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Investigating the molecular mechanisms underlying drought
resistance in Potato using genome and transcriptome editing
GUMMI SAINATH
Investigating the molecular mechanisms underlying
drought resistance in Potato using genome and
transcriptome editing
Gummi Sainath
Reg. Nr: 940111289130
Course code: 80439
July 2019
MSc Thesis Plant Breeding
Breeding for growth and development
Potato Group
Wageningen University and Research
Supervisor:
dr. CWB (Christian) Bachem
Examiners:
dr. CWB (Christian) Bachem
dr. Sara Bergonzi
Acknowledgements
I would like to express my gratitude to several people who graciously shared their precious time
to help me in this project. I would like to thank dr. CWB (Christian) Bachem for giving the
opportunity for pursuing the master thesis in Potato group. I thank Lorena Ramirez Gonzales
who was my daily supervisor for teaching me from pipetting to modular golden gate cloning.
With her outstanding weekly planning of experiments, I could complete this thesis with these
novel aims. I would like to thank dr. Sara Bergonzi for her guidance and extremely detailed and
precise comments and questions which have encouraged me to reflect critically in thesis
writing. My thanks also go to Li Shi, MSc who repeatedly advised me on stomata data analysis.
I would also thank MEP (Marian) Oortwijn for her practical questions while conducting
experiments and administrating the consumables for lab work. I would also thank Tammy
Soputro for her literary work in potato genome editing. Finally, my thanks go to corporate
companies Alphabet Inc. (for developing artificial intelligence search engine known as
“Google”).
Summary
Potato crop production exceeds 300 million tons annually and more than a billion people
consume potato tubers on a daily basis. Increasing potato tuber production under drought stress
is a challenge for potato farmers. In potato, achieving balanced canopy development and
eventual tuber yield is the best way for potato plant to avoid drought stress. Tuber yield is
regulated in a remarkably similar way to flowering via circadian rhythm under different light
durations. In previous studies, we have identified StCDF1 gene as a central regulator of
tuberization. An lncRNA StFLORE1 was detected in an opposite orientation covering the
second exon of the StCDF1 coding region.
From previous experiments, it is shown that knocking down StCDF1 increases drought
tolerance by upregulating the expression of StFLORE1. In order to understand the function of
StFLORE1, knockdown and overexpression transgenic plants were engineered. In this study,
the characterisation of StFLORE1 is revealed by investigating these transgenic lines. plants with
over expression of StFLORE1 were drought resistant as StCDF1 knockdown plants.
Subsequently, overexpression of StFLORE1 plants tuberized late by repressing the StCDF1
mRNA expression.
In addition to StCDF1, other four StCDFs genes have been revealed. Abiotic stress experiments
performed in tomato (Solanum lycopersicum) plants, showed high expression of SlCDF4.
Tomato SlCDF4 gene is StCDF2 orthologous gene in potato. In this study we aimed to knockout
the StCDF2 to study its role in abiotic responses. CRISPR/Cas9 (Clustered Regularly
Interspaced Short Palindromic Repeats/ CRISPR associated protein 9) genome-editing system,
was used to target mutation in the two exons of StCDF2. CRISPR/Cas9 construct with four
single guide RNAs (sgRNAs) was successfully developed and transformed in CE3027 diploid
potato.
Key words: Solanum tuberosum, Potato, Lnc-RNA, StCDFs, StFLORE1, drought tolerance,
transpiration, CRISPR/Cas9
Table of Contents
1. Introduction ........................................................................................................................... 1
2. Background ........................................................................................................................... 3
3. Materials and methods .......................................................................................................... 7
3.1. Functional role of StFLORE1 and its regulation in drought tolerance .................................. 7
3.2. CRISPR Cas9 site targeted mutagenesis of StCDF2 using Golden gate cloning system ....... 9
4. Results .................................................................................................................................. 10
4.1. Role of StFLORE1 transcript in drought regulation.............................................................. 10
4.2. CRISPR/Cas9 knockout of StCDF2 gene ................................................................................ 17
5. Discussions .......................................................................................................................... 19
6. Conclusions ......................................................................................................................... 21
Cited literature ......................................................................................................................... 22
Appendices ............................................................................................................................... 26
1
1. Introduction
Potato (Solanum tuberosum) originated from Andes mountains of South America. It belongs to
the Solanaceae family. Potato crop production exceeds 300 million tons annually and more than
a billion people consume potato on a daily basis. Quantity yielded from one hectare of potato
crop is two to four times higher compared to cereal crop (CIP - International Potato Center,
2018). Potato can reproduce asexually by vegetative propagation of tubers. Tubers are formed
by swelling of underground stems known as stolons. Despite of asexual reproduction potato
plants can be propagated by berries and seeds resulting from flowering. Tuber is the
reproductive and economic plant organ and widely studied (Friedman et al., 1997).
Potato farmers propagate seed tubers for crop production. Most of the commercially sown seed
tubers are tetraploid. Due to inbreeding depression and heterozygosity in potato, breeders face
severe challenges to attain homozygosity in the cultivars used for multiplication. This is the
reason why potato hybrid breeding is not yet commercialised (Hirsch et al., 2013). Increasing
crop production under water scarcity is a big challenge for potato farmers (Hodges, C. N et al.,
2010). In potato, achieving balanced canopy development and eventual tuber yield is the best
way for potato plant to avoid drought stress (Aliche E. B et al., 2017). The fact that potato is
highly heterozygous, breeding cycle is long, complicated due to polyploidy, self-
incompatibility and vegetative propagation, genome editing is the best way for increasing
drought tolerance. Genome editing is altering DNA on preset sites. At present, CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats) proteins can be used in targeted
genome editing that makes the breeding cycle shorter.
From functional studies on Arabidopsis flowering and potato tuberization, key genes have
identified for the development of drought tolerant potato varieties. In Arabidopsis under long
days, (12 to 16 hours of day light) CONSTANS (CO) regulation occurs after dawn in the light.
It is essential for the day-length-dependent promotion of flowering and is regulated by the
circadian clock component GIGANTEA (GI). GI interacts with FLAVIN KELCH F BOX1
(FKF1), this interaction stabilizes FKF1 and promotes the degradation of CYCLING DOF
FACTORs (CDFs). In short days (8 to 10 hours of day light), CO regulation degrades the CO
transcriptional repressors, so that CDFs, does not occur under short days because the mRNAs
of the light-dependent CDF degradation protein complex GI–FKF1 only rises around hours
after dawn. They are thus only expressed in darkness, and therefore the proteins do not form an
active complex. The CDF proteins are therefore present to repress CO transcription (Andrés, F
2012).
Under day light StCDFs family plays a crucial role in controlling the photoperiod pathway.
StCDFs repress flowering by downregulating StCO expression in leaves. StCO is degraded in
the dark by StCOP1 and in morning it is activated by photoreceptor StPHYB. However, StCDF1
gene is core regulator for vegetative and reproductive growth. StCDF1 plays crucial role in
tuber formation by a circadian cycle regulation. During long days, StCDF1 is degraded by
circadian complex protein, which leads to StCO proteins activate and as well as the activation
of StSP5G which represses StSP6A, generating early flowering. Whereas during short days,
StCDF1 is no longer degrade and is able to repress StCO protein which inactivates StSP5G,
2
leaving StSP6A active for increasing tubers formation (Abelenda, J et al., 2011, Kloosterman
et al., 2013).
