6-Benzylaminopurine

Cas Crispr associated gene

CodA Cytosine deaminase gene

CRISPR Clustered regularly interspaced short palindromic repeats

crRNA CRISPR RNA

DNA Deoxyribonucleic acid

GMO Genetically modified organism

HDR Homology directed repair

MS Medium Murashige and Skoog medium

NHEJ Non-homologous end joining

NAA 1-Naphthaleneacetic acid

PAM Protospacer adjacent motif

PCR Polymerase chain reaction

RNA Ribonucleic acid

RO Reverse osmosis water

sgRNA Single guide RNA

STU Single transcript unit

TALEN Transcription activator-like effector nucleases

tracrRNA Trans-activating crispr RNA

ZEA Zeatin

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1. Introduction

The Clustered regularly interspaced short palindromic repeats known as CRISPR is the

adaptive immune system that defends the prokaryotic cells from bacteriophages. CRISPR is

composed of 25–50 bp repeated sequences that are separated by interspaced unique sequences

called spacers. Spacers guide the CRISPR associated genes (Cas), that encode DNA

endonuclease genes, into their complementary target sites in a foreign invading (Mojica et al.

2005).

CRISPR was applied as a genome editing technique by synthesis a single guide RNA (sgRNA)

composed of 20 nucleotides and complementary to the target sequence and which can be

expressed together with Cas9 endonuclease in the host cell, where the sgRNA would direct the

Cas9 to the target site to create a double strand break.

The expression of Cas9 and the sgRNA together under control of Polymerase II promoter in a

single transcriptional unit (STU) construct allowed efficient transient expression of both Cas9

and sgRNAs with the same efficiency with the aim to introduce transgene-free genome editing

organisms that display a range of genetic diversity and traits that will be suitable for breeding

programs.

The inbred laboratory line Petunia V26 with blue flower is a promising variety in research and

commercial applications. Transient expression of CRISPR/Cas9 by agrobacterium can produce

a range of allelic diversity that goes without the GMO concerns about transgenes and provides

plenty of targets in the flavonoid pathway that can be used for research purposes or to produce

different colors of petals for market demand.

However, the obvious changes in the phenotypes outcome of gene editing by transient

expression, the significant modifications in the DNA level cannot be easily detected by

molecular tools. Therefore, a negative selection system was employed to allow mutant

genotypes to germinate and inhibit the non-mutants.

CodA is a cytosine deaminase gene that converts 5-fluorocytosine into 5-fluorouracil that

inhibits the growth and morphogenesis of plants in tissue culture. The application of gene

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editing by transient expression on CodA introduced a conditional negative selection marker

that allowed plants with mutated CodA to grow on media containing 5-fluorocytosine, which

would indicate the possibility of editing other genes in that cell as well. Furthermore, effective

regeneration conditions must be optimized to induce the cells disrupted by Cas9 to regenerate.

In this research, agrobacterium CRISPR/Cas9 based gene editing was established to target

CodA gene in transgenic Petunia V26 #1000 by transient expression, and the transformed leaf

pieces were regenerated amidst optimized conditions to produce a large number of plants that

could be screened for mutations.

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2. Literature Review 2.1 Early Events of Gene Editing Before CRISPR (B.C).

Gene editing is a process that modifies the genome of a living organism either by insertion,

deletion, or substitution and has been used to enhance and introduce economic traits (El

Mounadi et al. 2020). Since the beginning of agriculture on earth and before the field of

genetics was established, humans used to manipulate the genetic make-up of plants and animals

through selection breeding in order to improve yield and increase food production (Doebley et

al. 2005, Zhang et al. 2013). The result of repetitive cycles of selection introduced new

genotypes with superior traits of interest. However, it is unpredictable, demands ages, and costs

the plant its genetic diversity (Doebley et al. 1990, Louwaars 2018).

Genetic engineering through programmable endonucleases was developed to make plant

breeding faster and more predictable. Endonucleases were used to induce double strand breaks

in the targeted sequences, which would be confronted and restored by DNA repair mechanisms:

the non-homologous end joining (NHEJ) that can ligates the double strand breaks without a

homologous template and the homology directed repair (HDR) which requires a homologous

template to repair the damage (Pfeiffer et al. 1994, Moore and Haber 1996). Both DNA repair

mechanisms are involved DNA in recombination. However, NHEJ causes various insertions,

deletions, and substitutions in the target sequence which may result in different mutation.

Moreover, HDR is less efficient than NHEJ and can be manipulated by providing a template

sequence contained the target mutation (Puchta et al. 1996, Puchta 2004, Symington and

Gautier. 2011, Kim et al. 2019, Liu et al. 2019, Sansbury et al. 2019).

Three programmable endonucleases techniques were developed for accurate genome editing

in plants: Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and

the clustered regularly interspaced short palindromic repeats associated with endonuclease

Cas9 (CRISPR/Cas9). They function through targeting the sequence of interest and guiding the

endonuclease to create a double strand break.

Zinc finger nucleases composed of two domains: zinc finger DNA binding domain and a fused

nuclease domain, each domain attaches to opposite DNA strand, and both composed of two to

three β-layers and one α-helix. The β-layers containing two cysteine and two histidine Cys2-

His2 bind zinc finger and four other amino acids in the in the N-terminal region of the α-helix.

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The complex structure of zinc finger nucleases requires a significant long18 bp sequence to be

recognized for gene editing (Kim et al. 1996, Segal et al. 1999, Razin et al. 2012).

TALENs composed of two DNA-binding domains known as transcription activator-like

effectors (TALE) fused to FokI endonuclease. TALE composed of a central repetitive domains

(CRDs) sequence is flanked with two handed structures. Each domain is composed of 34 amino

acids and binds to one nucleotide in the targeted sequence. The left-handed domain composed

of two helix structures contain repeat variable diresidues (RVDs) that forms a loop with the

CRDs, and the right-handed domain wrappes around the structure to form a superhelix (Boch

et al. 2009, Moscou and Bogdanove. 2009, Christian et al. 2010, Deng et al. 2012, Mak et al.

2012).

