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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