In Arabidopsis, a Long non-coding RNA (LncRNA) named as FLORE1 repress, CDF5
expression and regulates flowering. FLORE1 is responsible for the tight regulation of CDF5
transcripts, acting through Natural Antisense Transcripts (NAT) (Henriques et al., 2017).
AtCDF5 is orthologous gene of StCDF1(table 1.1). Findings from our group revealed that a 1kb
lncRNA named as StFLORE1 was mapped on antisense end of StCDF1 gene. Drought stress
experiments revealed that StCDF1RNAi plants showed good resistance to drought but also
increasing expression in StFLORE1. In order, to study the functional role of StFLORE1, the
sgRNAs (single guide RNAs) were designed to inactivate the promoter region using CRISPR
Associated protein-9 (Cas9) (Ramirez GL et al unpublished).
In addition to CDF1, in potato other four CDFs genes have been revealed, StCDF1 clusters
StCDF2 and StCDF3, and in a separate group StCDF4 and StCDF5 (Kloosterman et al., 2013).
Abiotic stress experiments was performed in tomato plants, and SlCDFs genes expression were
quantified. Drought exposition in leaves and roots induced higher amount of SlCDF2 and
SlCDF4 in leaves whereas in roots, most of the SlCDFs genes showed an increased in gene
expression (Corrales et al., 2014). In the table below the names of the corresponding potato
orthologous is highlighted (table 1.1). It is interesting to note that SlCDF2 is StCDF1
orthologous and SlCDF4 is StCDF2 orthologous respectively in potato (table 1.1). This thesis
highlights the role of StFLORE1 transcript by phenotyping and genotyping of the mutants
generated using CRISPR/Cas9. It also covers the procedure used for CRISPR/Cas9
mutagenesis of StCDF2 gene.
S. tuberosum S.lycopersicum Arabidopsis
StCDF1 PGSC0003DMT400047370 SlCDF2 Solyc05g007880.2.1 CDF5 At1g69570
StCDF2 PGSC0003DMT400064695 SlCDF4 Solyc02g067230.2.1 CDF1 At5g62430
StCDF3 PGSC0003DMT400003359 SlCDF5 Solyc02g088070.3 CDF2 At5g39660
StCDF4 PGSC0003DMT400083080 SlCDF3 Solyc06g069760.2.1 CDF4 At1g26790
StCDF5 PGSC0003DMG400019528 SlCDF1 Solyc03g115940.2 CDF3 At3g47500
Table 1.1. Summarizing the homology of StCDFs genes among Arabidopsis, S.lycopersicum and
S.tuberosum.
3
2. Background
Plants have evolved a sophisticated transcriptional control to grow under severe abiotic stress
(Yamaguchi-Shinozaki 2006). One class of transcription factors that plays a crucial role is DOF
(DNA-BINDING ONE ZINC FINGER), whose members are known to be involved in
phytohormone responses, abiotic stress, metabolism regulation, photoperiodic regulation,
hormonal regulation, and other aspects of plant development as shown below in Figure 2.1
(Yanagisawa et al., 2002, Corrales et al., 2014, Wang et al., 2017). They are composed of 200–
400 amino acids and contain a very conserved DNA-binding domain located in the N-terminal
that includes a single C(2)-C (2) type zinc finger motif, which binds to T/AAAAG sequence in
the promoter of target genes (Yanagisawa, 2002; Rueda-Lopez et al., 2008). In contrast, the C-
terminal contains a transcriptional regulation domain with various functions (Pireyre M et al.,
2015).
Figure 2.1: A Dof protein domain structure contains the Dof domain (green), a nuclear localization
signal-NLS (purple) and the transcriptional activation domain (red). A serine-stretch is indicated in
yellow. The cysteine residues for putative coordination of zinc are shown in red letters in the Dof domain
amino acid sequence.
Redundant role of Arabidopsis CDF transcription factors in regulating flowering
Fornara F et al., 2009, studied the roles of CDFs genes in Arabidopsis flowering by generating
multiple mutants. Results from quintuple mutant (gi, cdf1, cdf2 ,cdf3, cdf5), indicate that CDF1,
CDF2, CDF3, CDF5 are largely epistatic to GI in the regulation of CO expression and the
control of flowering time. By combining different mutants and CDF1RNAi line they tested for
genetic redundancy between CDF1, CDF2, CDF3, and CDF5 because of predicted similarities
in their protein products and in their expression profiles. The cdf2 and cdf5 showed early
flowering when compared to wild type plants. In contrast, the single cdf3 mutant showed no
obvious alteration in flowering time. cdf2 cdf5 double mutant showed an additive effect when
compared to cdf2 and cdf5 single mutants. Crossing the CDF1RNAi transgene into the that
CDF2 and CDF5 double mutant produced a triple mutant flowering as early as the that CDF2,
and CDF5 double mutant. Finally, in a quadruple mutant that CDF1, CDF2, CDF3, and CDF5,
flowering was strongly accelerated. Based on above results they concluded that CDF1, CDF2,
CDF3, and CDF5 redundantly repress the floral transition.
4
Overexpression of Arabidopsis CDF3 gene led to the accumulation of metabolites and that
contributed to improved response to drought stress
Corrales et al., 2017 showed that the transient expression analysis indicated that CDF3 could
activate directly the expression of an abiotic stress regulated genes likely through the multiple
DOF binding sites localized in its promoter region, suggesting that CDFs might function as
upstream regulators of plant responses to abiotic stress. Metabolomics analyses of CDF3
overexpressing plants indicated the increased amounts of free amino acids such as proline and
sugars observed in 35S:CDF3 plants are factors that would aid the tolerance to low temperature
and drought stresses. Physiological studies have demonstrated that 35S::CDF3 plants exhibited
higher rates of photosynthesis and biomass under osmotic stress conditions than control plants.
CDF3 play a significant role in plant responses and tolerance to changing environmental
conditions. CDF3 is orthologous to StCDF5, and the role of the StCDF5 is not studied yet
(Table 1.1).
Role of three different alleles of the StCDF1 gene in drought response and tuberization
time
Besides the wild type (WT) allele StCDF1.1 that regulates tuberization under short day
conditions, there are two mutated alleles which allow the plants to tuberise under long day
conditions; StCDF1.2 with a 7bp insertion and StCDF1.3 with a 865bp insertion. In both cases
StCDF1.2 and StCDF1.3 (Figure 2.2) the mutated protein lacks the regulatory domain
increasing stability throughout the day (Kloosterman et al., 2013).
Figure 2.2: Structure of the three different alleles of the StCDF1 gene (Kloosterman et al., 2013). This
cartoon shows the three different alleles of StCDF1 with the three different domains (I, II and III). The
figure shows that StCDF1.1 has the third domain, while this is missing in StCDF1.2 and StCDF1.3 due
to an insertion resulting in a truncated and fusion protein (represented by the non-shaded part)
respectively.