CRISPR/Cas9 specificity is determined by artificial 20 nucleotides that guide the endonuclease

to its complementary target sequence. CRISPR lacks the complex structure of Zinc finger

nucleases and TALENs, and is much easier in construction, more efficient and can target

various and multiple sequences (Zhao et al. 2020).

2.2 A Brief History of CRISPR

CRISPR was accidentally discovered in E. coli in 1987 by Yoshizumi Ishino. Ishino and his

team were studying a gene responsible for conversion of alkaline phosphatase isozyme and

they identified unusual repeated sequences interspersed by non-repeated sequences of similar

length (Ishino et al. 1987). In 1993, similar series of repeated sequences separated by unique

sequences was found in Archaea Haloferax mediterranei (Valera et al. 1993), followed by other

analogous discoveries in different bacterial and archaeal species (Dudley et al. 2013, Hao Li et

al. 2014, Sola et al. 2015).

Remarkably, Ruud Jansen in 2002 showed that the interspersed sequences are mobile elements

and referred to this family of repetitive sequences as the clustered regularly interspaced short

palindromic repeats (CRISPR). Jansen identified four CRISPR‐associated (Cas) genes that

have a functional relationship with these repeats (Jansen et al. 2002). However, the function of

these repetitive sequences remained unknown until 2005 when Francisco Mojica discovered

homology between the spacers and transmissible genetic elements in bacteriophages.

Interestingly, these bacteriophages could not infect bacterial cells containing homologous

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spacers, which strongly suggested that CRISPR plays an immunity role against microbes

(Mojica et al. 2005).

Cas genes were identified as a DNA repair system by Makarova in 2002 who abandoned this

hypothesis in 2006. Makarova and her team suggested that Cas region composed of more than

20 genes that contained five core genes that produce DNA helicase and hydrolase, and others

produce exonuclease (Makarova et al. 2002).

In 2005, Vergnaud reported, after identifying 132 spacers, that only three spacers exist in the

common ancestors of Yersinia pseudotuberculosis, however, the most were specific to single

genotypes and not present in closely related genotypes, which suggested that the introduction

of new spacers is a frequent event, and the spacers are acquired from extrachromosomal origin

(Vergnaud et al. 2005). In the same year, Bolotin et al. (2005) suggested that the spacers are

traces of viral attacks, and they provide the cell with an immune system that produces antisense

RNAs complementary to the virus genomes, which together with CRISPR‐associated (Cas)

plays an unknown role in DNA degradation of the foreign genome (Bolotin et al. 2005). Similar

was suggested by Makarova et al. (2006) upon exploring the function of Cas gene products,

where 25 protein families were characterized, and CRISPR/Cas system was hypothesized as a

defense mechanism against invading bacteriophages, where the spacer of CRIPSR is used as

small interfering RNA (siRNA) that base paired with its homology region in the invading

genome leading Cas protein to degrade or shut down the foreign genome.

CRISPR defense mechanism was fully explored by Barrangou in (2007) who studied the

bacteriophage resistance in Streptococcus thermophilus. Barrangou and his team provided

evidence for Makarova’s hypothesis that both spacers and Cas genes work together as a defense

mechanism against invading bacteriophages, where he showed that spacers that are homology

to a sequence in the foreign invading DNA called protospacers, could prevent the virus

infection, however, a certain change in the spacer sequence resulted in cell susceptibility.

Furthermore, by adding a spacer to a susceptible bacterium, the cell gained resistance against

the virus that contain a homology protospacer to the inserted spacer, and by removing a spacer

from a resistance cell, the cell lost its resistance and became susceptible. On the other hand,

inactivating of Cas gene number 5, resulted in loss of bacteriophage resistance, which, together

with its structure that contained the sequence of nuclease, suggested that Cas5 is responsible

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for shutting down the foreign genome. However, inactivating Cas7 did not affect cell

resistance, and perhaps it is involved in the creation of new spacers (Barrangou et al. 2007).

In 2011, type II CRISPR system was discovered where the spacer composed of joint two RNA

molecules; the first molecule called CRISPR RNA (crRNA) that is complementary to the target

sequence, and the second molecule called trans-activating CRISPR RNA (tracrRNA) contained

a complementary region to the free end of crRNA and RNase III and Csn1 regions that are

required for the maturation of crRNA. The tracrRNA-crRNA complex was capable to guiding

the Cas DNA endonuclease into the target site (Deltcheva et al. 2011, Gottesman 2011).

A short 2-5 bp sequence motif was found adjacent to one end of the protospacers in the invading

genomes, named protospacer adjacent motifs (PAMs). These motifs were considered as part of

the repeated sequence of CRISPR (Horvath et al. 2008), until 2009 when Mojica and his team

discovered that PAMs sites are conserved and common in various CRISPR systems and

involved in protospacer recognition by CRISPR and the variation between PAM sequences

between species indicate that there is a motive preferable (Mojica et al. 2009). Moreover, PAM

was considered as the target site of Cas protein after the base pairing of the spacer to the

protospacer (Shiraz et al. 2013). In this regard, the absence of PAM sites in CRISPR locus,

prevents self-targeting of Cas9 to the spacer (Mali et al. 2014).

The spacers uptake mechanism is still not clearly defined. However, several studies addressed

the role of Cas1, Cas2, and Cas4 in spacer acquisition. The adoption process of spacers starting

by forming a stable complex of Cas1 and Cas4 that process the prespacers. Then Cas2 will take

place Cas4 creating Cas1-Case2-prespacer complex that can integrate into the bacterial genome

forming a molecular memory of traces infections which called clustered regularly interspaced

short palindromic repeats (Qiang Chen et al. 2021).

2.3 CRISPR/Cas9 Mechanism of Action

In 2012, the Nobel laureates Jennifer Doudna and Emmanuelle Charpentier designed a site-

specific genome editing technique that can introduce a double strand break into any targeted

sequence based on type II CRISPR system. The newly designed tool was achieved by

combining the 3′ of crRNA and the 5′ of tracrRNA into one single guide RNA (sgRNA) with

a stem-loop or hairpin-like loop structure. tracrRNA was trimmed to remove the excess

sequences of RNA, a short polyA tail of 5~12 nucleotides was added for molecule stability,

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and the gRNA was combined with Cas9 endonuclease, that contained homologous domains to

HNH and RuvC nucleases, which cleaves the the complementary DNA strand and the non-

complementary strand respectively, resulted in a blunt ends which made it simple in function

over the other Cas genes (Charpentier et al. 2012). See Figure 1 for the difference between 2-

part crRNA-tracrRNA gRNA and the sgRNA.

Figure 1. comparison between the structure of (A) structure of 2-part guide RNA in CRISPR system II. And (B) structure of single guide RNA used in CRISPR/Cas9 mutagenesis. Adopted from Turk et al. (2019). Agrobacterium tumefaciens was successfully used to transfer CRISPR/Cas9 system into plant

cells where Cas9 and the single guide RNAs used to be transformed separately, which resulted

in stable transgenic plants (Miao et al. 2013, Jiang et al. 2013, Feng et al. 2013, Belhaj et al.

2013). This elucidated the promising applications of CRISPR/Cas9 in research and breeding.

2.4 Petunia V26

The flowering plant Petunia V26 is a member of Petunia genus that is characterized by its

simple growth requirements and short lifecycle (Stehmann et al. 2009). Petunia V26 is a

laboratory inbred line that is considered as a model organism for molecular biology and

genetics studies (Vandenbussche et al. 2016). It has a well-known genetic linkage map and

characterized genes and mutations (Gerats and Vandenbussche 2005), can be propagated

sexually by crossing and self-pollination (Stuurman etal. 2004) and asexually by tissue culture,

protoplasts, and explant regeneration (van der Meer 1999. Stable transgenic lines can be

produced with leaf-disk transformation and Agrobacterium infiltration protocols (Horsch et al.

1985, Yang et al. 2000). V26 is a favorable variety that can be used in research to study flowers

and flowering. On the other hand, Petunia V26 has blue-violet flowers that are popular in all

markets and flower galleries and it is considered as a promising variety for gene editing

applications in flower colors (Vandenbussche et al. 2016). Since the genetic pathway of

flavonoids in petunia was identified (Krol et al. 1990, Chua et al. 1993, Mason et al. 2002), this

A B

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provides plenty of target sites which can be edited by CRISPR/Cas9 to produce Petunia V26

with different colors (Noda 2018).

In this regard, the suppression of the flavonoid 3′,5′-hydroxylase gene that produces the blue

anthocyanidin would result in red flowers due to the activation of the flavonoid 3′-hydroxylase

gene that produces red anthocyanidins (Esakaa et al. 2006, Xia et al. 2014). Furthermore,

suppression of both anthocyanidin producing genes would result in plant with white flowers

(Tanaka et al. 2004) that can be used as a platform for metabolic engineering studies.

For commercial applications, many GMO concerns would prevent the production of a stable

transgenic lines of petunia since the Cas9 gene and the sgRNA would integrate into the genome

causing unknown effects (Guiltinan et al. 2018). However, transient expression of both the

sgRNA and the Cas9 gene by agrobacterium can produce a transgene free Petunia that avoids

GMO concerns in many countries and develop new varieties that could be used for commercial

applications and research.

2.5 Transient Expression of CRISPR/Cas9 by Agrobacterium tumefaciens

In 2016, Xu Tang and his team created a single transcriptional unit (STU) vector for

CRISPR/Cas9 transformation. The new construct used Pol II promoter to express both Cas9

and sgRNAs (Tang et al. 2016), and self-cleaving hammerhead ribozyme (Haseloff and

Gerlach, 1988) that would cut the flanking regions adjacent to the sgRNAs and free them from

the mRNA transcript before translation. The Cas9 coding sequence was terminated by a

synthetic polyA tail to increase the molecule stability (Tang et al. 2016). See figure 2 for

schematic illustration of the single transcriptional unit (STU) for CRISPR/Cas9 system.

Figure 2. A single transcription system under control of Pol II promoter. Upon translation, RZ will free the sgRNA that guides the Cas9 to the target site. Adopted from Tang et al. (2016).

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The single transcript CRISPR/Cas9 construct allows efficient genome editing and improves

the spatiotemporal control of the construct’s component (Tang et al. 2016). Moreover, the STU

Cas9 system allows efficient transient expression of both Cas9 and sgRNAs, which would

introduce a transgene-free plant that could avoid the GMO concerns and develop new varieties

that could be used for commercial applications (Zhang et al. 2016, Guiltinan et al. 2018).

The optimizing conditions for transient expression of CRISPR/Cas9 in petunia by

agroinfiltration have not been characterized. The process should produce transgene-free cells

with homozygous mutants (Tang et al. 2016). The regeneration of the transgenic plants with

no detectable transgene does not allow effective selection for the successful mutant genotypes

by molecular methods. Thus, a selectable marker that enables the detection of edited plants

would be very useful (Näsholm et al. 2004, Zhang et al. 2016).

2.6 CodA – A Conditional Negative Selection Marker

CodA is a bacterial cytosine deaminase gene which converts the 5-fluorocytosine (5-FC) into

5-fluorouracil (5-FU) that inhibits the growth and morphogenesis of plants in tissue culture

(Tassi et al. 1986). Transforming of CodA into Petunia V26 can be used to evaluate the

efficiency of transient expression editing in Petunia V26 (Thomson et al. 2015). The successful

transient expression of STU construct containing sgRNA/Cas9 to target CodA, would produce

plants with mutated CodA that can be regenerated on media containing 5-fluorocytosine, which

would be used as a negative selection to remove plants with active CodA (Stougaard. 1993).

Thus, if Petunia V26 is active in editing CodA by transient expression, the possibility of editing

another target gene is high. Effective regeneration conditions must be optimized to regenerate

a decent number of plants from the infiltrated leaves to be screened for mutations.

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3. Research Objectives The goal of this research was to assess the efficiency of transient expression by Agrobacterium

tumefaciens of sgRNA/Cas9 (STU) construct in Petunia V26 by inducing a mutation in a

conditional negative selection marker that allows mutant genotypes to be regenerated on a

selective media.

The objectives of this research were to:

• Optimize the regeneration conditions for Petunia V26.

• Optimize the conditions for transient expression.

• Designing a selection system for cells disrupted by Cas9.