Homozygous StCDF1.3 allele negatively affects abiotic stress tolerance and phenotype of
plants were weak. Heterozygote StCDF1.1/StCDF1.3 plants look same as homozygous
StCDF1.3 plants after drought stress. This is an indication that StCDF1.3 has a non-functional
StFLORE1 as there was 865bp transposon insertion (Figure 2.2). StCDF1:OE(Overexpression)
lines have less expression of StFLORE1 (CE3130 plant). StCDF1:RNAi lines have high
expression of StFLORE1. Similar to Arabidopsis FLORE, the expression levels reveal that
StFLORE1 might repress StCDF1. StCDF1 reduces drought tolerance as StCDF1:RNAi plants
are drought tolerant. The assumption from these results reveal that StFLORE1 increases drought
tolerance. Homozygous StCDF1.3:OE and Heterozygote StCDF1.1/StCDF1.3 shows
interference with regulation of stomatal opening. CRISPR/Cas9 transformation of StFLORE1
was done in homozygote diploid CE3027 (1.1/1.1) (Ramirez GL et al unpublished).
5
CRISPR/Cas9 for knocking out plant genes
Compared to first generation genome editing reagents that use DNA-protein interactions using
CRISPR proteins is inexpensive and effortless. In creating knock out or knock in of a single
plant gene mutant, CRISPR is more precise with high editing efficiency compared to
Transcription activator-like effector nucleases (TALENs) and Zinc finger Nucleases (ZFNs)
(Zhang K et al., 2017). So, this precise and low-cost editing made CRISPR technology as Holy
Grail in functional plant genome editing. First CRISPR nuclease that was discovered in bacteria
Streptococcus pyogenes (sp). Sp Cas9 was used in eukaryotic genome editing after testing its
editing efficiency in prokaryote genomes (Begemann M et al., 2017). In the process of testing
SpCas9 in eukaryotes, type 2 Cas9 system is effective in achieving successful mutants. First
and most widely used applications of CRISPR- Cas9 is knocking out genes. Nuclease active
Cas9 creates a Double Stranded Break (DSB) at the sgRNA targeted locus. These breaks are
repaired by Homologous recombination (HR) which are used for introduction of new mutations.
When DSB is repaired by Non-homologous End Joining (NHEJ) process, indels are introduced
for production of frame shifts and stop codons, ultimately leading to gene knockout (Tycko, J
et al., 2016). For knockout experiments, indels are introduced within the functional elements
(e.g., cis-element of promoter, mature miRNA sequence). Then using Cas9 and a pair of gRNAs
for deleting a target non-coding fragment. After that indels are introduced they damage the
mRNA translation by deleting a fragment from the coding sequence for mutating the intron-
exon junction for generating a splicing variant (Ding, Y et al., 2016).
Research Aims
Genes involved in regulating drought stress and understanding of their potential role need
tremendous improvements (Anithakumari 2012).
From above reasoning role of DOF genes in abiotic stress responses is dynamic. Following
objectives were investigated in this master thesis project.
1. Characterization of StFLORE1 function in potato
2. What is StCDF2 role in potato? Is it also involved in tuberization? Is it involved in drought
stress response similar to its orthologous SlCDF4 ? Does StCDF2 repress flowering time similar
to its orthologue CDF1.
Hypothesis 1.StFLORE1 expresses in vasculature
2.StCDF1 mRNA is repressed by the StFLORE1 3.StFLORE1 plants are early in tuberization and possess drought tolerance
Outline
CRISPR Cas9 promoter knockout and overexpression of StFLORE1 transcripts in the CE3027
background (diploid genotype with late tuberising diploid genotype carrying homozygous
StCDF1.1 allele; Kloosterman et al., 2013) was used to investigate the involvement of
StFLORE1 in potato drought tolerance. In this thesis the size of promoter deletions were
genotyped. Based on the genotyping, morphological and physiological observations under
6
drought stress was investigated. For functional characterization of other StCDF genes, knockout
of the StCDF2 in CE3027 was carried out using CRISPR/Cas9 modular golden gate cloning
system. Procedure used for experiments are described in the materials and methods. Further the
outcome of the procedure is discussed in the results section. Relevance of these results deduced
from experiments was highlighted in discussion section by connecting it with ongoing research.
7
3. Materials and methods
3.1. Functional role of StFLORE1 and its regulation in drought tolerance
Detection of transcriptomic deletions of StFLORE1 knockout transcripts
Genomic DNA from plant leaves of 12-weeks-old plants were extracted. Genomic DNA of
knock out lines were subjected to PCR analysis using Dream Taq DNA polymerase (Thermo
Scientific) with primers Stflore1fw and Stflore1.rev (Appendix 1). PCR products were analysed
by Agarose gel electrophoresis for screening the allelic variations. PCR products with
homozygous mutations were purified using DNA clean & concentrator kit (Zymoclean). After
measuring the DNA concentration using Nanodrop, the amplified and purified fragments were
cloned into pGEMT Easy vector (Promega) and left overnight at 40C. The cloned mixtures were
used to transform chemically competent E. coli (DH5α). The transformed media was plated on
solid LB with Ampicillin and incubated at 370C for overnight growth. Transformed (white)
colonies were subjected to PCR analysis using Dream Taq DNA polymerase (Thermo
Scientific) with M13 Fw and M13 Rev primers. PCR products were analysed by agarose gel
electrophoresis to confirm the presence of >200bp fragment. The positive ones were grown in
10ml tubes of LB media and kept in the shaker for overnight at 370C. The plasmid DNA was
isolated using Qiagen QIAprep Spin Miniprep kit. Concentration of DNA was measured using
Nanodrop and sent for sequencing with M13 forward and M13 reverse primers.
Spatial localization of pStFLORE1: GUS
Transformed diploid CE3027 plants with pStFLORE1:GUS construct were grown and
multiplied in vitro on MS20 media (Murashige and Skoog). After 3 weeks of growth leaves,
stems and roots were suspended in GUS staining buffer (0.5mM Potassium ferrocyanide,
0.5mM potassium ferricyanide, 0.5M NaHPO4, 0.5 M EDTA, and 1mg/ml X-gluc) for >48
hours at 370C. Plant organs were washed in 80% Acetone for 1 hour and then suspended in 75%
Ethanol for 12 hours. GUS Stained leaf, root and stem samples were observed under light
microscope and pictures were taken. GUS stained stems were sliced using rotatory Microtome
and observed under light microscope at 40X zoom.
Transcript and gene expression analysis of StFLORE1 and StCDF1
To analyse the expression of StCDF1 and StFLORE1, leaves were harvested from 13 week old
plants grown in the greenhouse and stored in liquid nitrogen until RNA extraction. The leaf
material was grinded in liquid nitrogen and from total RNA was extracted using RNeasy mini
kit (Invitrogen, California, USA). Total RNA was treated with DNase I (Takra, Japan) and
cDNA synthesized either by superscript VI reverse-transcriptase (Invitrogen) using gene
specific primers, in the case of StFLORE1 or by iScript cDNA synthesis kit, in the case of
StCDF1. Synthesized cDNA was diluted and stored in -20°C. A real time PCR was carried out
using designed primer pairs StFLORE1.forward, StFLORE1.reverse, StCDF1.forward and
StCDF1.reverse (Appendix 1). Bio-Rad iCyceliQ machine was used for qrt-PCR. Using above
8
mentioned primer pairs, SYBR green dye and cDNA were mixed and diluted with water. In real
time PCR, NAC was used as a control. After normalization with Elf gene, StCDF1 gene and
StFLORE1 transcript were determined by Real-time PCR in Overexpression (OE) plants of
StFLORE1 CE3027, the CRISPR/Cas9 StFLORE1 and CE3027 use as a control. Data was
analysed using 2-ΔΔCt method and NAC gene was used as a reference (Livak and Schmittgen
2001). Data obtained was analysed in Microsoft Excel. Later data was visualised in PRISM
GraphPad.
Morphological effects of StFLORE1 knockout and overexpression
Plant height (cm), tuber number, stem diameter(mm2) and number of tillers were recorded at
mature stage after 15 weeks of transplantation. Chlorophyll content was recorded in two
different time points using MultispeQ v1.0 (www.photosynq.org). These two time points were
recorded before and after drought stress. Drought stress was initiated by restricting 50ml/water
for the transgenic lines for 20 days. After, initiating drought stress measurements of Chlorophyll
content were recorded for the second time point. In week 16 after transplantation dry weights
and fresh weights (mg g1) were measured. Pictures of transgenics phenotypes were
photographed using Samsung galaxy S9 plus. Data was recorded in Microsoft excel and
visualized in PRISM GraphPad.
Stomatal physiology in StFLORE1 knockout and overexpressor plants
Leaves of 4 weeks old plants were cut and submerged first into solution (MES/KOH buffer
favours force stomatal opening) for 2 hours. Treated leaves were cut along the midrib into two
halves. Half of one leaf let was submerged again in Abscisic acid (ABA) 10 uM, and the other
half was kept as a control. Sliced leaves were teared along the lower side of the leaf, a colourless
narrow border was visible on sliced edge of the lower epidermis of the leaves. This thin
membrane of the transparent layer is separated using forceps. Separated lower epidermis layer
was pictured under microscope using 40x magnification. Number of stomata per field area were
deduced from the pictures for calculating stomata density for leaf part treated with ABA. Length
and width of the stomatal aperture were also examined for calculating pore aperture ratio. To
indicate the sensitivity of stomata to ABA, pore aperture area between ABA treatment and
MOCK treatment were calculated. Pictures were further analysed in Image J software and
recorded in Microsoft Excel and visualized in PRISM GraphPad.
9
3.2. CRISPR Cas9 site targeted mutagenesis of StCDF2 using Golden gate cloning
system
Genomic DNA and plasmid extraction
Using CTAB protocol (Doyle & Doyle, 1987), genomic DNA was extracted from CE3027
leaves. Using QIAprep® Spin Miniprep Kit (Qiagen) plasmid was isolated from bacteria after
cloning.
Vector construction
Four sgRNAs were designed using openly available CRISPR-design web tool
(http://crispor.tefor.net/). To combine four different single guide RNAs (sgRNAs) in one vector
golden gate cloning system was used. Using BsaI-HF enzyme golden gate cloning was done
followed by a ligation reaction for amplification of the four gRNA individually in E. coli
(DH5α). Product from PCR after ligation reaction was analysed on gel for fragment size 164
bp. Then PCR product was purified using QIAquick PCR purification kit and measured
concentration using spectrophotometer (Nanodrop). In the next step four gRNAs were
recombined with pICH47732: NOSp: NPTII-OCST, pICH47742::35Sp:Cas9-NOST, the linker
pICH41780 and cloned in pAGM4723 in a single cut-ligation reaction with BbsI and T4-ligase
in E. coli (DH5α) (Thermo Fisher Scientific) (Weber et al., 2011). The PCR product was
analysed on gel for fragment size >1.5kb. Using level 2 primers (Appendix 1), the purified
product was sent for sanger sequencing. The secondary structures were predicted at 37oc.
(http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3). The vectors were
transformed in Agrobacterium tumafaciens (strain Agl1) using electroporation after proper
sequencing results. Colony PCR was performed using transformed A. tumafaciens using
primers Cas9_6.fwd and RB-F1.rev (Appendix 1). To confirm the presence of construct in E.
coli and A. tumafaciens the sequencing was done using level 2 primers.
Agrobacterium transformation in diploid potato
Diploid S. tuberosum CE3027 was used as background for transformation. Plantlets were
propagated in vitro on MS20. Prior to transformation plantlets were grown for four weeks.
Agrobacterium were propagated on LB liquid media and propagated at 280C for 48hrs.
Overnight culture was centrifuged and sterilized acetosyringone to obtain an OD of 0.6. One
hundred stems (without internodes) of four-week-old CE3027 plantlets were excised in sterile
condition. The 1.5ml PACM liquid (2g/L caseine hydrolysate, 1 mg/L 2.4-D, and 0.5 mg/L
kinetin in MS30 liquid media) was added to excised plantlets which were arranged on sterile
papers over R3B media (MS30 solid media with 2 mg/L NAA and 1 mg/L BAP). After 10
minutes of suspension in PACM liquid, the stems were dispersed on new R3B plate without
filter paper for Agrobacterium inoculation. After Agrobacterium inoculation plant lets were
placed in climate chamber for two days. Inoculated stems were transferred to SIM media (MS20
solid media, 1 mg/L Zeatin riboside, 200 mg/L cefotaxime, 200mg/L vancomycin and 100 mg/L
kanamycin).
10
4. Results
4.1. Role of StFLORE1 transcript in drought regulation
Spatial localization of pStFLORE1: GUS
To understand where the regulation takes place we fused the 2kb upstream region of both
StCDF1 gene and of StFLORE1 to a beta-glucuronidase (GUS) gene for histochemical
localisation. Fornara et al., 2009, mentioned that CDF1 expression is localised in the
vasculature tissues, roots and stems. From Henriques et al., 2017, research shows that FLORE
is localised in roots, stems and also vasculature. After performing GUS staining in the
transgenic pStFLORE1-GUS CE3027, signal was detected in vasculature, roots and stems, as
for FLORE (figure 4.1). Differently, pStCDF1: GUS Andigena (WT potato), showed that
StCDF1 is likely to be expressed in stems and leaves but not in roots. The procedure carried out
in transgenic pStFLORE1-GUS CE3027 is highlighted in Appendix 1. From this study, it
indicates that the StFLORE1 is co expresses with StCDF1 in leaves.
Figure 4.1: GUS activity in transgenic pStFLORE1:GUS. The activity is seen in a. roots, b. vascular
bundles, c. xylem vessels and d. phloem tubes.
a b
d c
11
Visualization of transcriptomic deletions of StFLORE1 Knock out transcripts
In order to know the function of StFLORE1, using CRISPR/Cas9 a functional knockout of this
1kb promoter region was performed using modular golden gate cloning. According to
Henriques et al 2017, CDF5 encodes a lncRNA named as FLORE. FLORE negatively regulates
AtCDF5 as natural antisense transcript. According to the previous results from RNA-seq data,
an antisense of StCDF1 is transcribed from the second exon of StCDF1 until the intron as show
in Appendix 1. In figure 4.2, the StFLORE1 transcript and four sgRNAs designed at four
different positions of transcript is highlighted. The knock out procedure and sgRNAs designed
for StFLORE1 knockout are highlighted in Appendix 1.
In this study, for screening transgenic candidates, DNA extraction was performed in 12 weeks
old CE30237 plants following by PCR to amplified 1.5kb of the promoter region and cloning
in pGEMT vector in order to sequence. In total 5 transgenic plants, 3 lines showed 700 bp
deletions and 1 line showed a 300 bp deletion in the promoter region of StFLORE1. We also
find 1 line with 86 bp deletion in the 3’UTR of the CDF1, this line was decided to exclude in
case StCDF1 is compromised. From sequencing results, we find that all of our transgenic plants
still have at least one allele with the promoter region intact. Based on this study, we could
understand that the deletions made by Cas9 in StFLORE1 mutants were divided into two groups
i.e. 300bp deletion and 700bp deletions.