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4. Materials and Methods 4.1 Plant material

Petunia V26 which is a laboratory inbred line with violet flowers and has been self-pollinated

over subsequence generations to get homozygous line. V26 #1000 is its transgenic derivative

expressing CodA under the 35S promoter.

4.2 Optimizing plant growth medias

Three different media were prepared by adjusting a mixture containing 4.4g Murashige and

Skoog salt mixture with vitamins (Duchefa Biochemie), 20 g sucrose, 1 mg folic acid, and 10g

glucose in 1000 ml RO water, to pH 5.7 using KOH. 8 g agar was added and phytohormones

in three combinations: 1 mg/L 6-Benzylaminopurine (BAP), or 0.1 mg/L 1-Naphthaleneacetic

acid (NAA) and 1 mg/L Zeatin (ZEA), or 1 mg/L ZEA. All media were autoclaved at 120°C

for 20 min. The media were left to cool to 55°C before adding 500 mg/L of Claforan, then

poured into Petri dishes (30 ml/plate). A serial dilution of 5-flurocytosine (500 mg/L) was made

in 8 plates of the optimized medium in a final volume of 30 ml.

4.3 Cas9 construction design

Two sgRNAs were designed by amplified a double stranded fragment using Primers GER1771

and GER1772 (Appendix, Table A5). The generated fragment was cloned into the STU Cas9

expression vector, which would be named pMOH2, using Golden Gate cloning in a thermo

ligation cycler (Appendix, Table A1) using a total volume of 20 μl of the reaction components

(appendix, table 2). Two pairs of Primers were design to detect mutations, GER1773,

GER1774, GER1775, and GER1776 (Appendix, Table A5).

4.4 Transformation

Transformation procedure of plasmid pMOH2 into both Escherichia coli DH5a and then, after

the verification of the construct, into Agrobacterium tumefaciens GV2260 was performed using

heat shock protocol. The agrobacterium strain GV2260(pMOH2) is referred to as MAT2.

4.4.1 For Escherichia coli DH5a

The competent cells, that were stored in 1.5 ml Eppendorf tubes in -80°C, were allowed to thaw

in ice for 20 minutes, followed by addition of 500 ng of the plasmid DNA for 30 minutes. The

tube was incubated for 45 seconds at 42°C (heat shock) then placed back in ice. 1 ml of SOC

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was added and the tube was left at room temperature for 1 hour to allow the bacteria to recover.

The Eppendorf tube containing the transgenic cells were spined at 7K for 3 minutes, 750 µl of

the supernatant was removed and the rest of the sample was resuspended and plated to LB plate

with 50 mg/L kanamycin for selection then incubated at 37°C for overnight (Hall et al. 2007).

4.4.2 For Agrobacterium tumefaciens GV2260

The competent cells, stored in 1.5 ml Eppendorf tube in -80°C, were allowed to thaw in ice for

10 minutes, followed by addition of 500 ng of the plasmid DNA for 5 minutes. The tube was

frozen in liquid nitrogen, followed by incubation for 5 minutes at 37°C (heat shock) then placed

back in ice. 1 ml of SOC was added to the cell suspension and the tube was left in room

temperature for 1 hour to allow the bacteria to recover. The Eppendorf tube containing the

transgenic cells were spinned at 7K for 7 minutes, 750 µl of the supernatant was removed and

the rest of the sample was resuspended and plated to LB plate with rifampin, kanamycin, and

carbenicillin (100 mg/L each) for selection then incubated at 28°C for three nights (Höfgen et

al. 1988).

4.5 Construct verification

To confirm the cloning of two sgRNAs into the correct position in the STU Cas9 expression

vector, the plasmid DNA was isolated from the competent cells growing on the selection plate

using boiling prep and, after verifying the construct, from a newly pure culture using miniprep,

followed by sequencing.

4.5.1 Boiling prep

A fair amount of bacteria was collected with a toothpick and resuspended in an Eppendorf

containing 50 µl cold STET-buffer (8% sucrose, 5% triton X-100, 50 mM Tris-Cl, 50 mM

EDTA, pH 8), 4 µl of Lysozyme (10 mg/ml) was added and the mixture was boiled for 50

seconds before centrifuging for 10 minutes at full speed in microcentrifuge. The pellet was

removed using a toothpick, 50 µl of cold isopropanol was mixed with the supernatant, and the

tube was incubated at -20°C for 10 minutes before centrifuging at full speed in microcentrifuge

for 10 minutes at +4°C. All supernatant was removed and the pellet was washed with 500 µl

cold 70% ethanol and centrifuged at full speed in microcentrifuge for 1 minute at +4°C. Ethanol

was removed and pellet was dried in vacuum for 5 minutes before resuspending in 20 µl TE

(10 mM, Tris-Cl, and EDTA, pH 8) (Quigley et al. 1981).

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4.5.2 Miniprep

Miniprep protocol was done using the GeneJET Plasmid MiniPrep Kit, by inoculating single

colonies of a fresh pure culture of pMAH2 into 5 ml tubes of L-broth with addition of 50 mg/L

kanamycin followed by shaking at 28°C or 37°C (for agrobacterium or E. coli respectively) for

overnight. On the next day, 5 ml of the bacterial suspension was spinned in a falcon tube at 4K

for 10 minutes, all supernatant was removed, and the pellet was resuspended in 250 µl

Resuspension Solution, followed by the addition of 250 µl Lysis Solution and 350 µl

Neutralization Solution, before centrifuging for 5 minutes at full speed in microcentrifuge. The

supernatant was transferred to the Thermo Scientific GeneJET Spin Column and centrifuged

for 1 minute at full speed in microcentrifuge, to allow the plasmid DNA to bind to the column.

The column was washed twice with 500 µl of Wash Solution and centrifuged for 1 minute at

full speed in microcentrifuge. The column was transferred into a new Eppendorf tube and 50

µl of TE were added and the tube was centrifuged for 2 minutes to elute the Plasmid DNA.

4.5.3 Restriction enzyme digestion

100 ng of the isolated plasmid DNA were digested with 0.2 µl EcoRV in a total volume of 20

µl of fast digestion buffer (Thermo Scientific™), followed by incubation for 15 minutes, before

separation on 0.8% agarose gel.

4.5.4 Sequencing

pMOH2 was sent to Macrogen Europe for sequencing.

4.6 Agroinfiltration of plant leaves

Agroinfiltration protocol was done according to (Yang et al. 2000), by inoculating one colony

of a new pure culture of MAT2 into 5 ml of L-broth solution without addition of antibiotics

followed by incubation at 28°C for overnight. On the next day, 2 ml of the bacterial solution

was spinned at 7K for 7 minutes, all supernatant was removed, and the pellet was resuspended

in 1 ml of (200 µM acetosyringone, 10 mM MgCl2, 10 mM MES, pH 6.0), then the sample was

diluted to O.D.600 = 0.500 and allowed to rest at room temperature for three hours. Small

pinpoints were made on the plant leaves and where the diluted sample was injected into the

leaf using 1 ml syringe.

21

4.7 Surface sterilization

Healthy leaves of Petunia V26 or V26 #1000 were collected in a sterile beaker. The leaves

were washed with 70% EtOH for 1 minute followed by a rinse in sterile reverse osmosis (RO)

water. The leaves were soaked in 1:4 commercial bleach for 10 minutes before rinsed several

times in sterile RO water. The middle veins and the bleached edges were removed from all

leaves which then were cut and placed abaxial side up on the media that contained 500 mg/L

Claforan and a serial dilution of 5-flurocytosine (25 - 500 mg/L).

4.8 DNA extraction

DNA extraction from plant leaves was performed using (Dellaporta 1983) procedure. The

procedure was carried out according to the following: 100 mg of leaf tissue was collected in 2

ml Eppendorf tube containing two steel beads. The tube was frozed in liquid nitrogen followed

by milling the sample to powder using (machine name and protocol). The result was incubated

in dry ice for 30 minutes, to increase the temperature of the sample, then resuspended in 600

µl extraction buffer (100 mM Tris-Cl, 50 mM EDTA, 500 mM NaCl, 20 mM 2-ME, 67 µg/ml

RNase, pH 7). 40 µl of 20% SDS was added to the resuspended solution then the sample was

incubated for 10 minutes at 65°C and mixed occasionally. 200 µl of 5 M KAc was added to the

lysate before spinning for 15 minutes at room temperature. The supernatant was mixed with

0.7 vol i-PropOH and incubated in dry ice for 15 minutes, followed by spinning the sample for

10 minutes at +4°C, which would allow the pellet to stick to the inner surface of the tube. The

supernatant was poured away, and the pellet was washed with 500 µl of 70% EtOH before

spinning the tube again for 10 minutes at +4°C. Then, the supernatant was removed carefully,

and the pellet was resuspended in 50 µl TE and left at room temperature for overnight.

4.9 DNA concentration and purity

The extracted DNA was detected on a 0.8% agarose gel containing 0.1 mg/L ethidium bromide.

The DNA concentration was estimated by comparing a serial dilution to a standard 50, 100 and

200 ng genomic DNA. The DNA concentration was measured using Nanodrop.

4.10 HinfI assay

HinfI endonuclease (Thermo Scientific™) was used to cut the isolated DNA and PCR

fragments of CodA, where 8 µl of DNA (12.5 ng/µl) was digested with a serial of quantities of

HinfI enzyme ranged from 0.2 µl to 2.5 µl (to optimize the enzyme digestion), in a total volume

22

of 20 µl that contained 10 µl Milli-Q water and 2 µl 10X fast digestion buffer and incubated

for 15 minutes to 24 hours (to optimize the incubation period).

4.11 PCR

PCR procedures were performed in a thermocycler (PTC-100 MJ Research) using a total

volume of 50 μl of the reaction components, (Appendix, Table A3) where the reaction was

carried out to amplify single guides RNA for STU vector construction using primers; Primer

GER1771 and Primer GER1772, (Appendix, Table A5) and which would be subjected for

Golden Gate ligation reaction to be cloned into pPOG4 plasmid. And a total volume of 25 μl

of the reaction component (Appendix, Table A4) where the reactions were carried out to screen

individuals’ DNA samples that were treated with Hinf1 digestion to verify gene editing after

agroinfiltration, using primers GER1773, GER1774, GER1775, and GER1776 (Appendix,

Table A5), and which would be subjected to T7 endonuclease assay to detect mutation. The

thermocycler amplifications were started by initial incubation and enzyme activation at 95 for

3 minutes followed by numbers of cycles of denaturation at 94°C for 45 seconds, primer

annealing at 50-54°C for 45 seconds and extension at 72°C for 45 seconds. The final hold was

at 10°C (Appendix, Tables A6 and A7).

4.12 Agarose gel electrophoresis (AGE)

The PCR products were separated on 2% agarose gel containing 0.1 mg/L ethidium bromide.

λ-Pst1 DNA size marker was used to standardize the molecular size of amplified bands.

4.13 Gel documentation

The genomic DNA, the plasmid DNA, and the PCR products were visualized and photographed

using the Quantity One 1-D Analysis Software, Bio-Rad Gel Documentation system.

4.14 Mutation detection

Detection of mutations was performed using T7 Endonuclease I-based Mutation Detection with

the EnGen® Mutation Detection Kit (NEB #E3321). The amplified fragments of CodA gene

using GER1773, GER1774, GER1775, and GER1776 primers were denatured before

reannealed again in order to create heteroduplexes mismatch between the mutant and

nonmutant fragments that can be digested by the T7 endonuclease enzyme resulting in two

fragments with different length that could be detected using gel electrophoresis.

23

5. Results A general problem in gene editing experiments is that it is difficult to obtain large numbers of

in vitro shoots after regenerating the transformed tissue. In order to increase shoot formation

frequency, the regeneration conditions were optimized allowing a large number of plants to

regenerate and to be screened for mutations. The transient expression of a sgRNA/Cas9 (STU)

construct was tested in petunia, where sgRNA targeted a conditional negative selection gene

where cells with mutant genotypes were germinated on a selective media.