Figure 4.2: Schematic diagram of 4 sgRNA that target 1 kb of StFLORE1 promoter. Blue boxes indicate
StCDF1 exons; grey boxes indicate promoters’ region and white boxes indicate UTR region. StFLORE1
promoter region sequenced from CRISPR/Cas9 plants cloned using pGEMT-vector. Wild type sequence
was alignment in the first raw as a control. Scissors images represent the position of the 4 sgRNAs
designed. Based on deletions mapped or number of base pairs deleted in 1kb StFLORE1 promoter the
knockout lines are divided into two section i.e. small deletions (300bp) and large deletions (700bp).
1.kb 700bp
SgRNA4 SgRNA3 SgRNA2 SgRNA1
500bp 300bp 100bp
StFLORE1-22
StFLORE1-35
StFLORE1-92
700bp
Deletions
700bp
Deletions
300bp
Deletions
12
Transcript and gene expression analysis of StFLORE1 and StCDF1
After the genotyping the CRISRP/Cas9 lines, the next question to investigate was to check the
StCDF1 mRNA expression in the StFLORE1 deletion lines. Henriques et al., 2017, showed
that FLORE and CDF5 transcripts cycle are antiphasic. They showed that this is due to high
expression of FLORE leads to low expression CDF5 and vice versa. Overexpression (OE)
plants of StFLORE1 CE3027 were also planted in the greenhouse with the StFLORE1
CRISPR/Cas9 lines and CE3027 wild type controls to investigate the antagonistic expression
and assess drought tolerance.
Figure 4.3a, shows that StFLORE1 transcript expression was high in StFLORE1:OE plants. In
300bp deletions of StFLORE1 CRISPR/Cas9 lines, the StFLORE1 expression is higher than
control and 700bp deletions StFLORE1. In figure 4.3.b, expression of StCDF1 mRNA in
StFLORE1 CRISPR/Cas9 lines is high compared to StCDF1 expression in StFLORE1:OE
plants and control. In figure 4.3, when the StFLORE1 transcript expression in control plants
was low, StCDF1 gene expression was high. From this study it clearly shows that StFLORE1
represses StCDF1. The results show that overexpression of StFLORE1 decreases the StCDF1
mRNA expression levels.
Figure 4.3 Relative expression of StCDF1(b) and StFLORE1(a) in two CRISPR/Cas9 pStFLORE1 lines
and two over-expression (OE) 35S: StFLORE1 lines by triplicate, using CE3027 as a control (star mark
(*) represents a statistically significant difference (p<0.05)).
13
Morphological effects of StFLORE1 under drought conditions
From the gene expression results of StFLORE1 and StCDF1, it is clear that the StFLORE1
would play a major role in regulating the drought response. According to Aliche E. B et al.,
2017, potato crop with balanced plant canopy and eventual tuber yield are essential traits for
plants to escape drought. In figure 4.4a, the vegetative growth (plant fresh weight) in
StFLORE1:OE lines was high compared to the StFLORE1 CRISPR/Cas9 lines and control.
From figure 4.4b the StFLORE1:OE plants were delaying in tuberization compared to control
and StFLORE1 knockout lines. Primarily, the OE of the StFLORE1 decreases the StCDF1
expression (figure 4.3) and this expression levels are responsible for delaying in tuberization
(Abelenda JA et al., 2013) (figure 4.4a&b). In Figure, 4.4c-e average number of tillers, plant
dry weight and stem diameter of OE lines is high compared to other plant lines. This indicates
that the StFLORE1:OE has balanced morphology under drought conditions. In figure 4.4g, the
StFLORE1:OE plants are tolerant and healthy compared to the control and deletion lines.
Ramírez, D. A et al., 2014 highlighted that the chlorophyll increment in potato plants is an
oxidative stress effect which reduces the plant yield. For confirming whether the drought
experiment has shown effect, the chlorophyll content was recorded, figure 4.4f clearly shows
that there is decrease in chlorophyll content in all plants except the 300bp deletion lines. In
figure, 4.4 c, g and h the plant height graph and pictures of plants and tuber yield is recorded
and captured for showing the variation in the CRISPR/Cas9 lines of StFLORE1. The plant
height and tubers of 300bp deletion lines tend to outperform compared to other lines. This is a
sign that DSB induced by multiple sgRNAs and Cas9 leads to large deletions and complex
rearrangements in the genome (Kosicki, M et al., 2018). In this study, phenotyping under
drought conditions revealed that the StFLORE1 positively regulates drought.
f g
14
300bp 700bp
f
d c
e
g
15
Figure 4.4. a) to h) In the graphs and pictures the over expression and knockout StFLORE1 lines were
under drought stress for 20 days, star mark (*) represents a statistically significant difference (p<0.05).
CE3027 was used as control under normal conditions. a. Tuber and plant biomass (kg), b. Average
number of tubers and number of weeks till tuberization, c. Plant height (cm), d. Average number of
tillers, e. stem diameter, f. Chlorophyll content, g&h. pictures of plants and tubers after 15 days of
drought treatment. wt. (CE3027) was used as control under normal conditions.
h
16
Stomatal physiology of StFLORE1
Phenotypic results under drought stress revealed that the StFLORE1 plays a key role in abiotic
response. As drought is a complex trait, in order to gather further indication stomatal anatomy
has been studied. Plants become sensitive to drought when there is increase in Abscisic acid
(ABA), because the hormone affects the stomatal opening which in turn reduces transpiration.
If the greater number of stomata are open, they respond quickly to drought stress (ABA
treatment), by conserving water leading to drought sensitive (Obidiegwu, J. E., 2015). In order
to check the stomata response to ABA, the pore aperture ratio has been calculated. In figure
4.5a, the pore aperture ratio of StFLORE1:OE plants tend to be lower compared to the control
and knock out plants (lower the ratio stomata were closed while responding to the ABA
treatment, which in turn decreases transpiration).
One sample t and Wilcoxon test of pore aperture ratio indicated that the p value is <0.0001, i.e.
data is highly significant, this method has been chosen because the populations cannot be
assumed to be normally distributed. StZEP gene is responsible for expression of zeaxanthin
epoxidase which play a major role in ABA biosynthesis and increase drought and salt stress
tolerance. Over expression of StZEP genes increases ABA levels in plants. ABA is responsible
for stomatal closure, this makes plants to transpire less and overcome drought in potato (P, G.,
Yang et al., 2019). The StZEP (figure 4.5b) gene expression and pore aperture ratio (figure
4.5a) in the StFLORE1:OE lines with low StCDF1 expression (figure 4.3b) reveal that
StFLORE1 regulates drought tolerance. Based on this study it is clear the StFLORE1 plays a
major role with its counter partner StCDF1 in stomatal dynamics. This is interesting to note
because the stomata size was way bigger in 300bp deletion lines compared to the 700bp deletion
lines. In Appendix 2, the pictures and the graphs associated with stomata size, stomata density
are attached.
Figure 4.5: a. Stomatal pore aperture ratio (One sample t and Wilcoxon test of pore aperture ratio indicate
that p value is <0.0001 i.e. data is highly significant); b. Relative expression of StZEP gene in two
CRISPR/Cas9 pStFLORE1 lines and two over-expression StFLORE1OE lines, using CE3027.
a b
17
4.2. CRISPR/Cas9 knockout of StCDF2 gene
Secondary structures of sgRNAs from sanger sequencing of in vitro
After studying the role of StCDF1 and StFLORE1 in regulating drought response, now it’s
necessary to check whether StCDF2 has role in regulating abiotic stress responses. To study
functional regulation of StCDF2, knockout experiment is performed in this study. Formation of
active complex sgRNAs plays a key role in cleavage efficiency of Cas9. Before applying the
sgRNAs in in vivo, pre-screening sequences of in vitro sgRNAs were developed. Liang et al
2016, highlighted that efficiency of sgRNAs can be checked by number of stem loops in the
secondary structure of each primer designed. In figure 4.6, the secondary structures of invitro
sgRNAs reveal that sgRNAs have >3 stem loops. Four sgRNAs complimentary to StCDF2, a
circadian clock TF with unknown function were tested. The sgRNAs were designed at DOF
binding domain targeting at 2 exons of the gene (figure 4.6). Two sgRNAs were designed at 5’
end, after the stop codon and two sgRNAs at 3’ end before the start codon (Appendix 3). These
positions were selected assuming that the whole gene could be deleted on a genome wide scale
or at least >80% knockout efficiency. Sequencing results of 4 sgRNAs from level 2 vector is
highlighted in Appendix 4. In this experiment, the four sgRNAs were cloned into level 2 E.coli
vector.
Figure. 4.6. sgRNA design for StCDF2 knockout. Predicted RNA structures with lowest free energy in
the in vitro experiments (Predicted RNA structures with lowest free energy in the in vitro experiments
(predicted for 22°C)).
18
CRISPR/Cas9 construct in Agrobacterium
The constructed CRISPR/Cas9 cassette in E.coli containing all four sgRNAs was transformed
in Agrobacterium. Transformed colonies were screened using PCR amplification and gel
electrophoresis were performed to check the fragment size. In figure, 4.6 eight colonies (C)
were loaded, out of C1 to C8, C7 and C8 were expected to be 1.5kb fragment size. Sequencing
results showed that C7 and C8 had Cas9 nuclease and kanamycin resistance.
Figure 4.6. PCR product of eight colonies of Agrobacterium transformed with CRISPR/Cas9 cassette
(1.5 kb Cas9 nuclease with other and four sgRNAs)
Agrobacterium mediated transformation in potato
Potato CE3027 plantlets were transformed using C7, Agrobacterium & the CRISPR/Cas9
construct. After transformation the plantlets were grown in R3B media for two days (Figure
4.7a). Later transferred to SIM media for regeneration (Figure 4.7b).
Figure 4.7 Plantlets in a. R3B b. Plantlets growing in SIM media after transformation.
a b
19
5. Discussions
StFLORE1 controls the stomata size and plant height by repressing the StCDF1
Kloosterman et al., 2013, highlighted that StCDF1 is master regulator of the tuberization. The
3’UTR end of StCDF1 locus showed that there is antisense transcription and this locus is named
as StFLORE1 (Ramirez et al., unpublished). To understand the functional regulation of
StFLORE1, overexpression and knockout lines were made. Deletions were mapped in
CRISPR/Cas9 StFLORE1 plants, the transcript size of the area targeted by the most external
sgRNAs was 1kb and two groups were divided based on the size of their deletions. Comparing
the 700bp and 300bp CRISPR/Cas9 StFLORE1 deletion plants possess variation
phenotypically, physiologically and molecularly.
In figure 4.4c, the plant height of 700bp CRISPR/Cas9 StFLORE1 deletion plants were 15cm
short compared to CRISPR/Cas9 StFLORE1 deletions. From figure 4.4h, pictures reveal that
tubers of 300bp deletions mapped have tremendous tuberization but 700bp deletions reveal
have few tubers and late tuberization. Pore aperture ratio and phenotype after drought after
response (Figure 4.1g, 4.5a) of 300bp StFLORE1 deletions reveal they are insensitive to drought
(figure 4.4g). In figure 4.3a&b the gene expression levels of StCDF1 and StFLORE1 were
static.
StCDF1 repress the StCO due to presence of WAAGY sites on StCO promoter (Abelenda et
al., 2016). StFLORE1 transcript possess also WAAGY sites as StCO (Appendix 1). We suspect
that in, 300bp deletions of StFLORE1 promoter CRISPR/Cas9 line (92), WAAGY sites that
were deleted might have a role in interacting with StCDF1 in order to repress StFLORE1, which
means it would be a line with StFLORE1 independent from StCDF1 regulation. Whereas, in
700bp deletions of StFLORE1 CRISPR/Cas9 lines most of the transcript region affecting
StFLORE1 transcription.
20
StFLORE1 increases drought tolerance and decreases tuber yield
Knocking out and over expression of StFLORE1, showed that it has a major role in controlling
the plant development. Previously, RNAi:CDF1 CE3027 plants showed late tuberization but
resistant to drought. In figure 4.4a&b, StFLORE1:OE CE3027 plants also showed similar
phenotype to RNAi:CDF1 CE3027 with higher values of plant fresh weight, better tuber yield
and drought resistant phenotype. Klepper, B et al., 1987 and Ohashi, Y et al., 2006 highlighted
that stem diameter, chlorophyll content and biomass are important traits to understand the
drought tolerance in cultivars. Further, observations from plants with StFLORE1:OE CE3027
like stem diameter, and chlorophyll content (figure 4.4e,f) reveal that they are better tolerant to
drought stress compared to CRISPR/Cas9 StFLORE1 knockouts and controls.
Phenotypically, drought response is shown, in order to deduce more indications from
physiology and molecular perspective. The StZEP (figure 4.5b) gene expression and pore
aperture ratio (figure 4.5a) in the StFLORE1:OE lines with low StCDF1 expression (figure
4.3b) reveal that StFLORE1 regulates drought tolerance. Gibberellin Acid (GA) regulates
tuberization process, but high levels of ABA are antagonistic for GA accumulation. OE:
StFLORE1 plants have increased levels of StZEP and possess drought tolerance with less tuber
yield (figure 3.4h&i) (Abelenda et al., 2013, Xu X et al., 1998, Roumeliotis, E et al., 2012).
StFLORE1 is localised not only in leaves but also phloem, xylem and roots (figure 4.1).
LncRNAs move from source to sink tissues i.e. leaves to root tips (Zhang, Z et al., 2019). This
indicates that StFLORE1 might systematically move through the phloem to roots. Phosphate
starvation usually occurs under drought stress due to high ABA levels. Phosphate starvation
signalling pathway is regulated by many transcription factors, miRNAs and transporters (Baek,
D et al., 2017). LncRNAs might move from source to sink under phosphate starvation
tuberization. Despite the fact that, different localization could mean is that StFLORE1 might
overlap (stem and leaves) but also separate (roots) functions. GUS staining is an indication,
however the reason for not finding the expression in stomata might be that the a 2-kb fragment
upstream of the 3’ transcriptional start site StCDF1 fused might have regulatory regions could
be even further than 2kb or even in introns or downstream .
21
CRISPR technology for investigating the role of StCDFs family
Previous results of our group highlights that expression levels of other StCDFs where
downregulated by StCDF1. In Appendix 2, StCDF1:OE plants reveal that StCDF1 expression
levels downregulates StCDF2 and StCDF3. In order, to understand the function of the genes in
detail, StCDF2 CRISPR/Cas9 knockout is partially performed in the thesis (highlighted in
methods and results sections).