5.1 Optimizing plant growth media

Optimization of regeneration was examined using six orders of media (Table 1). Each order

composed of three media in succession where the plants were placed two weeks on each

medium. 96 plates were used to optimize the suitable growth media for petunia V26

regeneration. V26 showed a remarkable growth on media orders no #5 (Figure 3a), and

produced callus in the presence of 0.1 mg/L auxin NAA together with cytokinin which did not

sprouted fine shoots on both ZEA and BAP on media orders no #1, #2, #3 and #4. However,

order no #6 killed the petunia leaf disks (Figure 3b).

Table (1). Orders of Media that used to optimize Petunia V26 growth condition.

# 1st Media 2nd Media 3rd Media

1 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L BAP

2 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L ZEA

3 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L /L BAP 1 mg/L BAP

4 1 mg/L ZEA + 0.1 mg/L NAA 1 mg/L ZEA 1 mg/L ZEA

5 1 mg/L ZEA 1 mg/L ZEA 1 mg/L ZEA

6 1 mg/L BAP 1 mg/L BAP 1 mg/L BAP

a b

24

Figure 3. a. V26 growing on media order no #5 and b. V26 growing on media order no # 6.

5.2 Construct Verification

Two sgRNAs were designed to target two Hinf1 target sites followed by PAM sites (Figure 4),

by amplified a 155 bp double stranded fragment contained BsaI overhangs, sgRNAs and region

homology to pPOG4 vector (Figure 5). The cloning of two sgRNAs into the correct position in

the STU Cas9 expression vector was confirmed using restriction cleavage mapping and

screened on 0.8% agarose gel (Figure 6a-c), followed by sequencing (Figure 7).

EcoRI GER1773> Hinf1 ATGATTTCTGGAATTCGGTCTCAAATGGCGAATAACGCTTTACAAACAATTATTAACGCCCGGTTACCAGGCGAAGAGGGGCTGTGGCAGATTCATCTGC 10 20 30 40 50 60 70 80 90 100 M A N N A L Q T I I N A R L P G E E G L W Q I H L Q -> codA AGGACGGAAAAATCAGCGCCATTGATGCGCAATCCGGCGTGATGCCCATAACTGAAAACAGCCTGGATGCCGAACAAGGTTTAGTTATACCGCCGTTTGT 110 120 130 140 150 160 170 180 190 200 D G K I S A I D A Q S G V M P I T E N S L D A E Q G L V I P P F V -> codA <GER1776 GER1775> Hinf1 GGAGCCACATATTCACCTGGACACCACGCAAACCGCCGGACAACCGAACTGGAATCAGTCCGGCACGCTGTTTGAAGGCATTGAACGCTGGGCCGAGCGC 210 220 230 240 250 260 270 280 290 300 E P H I H L D T T Q T A G Q P N W N Q S G T L F E G I E R W A E R -> codA AAAGCGTTATTAACCCATGACGATGTGAAACAACGCGCATGGCAAACGCTGAAATGGCAGATTGCCAACGGCATTCAGCATGTGCGTACCCATGTCGATG 310 320 330 340 350 360 370 380 390 400 K A L L T H D D V K Q R A W Q T L K W Q I A N G I Q H V R T H V D V -> codA TTTCGGATGCAACGCTAACTGCGCTGAAAGCAATGCTGGAAGTGAAGCAGGAAGTCGCGCCGTGGATTGATCTGCAAATCGTCGCCTTCCCTCAGGAAGG 410 420 430 440 450 460 470 480 490 500 S D A T L T A L K A M L E V K Q E V A P W I D L Q I V A F P Q E G -> codA <GER1774 Hinf1 GATTTTGTCGTATCCCAACGGTGAAGCGTTGCTGGAAGAGGCGTTACGCTTAGGGGCAGATGTAGTGGGGGCGATTCCGCATTTTGAATTTACCCGTGAA 510 520 530 540 550 560 570 580 590 600 I L S Y P N G E A L L E E A L R L G A D V V G A I P H F E F T R E -> codA

Figure 4. Cas9 targeted Hinf1 sites illustrated in pink and green, and flanked by 2 pairs of detection primers.

b aa

25

CAGGTCTCACGGAGCTGTGGCAGATTCATCTGCGTTTTAGAGCTAGAAATAGCAAGTTAA

AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGTCACCG

GACAACCGAACTGGAATCAGTCGTTTGGAGACCGA

Figure 5. Double strand fragment contained two guide RNAs amplified by GER1771 and GER1772 and contained GGTCTC is the BsaI recognition sequence; “N20” is the sgRNA01 target sequence; “N20” is the reverse complement of the sgRNA02 target sequence; The red letters are complementary to pPOG4. Bsa1 generated sticky ends.

Figure 6. a restriction mapping of pMOH2 constructs isolated from E. coli using boiling prep. Lane1, control pPOG4; Lane 2 to 5, four different isolates of the new construct where lane 2 shows the correct band size. b restriction mapping of pMOH2 constructs isolated from E. coli using miniprep. Lane 1 to 4, four different isolates of the new construct where lane 1 shows the correct band size; Lane 5, control pPOG4. c restriction mapping of pMOH2 constructs isolated agrobacterium tumefaciens using Miniprep. Lane 1 and 2, different isolate of the new construct shows the correct band size; Lane 3, control pPOG4. CGTGGGCTGAACGGAAACCATCCGGTGACAAAGCACCGACTCGGTGCCACTTTTTCAAG

TTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGACTGATTCCAG

TTCGGTTGTCCGGTGACAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGAC

TAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGCAGATGAATCTGCCACAGCTCCG

GTGACAAAAGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAG

ATCCTCAAACCTTCCTCTTCTTCTTAGGATCAGCCCTTGAATCACCACCGAGCTGTGAGA

GATCGATCCTAGTCTCGTA

Figure 7. DNA sequencing to confirm the successful construction of pMOH2.

c

M 1 2 3 4 5 M 1 2 3 4 5

a

M 1 2 3 M

b c

26

5.3 HinfI Assay

pMOH2 was designed to target HinfI cutting sites. The successful mutation would prevent

HinfI from cutting its site and result in a positive band after amplifying the flanking region of

the target sits with PCR. Optimization of HinfI endonuclease was examined by digesting the

total DNA using a serial concentration ranged from 0.2 µl to 2.5 µl and serial incubation periods

ranged from 15 minutes to 24 hours. The digested samples were screened on 0.8% agarose

before amplified by primers GER1773 and GER1774 and screened on 2% agarose gel (Figures

8 and 9). The results showed that HinfI digested the total DNA, however, the PCR results

showed that the different concentrations and incubation periods of HinfI did not cut its target

sites on CodA.

To confirm the result, the total DNA was digested with with 2 µl HinfI and incubated for 24

hours before amplified by primers GER1773, GER1774, GER1775 and the amplified

fragments (Figure 10) were digested with 0.2 µl HinfI for 15 minutes (Figure 11). The PCR

results showed that HinfI did not cut its target sites on CodA but it could cut the same target

sites on the amplified fragments, by primers GER1773, GER1774, GER1775, and GER1776,

with 0.2 µl for 15 minutes resulted in significant cut bands.

Figure 8. optimizing the incubation period for 2.5 µl HinfI. Lanes 1 to 7, amplified fragments by GER1773 and GER1774; Lanes 8 to 13, the digested DNA. The incubation period for lanes 1 and 7 was zero; for lanes 2 and 8 was 15 minutes; for lanes 3 and 9 was 30 minutes; for lanes 4 and 10 was 60 minutes; and for lanes 5 and 11 was 120 minutes; and lanes 6 and 12 were control DNA not treated with HinfI.

M 1 2 3 4 5 6 7 8 9 10 11 12

27

Figure 9. Optimizing the concentration HinfI after incubating for 24 hours. Lanes 1 to 7, amplified fragments by GER1773 and GER1774; Lanes 9 to 14, the digested DNA; Lanes 8, Master Mix; Lanes 1 and 9, control without HinfI; Lanes 2 and 10, contained 0.2 µl; Lanes 3 and 11, contained 0.5 µl; Lanes 4 and 12, contained 1 µl; Lanes 5 and 13 contained 1.5 µl; lanes 6 and 14, contained 2 µl; lanes 7 and 15, contained 2.5 µl.

Figure 10. Amplified fragments by GER1773, GER1774, GER1775, and GER1776. Odd numbers represent the DNA digested with 2 µl HinfI and incubated for 24 hours, and even numbers are their controls. Lane 1 to 6, amplified fragments by GER1773 and GER1776; Lane 7 to 12, amplified fragments by GER1774 and GER1775; Lane 13 to 18, amplified fragments by GER1773 and GER1774; Lanes 5, 6, 11, 12, 17, and 18 represent control DNA that were not transformed with pMOH2; the rest of the lanes are DNA isolated from transformed plant with pMOH2.

M 1 2 3 4 5 6 7 8 M 9 10 11 12 13. 14 15

M M 1 2 3 4 5 6 M 7 8 9 10 11 12 M 13 14 15 16 17 18

28

Figure 11. Digesting PCR products amplified by GER1773, GER1774, GER1775, and GER1776 with HinfI. Odd numbers were digested with HinfI, even numbers are their controls. Lane 1 to 6, amplified fragments by GER1773 and GER1776; Lane 7 to 12, amplified fragments by GER1774 and GER1775; Lane 13 to 18, amplified fragments by GER1773 and GER1774.

5.4 Mutation Detection

The amplified fragments of CodA by primers GER1773, GER1774, GER1775, and GER1776

was subjected to T7 Endonuclease, and was separated on 2% Agarose gel (Figure 12). The

results showed that T7 Endonuclease assay was not sufficient to produce cut bands and detect

mutations.

Figure 12. T7 endonuclease assay. Odd numbers were treated with T7 endonuclease and even numbers are their controls. Lanes 1 and 2, amplified fragments by GER1773 and GER1776; Lanes 3 and 4, amplified fragments by GER1774 and GER1775; Lanes 5 and 6, amplified fragments by GER1773 and GER1774; Lane C, T7 control sample.

M M. 1 2 3 4 5 6 M 7 8 9 10 11 12 M 13 14 15 16 17 18

M 1 2 3 4 5 6 M C M M

29

5.5 CodA selection assay

Transformed plants were regenerated on the optimized medium that contained an antibiotic to

prevent bacterial growth and a serial dilution of 5-fluorocytosine (Table 2) which is a

conditional negative selection marker to allow only transgenic cells to grow. Petunia V26 used

as a positive control and its transgenic derivative expressing CodA Petunia V26 as a negative

control. The result showed that transformed Petunia V26 that contained CodA and was infected

with MAT2 could differentiate on medium contained 62.6 mg/L 5-fluorocytosine (Figure 13).

Table 2. 5-fluorocytosine amount that used to detect the minimum concentration for growth and morphogenesis inhibition.

# Dilution% Amount (mg)

Volume (ml)

Concentration (mg/L)

1 100 15.00 30 500

2 75.0 11.25 30 375

3 50.0 7.500 30 250

4 25.0 3.750 30 125

5 12.5 1.875 30 62.5

6 7.50 1.125 30 37.5

7 5.00 0.700 30 25.0

8 0.00 0.000 30 00.0

30

Figure 13. Selective media contained 62.6 mg/L 5-fluorocytosine that allowed plants with mutated CodA to regenerate, indicated in red. A, Peunia V26 used as a positive control. B, Petunia V26 #1000 as a negative control. C, Petunia V26 #1000 infected with MAT2.

A

B

C

31

6. Discussion Leaf disks of Petunia V26 were regenerated on modified MS-Media that contained 10g/L

glucose and 1mg/L folic acid and either 1 mg/L ZEA or 1 mg/L BAP or 1 mg/L ZEA + 0.1

mg/L NAA. The manipulation of plant growth regulators indicated that medium containing

ZEA significantly showed superior effect over media containing BAP or ZEA+NAA. The

results suggested that the modified MS-Media that contained 1 mg/L ZEA could guarantee the

production of large number of fine shoots, which would be suitable for different application

required regeneration of leaf disks.