6. Conclusions
1. StFLORE1, regulates tuberization and transpiration by affecting abiotic stress responses in
potato.
2. Functional analysis of StFLORE1 plants show good tolerance to drought.
3. StFLORE1 plants tuberized late by repressing the StCDF1 mRNA expression.
4. Stomatal opening is influenced by StFLORE1 and it enables to plant to escape drought.
Recommendations
1. As the phenotypes of StFLORE1 showed variation, in further experiments using
CRISPR/Cas13 (“SHERLOCK”) would be alternative to CRISPR/Cas9 for RNA editing.
2. CRISPR/Cms1 (“CRISPR 3.0”) with RNP delivery for Gene editing in potato has good
potential in yielding commercial lines without unintended DNA integration.
3. For capturing the images of spatial localization of pStFLORE1: GUS in stomata confocal
microscopy would be an alternative.
4. Using X-ray computer Tomography (XRT / CT): Non-invasive 3D imaging of internal
structures for studying the tuber and stolon development would be better for plants
growing in the pots.
22
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26
Appendices Appendix 1
Sequence Orientation
Stflore1 TGTCCTAAATGAAGGAAAAGCA forward
Stflore1 TTTTGCCCTGCAAGCTAAT reverse
sgRNA BsaI Rev TGTGGTCTCAAGCGTAATGCCAACTTTGTAC
U6pEcoRIF GGAGAAAGGCGGACAGGTAT
Cas9_6.fwd ATCTCCCGAAGATAATGAGCAGAAG
RB-F1.rev GGATAAACCTTTTCACGCCC
ELF_forward GGAGCACAGGAGAAGATGAAGGAG
ELF_reverse CGTTGGTGAATGCGGCAGTAGG
CRISPR.cas 9 TTTTGCCCTGCAAGCTAAT Reverse
35S: pStFLORE CACCGGAGAGTGTAGTAGGATTT Fw
35S: pStFLORE CTACACTCTTCAGATCCCATTTG Rev
pSTFLORE:GUS CACCCTCATAAGTGGAGTAAGCCTTACGA Fw
pSTFLORE:GUS TCACTAATTATGTTGCTCGAATCCT Rev
NAC ATATAGAGCTGGTGATGACT Fw
NAC TCCATGATAGCAGAGACTA Rev
27
CDF1 qpcr GCAGAAATGCAGGGTAAAGC Fw
CDF1 qpcr GACACAAGAACCCGCTATGC Rev
SgRNA2.CDF2 CCATGAGCTCCAGAAGAATC CGG rev
SgRNA1.CDF2 CCGGATTCTTCTGGAGCTCA TGG fw
SgRNA3.CDF2 GTTCCAGTTAGTTACGTAGC TGG fw
SgRNA4.CDF2 TACGTAACTAACTGGAACCC CGG Rev
PDS8340 GAACCCTGTGGTTGGCATGCACATAC For sequencing
level 1
plasmids
PDS8534 TTTGTGATGCTCGTCAGGGG for sequencing
level 2 (NPTII)
PDS8535 CCCGAGAATTATGCAGCATTTT for sequencing
level 2 (Cas9)
PDS8536 TCATCAGTCAATTACGGGGCT for sequencing
level 2 (Cas9)
AL717 GCTTGGCATCAGACAAACCGG For sequencing
level 2
M13F GTAAAACGACGGCCAG for sequencing
cloning
product
CDF2.Cloning GAAGAACTGCCATAACTTT Forward
CDF2.Cloning TCCTCGTCATCATCCAGGT Reverse
28
StFLORE1, CRISPR/Cas9 knockout procedure
In order to do this, we created a CRISPR-Cas9 cassette targeting 1 kb of StFLORE1 promoter
using four RNAs guides, including important DOF-motifs site. To detect mutagenesis, we
amplified by PCR fragments containing guide1 and guide2, also another fragment from guide1
to guide 3 and finally a fragment containing the four guides. From 100 regenerated plants we
obtained 4 transgenic plants from which we amplified 1kb upstream the transcription site of
StFLORE1 to cloned them into pGEMT-vector and 10 colonies from each transgenic plant were
sent to sequence. From our results, in all the plants tested, at least one colony have the
StFLORE1 promoter intact from mutation. From this transgenic, we check that StCDF1 gene
was not mutated by our guides, since we found one transgenic StCDF1 was affected we
excluded from our results.
RNAseq read-mapping of the StCDF1 locus
RNAseq read-mapping of the StCDF1 locus. Red arrows indicate the location of positive strand reads
from the StCDF1 sense transcript and blue arrows indicate antisense RNASeq transcript reads
Method used for GUS staining
For pFLORE:GUS construct, a 2-kb fragment upstream of the 3’ transcriptional start site
StCDF1 was reverse complement and cloned into pENTR TOPO vector to generate the ENTRY
Gateway® clones, which was transferred to pkGWFS7 following the manufacturer’s
instructions. The pFLORE:GUS construct were introduced into into diploid potato CE3027.
The subcellular localization of transgenics lines were used for GUS staining as described
previously (Blazquez, 1997).
WAGY sites in StFLORE promoter and AtFLORE promoter
WAGY sites in StFLORE promoter and AtFLORE promoter by using online New PLACE (a
database for cis-acting Regulatory DNA elements)
https://www.dna.affrc.go.jp/PLACE/?action=newplace. Line with 300bp deletion is positioned
between 1000-700 bp, and lines with 700bp deletions are positioned between 0-700 bp.
StCDF1
StFLORE1
29
Appendix 2
Gene Expression of StCDF1, StCDF2 and StCDF3
Expression of StCDF1, StCDF2 and StCDF3 in p35S:CDF1.2 and wild type plants (CE3027) during
short days conditions.
Stomata pictures
The 92(300bp) line stomata size is big compared to the 22(700bp) lines
30
Stomata density
Stomatal density is an important parameter for estimating plant performance under drought,
higher the stomatal density better transpiration, this helps plants to escape drought (Bertolino
LT et al., 2019). In figure 3.5a, StFLORE1OE resulted in lower stomatal size hence higher
stomatal density compared to StFLORE1OE knockout plants. In figure 3.5a the 300bp
deletion plants have similar values compared to the control
Average stomata size
Mean stomata size of the StFLORE1 deletion lines show that 22(700bp) and 92(300bp). This
shows that StFLORE1 and CDF1 play a major role in controlling the stomata dynamics.
0
50
100
150
200
250
300
350
aba mock aba mock aba mock aba mock aba mock aba mock aba mock
207 224 22 35 53 92 control
35s Cas9 control
31
Appendix 3
StCDF2 alignment 1 130
PGSC0003DM ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA
PGSC0003DM ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA
Consensus ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA
131 260
PGSC0003DM CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT
PGSC0003DM CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT
Consensus CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT
261 390
PGSC0003DM ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT
PGSC0003DM ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT
Consensus ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT
391 520
PGSC0003DM GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGGTACTT TCACACTTTG TTATAAGAGT ATAATTTGTT GATTTTATTG TTAGTAATTT GTATGCGACT GTTGTTGACT
PGSC0003DM GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGG----- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGG..... .......... .......... .......... .......... .......... .......... ..........