By comparing the results of HinfI endonuclease assay with their controls (Figures 8, 9 and 10),

it is indicated that the various concentrations and incubation periods of HinfI endonuclease

could not show a significant effect on its target sites on CodA, however digesting the PCR

products of CodA with the minimum concentration and incubation period, used to digest total

DNA, resulted in significant cut bands (Figure 11), which intuitively suggested that HinfI target

sites (GANTC) on CodA were methylated. Similarly, was suggested by Sundaresan in 1991

upon studying the methylation sensitivity of HinfI endonuclease where he concluded that HinfI

was not capable to digest cytosine methylated DNA (Sundaresan et al. 1991). On the other

hand, Ashapkin in 2011 discovered that cytosine methylation in plant genome occurred at

CHH, CG, and CHG sequences, where H is A, T, or C (Ashapkin et al. 2011), which together

with Sundaresan’s conclusion clarified why HinfI lacked the ability to cut its target sites on

CodA (GATTC and GAATC) because they were cytosine methylated but was able to cleavage

the amplified fragments of CodA which were not methylated.

T7 Endonuclease was used to digest the mismatched nucleotide between the mutant and

nonmutant fragments. The results should indicate whether the mutation existed or not, however

T7 Endonuclease could not digest neither the mismatched nucleotide in its positive control

fragments nor the amplified fragments of CodA, which suggested that T7 Endonuclease assay

was not sufficient to observe the Cas9 mutations. Similar was proposed by a survey to study

the CRISP/Cas9 detection assays in 2018, where it was concluded that the accuracy of mutation

detection by T7 Endonuclease was 22%, due to its sensitivity to the length and types of the

mismatched nucleotides, flanking and secondary structure, and other relative features in the

targeted fragments (Pruett-Miller et al. 2018).

32

The presence of 5-fluorocytosine in plant media inhibited the regeneration and morphogenesis

of plants with active cytosine deaminase gene which converts the 5-fluorocytosine into 5-

fluorouracil and allowed plants that lack the inactive gene to differentiate and regenerate, which

was used as a negative selection to remove cells with active CodA and allow others with edited

or mutant CodA to regenerate. Transformed Petunia’s leaf disks with pMOH2 were placed on

media containing different amount of 5-fluorocytosine to detect the minimum inhibitory

concentration where 62.6 mg/L of 5-fluorocytosine showed a visible growth of transformed

Petunia with pMOH2 and inhibited non-transformed Petunia (Negative control). This result

indicated that plasmid MOH2 that contained sgRNAs to target CodA in Petunia V26 was

designed and targeted its sites successfully and proved that CodA can be used as a conditional

negative selection to detect mutations by CRISPR/Cas9 and other mutagens.

33

7. Conclusion

The efficiency of transient expression of sgRNA/Cas9 construct in Petunia V26 was

successfully assessed and proved that transgene-free varieties can be developed for commercial

applications.

sgRNA/Cas9 construct was designed to target a cytosine deaminase gene (CodA) that converts

5-fluorocytosine into the toxic compound 5-fluorouracil, to detect cells with an edited genome,

where cells with successful mutation could be detected on 62.5 mg/L 5-fluorocytosine. The

result revealed that CodA can be used as a conditional negative selection marker

The infiltrated Petunia leaves were regenerated amidst optimized conditions in order to produce

a large number of plants that could be screened for mutations. Large numbers of in vitro shoots

were regenerated on a modified MS-media containing 1 mg/L zeatin.

34

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9. Appendix

Table (A1), The Ligation cycles Program

Step Temp / period

10 cycles of;

Restriction Digestion

DNA Ligation

37 °C / 5 min

16 °C / 10 min

Restriction Digestion 37 °C / 5 min

Enzyme Inactivation 80 °C / 10 min

Final hold 15°C

Table (2A), Golden Gate ligation reaction

Component Amount

10x T4 ligation buffer 2 µl

pPOG4, 150 ng 2 µl

Purified PCR product 1 µl

Bsa1 restriction enzyme 1 µl

T4 ligase 1 µl

MQ water 13 µl

Total volume 20 µl

Table (A3), the PCR 1st reaction components.

Component Amount

pPOG4, 75 ng 1 µl

Primer GER1771, 10 pmol/µl 1 µl

Primer GER1772, 10 pmol/µl 1 µl

dNTP, 10 mM 1 µl

5x buffer 10 µl

Phusion polymerase 0.5 µl

MQ water 35.5 µl

Total volume 50 µl

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Table (A4), the PCR 2nd reactions components.

Component Amount

Genomic DNA, 12.5 ng/µl 8 µl

Forward primer, 10 pmol/µl 0.5 µl

Reverse primer, 10 pmol/µl 0.5 µl

dNTP, 10 mM 0.5 µl

DreamTaq enzyme 0.125 µl

10x DreawTaq buffer 2.5 µl

MQ water 13 µl

Total volume 25 µl

Table (A5). List of primers used in the study.

Primer Name Primer Sequence GC % Tm

GER1771 F-CAGGTCTCACGGAGCTGTGGCAGATTCATCTGCGTTTTAGA

GCTAGAAATAGCAAGTTAA 45 60

GER1772 R-TCGGTCTCCAAACGACTGATTCCAGTTCGGTTGTCCGGTGA

CAAAAGCACCGA 52.8 60

GER1773 F- ATTAACGCCCGGTTACCAGG 55 67

GER1774 R- AATCGCCCCCACTACATCTG 55 67

GER1775 F2- TCACCTGGACACCACGCAAA 55 70

GER1776 R2- TTTGCGTGGTGTCCAGGTGA 55 70

Tm Referees to Melting Temperature. GGTCTC is the BsaI recognition sequence; “N20” is the sgRNA01 target sequence; “N20” is the reverse complement of the sgRNA02 target sequence; The red letters are complementary to pPOG4.

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Table (A6), the PCR 1st reaction Program

Step Temp / period

Initial denaturation 96°C / 2 min

14 cycles of;

Denaturation

Annealing

Extension

94°C / 30 sec

55°C / 30 sec

72°C / 30 sec

Final extension 72°C / 2 min

Final hold 15°C

Table (A7), the PCR 2nd reaction Program

Step Temp / period

Initial denaturation 95°C / 3 min

35 cycles of;

Denaturation

Annealing

Extension

94°C / 1 min

55°C / 30 sec

72°C / 45 sec

Final hold 10°C