521 650
PGSC0003DM TCCTATTGGA CTCTGCTTCG CCTCATTCCG GAGCATTTCC CTAGGGTTTT GAATCAGAGA TAACCAATTG AGTTACTAAG ATTAATCCAG TCAACCAATT GAGCTAAGAA TAATCCCGTC AATCAATTAA
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
651 780
PGSC0003DM GTTACTAAGA TTTCCCTAGA TTAAATTAAT TCCTTTGAAG GAAAGACGGT TAAATTTATC CATGTTTTGA TTAGGTTAGG TAGTCACAAA TTTCTCTTAA TTGGGATTCC TTGTTTTCTT CGTGTTCTCA
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
781 910
PGSC0003DM ACATTTTGTG TTTGGTATGA GGGAGGATAT TTTCATGGAA AATGTGTTTT TTGAAAAACA AGTTGGTTTC TTACTTATTT TTTAGTGGTT TGGTAAGTAA GCAAAAATAT GTTATTTGTA GAGCATTTAT
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
911 1040
PGSC0003DM ATGTAATCAT GCAAAACAAT ATAGGAGTGG TGGGATGGTC AAGGGATAGG GGCGGGGTGC GTTGGTGGGC AGTGAGTGTA CGATAAACAT AGAAGTTCAC TTGTGGAACT TGTTTTCCCT ACTTACGTTA
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1041 1170
PGSC0003DM AGGAAGTCGT TTTCCTCGTT TTTAAGGAAA ATATCCATAA TTTTGTTCAA CCAAACATGG AAAGATTTTC CTCCATACCA AGAACACCAT TAGCTCCTTA TCTCCATGAA TGCTTTTTCA TTTTGAAATG
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1171 1300
PGSC0003DM GAAAGTTATT AATTTTACAA TGGATATAAC TAGGCTCTGT AATCTAATTT TTAAGTGTCA GTGCAAATTA ACATGTTATA GAAGGTTGCT TGTTGTAAAA ATATATTTTA ACTGTCAATT TATAGAAGTT
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1301 1430
PGSC0003DM CAACTATTTT ACAGTTGGAA GGAGATTGAT TAGAGTTCAA TTTTAATCCA GCAACGGAAC AATCATTGTT TAACAACTTT CACATCTCTA TAACTCAGAT TTGTTTTACT TTTCTATGTT GAGTTCTTGT
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1431 1560
PGSC0003DM AATGGGCAAT CTATTTGTGC AAAAAAAGAG TAGTTTAATT GTAAGACTTG CTAAATGAGA AACTTTGACT TTATACCTTT TATTTGTGTA ATGATACCCT TCGGGGTGGC CCAATGGTTT GGGCTTTGGG
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1561 1690
PGSC0003DM CTTGGGACTT CTATGTTGGA GGTCTCAAGT TCGAAACCCC TTGCCAGCGA AAGCAAGGGG TTTGCCTTCT GGGTCGAGCT CATTGCACCA GGCTTGTCTA GTGCGGGTTA CCTCTTCTAT GTAGTTTGCG
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1691 1820
PGSC0003DM AGCTATTGCA TAGGAGCGGG AGTTTTACCC TGTGAGTACC CAAAGGGTAG CGGTTGCGGA TTTCCCTTGT CATAAAAAAA TAATAATTTG TGTAATGATA TGAATACTCT AATTTCCACA TTCAGTTGGA
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1821 1950
PGSC0003DM TGCAGATACT TATTTTGTTA TGCACTCGTA TCCTTAATTT GACATGGTTA GTCCCTTCAT GATATATAAT CTACTATCAG TTTTGTTTTA CCTTTGGCCT CTCAATATTT CTCTCTACAA CTTTCACATG
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
1951 2080
PGSC0003DM CTTCTTACCG ACGGAAAAGC TTGGTTGTCT CAATAGTGAT AAAAGACTAG TGAAATCTCG TTGAACCTTT TCTTTTGAGA TCTCCCTAAG TTTCTAATAA TTTCATTAGT TTAAAAGAAA ATTTTACTGA
PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
2081 2210
PGSC0003DM CTCTATTTAC TACTCTGTGA AATGCATCAC GCGTACAGGA TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG
PGSC0003DM ---------- ---------- ---------- ---------A TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG
Consensus .......... .......... .......... .........A TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG
2211 2340
PGSC0003DM AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA
PGSC0003DM AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA
Consensus AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA
2341 2470
PGSC0003DM TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG
PGSC0003DM TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG
Consensus TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG
2471 2600
PGSC0003DM TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC
PGSC0003DM TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC
Consensus TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC
2601 2730
PGSC0003DM CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT
PGSC0003DM CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT
Consensus CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT
2731 2860
PGSC0003DM AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG
PGSC0003DM AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG
Consensus AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG
2861 2990
PGSC0003DM GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG
PGSC0003DM GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG
Consensus GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG
2991 3120
PGSC0003DM TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC
PGSC0003DM TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC
Consensus TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC
3121 3250
PGSC0003DM ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG
PGSC0003DM ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG
Consensus ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG
3251 3380
PGSC0003DM TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA
PGSC0003DM TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA
Consensus TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA
3381 3510
PGSC0003DM GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA
PGSC0003DM GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA
Consensus GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA
3511 3640
PGSC0003DM GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT
PGSC0003DM GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT
Consensus GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT
3641 3770
PGSC0003DM AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA
PGSC0003DM AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA
Consensus AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA
3771
PGSC0003DM AATTAAA
PGSC0003DM AATTAAA
Consensus AATTAAA
32
Appendix 4
Level 2 sequencing results
>190627-004_K23_1D5CPAA025_F12.ab1.guides 1297
GCGGTGAGATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCC
AAGAAGAAGAGGAAGGTGTGAGCTTGTCAAGCAGATCGTTCAAACATTTG
GCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTA
TCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAA
TGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATT
ATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATA
AATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGACGCTACTAGAAT
TCGAGCTCGGAGTGATCAAAAGTCCCACATCGATCAGGTGATATATAGCA
GCTTAGTTTATATAATGATAGAGTCGACATAGCGATTGCCATGAGCTCCA
GAAGAATCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG
TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCA
GCTTTCTTGTACAAAGTTGGCATTACGCTTTACGAATTCCCATGGGGAGT
GATCAAAAGTCCCACATCGATCAGGTGATATATAGCAGCTTAGTTTATAT
AATGATAGAGTCGACATAGCGATTGCCGGATTCTTCTGGAGCTCAGTTTT
AGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA
AAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACA
AAGTTGGCATTACGCTCAGAGAATTCGCATGCGGAGTGATCAAAAGTCCC
ACATCGATCAGGTGATATATAGCAGCTTAGTTTATATAATGATAGAGTCG
ACATAGCGATTGGTTCCAGTTAGTTACGTAGCGTTTTAGAGCTAGAAATA
GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGGGGCACCGA
GTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTAC
GCTTGGGGAATTCCTCGAAGGAGTGATCAAAAGTCCCAATCGATCAGGTG
AATTTAGCAGCTTAGTTTATAAAGGAAAAAGTCCAAAACCGATTGGCGAA
ACAACTGAACCCCTTTTAAACAAAAAAACAAAATTAAAAAAGGGTTTCCC
TTTCATTTAAAAAGGGGCCCAAGGGGGTTTTTTTTTAAACCCCTTTTTTT
TTTGAAAAATGGGTTATCCCAGGAGAAAGAGTCCTTTTTCCCCGGGA